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2001 Hippocampal theta-related properties of the basal ganglia
Hallworth, Nicholas E.
Hallworth, N. E. (2001). Hippocampal theta-related properties of the basal ganglia (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/12950 http://hdl.handle.net/1880/40798 master thesis
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Hippacampal Theta-Related Properties of the Basal Ganglia
by
Nicholas E. Hallworth
A THESIS
SUBMlTTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
DEPARTJMENT OF PSYCHOLOGY
CALGARY, ALBERTA
JANUARY, 2001
O Nicholas E. Hallworth National Liimry Bibliothmue nationale 1*1 ofcam& du Canada Acquisitions and Acquisitions et Bibliographic Sewices services bibliographiques 395 Wellington Street 395. nm Wellington OttawaON KlAON4 O(Lawa0N KlAW Canada Canada
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The author retains ownershtp of the L'auteur conserve la propndte du copyright in this thesis. Neither the droit d'autew qui protkge cette these. thesis nor substantial extracts fiom it Ni la these ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent etre imprimis reproduced without the author's ou autrement reproduits sans son permission. autonsation. Abstract
Using the electrical activity recorded fiom these structures as a guide, the present study examined interactions between the hippocampal formation and the basal ganglia.
In urethane anaesthetized rats, extracellular recordings were made for cells located in the basal ganglia during hippocampal electroencephalographic desynchrony (LIA) and spontaneously occurring and/or sensory induced hippocampal electrencephalographic synchrony (theta). A large proportion of these neurons were considered to have discharge profiles that were related to the simultaneously recorded hippocampal field state.
The routes through which theta-related cells of the basal ganglia interact with the hippocampus were assessed. Further investigation revealed two distinct populations of theta-related cells in the basal ganglia: those that either contribute or monitor ascending sensory information which acts to synchronize the field activity recorded fiom the hippocampus, and those that receive descending information from the septohippocampal
system which potentially could influence motor output. Acknowledgements
First and foremost, I must acknowledge Dr. Brian Bland Simply put, he has made ". ..an electrophysiologist out of me. ..". Dr. Bland's enthusiasm for theta research is infectious, and the dedication he has for his work makes him an extraordinary scientist,
He has been a great mentor and friend Cheryl Bland has also been a tremendous inspiration. Her generosity, opemess, and warmth are incomparable, making my experience both inside and outside the laboratory a joy. Together, the Blands' loyal and dedicated, but also celebratory, attitude has profoundly influenced the manner in which I approach every day.
I had the fortune to interact wrth many significant individuals throughout my time in the Bland laboratory. From the beginning I received thorough training from Darren
Scarlett and Dr. Yasuhiro Hanada Later, additional help came from Drs. Kiyohisa
Natsume and Jan Konopacki. The techniques I gleaned fiom these scientists were invaluable.
Analytical help came from a number of sources. I am indebted to Dr. Charles
Scialfa for his patience and willingness to discuss exmental designs and methods. I received additional help in this area from Dr. Lome Sulsky and Matt Scott. I was very fortunate to have Dr. Jos Eggemout as an instructor. He was always ready to discuss my results and suggest analytical options. I gratefully acknowledge Dr. Yoshiki
Kaneoke for the provision of the burst and oscillation detection algorithms. Fortunately,
I was able to review and compare these techniques with Dr. Kaneoke, Peter Magill, and
Dr. Mark Bevan I am indebted to Dr. Cam Teskey for having read and revised my manumipt.
Furthermore, Dr. Teskey was a most valuable source of criticism, always forcing me to consider my work fiom different viewpoints. Drs, Bob Sainsbury, Marshall Wibo, and Richard Dyck provided similar services, challenging me to cogtemplate my findings within a wider neuroscientific context. I was always able to discuss my work with Audny
Dypvik, Marie Monfils, Craig Brown, Isaac Bogoch, Sam Lazareck, Ryan Cooper, Greg
Neehly, Danen Fogg, Chris Wright, Peter Rilstone, and Parneet Cheema. These conversations were always fruitfid and motivating.
Finally, I would like to acknowledge my parents, Michael and Annette Hallworth.
Your Love and support are immeasurable. Thank you This work is dedicated to my grandparents, Herbert and Beryl Hallworth.
Without you, none of this would be possible.
PH-SUM complex ...... 27 Bfstinstem ...... 28 Pharmacology ...... 29 Hippocampa1 field activity ...... 29 Hippocampal cell activity ...... 30 Medial septum ...... 32 PH-SUMcomplex ...... 34 Brainstem ...... 35 Function of hippocampal theta ...... 35 The basaI ganglia ...... 37 Structure and fiber connections of the basal ganglia ...... 38 Anatomy of the caudate-putamen ...... 39 Anatomy of the globus pallidus ...... 39 Anatomy of the substantia nigra ...... 40 Pars reticdata ...... 40 Pars compacta ...... 41 Circuitry of the basal ganglia ...... 41 Afferent connections ...... 41 CorticomiataI projections ...... 41 Thalamostn'atal projections ...... 42 Nigrostriatal projections ...... 42 Other ascending afferent projections ...... 43 Efferent connections ...... 43 Striatopllidd projections ...... 43 Striatonigral projections ...... 44 Connections of the subthdairnc nucleus ...... 44 Projections fiom the globus pallidus ...... 45 Projections fiom the enteropeduncular nucleus and the pars reticulata of the substantia nigra ...... 45 The direct and indirect pathways ...... 46 The basal ganglia aad motor control ...... 47 Unit electrophysology of the basal ganglia ...... 47 Interactions between the basal ganglia and the ascending hippocampal synchronizing systems ...... 51 Anatomy ...... 51 EIectrophysioIogy ...... 53 Objectives and hypotheses ...... 56
Methods-Experiment one ...... 59 Objectives ...... 59 Subjects ...... 59 Surgery ...... 60 Data acquisition ...... 63 Data analysis ...... 67
Results-Experiment one ...... n viii Histology ...... 77 Classification ...... 78 Distriiution ...... 127
Discussion-Experiment one ...... 135
Methods-Experiment two ...... 153 Objectives ...... 153 Subjects ...... 154 Surgery ...... 154 Data acquisition ...... 162 Data analysis ...... 164
Results-Experiment two ...... 164 SN stimulation ...... 164 GP stimulation ...... 171 CPu stimulation ...... 178
Discussion-Experiment two ...... 188
Methods-Experiment three ...... 195 Objectives ...... 195 Subjects ...... 196 Surgery ...... 197 Data acquisition ...... 198 Data analysis ...... 199
Results-Experiment three ...... 200 Histology ...... 200 Control experiments ...... 200 Effects of procaine suppression of MS on discharge properties of CPu neurons ...... 202 Effects of procaine suppression of MS on discharge properties of SNR neurons ...... 214 Effects of procaine suppression of MS on discharge properties of neurons outside of basal ganglia ...... 215
Discussion-Euperiment three ...... 222
General Discussion and Future Considerations ...... 235
Literature Cited ...... 247 List of Tables
Table 1. Number and discharge properties of substantia nigra neurons
in the urethane anaesthetized rat...... 128
Table 2. Number and discharge properties of globus pallidus neurons
in the urethane anaesthetized rat ...... 129
Table 3. Number and discharge properties of caudate/putamen neurons
in the urethane anaesthetized rat ...... 130
Table 4. Number and discharge properties of neurons outside basal ganglia
in the urethane anaesthetized rat ...... 13 1 List of Figures
Figure 1 . Hippocampal EEG activity and accompanying FFT analysis ...... 4
Figure 2 . Anatomy of the hippocampal formation ...... 8
Figure 3 . Classification scheme for theta-related cells ...... 19
Figure 4. Analyses used to characterize cell discharge pattern ...... 71
Figure 5 . Locations of recording sites in the CPu and surrounding areas ...... 79
Figure 6. Locations of recording sites in the GP. ventral thalamus. and
surrounding areas ...... 81
Figure 7. Locations of recording sites in the SN .ventral thalamus. and
surrounding areas ...... 83
Figure 8. Representative data and analyses for a theta ON periodic cell ...... 89
Figure 9 . Representative data and analyses For a theta ON non-periodic cell ...... 95
Figure 10 . Representative data and analyses for a theta OFF periodic cell ...... 100
Figure 1 I . Representative data and analyses for a theta OFF non-periodic cell ..... 105
Figure 12 . Representative data and anaIyses for a non-related periodic cell ...... 111
Figure 13 . Representative data and analyses for a non-related non-periodic cell .... I16
Figure 14 . Representative data and analyses for a sensory activated
non-periodic cell ...... 120
Figure 15 . Representative data and analyses for a sensory inactivated
periodic cell ...... I24
Figure 16. Degree of periodicity as a hction of theta-relatedness for
basal ganglia neurons ...... 133
Figure 17. Experiment two, MS inactivation placements ...... 156
xi Figure 18. Experiment two, basal ganglia inactivation placements ...... 160
Figure 19. Hippocampal field activity in response to SN stimulation,
before, during, and after MS inactivation . .. . ,...... 166
Figure 20. Hippocampal field activity in response to SN stimulation,
before and during GP inactivation ...... , ...... 169
Figure 2 1. Hippocampal field activity in response to GP stimulation,
before, during, and after MS inactivation...... 173
Figure 22. Hippocampal field activity in response to GP stimulation,
before and during SN inactivation ...... - . . . 176
Figure 23. Hippocampal field activity in response to CPu stimulation,
before, during, and after MS inactivation ...... 180
Figure 24. Hippocampal field activity in response to CPu stimulation,
before, during, and after SN inactivation ...... 183
Figure 25. Hippocampal field activity in response to CPu stimulation,
before, during, and after GP inactivation ...... 186
Figure 26. Representative data for a theta ON non-periodic CPu cell
before, during, and after chemical inactivation of the MS ...... 205
Figure 27. Representative data for a theta ON periodic CPu cell
before, during, and after chemical inactivation of the MS ...... 208
Figure 28. Representative data for a theta ON non-periodic CPu cell
before, during, and after chemical inactivation of the MS ...... 2 11 Figure 29. Representative data for a theta ON periodic SNR cell
before, during, and &chemic;il inadvation ofthe MS...... 216
Figure 30. Representative data for a tbeta ON non-periodic VL cell
before, during and after chemical inactivation of the MS ...... 220
Figure 3 1. Model of connections between theta-related basal ganglia cells
and the hippocampal synchronizing system ...... 238 List of Abbreviations d09/digital A/D anterior-posterior AP anteroveatral thalamic nucleus AV auto-correlation histogram AC brtrstiness index BI caudate-putamen CPu corn ammonis field 1 CAI cornu ammonis field 2 CA2 comu monis field 3 CA3 comu ammonis field 4 CA4 cross-correlation function CCF deep rnefencephdic nucleus DpMe dorsal-ventral DV dorsomedial-posterior hypothalamus DMPH electroencephalograph EEG enteropeduncular nucleus EP entorhid cortex EC fast Fourier transform FFT hdusstriati FStr globus pallidus GP hippocampal large irregular amplitude activity LIA hippocampal low voltage fast activity beta hippocampal theta rhythm theta interspike interval IS1 lateral L medial geniculate nucleus, dorsal MGD media1 geniculate nucleus, medial MGM medial genicdate nucleus, ventral MGV medial I emniscus ml medial mamrniilary nuclei MM medial raphe nucIeus MR medial septum MS membrane potential oscillation MPO methyl4phenyI- I ,2,3,6-tetrahydrop~dine MPTP Pearson correhtion coefficient lilo pedundopontine nucleus PPT posterior hypothaIamus PH reticdaris pontis oraIis RPO reticular thalamic nucleus Rt standard error about the mean SE stimulation isolation unit Sru substantia inn- SI substantia nigra SN substantia nigra pars compacta SNC substantia nigra pars reticulata SNR subthalamic nucleus STN supramammillary region SUM ventral posterolaterai thalamic nucIeus VPL ventral posteromedial thalamic nucleus VPM ventrolateral thalamic nucleus VL ventromedial thalamic nucleus VM Introduction
L Overview
Arguably the most important discovery ma& through neuroscience research thus far is that different regions of the brain are specialized for carrying out different functions. Nevertheless, as neuroscientists keep subdividing the brain, one question looms ever larger. how do these disparate functional zones communicate with each other?
Indeed, in the opening chapter of their highly read Introductory Neuroscience textbook,
Kandel et al. (2000) state that the brain may be thought of as containing two broad maps, one for sensory perceptions and another for motor commands. Yet "[tlhe two maps are interconnected in ways we do not hlly understandn
One of the most useful tools which can be employed to identi@ how large groups of neurons communicate with each other is the electroencephalograph (EEG). The EEG represents the summated electrical activity of a large number of cells (also known as field potentials) measured from a specific focus fiom the skull or in the brain (Westbrook,
2000). EEG recordings have been used to elucidate how the cortex and thalamus "talk" to each other during sleep and wake states (Steriade et d, 1993). EEG recordings have also revealed how different portions of the oEactory system communicate with each other so that odors are recognized (Freeman,1987). Local field potential recordings have been used to explain in part the "binding problem": how different regions of the visual system interact such that objects in the visual environment can be recognized as separate entities
(Singer and Gray, 1995). IL Hippocampal electrwncephalqijrapbic activity
Some of the most prominent EEG activity in the mammalian brain can be recorded fiom the hippocampus. This activtty is notable as the signal recorded often contains periods of robust synchrony; that is, high ampIitude, relatively low frequency, nearly sinusoidal oscillations. The desynchronous periods recorded from the hippocampus are characterized by high frequency, fluctuating amplitude, irregular activity (Klemm, 1976; Steriade et al., 1990; Stumpf, 1965; Leung et d., 1982; Bland,
1986).
At least three distinct classes of hippocampal EEG have been descn'bed @land,
1986; Kiernm, 1976; Leung et al., 1982; Leung, 1992). The first type, rhythmical slow activity, or hippocampal theta rhythm (theta), corresponds to hippocampal synchronization. Theta is in fact one of the largest amplitude rhythmicities produced by the normal mammalian brain (BIand and Whishaw, 1976). &synchronized patterns are characterized as either large irregular amplitude (LIA) or low voltage fast (beta) activity.
At a behavioral kevel, theta has been subdivided into two types. Type 1 theta is related to mobility and can be recorded from the himpalformation while the animal engages in so-called "voluntary" forms of movement such as nmning walking, swimming, rearing, jumping, postural shifk, head movements, etc. (Vandemoif, 1969;
Bland, 1986). Type 2 theta is related to immobility and can be seen in the hippocampal field activity of an animal which is immobile yet processing relevant sensory information such as tones, whistles, Light flashes, stroking of fur, and preser~ceof other animals
(Kramis et aI., 1975; Sainsbury et al-, 1987; Sainsbuxy and Montop, 1984; BIand, 1986).
LIA is recorded from the hippocampus when an animal engages in socalled "automaticn forms of motor behavior such as grooming.,licking, chewing, face washing, etc.
(Sainsbury, 1970; Vanderwolf, 1969; Bland, 1986).
The two types of theta can be fiuther distinguished at the pharmacological level.
Type 1 theta is thought to be produced by a serotonergic system (Vanderwolf and Baker,
1986). It cmot be abolished by large doses of atropine sulphate, but it is abolished by anaesthetics such as ethyl ether, urethane, and pentobarbital (Bland, 1986). Type 2 theta is thought to be produced by a choIinergic system (BIand et al., 1984; Kramis et aI.,
1975). I? is sensitive to cholinergic antagonists such as atropine sulphate and scopolamine, but resistant to anaesthetics such as ethyl ether, urethane, and pentobarbital
(Bland, 1986). Although the desynchronized high frequency beta activity found in the hippocampal EEG has its own limited behavioral and pharmacological correlates, the scope of this thesis is limited to the LIA and theta hippocampal EEG states.
An example of EEG activity recorded fiom the hippocampus which shows the spontaneous cycling between desynchrony (LIA)and synchrony (theta) can be seen in the upper portion of Figure 1.
LU. Structure and fiber connections of the hippocampus i Anatomy of the hippoearnpal formation
Based on phylogenetic and cytoarchitectonic data, the cortex of the mammalian telencephalon can be subdivided into the alIocortex and the neocortex. The allocortex can be fWersubdivided into the archicortex, or hippocampal formation, and the paleocortex
Anatomists were first attracted to the hippocampal formation because of its unusual appearance deep within the human brain: when dissected carefully it resembles a 4 Figure 1. Upper Portion. EEG activity recorded fiom the hippocampal formation ofa urethane mesthehzed rat showing the spontaneous cycling between desynchronized
(LIA) and synchronized (theta) pattern. This signal was filtered with a half amplitude
law fdter setting of 1 Hz and a half amplitude high filter setting of 35 Hz. Tbis excluded
high frequency signals from the recording.
Lower Portion Respective FIT analyses used to distinguish between LIA
and theta field activity. The (approximately) 4 second long segment of desynchronous
activity outhed as LiA (top left of signal depicted in the upper portion of this tigure)
produced a power spe&um with a peak at 1.3 Hz The 4 second long segment of
synchronous activity outlined as theta (bottom right of signal depicted in the upper
portion of this figure) produced a power spxtmm with a peak at 3.9 Hr
6 seahorse lying along the inferior horn of the lateral ventricle. By the end of the 19& century the Golgi technique had revealed its magnificent laminar organization, which is simpler than other regions of the cerebral cortex. Because of this, subsequent work has clarified the anatomical organization of the hippocampal formation to a greater extent than any other cortical area The majority of information presented here will come from five of the more recent reviews which address the major aspects of the morphology of the hippocampal formation (Schwerdtieger, 1984; Swanson et al., 1987; Amaral and Witter,
1989, 1995; Lopes de Silva et d., 199 1).
In mammalian non-primate brain, the hippocampal formation is an elongated structure with its longest axis running tiom the septa1 nuclei rostrally to the temporal cortex ventrocaudally. Along this axis, the hippocampal formation consists of two interlocking C-shaped cell layers: the granular cell layer of the dentate gyrus and the pyramidal cell layer of the cornu mmnonis (hippocampus proper).
The hippocampal formation can be divided into four relatively simple cortical regions. These include the dentate gyrus, the cmuammonis (which can be subdivided into four subfields, namely CAI, CA2, CA3, and CA4), the subicular complex (which can also be divided into three subdivisions: the subiculum, presubiculum, and parasubicuium), and the entorhinal cortex.
One can think of the hippaxmpaI formation as being comprised of a number of cytoarchitectonically stereotyped slices that are stacked up to form the long (or septo- temporal) axis of the structure (Andenen et al,, 1971). Within each slice, along the length of this axis, the major divisions of the hippocampat formation are linked by unique and largely connections. The dentate gyms receives its major input fiom 7 the entorhinal cortex via the so-called perforant pathway. In turn, the granule cells of the dentate gyrus project via their mossy fibers to the CA3 field of the comu ammonis.
Pyramidal cells of the CA3 field give rise to collateral axons that terminate within CA3 as association connections and aIso provide the major input to the CA1 field of the hippocampus, the so-called SchafEer collaterals. This intrinsic circuitry is described as the trisynaptic pathway (see Ieft hand side of Figure 2). From CAI, axons reach the pyramidal cell layer of the subiculum, from where the majority of hippocampal output is directed.
Within all fields of the hippocarnpus a large number of interneurons are present.
These interneurons give rise to widespread axonal arborizations. Thus. not only is each slice (or lamella) interconnected by the trisynaptic pathway. but each slice also exhibits a longitudinal (septo-temporaI) and transverse (perpendicular to the longitudinal &.is) organization (Andersen et al.. 1971). Ergo, interaction among cells goes in both axes of the hippocampus.
The interlocking C-shaped morphology of the hippocampus provides a layered cytoarchitecture within each lamella as shown on the right hand side of Figure 2. The
most dorsal layer is the alveus: it consists of the axons of pyramidal cells and incoming
fibres. Beneath this is the stratum oriens. This layer consists mainly of the basal
dendrites of the pyramidal celIs. Next, the stratum pyramidale is formed by the densely
packed cell bodies of the pyramidal cells. As mentioned above, the pyramidal cell layer
is divided into cytoarchitecto~cfields CAI, CA2, CA3, and CA4. The smallest
pyramids are found in CAI and the largest in CA4. Field CA4 ties within the hiIus of the Figure 2. Anatomy of the hippocampal formation
Le# Hand Side. A horizontal section taken perpendicular to the septotemporal axis (lamella) depicts aspects of the trisynaptic pathway.
Right Hand Side. Within each lamella, the hippocampus and dentate gyrus
contain distinct cytoarchitectonic Iayers.
I0 dentate gyrus (described in more detail below). Still moving ventrally, the stratum radiatum is characterized by few cell bodies but many fiber systems, the most important of which is formed by the Schaf6er coilaterals running fiorn the CA3 to CAI field of the hippocampus. Also in this layer are the large apical dendrites of the pyramidal cells, and the axons of dentate granule cells. The next layer, called the stratum lacunosum moleculare, contains axons fiom the perforant pathway which terminate peripherally along the apical dendrite branches of the pyramidal cells. The dentate gyms consists of three layers, which curve to form the upper and lower blade of the inner of the two interlocking C's. The top (and bottom) layer is a molecular layer which contains the terminals of the dorsally (top layer) and ventrally (bottom layer) projecting dendritic branches of the dentate granule cells. These ceils form the stratum granulare of the dentate. The hilus resides in between the upper and lower blades of the stratum granulare. The closely packed granule cells of the strahlm granulare project their avons into the hilus to form a thick bundle which innervates pyramidal cells in CA3 (the so- called mossy fibers).
In addition to the perforant path running from the entorhinal cortex to the dentate gyrus, there are two other major inputs to the hippocampal formation: 1) the medial septum projects to the hippocampus proper and the subiculurn, and 2) intrinsic cornmisurd fibers originating in the contralateral hippocampus terminate in the hippocampus proper and the hilus. 11 ii. Afferentiefferent circuitry extrinsic to the hippocampus proper and the dentate gYms a) Entorhhal cortex
Of all projections, the entorhinal cortex (EC) provides the most prominent input to the hippocampus in the fonn of the perforant pathway. Entorhinal efferents arise mainly fiom layen 2 and 3 of the medial and lateral EC. The perforant path projects both ipsi- and contralaterally, and terminates most densely in the molecular layer of the dentate gyms, but also in the cornu amonis and subiculum. From the dentate gyrus, neural activity is processed along the trisynaptic pathway of the hippocampus and is ultimately sent back dong a major efferent fiom the subiculum to the EC. This major efferent is a reciprocal connection. It terminates mainly in the medial EC and is strictly ipsilateraI in rats (Swanson and Cowan, 1977). b) Subiculum
While the EC is the portion of the hippocampal formation that provides the major inputs to the hippocampus, the subicular complex is generally thought to be the major output structure of this formation However, there is some disagreement as to what components of this complex should be considered to be on the output side of the
hippocampal formation, and which components should be considered to be on the input
side of the hippocampal formation. The question of whether the pre- and parasubiculum
should be considered links along the unidirectional trisynaptic pathway has never been
fully addressed Because these areas do not receive robust projections fiom CAI, and
because they receive a Wyheavy neocortical and thalamic input, the pre- and t2 parasubiculum have recently been judged to be hippocampal input structures, and as such differ fiom the subiculum (Amaral and Witter, 1995).
As outlined above, neuronaI activity travels along the trisynaptic pathway and projects in a roughly topographical manner fiom the CAI pyramidal cell layer to the subicular pyramidal cell layer. From here, projections are generated to a number of cortical and subcortical regions. A reciprocal connection is shared with the input side of the hippocampal formation; the EC. Other significant efferent connections include the medial prefrontal cortex, the septal complex, the mammillary nuclei, and the nucleus accumbens.
C) Septum
The sewregion is found between the anterior horns of the lateral ventricles and
courses to form a part of the anterior bundle of the preoptic region of the hypothalamus.
It can be parhtioned into four major divisions (Swanson and Cowan, 1979): 1) the lateral
septal nucleus, 2) the medial division consisting of the medial septal nucleus and the
nucleus of the diagonal band of Broca, 3) the posterior division consisting of the
septofimbrial and triangular nuclei, and 4) the ventral division consisting of the bed
nuclei of the stria terminalis and the small bed nudeus of the anterior cornmisure. Only
the medial septal nucleus and the ventral limb of the diagonal band of Broca are relevant
to this discussion. Hereafter they are considered a single complex and will be referred to
more simply as the medial septum (MS)-
The septum and hippampa1 formation share many reciprocal comectiom which
have been studied extensively (for a review see Swanson et d., 1987). The MS is
thought to be a critical relay point for ascending si@ to the hippocampus. In 1987, 13 Nyakas et al. reported two principal fiber systems that project hmthe MS to the hippocampus. These inputs leave the septum via the fhnbrial and fornix pathways. The first fiber system consists of thick, coarse fibers with large terminal boutons. This system innervates CA3, CAI, and the hillus. The second system has delicate, thin fibers with numerous en pawant varicosities. This system terminates most densely in the CAI sub- pyramidal zone. As will be discussed below, these two systems are thought to mediate different neurochemicals; one cholinergic, the other GABAergic.
Efferent projections fiom the hippocampal formation to the septum are also extensive. They arise from neurons along the entire longitudinal axis of the hippocampus proper and the subiculum (Lopes de Shet al., 1991). These fibers terminate mainly in the lateral septal nucleus in the rat. CA3 sends bilateral projections whereas CAI and the subiculum only send ipsilateral efferents to the septal region.
The MS also projects to both the EC and the subiculum (Jakab and Leranth,
1995). d) Diencephalic region
There are a number of reports which suggest that certain cell groups in the diencephalon send their afferent projections to the hippocampal formation (Wyss et al.,
1979; Riley and Moore, 1981). The hypothalamic region has distinct inputs to the hippocampal formation These arise fiom the dorsomedial hypothalamic nucleus, the lateral and posterior hypothalamic pH)areas, the ventral premamillary nucleus, and the supramammilIary region (SUM), as well as from parts of the lateral and medial mammillary (MM) nuclei. Hereafter, these diencqhalic nuclei are interchangeably referred to as the PH-SUMcornpiex 14 Direct afferents from the PH-SUMto the hippoampus terminate mainly on dentate granule cells @ent et al., 1983), but also in all fields of the hippocampus proper
(Ter Horst and Luiten, 1986,1987).
In 1992, Vertes injected anterograde tracers into the SUM and showed that this area projected heavily to the MS. In 1995, Vertes et al. did a similar tracer study for the
PH and found that it had similar projections to the MS, and also additional projections to a number of thalamic nuclei. e) Brainstem
Much of the anatomical data which detail the projections from the brainstem to the hypothalamus, the septum, and the hippocampal formation have been reviewed by
Vertes (198 1, 1982, I988), Vertes et al. (1993), and Fortin (1995). These authors concluded that a system which acts to synchronize hippocampal fieid activity originates in the pontine reticular formation, specifically in the nucleus redcularis pontis oralis
(RPO) and the peduncdopontine nucleus (PPT). Furthermore, a desynchronizing input arises from the medial raphe nucleus (MR).
Although direct brainstem afferents to the hippocampus hrnthese struchues have been demonstrated (Wyss et d., 1979; Riley and Moore, 198 l), the most significant brainstem influences on the hippocampus are thought to be conveyed mainly through the septum and hypothalamus. Indeed, using an anterograde tracer, Fortin (1995) showed that the RPO projects to the PH while the PPT projects to the SUM and septum (the RPO and PPT were also shown to be reciprocally ~0~eCted).Both nuclei also send projections to the intralaminar thdamic nuclei and the PPT sends projections to the medial thalamic nuclei. 15 iii. Ascending synchronizing system
This thesis considers the hippocatnpal formation inputs described above as a system whose components influence hippocampal EEG. A pathway of ascending input which serves to synchronize the hippocampal EEG originates in the pons, ascends and synapses with the PH-SUM,projects through to the MS, and finally innervates the hippocampus. The nomenclature adopted in the literature to refer to this pathway is the ascending brainstem hippocampal synchronizing system. From the hippocampus, synchronized activity is relayed to hippaampal efferents (such as the subiculum).
Details on how this system influences hippocampal field activity are presented below.
IV. Electrophysiology of hippocampal field activity
L Historical perspective
Although Jung and KornmulIer (1938) were the first to make reference to a rhythmical field potential recorded from the hippocampus of rabbits during stimdation of their peripheral nerves, it was not until I954 that a systematic description of the field activity was made by Green and Arduini. This paper has become the benchmark for the beginning of intense study of hippocampal theta rhythm because it was an extensive study that used thm animal species: rabbits, cats, and monkeys. They termed the rhythmic, sinusoidal-like field activity theta since its frequency was simiIar to the theta range (4 to 7 Hz) defined for newortical regions. Theta fkquencies in fieely moving animals can range from 3 to 12 Hz, thus the term theta is somewhat of a misnomer but is so well entrenched that it continues to be used.
AAer the publication of the Green and Arduini paper (1954), theta researchers pursued two separate though not necessarily independent lines of research One approach 16 was to conelate theta with various behavioral or psychological concepts. The other approach was to study the physioiogy of the theta rhythm per se. These studies were
concerned with the topography of theta within the hippocampal formation and the cellular
mechanisms and afferent systems involved in theta generation.
ii Theta profilea and mechanisms of theta generation
a) Depth profiles
The amplitude and phase of theta field activity recorded differs as one passes a
recording electrode through the laminar structure of the hippocampal formation. This
technique of recording the field activity while moving dorso-ventrally through a structure
is referred to as recording a voltage depth profile. Winson (1976a) was the first to record
a hippocampal depth profile using c&ed rats. As he lowered his recording electrode,
Winson found theta in the dorsal region of the stratum radiatum, but then a null zone
where no theta was recorded, and eventually a second source of theta ventral to the
hippocampal fissure. The theta recorded from this second area was 180" out of phase
with the theta found above the stratum datum, and was approximately twice the
amplitude. Other researchers have confirmed these mukts in urethane anaesthetized rats
and cats (Green and Rawlins, 1979; Bland et al., 1979). Using more precise techniques,
these depth profiles suggest that there are two zones of theta generation, one located in
the stratum oriens of CAl, and a second located in the stratum moleculare of the dentate.
However, it was fond that the depth prome in freely moving rats was different
fiom that recorded in anaesthe$&d ads(Winson, 1974,1976b; Bland and Wishaw,
1976). Again, two theta generators were found in the same regions, but the phase shift
between them was gradual (over 400 pm) rather than rapid, and there was no null zone 17 beneath the -dal cell layer. This was later verified by Feenstra and Holscheimer
(1979). A similar profile has been confirmed in rabbits and cats (Bland et ai., 1975;
Bland et al., 1979, respectively).
Although multiple attempts have been made to explain these contradictions
(Feenstra and Holscheimer, 1979; Buzsaki et al., 1983) the commonly accepted explanation was originally proposed by Leung (1984% 1984b). Briefly, he demonstrated in a freely moving animal that activation of type 2 theta by itself produced the depth profile with a rapid 180° phase shift When activation of the type 1 system was added during the occurrence of type 2 theta, a graduaI phase shift was seen. Thus, it is the relative contributions of the type 1 motor system and type 2 sensory system which determine the type of shiR Leung (1984b)also provided a mathematical model which explained the differences seen in the phase shift across different preparations. These mechanisms have been thoroughly examined using both lesion (Whishaw and Sutherland,
1982) and pharmacological (Buzsaki et al., 1986) techniques. b) Mechanisms underlying theta generation
In addition to receiving major extrinsic inputs from the ascending synchronizing system, a number of cells within the hippocampus possess intrinsic oscillatory properhes.
The term membrane potential osciUatioas (MPOs) is used to designate these oscillations that arise in hippocampat cells as a result of the intrinsic properties of membrane currents
Dung and Yim, 1991; Bland and Colom, 1993). There have been a number of reports of the occurrence of MPOs (for a review see Bland and Colom, 1993; Bland, 2000).
The issue of how extrinsic and in-c properties of the hippocampus and ascending synchronizing system interact such tbat theta rhythm occurs remains to be Illy 2 8 resolved. One mechanism was initially presented by Bland and Colom in 1993 and firrther refined by Bland in 2000. A temporal sequence of changes in so-called "theta- related" cellular acti-fity occurs, on average, 500 msec before the transition of the hippocampal EEG to a synchronized state. During this sequence, extrinsic synaptic
inputs both initiate and synchronize MPOs produced intrinsically by hippocampal neurons. It is this synchronization of MPOs that manifests itself as extracelIdar theta
iii Cellular correlates of hippocampal theta
As stated above, the generation of hippocampal theta is dependent on changes in
"theta-related" cell activity both in the hippocampus and in the ascending system nuclei.
How is a "theta-related" cell defined?
One way in which we can assess if and how different regions of the brain are
communicating with each other is by assessing whether neuronal activity across these
regions is correlated. Thus, a theta-related cell is one whose discharge rate and/or pattern
exhibits some sort of relationship with the accrued extmcel1uIar activity of the
hippocampus.
In 1987, Colom and Bland developed a cell classification scheme used to describe
in what fashion a neuron's discharge profile was related to the hippocampal field state.
Figure 3 illustrates this classification scheme. The top mce of Figure 3 provides an
analog of hippocampal field activity showing LIA (the desynchronized activity at the
begbring and end of the trace) and theta activity (the synchronized activity in the middle
of the trace). Using this system, theta-related cells can be classified as either theta ON or
theta OFF. Theta ON cells, as their name suggests, have a higher discharge me during
theta than during LIA. Theta OFF cells, as their name suggests, have a higher discharge 19 Figure 3. A diagram depicting the classification scheme used for theta-related cells (Colom and Bland, 1987). The top line represents an analog trace showing hippocampal desynchrony and hippocam@ synchrony. The top two cell lines depict the discharge patterns of phasic and tonic theta ON cells during simultaneously recorded hippocampal field activity. The bottom two cell lines depict the discharge patterns of phasic and tonic theta OFF cells during simultaneously recorded hippocampal field activity.
2 t rate during LiA than duriog theta. In addition, theta-related cells can be subclassified depending on their pattern of firing. Phasic cells fire rhythmically and in a constant phase with the theta wave. Tonic cells simply fire in a periodic or non-periodic non- bursting manner. A neuron which does not have a discharge profile which shows any of these types of relationships with the hippocampal field state is considered non-related
These cell classifications have been documented in both anaesthetized and freely moving anhais, and apply to neurons within and extrinsic to the hippocampal formation.
a) Hippocampal formation
Although the classification scheme described above was first developed for
neurons within the hippocampus in 1987, earlier studies had documented the existence of
theta-related cells in the hippocampus. Rhythtmcal neurons throughout all fields of the
corn ammonis and dentate were reported as early as 1955 (Green and Machne) and by a
number of groups throughout the sixties (Green et al., 1960, 1961; Noda et al., 1969).
Non-rhythmic theta-related cells were first documented by Macadar et al. in 1970.
Garcia-Sanchez et al. (1978) demonstxated that about half of the non-rhythmic theta-
related cells they recorded from the hippocampus still showed a degree of phase-locking
with the theta field activity.
O'Keefe and Dostrovsky (1971) were the first to record rhythmically bursting
theta-related cells in the hippocampus of the freely moving rat The discharges of these
cells were conelated with motor behavior. It was not until 1982 that Sinclair et al. made
an attempt to distinguish hippocampal theta-related cells during type 1 (movement-
related) and type 2 (immobility-related) thaa It was reported that, although theta-reIated
cells did maintain their relatedness during type 2 theta, these cells always discharged at 22 higher rates during type 1 theta, even at equivalent frequencies of field activity. These findings were corroborated by Bland et at, in 1983. En addition, McNaughton et al.
(1983) showed that theta ON cells increased their discharge rates asymptotically as a
function of the velocity of the type I theta behavior being made. This was replicated for theta ON cells by Mizmori et al. (1990) and, in addition, they demonstrated that theta
OFF cells showed the opposite relation.
b) Medial septum
Petsche et al. (1962) originally studied the relationship of MS cells to
hippocampal field activity. This group reported two types of neurons in this region and
classified them as A- and B-units. A-units discharged randomly. B-units fired in
rhythmic bursts whch were phase-locked to the ongoing theta being recorded During
LIA, B-units fired irregularly. Out of this paper evolved the notion of the MS as the
"pacemakern for hippocampal theta activity. This pacemaker hypothesis has come under
considerable scrutiny and no longer appears to be tenable primarily because theta can be
recorded from the septally deaffemted hippocampus in vivo (Coiom et al., 199 1) and in
vitro (MacIver et al., 1986; Konopacki et al,, 1987).
Gaztelu and Buno (1982) also found distinct discharge profiles for MS cells as
related to the hippocampal field state. They reported three types of cell discharges. Type
1 cells discharged rhythmically in phase with hippocampal theta Type 2 cells discharged
non-rhythmically but only during the theta state, whereas trpe 3 cells discharged non-
rhythmically but during all hippocampal field states. Ford et al, (1989) also recorded
cells in the MS, and were able to demonstrate that the cells in this region could be
classified using the criteria for theta-related cells in the hippomnpus. That is, cells were 23 classified as theta ON or theta OFF with phasic and tonic subdivisions for both. This work was supported by results fiom Colorn and Bland in 1991, in which pairs of MS neurons were recorded Of the 143 cells recorded, 133 (93%) were theta-related and classified as theta ON or theta OFF. Because MS theta-related cells could be classified in a manner similar to hippocampal cells, it was suggested that the cell types of Colom and
Bland's classification scheme may be a generd characteristic of the organization of the ascending system. c) PH-SUM complex
Considering the inclusion of the PH-SUM in the ascending hippocampal synchronizing pathway, it is not surprising that Kirk and McNaughton (1991) demonstrated the presence of phasic theta ON cells in the SUM. This work was developed such that theta-related cells were found throughout the caudal diencephalon, with interesting regional distributions of ON and OFF subtypes throughout the PH, SUM,
MM,central medial. nucleus of the thalamus, and the dorsomedial hypothalamic nucleus
(Bland et al., 1995; Kirk et al., 1996). d) Brainstem
Given the importance of the RPO and PPT nuclei in the ascending synchronizing pathways, there are a number of studies relating cefldar activity in these areas to theta generation Vertes (1977,1979) identified a population of cells in the RPO of the freely moving rat which discharged in a tonic pattern selectively during the theta-associated states of walking and rapid eye movement sleep. Similar findings were also reported by
Siege1 et al. (1977)- In 1991, Nunez et al, investigated the discharge properties of RPO cells in urethane mesthetized rats. Wall the neurons recorded, 64% increased their 24 £iringrates, 2 I% decreased their firing rates, and 15% did not alter their firing rates during the occurrence of theta elicited by sensory stimdation. All neurons investigated were of the tonic sub-typetype
These &dings were further expounded upon by Hanada et al. in 1999, who recorded theta-related cells throughout the pontine region. Similar proportions of theta
ON, theta OFF,and non-related cells were found mostly in the RPO,but also in the PPT and MR. e) Entorhinal cortex
As mentioned above, the EC provides a major reciprocal connection with the hippocampus. Despite this, a paucity of research has been dedicated to establishing a relationship between cellular activity in this structure and the hippocampal EEG. Only one study has reported EC neurons with discharge profiles related to the ongoing hippocampal field activity (Dickson et al., 1995).
I) Other areas
Although it is not generally included as one of the ascending hippocampal synchronizing system nuclei, the cinguIate cortex appears to have some interactions with the hippocampal EEG. This cortex receives projections hmboth the MS and the subiculum (Saper, 1984). In 1988, Colom et al. reported that neurons recorded from this structure could be classified as theta-related
It has been shown that the superior colliculus is w~ectedwith a number of nuclei in the ascending synchronizing system, including the RPO (Fortin , 1995), the PPT
(Beninato and Spencer, 1986), and the PH (Rieck et d., 1986). In 1999, Natsume et aI. 25 demonstrated that 8 1% of neurons recorded from this structure were related to the hippocampai field state. All of these could be sub-classified as tonic ON or tonic OFF.
V. Extrinsic influences on theta Cield and theta-related celluhr activity i. Electrical stimulation
In their classic paper, Green and Arduini (1954) demo- that electrical stimulation of the brainstem reticular formation, medial thalamus, and medial hypothalamus resulted in generation of theta field activity in the hippocampus of anaesthetized animals. A similar phenomenon was demonstrated in freely moving animals in 1966 when Yoshii et al. stimulated the hypothalamus of rats and produced hippocampal theta, This study also confirmed the observation that the frequency of elicited hippocampal theta increased, within hits, as the intensity of stimulation increased In these studies, elicited theta fiquencies routineIy decreased near the end of a stimulation period.
The first series of investigations to thoroughly characterize theta elicited by
electrid stimulation was performed on klymoving rats with stimulation electrodes
implanted in the dorsomedial-posteriorhypothalamus @MPH) (Bland, 1971; BIand and
Vanderwolf, 1972). StimuIation of this region produced head movements, poshd shifb,
running, walking, or jumping: all of which were associated with hippocampal field
activity. Furthermore, the investigations demonsbated that increasing intensities of
electrical stimulation produced increasing intensities of motor behavior. For example, for
a rat placed in a nmning wheel, increasing intensities of stimulation dtedin inmasing
mgspeeds- 26 Since these reports, a number of investigations have revealed that eldcal stimulation of separate components of the ascending system has a synchronizing effect on the hippocampal EEG. These results are presented below. a) Medial septum
Considering the position of the MS in the ascending system, it should be no surprise that electrical stimulation of this structure results in synchronized hippocampal field activity (Ball and Gray, 1972; Kramis and Routtenberg, 1977; Kramis and
Vanderwolf, 1980; Scarlett and Bland, 1997). It has been firmIy established that electrical stimulation of the septum results in hippocampal field activity. However, there is an important difference between theta elicited by electrid stimulation of the MS and theta elicited by stimulation of other structures. In the latter case, theta is elicited by high frequency stimuIation (100 Hz) and as the intensity of the applied current increases, so does the frequency of theta. Only with septal sdmdation does each pulse of stimulation produce a corresponding wave of hippocampal synchrony. That is, the elicited theta
frequency is identical to the frequency of the applied current: S Hz stimulation produces 5
Hz theta, 8 Hz produced 8 Hz, and so on. One caveat is that stimulation kquencies must be within the range of normal physiological theta frequencies. High stimulation
frequencies (above 12 Hz) produce inegular hippocampal field activity (Ball and Gray,
1971).
In 1972, Bland and VanderwoIf showed that stirnuhion of the DMPH modulated
motor behaviors correlated with type 1 theta It appears that, for electrical stimulation of
the MS, simultaneously cnxarring behavior is disassociated from the hippocam@ field
activity. Kramis and Routtenberg (1977) and Kramis and Vanderwolf(l980) have 27 shown that septal stimulation can evoke hippocampal theta during both movement and immobility: behaviors normally correlated with the occurrence of hippocampal LIA
(immobility, grooming, chewing, etc.) continued during elicitation of hippoampal theta
As stated above, theta-related cells in the hippoampus are active during both voluntary movement and sensory processing in fieely moving animals (Sinclair et al.,
1982; Mizumori et al., 1990). There have been no published experiments which investigated the influence electrical stimulation of the septum might have on hippocampal theta ON and theta OFF neurons. This is qrising since a study of this type might possibly elucidate the dissociation between MS stimulation induced hippocampal theta and behavior. b) PH-SUM complex
The posterior hypothalamic region has been shown to be an important nucleus in the ascending hippocampal synchronizing system, High ihquency trains of stimulation
applied to this structure produce hippocampal theta activity and increasing intensities of
stimulation cause corresponding increases in theta fkquency (Green and Arduini, 1954;
Bland, 1971; Bland and Vandmo& 1972; Kramis and Vanderwolf, 1980; Oddie et al.,
1994; Oddie et al., 1996; Scarlett and Bland, 1997).
Hypothalamic stimulation also influences the behavior of freely moving rats
(Bland, 1971 ;Oddie et al., 1996).
PH stimulation induced theta can be modulated by MS stimulation (Scarlett and
Bland, 1997). That is, when the PH and MS are stimulated simultaneousiy, the kquency
of the stimulus induced hippocampal theta matches the fbpency of the pulses of the MS
stimulation, not the PH stimulation. MS stimulation is capable of modulating PH- 28 induced fkquencies upward or downward Furthermore, the addition of MS stimulation during PH stimulation increase the amplitude ofthe stimulation induced hipP-mPal theta. In keeping with the suggestion that the PH-SUM is a vital complex in the ascending system, electrical stimulation of this structure results in intense activation of theta ON cells in the hippownpus (Colorn et al., 1987; Smythe et al., 199 I), MS (Bland et al, 1990; Bland et al., 19941, and the entorhid cortex (Thckson et al., 1995). Theta
OFF cells in these structures are inactivated by PH stimulation. c) Brainstem
The origin of the ascending hippocampal synchronizing pathway was originally localized to the RPO based on electrical stirnutation techniques (Macadar et al., 1974;
Vertes, 198 1). In 1994, Oddie et d. showed that the fkequency of theta induced in the hippocampus was related to increasing intensities of RPO stimulation in a linear positive manner.
Bland et al. (1994) demonstrated that eled~icaIstimulation of the RPO resuIted in activation of theta ON cells in the MS iu addition to eliciting hippocampal theta field activity, Interestingly, upon eiectxical stimulation of the RPO, PH cells increased their tonic discharge rate, while celIs of the SUM and MM did not change their discharge rates, but changed their patterns to the rhythmically bursting pattern characteristic of phasic theta ON cells (Kirk et al., 1996). To date, there have been no studies on the influence of eIectricaI stimulation in the pons region on theta-related cells in the hippocampus. 29 Interestingly the MR appears to have a proninent role in a hippocampal desynchronizing system. A number of studies have shown that electrical stimulation of this structure produces desynchony in the hippocampal EEG (Macadar et al., 1974;
Vertes, 1981, 1982,1988). In 1981, Vertes clearly showed in the uretlme anaesthetized rat that this site was the only site within the brainstem that produced desynchronized patterns in the hippocampal field recording. ii. Pharmacology a) Hippocampal field activity
Some of the pharmacology underlying theta field activity has been alluded to in previous sections. Again, pharmacologically, theta activity can be divided into two groups which correlate with behavioral states (Bland, 1986). In 1975, Kramis et al. were the first to demonstrate in rats and rabbits that type 2 (immobility-related) theta was abolished by injections of the acetylcholine muscarinic antagonist atropine sulphate.
Type I(movement-related) theta was unaffected by this treatment Thus, it has been suggested that type 2 theta is mediated by acetylcholine, specifically by muscarinic receptors. A wealth of data confirms this view muscarinic antagonists, such as atropine sulphate and scopolamine abolish type 2 theta, whereas agonists such as carbachol and physostigmine evoke type 2 theta (for a review see Bland, 1986).
Animals produce hippocampal theta during sensory processing prior to (type 2 tteta) and coincident with (type 1 theta) voluntary movement (Oddie and Bland, 1998).
Although type 1 theta has been reported to be sensitive to anaesthetics (such as urethane), it should not be assumed that this type of theta can not be recorded in acute preparations.
The hippocampal theta generated in animals anatsthetized with urethane is reported to be primarily type 2, cholinergically-mediated theta (Bland and Colom, 1993). Indeed,
Stewart and Fox (1989a) demonstrated that a small atropine-resistant (type 1) component of theta was detectable in urethane anaesthetized rats treated with atropine.
Type 1 theta defied pharmacological classilication for many years. The contribution of a number of neurotransmitters (including dopamine) was eliminated before work performed in the eighties and early nineties demonstrated that this theta is mediated by serotonin (Vanderwolf and Baker, 1986; Vanderwoif, 1988; VanderwoIf et d., 1989; Peck and Vanderwolf, 1991). b) Hippocampal cell activity
Experiments which have examined the pharmacoiogy of theta-related cells in the hippocampus have drawn conclusions similar to those based on the pharrnacolo~of hippocampal theta field activity. In 1988. BIand and Cotom showed that administration
of cholinergic agonists. such as carbachol and eserine, elicited hippocampal theta activity
and activated phasic and tonic theta ON cells in the hippocampus of urethane
anaesthetized rats. The subsequent administration of a cholinergic antagonist, such as
atropine suiphate, abolished both the theta field activity and accompanying cellular
activation. In addition, nicotine was found to result in temporary synchronization of the
hippocampal field recording, but had no effect on theta ON cell activity.
In 1989, Bland and Colom performed a similar study but looked at theta OFF
cellular activity. They found that administration of es&e produced a reduction in the 3 1 discharge rate of phasic theta OFF cells and abolished tonic theta OFF discharge. The subsequent administration of atropine abolished all theta field activity previously elicited by eserine, hypothalamic stimulation, or sensory input. Yet, these last two treatments were still able to depress theta OFF cell discharge rates, even though theta field activity was not elicited Thus, it appeared that theta OFF cells were associated with ongoing cholinergically elicited field activity but were still inhibited by ascending inputs in the absence of cholinergically induced theta field activity- This suggests that while hippocampal theta OFF cells are involved in theta circuitry, they are not directly mediated by cholinergic synapses. Indeed, many working models of hippocampal theta generation now consider hippocampal theta ON cells as excitatory projection cells receiving cholinergic innervation from the MS, and hippocampal theta OFF cells as inhibitory interneurons receiving inhibitory GABAeric innervation from the MS (for a review see Bland, 2000). This will be fintber discussed in the next section.
In freely moving rabbits, a selective abolition ofthe discharges of hippocampal theta ON cells during type 2 (immobility-related atropine-sensitive) theta was observed upon the administration of atropine (Bland et al., 1984). The same theta ON cells continued to discharge rhythmically during type 1 theta, yet the number of discharges was reduced Buzsaki et al. (1983) have also reported the persistence of rhythmical cell . . discharge in the klymoving rat following admmstmtion of atropine. They firher reported significant reduction in the cell discharge rate following cholinergic blockage, suggesting that the cells received input hmtwo distinct theta systems. 32 c) Medial septum
The cholinergic nature of the septohippocampd projection has received strong suppon Lesions of the septum result in a loss of acetylcholinesterase staining in hippocampus and demases in hippocampal choline acetyltransferase activity (Lewis and
Shute, 1967). Stimulation of the MS causes release of acetylcholine in the hippocampus
(Dudar, 1975).
The septum not only provides much of the cholinergic innervation to the hippocampus, but many septal neurons are also cholinoceptive. In the fieely moving rat, cholinergic activation of the septum, via microinfbion of carbachol, elicited type 2 theta which could be subsequentIy abolished by microinfirsion of atropine (Monmaur and
Breton, 199 1). This atropine treatment did not disrupt theta accompanying voluntary movement. Lawson and Bland verified these results in 199 1. They also performed a pharmacological manipulation particularly relevant to this thesis: they used the local anaesthetic procaine hydrochloride to assess inactivation of the septohippocampal pathway. Procaine hydrochloride results in the reversible inactivation of neurons through sodium channel blockage (Maipeli and Schiller, 1979). Application of this drug abolished both types of theta in the freely moving rat. During the recovery period following septal inactivation, theta Erequency recovered quickly as compared to theta amplitude. Smythe et al. reported similar dtsearlier (199 1) for the acute preparation.
With respect to MS tmitactivityytwo early papers by Petsche et al. (1962) and
Stumpfet al. (1962) supported the idea of a cholinergic mechanism involved in septohippocampal rhythmicity Stewart and Fox (1989by 1989c) demonstrated two distinct populations of rhythmically bursting neurons in the MS of udmeanaesttdzed . - 33 rats. Following the intravenous -tion of atropine, the majority of neurons in the MS continued to burst in a phasic manner at theta field frequencies, while a smaller number of neurons lost their rhythmic discharge pttern which accompanied the loss of hippocampal theta activity. The data suggested that rhytbmic cells of the MS are composed of at least two distinct types, and that both may contribute to the production of theta in the hippocampus. Indeed, as mentioned above, Smythe et al, (1992) proposed that excitatory cholinergic and inhriitory GABAergic projections originating in the MS act synergistically to modulate the synchronizing activity from the ascending pathways.
One study that investigated the effects of manipulating the septohippocampal
GABAergic projection has provided further support for these ideas. In 1996, Bland et al. microinfused the GABA-A antagonist muscimo1 into the MS while recording hippocampal theta and hippocampal theta ON cell discharges spontaneously and in response to electrical stimulation of the PH, in urethane anaesthetized rats. Briefly, while the amplitude of spontaneous and electrically induced theta progressively deteriorated following the microinfusion, the frequency of theta remained static while it was observable. Correlated with this progressive loss of theta was a decrease in theta ON ce1I discharge rate. PH stimulation was no longer abIe to elicit theta during this period of maximal drug effect
Theta-related cells in the hippocampus are also affkcted by rnicroinhsion of procaine into the MS. In 1991, Smythe et al. examined the effects of this treatment on hippocampal theta-related cell discharge profiles. This drug treatment reversibly abolished spontaneousIy occurring theta, tail pinch (sensory stimulation) induced theta, and theta which had been previously elicited by stimulation of the PH Both the 34 frequency and amplitude of the hippocampal theta decreased quickly, but showed a progressive recovery after one burUCDuring the period of muidsuppression of the
MS by the procaine, hippocampal theta ON cell discharges were reduced to zero in most cases. Recovery of cell discharge rates and patterns (for phasic ON cells) padeled the fmt appearance of spontaneous theta in the hippocampal fieid recording. Conversely, during the period of maximal effkct of the drug, hippocampal theta OFF cells remained discharging at their pre-procaine LIA levels. The inhibition of hippocampal theta OFF cells normally produced by PH stimulation was also abolished during this time. d) PH-SUM complex
In 1994, using pharmacological methods, Oddie et al. assessed how the PH-SUM complex related to the rest of the ascending system. In the urethane anaesthetized rat, the microinfbsion of procaine into the region of the PH and SUM nuclei totally abolished spontaneously occurring theta, theta produced by a tail pinch, theta previously elicited by electricai stimulation of the PY and theta previously elicited by eIectricai stimuIation of the RPO. All hippocampal theta bad recovered after one hour. Upon microinfirsion of dachol into these structures, theta was continuously generated in the hippocampus.
Bland et al. (1994) demonstrated in urethane anaesthetized rats that, in addition to eliciting continuous periods oftheta in the hippocampus, the microinfusion of carbachol into the PH also resulted in the activation of theta ON cells in the MS. Conversely, suppression of hippocampal theta via PH procaine significantly reduced the discharge rates of all theta ON cells in the MS. In addition, procaine in the PH abolished the rhythmcity of all phasic theta ON cells recorded. 3 5 In 1996, Kirk et al. recorded single unit discharges hmthe PH, SUM, and MM during theta elicited by RPO stimulation, and after the microinfusion of procaine into the
MS. While RPO stimulation could no longer elicit hippocampal theta after this drug treatment, the RPO-elicited discharge patterns of all PH tonic theta ON cells and dl SUM phasic theta ON cells survived this treatment. In fact, the discharge rate of PH cells and the frequency of burst discharge of SUM cells during RPO stimulation increased after the infusion In contrast, this drug treatment abolished the RPO-elicited rhythmic discharge patterns of MM neurons, and decreased their firing rates. The conclusions of the study were that wMe the PH-SUM complex form part of the ascending hippocampal synchronizing system, the MM receives rhythmic input descending from the septohippocampal system. In addition, the PH-SUM complex receives descending inputs that depress their discharge rates during hippocampal theta e) Brainstem
A mapping study of the brainstern, using microinjections of carbachol, revealed that both the RPO and PPT provide choIinergic input used to generate hippocampal theta
(Vertes et d., 1995). Furthermore, mionof carbachol into this region results in intense activation of phasic and tonic theta ON cells in the MS (Bland et al., 1994). Interestingly, no chohergic antagonists have been applied to these structures
M. Function of hippacampal theta
In terms of functional significance, the role of hippocampat theta remains controversia1. Theta was ori@y thought to be with behavioral arousal and attentional processes (Green and Arduini, 1954; Grastyan et aL, 1966). These ideas were developed and led to the hypothesis that theta is involved in learning and memory (Adey, 36 1962; Elazar and Adey, 1967; Bemet, 1971,1973; Givens, 1996). Lesions tbat abolish theta without destroying the hippocampus interfere with spatial learning in rats to the same degree as complete hippownpa1 lesions (Stewart and Fox, 1990; Turnbull et al.,
1994). Conversely, conditions of memory impairment may be alleviated by treatments which activate theta (Givens and Olton, 1995). Short duration changes in the amount of synchronization in the hippocampal EEG have been shown to be associated with recognition memory in humans (Burgess and Gruzelier, 1997). Further support for this hypothesis is provided by the observation that stimulation of hippocampal afferents with patterns sirmlar to those of naturally occurring theta is optimal for inducing long-term potentiation (Pavlides et a]., 1988; Holscher et al., 1997; Otto et al., 199 1; Chnstie et a].,
1995).
O'Keefe and Nadel(1978) have proposed that the hippocampus processes spatial information by forming a cognitive map of the environment. Work has shown that the firing of place cells, the electrophysiological correlate of location within the cognitive map (Wiener et aI., 1989), shifts with respect to the phase of ongoing theta (O'Keefe,
1993; O'Keefe and Reece, 1993; Hirase et al., 1999).
In 1998, Sainsbury suggested a sensory-inhibition theory of function for hippocampal theta. Based on the observation that some species display long trains of type 2 (immobility-related) theta without making a subsequent movement, it was argued that the most parsimonious hypothesis regards theta as representative of general inhibition of the hippocampus and related structures as they are not being used during motor behavior or alert immobility. 37 A final category of theories relating theta rhythm to behavior suggest that it is involved in sensorimotor integration (Kornisaruk., 1977: Bland, 1986; Bland and Colom,
1993; Lawson and Bland, 1993; Miller, 199 1; Oddie and Bland, 1998; Sinnamon, 2000).
Sensorimotor integration implies that theta rhythm is functionally related to the coordination of sensory input with motor output of behaviors. Simply put, during the production of adaptively appropriate motor activity, theta serves to provide voluntary motor systems with continually updated feedback of their own performance relative to changing environmental (sensory) conditions (Bland, 1986; Oddie and Bland, 1998).
This final theory begins to address, at one level concerning a limited number of motor behaviors, the problem of interaction between sensory and motor systems posed by
Kandel et aI. (2000) in the first chapter of their te?ctbook.
Despite the lack of consensus on the role of hippocampal theta, it is evident that useN information might be gathered fiom experiments designed to investigate possible interactions between hippocampal theta systems and loci of motor control in the brain
(motor cortex, cerebellum, basal ganglia, etc.).
In the broadest sense, using the field activity recorded fiom the hippocampus as a valuable guide, this thesis is an attempt to determine how the hippocampus communicates with the motor systems of the brain, specifically the basal ganglia
VIL The -1 Ganglia
The basal ganglia consists of a number of large subcortical nuclei that participate in the control of movement (Cote and Crutcher, 199 I). The striatum (caudate nucleus and putamen) receive their primary input hmthe cerebral cortex These synapse with the subthalamic nucleus, the globus pallidus, and the substantia nip. Output is 38 primarily directed fiom these nuclei to the prefrontal, premotor, and motor cortices via the thalamus. The primary role of the basal ganglia is hypothesized to be the initiation of internally generated movements and the regulation and monitoring of ongoing movements.
VTIL Structure and fiber connections of the basal gangiia
In fact, the term basal ganglia has no precise limitations or definitions, and it has been used in many different ways throughout the years. From a developmental point of view, the term refers to the striatal and pallidal components of the basal telencephalon
(Srneets et al., 2000). In mammals, these structures include the caudate nucleus, putamen, nucleus accumbens, olfactory tubercle, globus pallidus, and ventral pallidurn.
Frequently, other forebrain and midbrain structures, such as the subthalamic nucleus, ventral tegmental areas, and substantia nigra are also included, merely because they are closely related to the striatopallidal circuitry. The striatopallidal system can be divided into do& and ventral aspects. In most mammals, the former consists of the striaturn proper, also known as the caudate-putamen, the globus paIlidus, and the enteropeduncdar nucleus. The latter consists of the nucIeus accumbens, part of the olfactory tubercle, and the ventral pallidurn. The focus of this thesis will be on certain nuclei within the dorsal system and other related structures. Speafically, theta-related properties of the caudate- putamen, globus pallidus, and substantia aigra will be considered. The anatomy and co~ectivityof each of these stnrctures will be discussed in greater detail below. The information presented here is sunvnarized hmthree comprehensive reviews on the topic
(Heimer et al., 1995; Smeets et d., 2000; MeMer, 1981). 39 i Anatomy of the caudateputamen
The dorsal striatun, or caudate-putamen lies within the ventromedially facing concavity of the external capsule. It is bound medially by the lateral ventricle, bed nucIeus of the stria terminalis, and globus pallidus, This structure is large and rotund rostrally stnd narrows in its caudal pouts. The caudate nucleus and putamen are separated
From each other by the internal capsule in many mammals, including cats, dogs, and primates. In rats and many other mammals however, the poor development of the internal capsule precludes the ready identification of the homologues of the caudate nucleus and the putamen Thus, it is preferable to refer to this entity in the rat as the caudate-putamen (CPu).
A characteristic constellation of neuronal classes typifies the CPu A very high proportion of these cells in the rat are of the so-called spiny variety. These spiny cells may be small or large bodied Spiny neurons give rise to both axons that leave the striaturn and to profbe local axons. Hence, spiny neurons have characteristics of both projection neurons and intemeurons. The large number aad dense packing of these cells gives rise to the homogeneous appearance of the CPu.
The remaining cells in the CPu of the rat are of the so-called aspiny variety. Most evidence favors the view that most, if not all, of these neurons represent several classes of intemeurons. ii. Anatomy of tbe giobus pallidas
The primate globus pallidus can be subdivided into an external and intd segment. There seemed to be little evidence for such a subdivision in rodents and other non-primates until it was recognized that at least part of what is usually referred to as the 40 enteropeduncular nucleus group represents the homologue of the primate intd pauldd segment. There is some distance between the two parts of the pallidal complex in the rat. Thus, the convention remains to apply the term globus pallidus (GP) only to the larger external component of the pallidal complex in the rat and to retain the term enteropeduncular nucleus (EP) for the smaller intracapsular component of the pallidaI complex. The EP extends medially to the point where it invades the lateral part of the hypothaIamus. The GP extends in a continuous fashion below the anterior cornmisure toward the base of the brain in the region of the olfactory tubercle.
The majority of neurons in the pallidurn are large and fbsiform, while small neurons are rarely seen. Most of the neurons are projection neurons. iii. Anatomy of the substantia aigra
The substantia nigra (SN), which is located on the dorsaI side of the pes pendunculi, is traditionally divided into a dorsal cell-rich region, the pars compacta
(SNC),and a more voIuminous ventral part, the pars reticulata (SNR),which characteristically has a much lower density of cell bodies. In the rat, the rather thm and welldefined lamina of dopaminergic cells (SNC)is located dorsal and adjacent to the cell-sparse part (SNR). a) Pars reticslata
The SNR shares many histological fatures with the internal segment of the globus pallidus (the EP) and, because both structures represent the output side of the basal ganglia, they are often considered the same entity. The SNR, like the pallidal areas, contains large neurons with long smooth dendrites. 41 b) Pars compacta
The SNC is part of a continuum of dopaminergic cells in the mesencephalon that also includes cells in the ventral tegmental area, and cells in the retrorubral area The
highest density of these dopaminergic cells is in the SNC. The SNC has been termed the
"black zone" because SNC neurons contain the dark staining neuro-melanin pigment
IX. Circuitry of the basal ganglia
Working out the co~ectivityof the basal ganglia has been the focus of a large
body of research (for general reviews see Mehler, 198 1; Nauta and Domesick, 1984; for
evolutionary and species specific reviews see Heimer et aI., 1995; Reiner et d., 1998;
Smeets et al, 2000; for a review of synaptic organization see Smith et al., 1998).
As suggested above, the striatum is the major recipient of inputs to the basal
ganglia fiom the cerebral cortex, thalamus, and brainstem. Its neurons project to the
pallidaI complex and substantia nigra. Together these nuclei give rise to the major output
projections fiom the basal ganglia The extrinsic and intrinsic connections of the bad
gangiia are given in greater detail in the next sections.
i. Afferent connections
a) Corticostriatai projections
Cortical projections reach the striatum in a rather direct fashion, such that dl
regions of the neocortex project to the CPu in a pattern that by and brge preserves the
topology of the cortical mantle. However, the mosaic of these projections is not strictly
defined, such that few parts of the CPu are projected upon by a single cortical area
Cortical afferents are generally excitatory and use glutamate as their tmnmhr- 42 While neocortical afferents kateall regions of the striaturn, affemts from the docortex, most notably the EC and subiculum, terminate preferentially in the ventral striaturn (Groenewegen et al., 1987; McGeorge and Faull, 1989; Amaral and Witer,
1995). The docortex bilaterally projects in a topographical manner to a ventral striatal territory which includes the olfactory tubercle, the nucleus accumbens, and a large ventromedial aspect of the Cmt. This important connection between the "motivation" and "action" centers will be discussed in greater detail below.
In addition, an amygdalostxiatal projection exists, such that some inputs fiom the amygddo-hippocampal transitive area bilaterally innervate areas throughout the CPu
(Canteras et ai., 1992; Brog et al., 1993). b) Thalamostriatal projections
The thalarnostriatal projections are a major source of more or less topographically organized striatal afFerents that originate primarily in the midline and intralaminar nuctei of the thalamus. These projections also include afferent5 fiom areas such as the ventral thalamic nuclei, medial genicdate body, and the latd posterior and posterior thdamic nuclei. The thalamostriat. projections appear to provide the brainstem with a direct link to the basal ganglia. The thalamic projections to the striatum are primarily ipsilateral, excitatory, and presumed to be glutamatergic. c) Nigrostriatal projections
The dopaminergic cells located in the SNC, ventral tegmental area, and retrorubrai area provide a massive innemtion ofthe striaturn. These mesostriatal dopamine projections appear to be mainly ipsiiral but with a minor contralateral contriiution. The cells of the SNC project throughout the CPu, and very little to the 43 accumbens and olfactory tubercle. On the other hand, cells in the ventral tegmental area also project throughout the CPu, but provide a wealth of afferent9 to the accumbens and olfactory tubercle. d) Other ascending afferent projections
Several brainstem areas project to the striaturn as well as to other parts of the basal ganglia. Mainly ipsilateral serotonergic fibres, primarily fiom the dorsal raphe nucleus, reach the CPu, EP, and SNR Other significant afferents come from the pons region Evidence suggests that the PPT provides a fairly dense cholinergic input to the
SNR and a modest input to the SNC. ii. Efferent connections
The information flowing into the striatum fiom several sources is subsequently relayed through three closely related structures, the GP, EP, and SNR. Amongst these, the EP and SNR form a more closely related pair. Chemically, all structures receive dense GABAergic innervation fiom the CPu, but only the EP and SNR also receive a dense substance P and dynorphin innervation. Conversely, while the GP receives a dense enkephaiinergic projection fiom the CPy the EP and SNR do not Mythe GP projects densely to the subthalamic nucleus (an important relay point discussed in greater detail below), whereas the EP and SNR's projections to this structure are less dense. Finally, and most importantly, the EP and SNR contain the cells of origin for the major output routes of the basal ganglia a) Strhtopallidal projections
The efferent connections of the stn'atum are established exclusively by delicate axons passing ~ediallyfiom their origin that converge on the pallidal structures much 44 like the spokes of a wheel. These bundles pass through the outer (GP)and inner (EP) segments of the pallidus and continue in part to the SN. The striatopalIida1 projdon is organized in a fairly orderly radial pattern, As mentioned above, while projections from the striate terminate massively in both the GP and EP, they can be delineated phamacologically: substance P and dynorphin containing fibers reach the EP, enkephalinergic fibers onIy reach the GP.
In addition, the majority of striatopallidd projections express D2dopamine receptors, b) Striatonigral projections
Most of the remaining projection fibers hmthe CPu impinge on the SN. interestingly, it appears as though most, if not all, striatonigal fibers are not within an independent system. That is, these fibers are actually the attenuated and largely un-myelhated end-stretches of striatal efferents that have given off collaterals to the pailidus at more proximal points in their course. This suggestion has been qualified pharmacologically: only those axons which continue from the GP to the EP and SN express both substance P and dynorphm mRNk
Furthermore, the majority of striatonigraI projections express Dldopamine receptors. c) Co~ectionsof the subtbdaimc nocleus
The subt)lalamic nucleus (STN) plays a pivotal role in basal ganglia organkition
This nucleus lies just below the thaIamus and above the anterior portion of the SN. The
STN receives a massive projection hrnthe GP and ia turn projects massively to both pallidsegments. Reciprocal connections have also been established with the SNR and 45 PET. Additional flerents to the STN come from the cerebral cortex, certain ttralamic nucIei, and the SNC. Finally, the STN may even innervate the CPu d) Projections from the globus pallidus
One the most prominent projections fiom the GP (to the STN) was mentioned above. Other important targets for the GP include the EP and the SNR, and to some degree the SNC. Projections originating in the sttiatum and in the GP appear to converge in the EP and SNR. In addition to these intrinsic basal ganglia projections, efferents fiom the GP innervate the superior colliculus, the reticular and mediodorsal thalamic nuclei, and the lateral habenula. e) Projections from the enteropeduncular nucleus and the pan reticulata of the su bstantia nigra
It is convenient to discuss the efferents of the EP and SNR together as already indicated, overall projections are similar. The major outflow of the basal ganglia is directed through the EP and the SNR, which send many collateral axon branches to both the thalamus and the brainstem. Major common targets include the ventromedial and ventmlateraI thalamic nuclei, the centromedian-paraf~scicularthalamic comptex, the mediodorsal thalamic nucleus, the superior colliculus, and the PPT. In additon, the EP projects to the lateral habenula In the rat, the euteropeduncular and nigral projections overlap.
It has been suggested that the SNR output can be divided into nigrothalamic,
nigrocollicuiar, and nigrotegmental IameUae, providing the basal ganglia with a number
of parallel output channels to match the notion of parallel input from the cortex 46 ii. The direct and indirect pathways
In 1989, Albin et al. formdated a unifying mode1 of the functional organkition of the basal ganglia This model, which has been expanded and ekborated by other groups
(for a review see Smith et d., 1998), is based on the so-called "direct" and "indire&' pathways of cortical information flow through the basal ganglia
Briefly, the two output nuclei of the basal ganglia, the internal pallidal segment
(the EP in the rat) and the SNR, provide continuous levels of inhibition to their target cells in the: thalamus and brainstem. This inhibitory output is thought to be moddated by two parallel pathways. In the direct pathway, information passes from the striatum
directly to the output nuclei along GABAergic, inhibitory fibers. The indirect pathway
passes first to the external pallidal segment (the GP in the rat) and from there to the STN
in a purely GABAergic pathway, and finally fiom the STN to the output nuclei in an
excitatory glutamatergic projection.
By virtue of the neurotransmitters and basaI activity of neurons in these networks,
activation of the direct and indirect pathways produces fimctionally opposite effects in
neurons of the target nuclei of the basal ganglia Again, the neurons within the SNR and
EP maintain a tonic level of inhibitory output When excitatory (glutamatergic) inputs
activate the stria- the output nuclei are briefly suppressed via the direct pathway, thus
permitting their tbalamic and brainstem targets to be activated. Conversely, via the
indirect pathway, activation ofthe striatum leads to inhilition of the GP, leading to a
release of inhriition of the STN, leading in turn to activation of the output nuclei, which
transiently increase their inhibition of their thaIamic and brainstem targets. 47 Furthermore, the two striatat output pathways are affected differently by the dopaminergic projection from the SNC. It had already been indicated that striatal neurons that project directly to the output nuclei have D ldopamine receptors that facilitate transmission while those that project indirectly have D2-dopamine receptors that reduce transmissioa. Although their synaptic actions are different, the dopaminergic
input (fiom the SNC) to the two pathways produces the same effect. By exciting the
direct pathway and inhibiting the indirect pathway' these dopaminergic cells reduce the
inhibition that basal ganglia output nuclei transmit to their targets. Therefore,
dopaminergic cell death in the SNC, as occurs in Parkinson's disease, leads to serious
motor disturbances.
X. The basal ganglia and motor control
That the basal ganglia is involved in controlling movements first emerged ftom
clinical observations (DeLong, 2000). Post-mortem examination of patients with
Parkinson's Disease, Huntington's Disease, and hermilismus revealed pathological
changes in basal ganglia nuclei. In tfie simplest view, the poverty of movement ia
disorders such as Parkinson's results horn over-activity of the indirect pathway, whereas
excessive movement in disorders such as Huntington's represents over-activity of the
direct pathway (Graybiel, 2000). SingIe cell recordings of neurons throughout the basal
ganglia have provided insights into the exact nature of this structure's association with
motor control.
i. Unit electrophysiology of the basal ganglia
The electrophysiologicalcharacteristics of basal ganglia neurons have been well
documented in both normal and disease states. The contribution of basal ganglia cell 48 activity to motor output has been most intensely studied in non-human primates. The paradigm most often used entails recording fiom neurons whiIe animals respond to triggered stimuli. The onset of rapid, stimulus-triggered limb movements is preceded first by changes in neuronal firing in the major circuits of the cortex and only later in the basal ganglia (Cote and Crutcher, 199 1).
Prior to the execution of a specific motor act, such as wrist flexion or extension, a low proportion of stn'atal neurons fire rhythmically (Lebedev and Nelson, 1999). This proportion is greatly increased in doparnine depleted animals (Raz et al., 1996). Almost all of these neurons exhibit a change in rhythmic frequency in the moments immediately before the animal makes a movement. For about half of these neurons, ths pre- movement change in rhythmicity is dependent on what type of movement (flexion or extension) the animal is going to make.
For cells throughout the pailidal complex, significant correlations have been obtained between neuronal firing and the duration, amplitude, and velocity of a movement (Georgopoulous et al., 1983; Anderson and Turner, 1991). Fillion (1979) and
Fillion and Trernblay (1991) have shown that while tonicaily active neurons throughout the pallidal complex in the normal monkey are not driven by any common input, the same neurons have related burst firing patterns in the Parkinsonian (induced via nigr0striata.I pathway lesions) monkey.
Nini et al. (1995), Boraud et al. (1998), and Raz et al. (2000) have shown that
neurons in the external pallidal segment (the GP in rats) decrease their firing rates after 1-
methyl-4-phenyi-I2J,6-t~dropyridine (MPTP) treatment. (Animals repatedly
administered MPTP develop Parkinsonian symptoms). Furthermore, the discharges of a 49 high number of cells in this structure become rhythmic and cross-correlated with one another after this treatment. These properties can be reversed by administration of Ievo- dopa (Boraud et aL, 1998).
The STN contains cells with firing pattern which are related to the planning and initiation of movement (Georgopoulous et al., 1983; Cheruel et al., 1996). Interestingly, neurons in the STN have been shown to have a spontaneously rhythmic firing pattern in both in vitro and in vivo preparations (Bergman et al., 1994; Bevan and Wilson, 1999;
Magill et al., 2000). This structure has been intensely studied because it appears to play a central role in Parkinsonian tremor generation (Plenz and Kitai, 1999). Indeed, its lesion or blockade in animals with chemically induced Parkinson's Disease attenuates akinesia, rigidity, and tremor (Bergman et al., 1990; Aziz et al., 199 1). In monkeys treated with
MPTP, the percentage of rhythmic STN neurons increases as does their mean firing rate
(Bergman et al., 1994).
In monkeys, during the execution of a specific motor act, the normally high rate of spontaneous discharge in movement-related neurons in the internal pallidal segment
(the EP in rats) becomes even higher in the majority of cells, but in some it decreases
(Cote and Crutcher, 1991; Dorrnont et al., 1997). Output neurons that exhibit decreases in discharge rate may play a crucial roIe in movement by disinhl'biting the thalamus and facilitating corticaUy initiated movements (Cote and Crutcher, 199 1). Populations of neurons that show increases in discharge rate would have the opposite effect, plausibly further inhibiting thalamocorticd neurons and thus suppressing antagonistic or competing movements. 50 Following MPTP treatment, cells within the internal pauldal segment of monkeys increase their firing rates @ergman et al., 1994; Nini et al., 1995; Boraud et al,
1998; Raz et al., 2000). Furthennore, like cells in the external segment, these cells become rhythrmc and their discharges are cross-correlated This effect is not as effectively reversed by levo-dopa treatment (Boraud et al., 1998).
In the cat, singIe cells in the SNR show increases in activity during the presentation of auditory and visual stimuli that may or may not result in a head movement (Joseph and Boussaoud, 1985), Using extracellular single cell recordings in the awake immobilized rat, Ruskin et d. (1999) found that tonically active neurons in the
SN experience slow oscillations in their finng rates in the second-to-minutes range. The authors suggested that slow firing rate oscillations may act to coordinate neuronal activity
responsible for motor sequences.
Evidently, how the basal ganglia are specifically related to motor control is still a
point of contention. As it has been shown that, in response to visually guided tracking
tasks, cells in basal gangIia discharge Iater than cells in motor cortex, and cells in basal
ganglia output centers discharge more seiectively in relation to limb movements than for
the activation of specific muscle groups, it has been suggested that the basal ganglia do
not pIay a significant role in the initiation of stimulus-triggered movements and do not
specify directly the muscular forces necessary for the execution of movement. It is
possible however, that the basaI ganglia may be invoIved in the initiation of internally
generated movements. This possibility is supported by the dinginability of
Parkinson's Disease patients to initiate movement. Alternatively, as was alluded to
above, it has been proposed that these structures selectively facilitate some movements 51 and suppress others. Finally, and perhaps most relevant to this thesis, the basal ganglia may compare commands for movement hmmotor fields with sensory feedback firom the evolving movement
The neurons of the basal ganglia clearly have discharge properties which are in some fashion related to movement initiation and maintenance, and these properties have been shown to vary with the disease state of the subject, Yet, no one has investigated this reIationship within the context of hippocampal synchrony, despite the long known correlation between voluntary movement and theta. Is there any more preliminary data which might suggest hippocampal theta and the basal ganglia are related?
XL Interactions between the basal ganglia and the ascending hippocampal synchronizing systems i. Anatomy
The principle connections between the basal ganglia and the ascending systems were discussed in the section on basal ganglia connectivity. The major interactions will be emphasized here, and potential routes of information flow between the two systems will be discussed
Probably the most important, and certainly the most well studied, co~ection between the hippocampus and the basal ganglia is established through the nucleus accumbens. In 1980, Mogenson et al. reviewed a body of literature which suggested a firnctional interface between the limbic system (of which the hippocampus is a major component) and the motor system (of which the basal ganglia is a major component).
According to this work, the nucleus accumbens is a key structure functionally linking
". .. motivation and action". This basal ganglia structure appears to receive direct 52 projections fiom the hippocampal fomation, in particular the CAI field, the EC, and the subiculm (Amaral and Witter, 1995; Phillipson and Griffiths, 1985; Groenewegen et al., 1987). In turn, fibers from the core of the nudeus accumbens project to the EP and parts of the mesencephalic dopamine system (ventral tegmental area and SNC) (Swanson and Cowan, 1975; Heimer et al., 1995). The body of literature proposing a hippocampal- accumbens-substantia nigra neural pathway was aIso reviewed by Lopes de Silva et aI. in
1985.
Fibers originating in the hippocampal formation might also impinge on the CPu
Using anterograde tracers in the rat, it has been shown that, in addition to projecting directly to all aspects of the nucleus accumbens, the major hippocampal formation output structure, the subiculum, also projects directly to aspects of the CPu (Groenewegen et al.,
1987). The subicuiar projections were concentrated mostly on a ventromedial temtory of the CPu, with caudal projections predominantly arising fiom the ventral subiculum, and rostral projections predominantly arising hmthe dorsal subiculum.
In addition, using retrograde Iabeling techniques in rats, the major hippocampal formation input structu~,the EC, has been shown to project to a roml and medial portion of the striatum (McGeorge and Faull, 1989).
"Lower" nuclei in the ascending hippocampal synchronizing pathway also project to the basal gangha Fortin's 1995 anterograde tracer analysis of the brainstem corroborated that ventrolateral areas of the PPT project heavily to the midbmin dopaminergic groups. The SNR also receives light to moderate projections kom this nucleus. Furthermore, the PPT projects dimdy to tha1mostriata.I neurons (Heimer et al.,
1995). 53 In turn, routes exist such that infomation can flow fiom the basal ganglia to nuclei of the synchronizing system Both the STN and SNR share reciprocal connections with the PPT. In addition, Scatton et al. (1980) showed that dopaminergic fierents from the SNC innervate the hippocampus via a route that courses through the septum.
However, this projection may not be relevant: both type 1 and type 2 theta persist in the complete absence of brain dopamine (Whishaw et ai., 1978). Thus, basal ganglia interactions with the hippocampus are not mediated through dopaminergic pathways.
Another possible route, which attracted much attention in the eighties, implicates the lateral habenula and the raphe nucleus, As mentioned above, both the GP and EP project to the habenula (Heirner et a]., 1995). It has been hypothesized that this structure influences the hippocampus through a disinhibitory habenulo-raphe-hippocampal pathway (Sabatino et a!., 1986,1987). FindIy, one of the major targets of the output nuclei of the basal ganglia is the superior colliculus. The GP also projects to this structure. As mentioned above, the superior colliculus has close ties with the ascending synchronizing system: tonic theta ON and theta OFF neurons have been recorded fiom this structure (Natsume et al., 1999). ii Electrophysiology
In the mid and late eighties, a series of experiments performed by an Italian group investigated the effects of electrical stimulation of a number of basal ganglia structures on the hippocampal field state (Sakino et al., 1985,1986, 1989). These groups showed that electrical stimulation of the caudate nudeus and the SNC induced robust, regular 4 to
7 Hz hippoampal theta. Interestingly, electrical stimulation of the internal segment of the pallidus (the EP in rats) did not induce theta. In all cases, pulses of stimulation were 54 delivered at £hquencies more like those used to elicit theta hmelectrW stimulation of the MS (530 Hz) than those used to elicit theta from other structures. In an attempt to map through which mute(s) these stimulations evoked their effects, the MS was electrolytically ablated in a number of preparations, Both caudate and SNC stimulation failed to have any effect on epilepbform activity in the hippacampus following MS lesions; stimulation of these shructures no longer evoked hippocampal theta In other preparations, the caudate was ablated while the SNC was being stimulated, and vice versa Caudate stimulation effects were unchanged by the destruction of the SNC; SNC stirnutation effects were unchanged by destruction of the caudate. The authors suggested that caudate and nigrai influences on the hippocampus are mediated by different pathways which must converge at some point below the MS.
Because neuronal discharges in certain basal ganglia structures are spontaneously
rhythmic or bmerhythmic in Parkinsonian animals, a number of studies have attempted to relate these cell discharges to rhythmic cortical EEG.
In a pair of recently published abstracts, Allers et al. (2000) and Walters et al.
(2000) established a relationship between discharge patterns of basal ganglia neurons and
cortical EEG. It should be noted here that cortical and hippocampal EEG are inversely
related. That is, synchrony in the hippocampus generally entails desynchrony in the
cortex, and vice versa (Sarnsbury 1998; Bland and Oddie, 1998).
In the first study, a small proportion ofneurons within the STN were fmdto
have rhythrmc changes in their firing rates which were conelated with the occurrence of
short bursts of cortical theta rhythm. This proportion increased foliowing administration
of dopamhergic agonists. In the second study, a small proportion of neurons within the 55 GP were found to have discharge rates which oscillated during cortical theta The kquency of these rhytbmic changes in firing rate matched the frequency of the cortical theta field activity. Dopaminergic agonists increased this proportion, and shifted the phase relationship of GP neuronal firing rate and theta field oscillations closer to zero degrees-
Magill et al. (2000) recorded fiom neurons in the GP and STN of urethane and ketamine anaesthetized rats while simultaneously recording EEG fiom the frontal cortex
During both anaesthetic regimes, STN neurons had periods of burst activity in their spike trains. During periods of synchronous slow wave activity (principal frequency component approximately 1 Hz), the rhythmical cortical oscillation was mirrored in the spontaneous firing patterns of STN neurons. Burst firing was periodic and precisely phase-locked with slow wave activity. In addition, STN bursts could be subdivided into
"miniature bursts* that were phase-locked to spindle sequences (7 to 14 Hz) superimposed on the slow wave activity. Spontaneous cycling to desynchronized patterns in the cortical EEG was always reflected in STN neurons: they adopted an irregular, single-spike firing pattern during these periods. Both sensory stimulation (in the form of a hindpaw pinch) and spreading cortical depression (induced via topical application of potassium acetate) induced desynchronous patterns in both the cortical
EEG and STN cell discharges. Furthermore, these neurons increased their firing rates during these periods.
Similar observations were made for GP neurons during the ketamine anaesthetic
preparations- A high proportion of GP neurons expressed a significant oscillation in their
spike train with a frequency that was similar to the concurrent slow osciIlation that was 56 recorded fiom the fiontal cortex The burst discbarges of pallidal neurons were ocassionally divided into "miniature burstsn in time with coincident spindles. Again, desynchronized cortical EEG patterns, that occurred spontaneously or were induced with sensory stimulation of spreading cortical depression, were associated with a loss of bursting activity and either an increase or decrease in firing rate.
However, cells recorded from the GP did not show the same properties during the urethane anaesthetized preparations. Using this regime, GP neurons discharged single spikes in a regular (periodic) manner even when slow wave activity occurred simuItaneously in fiontal cortex. However, GP neurons experienced a significant increase in their firing rates during periods of cortical desynchronization induced by sensory stimulation. In addition, GP neurons recorded under these conditions were significantly more active than those recorded during ketamine anaesthesia for both cortical EEG states.
The authors explained the differences in cell behavior between anaesthetic regimes by suggesting that the weaker STN activity under urethane anaesthesia was insufficient to relay synchronous information fiom the cortex to the GP such that these neurons could be driven in a bunting manner.
XIL Objectives and hypotheses
Anatomical, electrophysiological, and behavioral evidence suggests a possfile relationship between the basal ganglia and the hippocampus. Further examination of whether or not these regions of the brain communicate with each other is warranted, so that the potential role the hippocampus might have in motor control is clarified. The 57 most valuable tool which can be used to assess whether these regions 'W'to each other is the hippocampal EEG.
The objective of the first proposed experiment is to establish whether there is any relationship between the activity in the basal ganglia and the hippocampus. An attempt will be made to correlate the dynamic cellular discharge properties of neurons in the basal ganglia with patterns in the hippocampal field recording. Based on the evidence presented above, it is hypothesized that such a relationship does exist: structures that are
connected to the basal ganglia (such as the superior colliculus and PPT) contain theta-
related neurons; neurons in the basal ganglia discharge periodically in the theta frequency
range; and neurons in the basal ganglia have discharge properties which are related to the
EEG recorded fiom the fiontal cortex,
If any relationship is established between basal ganglia neuronal discharges and
hippocarnpal theta rhythm, the routes through which these regions communicate must be
elucidated. This is the general objective of experiments two and three.
The second experiment will test whether the basaI ganglia sends ascending input
to the hippocampus via the hippocampal synchronizing systems. Basal ganglia structures
will be electrically stimulated in an attempt to elicit hippocarnpal theta. The route
through which these putative synchronizing aff'travel will be investigated by means
of chemical blockade within the basal ganglia and at the level of the MS. This
experiment replicates, in part, work done by Sabatino et al. ( 1985,1986, 1989). Based on
their findings it is hypothesized that Stimdation of certain basal ganglia structures (CPu
and SN) will have a synchronizing effect on the hippocarnpal EEG which can be
reversibly blocked by microidusion of the Iwal anaesthetic procaine hydrochioride into the MS. In addition, it is hypothesized that hippocampal synchrony elicited by stimulation of bad ganglia input nuclei (CPu) can be blocked by microinhion of procaine into basal ganglia relay (GP, SNC) and output (SNR) nuclei. In turn, it is hypothesized that hippocampal synchrony elicited by stimulation of relay nuclei (GP) can be blocked by microinfusion of procaine into output (SNR) nuclei, but hippocampal synchrony elicited by stimulation of output nuclei (SNR)can not be blocked by microinfusion of procaine into relay (GP) nuclei.
The third proposed experiment will attempt to assess whether the ascending hippocampal synchronizing systems send descending information via the septohippocampai system to the basal ganglia The discharge profiles of theta-related cells recorded within the basal ganglia will be assessed following microinhion of procaine into the MS such that hippocampa1 theta can no longer be elicited with sensory stimulation. If the basal gangfia do indeed lie "below" the septohippocarnapl system in the ascending pathways this drug treatment should have no effect on theta-related discharge profiles, as was the case for theta-related cells in the PH and SUM (Kirk et al.,
1996). Conversely, if the basal ganglia is put of a descending efferent system which courses through the synchronizing pathways and projects fiom septohippocamapl neurons, this drug treatment should abolish theta-related neuronal behavior in the basal ganglia, as was the case for theta-reIated cells in the hippocampus (Smythe et al, 199 I) and the MM (Kirk et al., 1996). Based on the supposition that the basal ganglia might form part of an output route through which the hippocampus interacts with motor systems, it is hypothesized that theta-related neurond discharge profiles in the basal ganglia will be disrupted by this drug treatment 59 The basal ganglia structures investigated will be the CPu, GP, and SN (both
SNC and SNR). In this manner, a representative number of cells can be characterized in input, relay, and output nuclei of the basal gangh The experiments will be performed in urethane anaesthetized rats and will lay the foundation for hrther investigations in the fieely moving animal. Successfid completion of the proposed research will contnhte to our understanding of the mechanisms of hippocampal oscillation and synchrony, and will provide some insight into the route by which the hippocampus might affect motor systems.
Methods-Experiment One
L Objectives
The purpose of the first experiment was to assess whether there is any relationship between hippocampal field states and cellular activity in basal gangha structures.
Specifically, the discharge profiles of neurons witbin the SN, GP, and CPu were characterized in relation to the simultaneously recorded hippocampal field activities of theta and LIA
IL Subjects
Data were obtained from 107 male Long Evans rats, each weighing between 125 and 150 g, supplied by the Life and Environm& Sciences Animal Care Facility at the
University of Calgary. Animds were housed in groups of up to four in clear plycarbonate cages, and were given rat Laboratory Diets food and water ad libifwn. The light cycle for animal housing was a normal 12 hours on (at 8 am)/ 12 hours off (at 8 pm).
Up to 3 animals were used per day, with experiments Iasting between 3 and 8 hours. All 60 experiments commenced during the light-on cycle and the majority were completed within the same cycle.
III. Surgery
For each experiment, one animal was selected from its cage and initially anaesthetized in aa airtight chamber with a mixture of halothane (MT.C.
Pharmaceuticals) in oxygen (2.5% minimum alveolar concentration) flowing through it at a rate of 2.5 Lpm. Once suitably anaesthetized, the animal's chest and head were shaven, following which it was rapidly transfemd to a surgery table, where anaesthetic was readministered to the animal through a hose and facepiece.
Once the animal was llly anaesthetized (did not respond to foot pinch), an upper thoracic incision was made. Using blunt dissection, the left jugular vein was located and separated fiom its adhering tissue. A small anguIar incision was made in the vein, through which a beveIed cannula (polysylastic tubing with an outer diameter of 0.065 inches) was inserted and secured in place with suturing silk At this point, halothane anaesthesia was discontinued and the animal was switched to urethane anaesthesia (0.8 gfkg) administered in approximately 0.05 mL aliquots through the jugular cannula
The animal's trachea was then exposed through blunt dissection. A small angular
incision was made in the trachea, through which a stiff pIastic tube (outer diameter 0.085
inches) was inserted, allowing the animal to breathe without compIications. This trachea
tube was secured with suturing silk, the thoracic incision was then sutured, and the
animal was placed in the stereotaxic appratus.
Rats were prepared for stereotaxic swgqin the standard manner. A mid-sagttal
incision was made in the animal's scalp to rev4bregma and lambda The skin was tied 61 back to the ear bars with suturing silk, and the periosteum was retracted. Dorsal- ventral @V) coordinates for bregma and lambda were obtained, and the skull was leveled to horizontal. Anterior-posterior (AP) and lateral (L) coordinates for bregrna were obtained as it was used as a reference point for the stereotaxic coordinates. The coordinates for the hippocampal field recording (reference) electrode were always -3 -3 mrn AP and 2.2 mrn L. This position was determined, marked on the skull. and a hole (3 mm in diameter) was drilled at this site. The coordinates for the single unit recording
(roving) electrode were -5.4 mm AP and 2.4 mm L for the SN; -1.3 rnm AP ranging from
3.1 to 3.7 mm L for the GP; and 1.7 to -0.8 mm AP and 3.0 to 4.2 rnm L for the CPu.
Cellular activity was always recorded in the opposite hemisphere from the hippocampal
field activity. Depending on the investigation, one of these positions was determined.
marked on the skull, and a hole (3 mrn in diameter) was drilled at this site.
Additionally. one smaller hoIe (1 .S mrn in diameter) was drilled at approximately
0 mm AP and 2 mrn L, and another hole of the same size was drilled at approximately 2
mm AP and I rnm L (both holes were always ipsilateral to the reference electrode holes).
A jeweler's screw was fastened in the first hote, and an uninsulated tungsten electrode
was placed into anterior cortex in the second hole. This electrode served as an indifferent
electrode, and was attached to the jeweler's screw with dental acrylic. The frame of the
stereotaxic apparatus was connected to ground Using a KOPF 620 servomotor
micromanipulator, the reference electrode was then lowered (at a rate of 10 pdsec) to a
depth of approximately 2.3 mm DV into the hippocampus, From this point in the
investigation, and throughout all data acquisition, urethane anaesthesia levels were
maintained such that spontaneous cychg between hippocampal LIA and theta field 62 states occurred. Upon establishing a maximum amplitude of the theta signal, typically in the upper blade of the stratum moleculare layer of the dentate gyrus, the electrode was cemented to the jeweler's screw with the dental acrylic.
The tungsten microelectrode which recorded extracellular field activity was constructed in the laboratory. Briefly, a piece of tungsten (diameter 0.005 inches) no longer than 1.5 inches was threaded into an insulated holder so that about a half-inch of tungsten remained exposed The holder consisted of an 2 ioch long piece of 26 gauge metal tubing, insulated with shrink tube, with a male subminiature connector clamped to one end to facilitate the attachment of the completed electrode to the amplifiers. The tungsten tip of the eIectrode was sharpened electrolytically (saturated potassium nitrite solution), and the tungsten was insulated with a Kynar solution until it reached a resistance of at least 8 megohms. Just prior to being lowered into the hippocampus, the tip of the electrode was again etched electrolytically so that the resistance at the tip was
lower than 1 megohms. The electrode serving as an indifferent was constructed in a similar manner, but this tungsten was left uainsulated and tbe tip was not etched prior to
its placement.
Once the hippcarnpal recording electrode was secured with the dental acylic, the
animaI was permitted to sit without perturbation for about one half hour so that the
hippocampal signal could stabiIize. Using the micromanipulator, the tmit recording
electrode was then lowered into either the SN, GP, or CPu Cells were recorded hmthe
SNC at depths between 6.7 and 7.1 mrn DV, and fiom the SNR at depths between 7.1 and
8.4 mm DV. GP nmnswere recorded at depths between 4.8 and 7.5 rnm DV. CPu
neurons were recorded starting at depths of 3.2 mm DV and proceeding to depths of 6.8, 63 7.0, or 7.6 mm DV, with deeper recording deptbs corresponding to more posterior coordinates.
Glass extracellular singIe-unit recording electtodes were constructed fiom borosilicaie capillary tubing (outer diameter 1.7 mm) pulled on a Narishige vertical pipette puller. The tubes were back-filled with a 0.5 M sodium acetate solution mixed with 2% Pontamine Sky Blue. The tips of the electrodes were broken under visual guidmce to approximately 1 pm in diameter.
Roving electrodes were lowered through the bmin at a rate of 10 Cun/sec, slowing to a burst approach of 1 pdsec when isolating cells. Shank length on these electrodes was abut 10 mm, resulting in negligible compression of the brain.
Following data acquisition, which could take between 3 to 8 hours, tip locations were marked by pulsing 50 paaps of current for i5 minutes (5 minutes anodal, 5 minutes cathodal, 5 minutes modaI) through the glass electrode such that the sodium acetatdPotassium Sky Blue solution inside was iontophoreticalIy ejected out the electrode tip to stain the brain tissue.
At this point, the animal was heady anaesthetized and moved to a perfuson table. Here, the animal was overdosed with the utethane and perfused intracardidy first with saline (0.9%) and then with a mixture of 10% formalin and saline. The brain was removed and stored m a 30% sucrose-10%formalin mixture at 0-5 "C.
IV. Data Acquisition
The leads f~ recording electrical activity fiom both the reference and roving ektrode ampliliers were led iaSo two Grass PSI 1 preamp~ersersOne preamplifier isolated field activity while the other isolated unit activity. The latter had a signal gain of 64 20000X, with filter settings of half amplitude low at 300 Hz and half amplitude high at
1 KHz. All fiIters were first order analog filters with rates of decay of 6 dB. This
'harrow band" fdter configuration allowed only high fiequency signals to pass, and eliminated 60 Hz noise and artifact derived from tissue movement. As action potential durations are on the order of 0.5 to 2.0 msec (500-2000 Hz), this preamplifier captured the properties of basal ganglia cell discharge considered relevant to this investigation.
The other preamplifier had a signal gain of 300X, with a half amplitude low filter set to 1
Hz and a half amplitude high filter set to 1 KHz. The field signal was additionaliy filtered through a model 7D Grass polygraph with a half amplitude low filter setting of 1
Hz and a half amplitude high filter setting of 35 Hz. This allowed only low frequency signals to pass through. These filter settings allowed recording of hippocampal theta, which occurs in the frequency range of 2.5-7 Hz in the urethane anaesthetized rat, and recording of hippocampal LIA, which occurs in the fiequency range of 0-25 Hz. Other frequencies of the hippocampal EEG, such as beta and gamma activity, were excluded
from the recording.
To aid in electrode placement, the signals passing out of the preamplifiers were
led through an audio monitor (Grass AM5). When lowering the reference electrode, activity of cells in the neocortical layers overlying the hippocampus, cells within the
cornu ammonis layer in the hippocampus, and cells within the dentate gym could be
heard: these aU aided in optimizing position. This audio amplifier was aIso crucid for
isolation of basal ganglia neurons.
Activity fiom both electrodes was displayed on a Tektronix digital storage
oscilloscope for observation throughout an investigation. All signals plus additional oral 65 commentaries were recorded oa VHS cassette tapes using a TEAC XR-30,7-cham1
FM tape recorder. Data were subsequently transferred to paper hardcopy using the polygraph for off-line scrutiny of the signals.
For each investigation, once a basal ganglia neuron was isolated and considered stable (no change in its signal to noise ratio for a Wumof five minutes), a cell recording protocol was commenced,
Hippocampal synchrony occurred spontaneously in the urethane anaesthetized rat if the level of anaesthetic was carefully controlled (aliquot intravenous adrmnistration).
Maintained at optimal levels of anaesthesia, rats typicalIy cycled between LIA and spontaneous theta activity, each lasting for periods of 30-45 seconds or longer. At deeper levels of anaesthesia, the field activity consisted mostly of Llk At these levels, a light tail pinch (operationally defined as sensory stimulus) produced theta field activity for the duration of the pinch and sometimes elicited a subsequent cycle of spontaneous theta.
Depending on the anaesthetic depth of the animal, tail pinch induced theta was often higher in frequency than spontaneous theta. In order to characea ce!l's discharge rate and pattern in relation to the ongoing hippocampal field activity, an attempt was made to record the cell during LZA, spontaneous theta, and tail pinch theta It should be noted that on some occasions an animal remained too deeply anasthetized such that ody tail pinches were able to induce theta and no spontaneous theta was recorded In general, enough data could be obtained to establish a neuron's discharge profile with 20 minutes of recording, and a number of cells could be recorded fiom one animal within an 66 Hard copy output of all data was produced and smtinized off line, and data segments were selected for subsequent analysis. For each neuron, a minimum of two samples (or trials) of the cell's discharge behavior for each hippocampal field state
(except spontaneous theta when it was unattainable) were analyzed. Trials were selected based on how well they represented the cell's discharge behavior, with the order of seiection being random. For example, for one cell, all the LLA trids selected for analysis might have chronoiogically preceded all the tail pinch theta trials selected, whle for another cell, two taiI pinch theta trials might be seIected for analysis that chronologically occurred between a spontaneous theta trial selected for analysis and an LIA trial selected for analysis. A11 trials chosen for analysis were at least 4 seconds long.
Selected data segments were then digitized using a personal computer running data acquisition and analysis software (DataWave Technologies). MI analog signals were digitized through an &bit analog/digital (AD) converter. The hippocampal field signal had an AD gain of 8, and was sampled at an effective AD fhquency of 133 Hz, with a sampledbuffer size of 512. Using this software,buffers bad to be filIed in order for any field data to be acquired. Thus, a minimum of (512 sampled133 sampledsec)
3.85 seconds of hippocampal field data had to be acquired, and the length of all acquired data segments was necessarily always a multiple of this value.
Cell activity was digitized with an ADgain of 1,2,4, or 8 depending on the size of the discharge. For every neuron, thresholds (based on discharge parameters such as width, height, peak and valley times) were defined, and any events in the unit signaI that exceeded these thresholds were considered "spikesn (cell discharges). The spikes were sampled at an A/D frequency of 1.6 KHz and de£inedby 32 points. Field and cell 67 discharge signals were acquired simultaneously, yet the spike sampling protocol was not limited to filling buEers. Ergo, if data were acquired for a period of time longer than the time required for one field sampling buffer to be filled, but not long enough for the next buffer to be filled (for exampIe 5 seconds), the length of the acquired field data would be different fiorn the length of acqured spike data (in this case 3.85 seconds of field data and 5 seconds of spike data wodd be acquired).
V. Data Analysis
For each acquired data segment, the hippocampi field state was characterized and classified as either theta or LIA. A number of indices were used to help distinguish the two states, but the most usefbl information was derived when data segments were subjected to a frequency analysis using a fast Fourier transform (FIT).
Frequency analysis of a signal involves resolving the signal into its frequency components. A representation of the relative strengths of all frequencies present in a signal is called the spectm of the signal. The basic mathematical representation of periodic signals, of which hippocampal theta is one, is the Fourier series, wbich is a linear weighted sum of harmonically related sinusoids. The sum of the components will reproduce the originaI signal. Thus, Fourier transformation of a recorded sample of hippocampal field activity alIows one to determine the relative strengths of the predominant peak frequencies which make up the signal. The bottom portion of Figure 1 illustrates the spectral analyses of two distinct portions of a one minute long segment of hippocampal field activity. The x-axis of each specmm consists of the varying fiequencies of sinusoids which make up each signal; the y-axis consists of their respective powen (power = amplitude2). A number of valuable indices can be obtained 68 from these spectra which help distinguish the LIA field state from the theta field state.
Most importantly, the spectrum on the right has a very prominent peak at 3.9 Hz while the spectrum on the left does not have a single prominent peak per se, but a number of peaks. For this latter spectrum, the y-value (power) is at its highest point for a corresponding x-value (frequency) of 1.3 Hz Thus, the principal frequency component of the signal analyzed on the right lies within the theta tkequency range of 2.5-7 Hz
(urethane anaesthetized rat) while the principal frequency component of the signal analyzed on the left does not (it does however lie within the LIA fiequency range of 0-25
Hz). Because of the overlapping frequency ranges of LIA and theta, it is entirely possible that a signal's most prominent frequency component lies within the theta range yet the signal is not considered to be theta. Care must be taken in analyzing the relative prominence of such peaks. As seen in Figure I. theta signals produce spectra with very clear predominant frequency components (singular, discernible peaks), LIA signals do not (many peaks. lower power). However, it should be stated here that. because of the half amplitude low filter setting of 1 Hz used in this experiment, signals with principal components around this frequency will be depressed, and their power values in respective
FFTs will be somewhat underrepresented.
Another indicator derived from the FFT which can aid in classifying hippocampal field states is a "percent total powei' value. That is, how much of the total power of the obtained spectrum do sinusoids with frequencies in the theta range contribute? For the spectrum on the right of Figure 1,82% of the totd power of the spectrum lies between
2.5 and 7 Hz For the spectrum on the left, only 28% of the total power lies within the same range. 69 la a number of cases it was necessary to determine whetfier different theta trials had different principal frequencies. Within the data acquired for one neuron, in order to determine whether spontaneous theta and tail pinch induced theta had comparable frequencies, the peak fkquencies (provided by FFT) across aH spontaneous theta trials were averaged and compared to the average peak frequency across all tail pinch theta txials. A standard error about the mean (SE) for each average was calculated, and error bars consisting of +/- 2 SEs were constructed lf these error bars overlapped for spontaneous and tail pinch theta, these hippocampal field states were considered equivalent
The first, and potentially most usem, variable used to classitjr cells in relation to the now defined hippocampal field states was their discharge rates. Using the DataWave software package, a firing rate was provided for each segment of discharge data acquired.
The unit firing rates for all segments were then used to calculate a mean firing rate and
SE across all LiA trials, across all spontaneous theta trials, and across all tail pinch theta trials (again, spontaneous theta trials and tail pinch theta triaIs were grouped if these conditions did not differ in mean fkquency). Standard error bars (+/- 2 SE) were constructed about the mean cell discharge rate for each field state. For any hippocampal field states with overiapping enor bars, eel1 discharge rates were considered equivalent
This type of analysis was used extensively and will be discussed in further detaiI beIow.
The second variable used to class* neurons visd-vis the hippocampal EEG was their discharge pattern The burst and oscillation detection algorithms of Kaneoke and
Vitek (1996) were used to objectively and quantitative1y determine the firing pattern of these celIs. These techniques have recently been used in a number of studies, particularly those concerned with basal ganglia neuron kgproperties Boraud et al., 1998;
Ruskin et al., 1999; Magdl et al., 2000; Duque et al., 2000). Kaneoke and Vitek define
"...burst as a period in which a siguficantly higher number of discharges occur compared to other periods in a spike train, while oscillation is defined as a pattern of neuronal activity in which discharges occur periodically." They have developed different algorithms to detect these distinct (but not mutually exclusive) patterns of neuronal discharge in a given stretch of acquired data
Their method of determining whether a unit signal can be considered to contain burst periods is based upon detecting changes in cell discharge rate over small intervals, which they refer to as discharge density. By choosing a proper interval for each stretch of data, the discharge density should follow a Poisson process unless a cell has bursting activity. Using their method, each acquired segment of unit activity was divided into a number of small, equal intervals. The length of each interval (t) is the inverse of the mean firing rate for the data sample. A discharge density histogram (see Figure 4) was then constructed. The histogram shows the discharge density of each interval. That is, the number of intervals containing no cell discharge was plotted on the extreme left (bin
0); next to it is the count for intervals with one cell discharge in it, and so on. Aen, when spikes in a sample of data occm at random, the discharge density distribution may be expected to follow a Poisson distriiution with a mean of 1 (many intends with one spike, fewer with 2, and so on). For a section of data that shows bursting activity, one would observe many intervals with no cell discharges, and many intervals with a high discharge density. For each trial, the distriiution of the obtained histogram was checked to see whether it was sigmLicantly different (~4.05)by Chi square test from a Poisson 71 Fip4. Analyses used to characterk cell discharge pattern
UppPortion Representative unit activity (200 msec long) recorded from a neuron classified as periodic. This cell fkd at a regular pace, with a fairly uniform interval of approximately 32 msec between each discharge.
Bortom Portion Analyses used to class* this cell as periodic. Analyses were performed for an (approximately) 4 second tong segment of data containing the 200 msec long segment depicted in the upper portion of this figure. The discharge density histogram, AC histogram, and Lomb periodograrn were all provided by Kaneoke and
Vitek's (1996) analysis software. The ISI histogram was provided by DataWave software. This segment of data was not considered to contain any burst periods as revealed by the distributions of both the density and IS1 histograms. These distributions are in fact characteristic for a regularly discharging ceIL Indeed, this segment of data was considered to contain periodic discharge activity; the significant peak in the Lomb periodogram dknsthe prominent oscilhtion apparent in the AC histogram. The h horizontat line in this and subsequent Lomb periodograms represents the 0.05 signtficance Ievel. This pkidentifies a principal frequency component of 28.6 Hz in the AC histogram.
n distribution with a mean of 1, and whether the distribution was sipficantly positively skewed. When the distribution of discharge density for an acquired data sample met both criteria, that trial was considered highIy Likely to contain burst periods.
This burst detection method has the advantage of allowing one to quad& the degree of LLburstiness"in a spike train The smaller the value oft, the greater the probability of obtaining a density histogram which tends to follow a Poisson distribution.
A cLBurstinessIndex" (BT)can be used to assess the degree of bursting in a trial: BI=lltm, where tm equals the smallest interval at which the discharge density histogtam of a trial is still significantly different fiom a Poisson distribution Thus, the larger BI is, the greater the likelihood of burst periods in a stretch of data
Using software provided by Dr. Kaneoke, each segment of acquired unit data was subjected to this analysis. A discharge density histogram was first constructed using interval lengths which were 10 times the inverse of the mean firing rate of the data.
Again, the longer the interval used to analyze the data, the greater the probability of identifying burst periods (but the lower the BI due). Lfa spike train did not show burst periods at this length of interval (density histogram distribution did not differ from a
Poisson distriiution with a corresponding mean of lo), the trial was assigned a BI "bursting" when its BI 2 0.5. 74 Interspike interval (ISI) histograms were also produced for each stretch of data as an additional verification of inferences drawn from the Kaneoke and Vitek burst detection method. An IS1 histogram indicates the frequency of occurrence of intervals of different size in a stretch of cell discharge data perkel et al., 1967). All intervals between spikes which were shorter than 5 msec (a 5 msec bin width was used here) were counted in the extreme left bin, all intervals between spikes which were between 5 and 10 msec in length were counted in the next bin, and so on up to interval lengths of 95 to 100 msec (see Figure 4). For a stretch of data containing burst periods, one would expect to obtain a high count of short intewals (the intraburst intervals) and a high count of long intervals (the interburst intervals). The IS1 histogram method is a usell method for assessing whether a spike train has burst periods, but has its limitations (Kaneoke and Vitek, 1996), and can not provide a quantitative index (such as BI) for the degree of bursting in a trial. These analyses were performed for every trial in every neuron. For each cell (after verification with the IS1 method), the percentages of LIA, spontaneous theta, and tail pinch theta trials which were considered bursting (BI 2 0.5) were noted Additionally, a mean BI +/- 2 SE was calculated for each hippocampal field state. Overlapping error bars indicated no difference in degree of bursting. A discharge density histogram distn'bution sigdicady different from a Poisson distribution is obtained when a spike train contains burst periods; it is also obtained when the unit osciUates, or iim in a periodic pattern Such a neuron would produce a discharge density distniution which would dBer from a Poisson distribution as it would be quasi-normal with a mean of 1, That is, if t is the inverse of the mean firing rate for a 75 neuron, a cell that discharges at a regular pace with a dorminterval between each spike would produce a discharge density of 1 spikel intenml. This situation is depicted in Figure 4. The periodicity, or regularity, of the neuron's discharges are clear: they occur, on average, every 32 msec. This is expressed as a quasi-normal distribution with a mean of 1 in the density histogram, and as a quasi-normal distniution with a peak at the bin for 30 to 35 msec intervals in the IS1 histogram. CeUs such as this one are not classified as having burst periods however, as the skewness of their discharge density distribution is found not to differ significantly fiom zero (while bursting neurons will have positively skewed density distribution). A spike train was considered periodic when its auto-correlation (AC) histogram revealed statistically significant periodicity. In an autocorrelation, a series of data is correlated with itself as the data is shifted in time against the original. To construct an AC histogram, one count (y-axis) is added every time two events (one spike in the original signal, one spike in the duplicate signal) occur within a prescribed bin width for the corresponding time (x-axis). An example for periodic spike data can be seen in the bottom portion of Figure 4. For each spike train, the software furnished by Dr. Kaneoke provided outlines of AC histograms constructed with bin widths of 10 msec, over a total lag period of 500 msec. The significance of periodicity in an AC histogram was estimated by the Lomb periodogram (Press et al., 1992), with the analyzed Erequency range between 0 and 50 Hz As this periodogram is in essence a power spectrum of the AC histogram, any prominent kquencies within the histogram will be revealed (see Figure 4). For these FFTs, the y-axis is relative power which ranges from 0 to 1. ReIative power is caIcuIated by dividing all y-values by the maximum y-value (maximum 76 power of most prominent peak). This Lomb penodogram provides a test of significance of the most prominent peaks against the null hypothesis: a power spectrum derived fiom an AC histogram of random independent Gaussian celI discharges. In this study, it was postulated that a spike train bad a periodic discharge rate at the frequencies detected when the null hypothesis was rejected at the 0.05 level (Ruskin et al., 1999). It should be noted that data derived fiom this analysis were only considered viable if the putative piodic interval of a spike train was shorter than 500 msec (2 spikedsec) as only 500 msec of each trial were analyzed For the discharge density and AC histograms produced, the absolute values of the y-axis are not considered important It is the relative shapes of these distributions that are relevant; thus, Dr. Kaneoke's software does not provide y-axis values. The auttxonelation and Lomb periodogram analyses was performed on each acquired trial for each cell. Any neuron which had any trials showing a significant peak in the periodogram was considered to be periodic. Within a neuron, the percentage of LIA trials versus the percentage of theta trials (both spontaneous and tail pinch grouped) that showed periodic firing was noted One final measure was obtained to assess how a neuron's discharge pattern might be related to the ongoing hippocampal EEG. A cross-correlation function (CCF) was obtained for each trial, for each neuron. Digitized cell and field activity were split into 3.85 second sections (thus, any spike data acquued beyond the last filled field sample buffer was discarded), unit activity was convolved with a Gaussian kemeI (standard deviation of 15 msec) and lagged for +l second and -1 second about the peak positivity of the field signal. For each moment of lag, the two signals were cross-correIated using a 77 frequency domain algorithm (Press et al., 1992),and a standardized Pmonconelation coefficient (rho) was calculated. The resulting numbers were plotted as y-values for each x-value of lag time. A flat CCF indicates little correlation between spike events and the field signal. An oscillatory CCF indicates weak or strong (depending on the rho values) correlation between spike events and the field signal. If a trial could be split into a number of 3.85 second sections, the average CCF over all sections was obtained For each trial, the shape and maximum rho value for their respective CCFs were recorded. Finally, electrode placement was analyzed Once the brain had been stored for at least 24 hours in the sucrose-formalin mixture, the brain was blocked and mounted into a Leica 1800 cryostat, where 30 pm thick coronal sections were sliced. Brain slices were mounted on slides and, under a microscope, cell recording positions were assessed by identifying blue dots (Pontamine Sky Blue) andlor tapering electrode tracks. Results-Experiment One I. Histology In total, cell discharge profiles were established for 175 neurons. Data obtained permitted the broad distinction of hippwampal theta-related and non-related basal ganglia neurons. Ofthe 175 neurons recorded, 121 (69%) were classiiied as being theta- related, while 54 (3 1 %) were classified as being non-related. Of the 175 recordings, 45 were made in the SN. Pontamine Sky Blue dots for 16 recording sites were localized histologically to be in this structure, one unstained site was calculated in a track relative to a blue dot, and the locations of 28 unstained sites were 78 determined Eom histologically located tracks and electrode depth measures. Another 45 recordings were made in the GP. Blue dots for eight recording sites were localized histologically to be in this structure, while the locations of 37 unstained sites were determined bm histologically located tracks and electrode depth measures. Sixty-two neurons were recorded hmthe CPu. Twenty-four of the recording locations were stained, one unstained site was calculated in a track relative to a biue dot, and the locations of 37 unstained sites were determined fiom histologically located tracks and electrode depth measures. Finally, blue dots were located for 22 recording sites outside basal ganglia nuclei, and one site was calcuIated in a track relative to one of these. These recording sites were predominantly located in ventral aspects of the thalamus. Others were in areas just peripheral to the SN, GP, and CPu. Diagrammatic reco~ctionsof the locations of the 70 stained recording sites and 3 recording sites calculated in tracks relative to these are presented in Figures 5,6, and 7. 11. Classification Potentially, the analyses performed codd have revealed that basal gangiia neurons were related to the hippocampal field state in a number of ways. This was not the case. Most strikingly, not a single trial for any neuron could reliably be determined as showing bursting characteristics based on Kaneoke and Vitek's (1996) algorithm and the ISI histogram analysis. Thus, no distinction between theta related cells and non-related cells within the SN, GP, or CPu can be made using these criteria A number of neurons did produce spike trains that contained a significant periodicity, as revealed by the auto-correlation and Lomb periodogram analyses. However, for many neurons, there was no consistent pattern in the occurrence of the 79 Figure 5. Diagtammatic recwstructions of the histological Iocatiom of recording sites in the CPu and nurounding areas. CircIes represent cells classified as theta ON, squares represent cells classified as theta OFF, and triangles represent cells ciassified as non-related These symboIs were filled if the cell was classified as periodic. Horizontal sections were modified fkom Swanson Maps (1992). 81 Figure 6. Diagrammatic reconstrwtions of the histological locations of recording sites in the GP, ventral thalamus, and surrounding areas. Circles represent cells classified as theta ON, squares represent cells classified as theta OFF, and triangles represent cells classified as non-related These symboIs were filled if the cell was classified as periodic. Horizontal sections were modified htn Swanson Maps (1992). 83 Figure 7. Diagrammatic reconstructions of the histological locations of recording sites in the SN,ventral thaIamus, and surrou~dingareas. Circles represent cells classified as theta ON, squares represent ceIls classified as theta OFF, and triangles represent cells classified as non-related. These symbols were filled ifthe cell was classified as periodic. Horizontal sections were modified hmSwanson Maps (1992). 85 periodic trials as related to the hippocampal field state (this is discussed in greater detail later). The cross-correlation analysis performed produced similar results. Of 175 neurons analyzed, only 27 produced one or more spike trains which showed any correlation with the phase of the hippocampal field activity. When this did occur, it was often only one trial per cell which showed any oscillation in its CCF. Furthermore, there was no consistent pattern in the occurrence of the crosscorrelated trials across hippocampal field states. Oscillatory CCFs revealed only weak to moderate cross- correlations, with functions very rarely passing +/- 0.2 rho and never reaching +/- 0.4 rho. Finally, there appeared to be no consistent preferential phase at which these CCFs crossed the 0 lag time line (across all cross-correlated neurons, there was no preferential phase of the theta wave at which discharges occurred). A definite distinction between theta-related and non-related neurons was provided by the rate analyses. A clear distribution of neurons had discharge rates which were reiated to the state of the hippocampal EEG, while another distribution had discharge rates which were not related to the state of the hippocampal EEG. Within the so-called theta-related group of neurons the manner ia which a neuron's discharge rate was related to the hippocampal field activity merserved to sub-c~assifythese neurons. Each neuron categorized as theta ON had a mean discharge rate during the LIA field state which was lower, by at least 4 SEs, than its mean discharge rate during the theta states (both spontaneous and tail pinch induced theta if both were obtainable). Each neuron categorized as theta OFF had a mean discharge rate during the LIA field state which was higher, by at least 4 SEs, than its mean discharge rate during 86 the theta state(s). Neurons within each of these groups may or may not have had one or more acquired trials in which the spike train was analyzed to be periodic. Thus, any theta ON or theta OFF neuron could be WershIassified as periodic if any of its analyzed spike trains produced an oscillatory AC histogram and subsequent significant peak in the Lomb periodogram. If no trials produced such a peak, the neuron was classified as non- periodic. Ergo, within the theta-related group, a cell could be potentially classified in one of four subgroups: theta ON periodic, theta ON non-periodic, theta OFF periodic, and theta OFF non-periodic. Cells were considered non-related if they did not display significant differences in cell discharge rates between the hppocampd LIA and field state(s). Two non-related neurons located in the Chand one non-related neuron located in the ventral posteromedial thdamic nucleus (VPM), presented some interesting exceptions to this rule. Two of these (one CPu neuron aud the VFM neuron) were considered sensory activated. Conditions for sensory activated classification were outlined by Hanada et d. in 1999: 1) there is no significant difference in discharge rate between spontaneously occurring theta and LM, but there is an increase in discharge rate in the tail pinch condition relative to the other states; and 2) theta field frequencies during spontaneously occurring theta and tail pinch induced theta were equivaIent as shown by overlapping error bars. The other CPu neuron was considered sensory inactivated. Hanada et al. also descni the criteria for classifjing a neuron in this group: 1) there is no significant difference in discharge rate between spontaneously occuning theta and LIA, but there is a decrease in discbarge rate in the tail pinch condition relative to the other states; and 2) theta field fiequencies during spontaneously occurring theta and tail pinch induced theta were considered equivalent. The non-related neurons in the SN and GP were all classified as non-sensory: cell discharge rates were considered equivalent amss LIA, spontaneous theta (if it was recorded), and tail pinch theta field states. In the same way as theta-related neurons could be, non-rebted neurons could either be periodic or non- periodic. Thus, witbin the non-related group, a ceII could be potentially classified as one of six subtypes: non-related periodic, wn-related non-periodic, sensory activated periodic, sensory activated non-periodic, sensory inactivated periodic, and sensory inactivated non-periodic. However, no sensory activated non-periodic neurons were recorded, and the sensory inactivated neuron that was recorded was periodic. Thus, for this study, only four of these potential classifications will be discussed. Sample recordings and representative analyses for each category of cell are presented in Figures 8 to 15. The top panel of Figure 8A shows the dynamic cell behavior of a representative theta ON periodic cell (Iocated in the CPu) during a transition from hippocampal LIA to tail pinch induced theta. An increase in cell discharge rate is clearly apparent. The three bottom panels display representative acquired hals (ody 3.85 seconds displayed, although trials may have been longer) from which data were obtained and used to classify the neuron, Again, a lower cell discharge rate dhgthe representative LIA trid (7.8 spikes/sec) as compared to the theta trials is readily apparent. This neuron had a mean fhing rate of 7.7 +I- 0.1 splkes'sec over all LIA trials. There does not appear to be any difference in cell discharge rate between the seemingly lower fkcpency spontaneous (22.6 spikes/sec) and higher kpency tail pinch induced (21 spikedsec) theta trials presented. Meed, the cell discharge rates for these field states were considered 88 equivalent as there was overlap between the error bars const~~ctedaround the mean discharge rate over all spontaneous theta trials (18.2 +/- 8.8 spikes/sec) and the enor bars constructed around the mean discharge rate over all tail pinch theta trials (22.5 +/- 2.2 spikesisec). As the cell firing rate is high, the periodic discharge pattern of this neuron is difficult to distinguish in the presented traces, but is revealed in the accompanying analyses seen in Figure 8B. The analyses used to classify this theta ON periodic neuron are shown for the presented LI& spontaneous theta, and tail pinch theta trials in respective rows. The first column of analyses are the msused to distinguish the field states. Prominent peaks are seen for the theta trials but less so for the LIA trial. The LIA trial depicted here had a principal fiequency component of 1.6 Hz. The apparent difference in frequency between the spontaneous theta trial and tail pinch theta trial presented in Part A is contimed in the bottom two FFTs of Part B. The spontaneous theta shown has a main fiequency of 3.4 Hz, while the tail pinch theta occurred at 4.4 Hz Furthermore, the tail pinch theta shorn here might be considered more robust than the spontaneous theta as the peak in its FFT is taller and better dehned. This implies that most of the power in the tail pinch signal was contributed by a pure 4.4 Hz sinusoid There appears to be a slightly more scattered distriiution of power for the spontaneous theta signal, suggesting that a number of sinusoids were summed to create the observed signal. The mean principal kquency over all spontaneous theta trials for this neuron was 3.5 +/- 0.3 Hz. The mean fiequency over all tail pinch induced theta trials was 4.6 +/- 0.3 Hz. Representative analyses used to classifjl this neuron as periodic are displayed in the second and third columns of Part B. Scrutiny of the AC histogram (second colum) 89 Figwe 8A Top PaneL Dyaamic behavior of a theta ON periodic cell recorded hm the CPu during the transition from LM to tail pinch induced theta. Bonorn Panels. Representative segments of cell discharge during LIA, spontaneous theta, and tail pinch theta hippocam@ field states used to classify this cell as theta ON periodic. Respective field and unit amplitude scales apply to each segment. Figure 8B. Corresponding analyses used to distinguish hippocmpaI fieId states and classify this cell as periodic. The FFT analysis for each respective hippocampal field data segment in Part A is shown in the first column. The AC histogram and accompanying Lomb periodogram far each respective segment of cell discharge activity in Part A are sbown in the second and third columns. lhscell was classified as periodic as the Lomb periodogram for the tail pinch theta segment shown in Part A contained a significant peak. The last colurnn depicts the CCFs constructed for the respective lmit and fieId activity shown in Part A. - 1sec LIA Tail pinch theta - - - 1S8C lsec lsec LIA Spontaneous Tail pinch theta theta 92 for the spike data presented for the LIA trail in Part A reveals no apparent periodicity or oscillation in the occurrence of the peaks. This is corned by the respecttve Lomb periodogram (third column); the spectrum of the LIA auto-correIation "signal" has no peaks which are si@cantIy different from those expected for random, non-periodic data Although the AC histogram for the spontaneous theta trial appears to have some oscillation in it, spectral analysis reveals no significant principal frequencies. The AC histogram for the unit discharge data presented for the tail pinch theta trial in Part A has an even greater degree of periodicity in the occurrence of its peaks. Indeed, for this trial, the spectrum of the AC histogram did contain a prominent peak which significantIy (p4.024) exceeded tbe level one would expect for a spectrum derived fiom the AC histogram for random, non-periodic data. Thus, for this trial, the neuron was considered to discharge in a periodic manner, and as such, was classified as periodic. The principal frequency of the periodic activity in this tail pinch spike train was 19.5 & quite different i?om the principal frequency of the simultaneously recorded theta wave. This lack of cotrefation between the spike train periodicity and the hippoampal field state is fUrtber confumed by the CCF generated for this tn'al (fodcolumn). There is no sigmficant oscillation in the CCF for this trial, or for either the spontaneous theta or LIA trials. It should be restated that no neurons analyzed bad discharge profiles which contained periods of burst activity. Therefore, any periodicity revealed in a spike train entails that the neuron discharged single spikes (and not rhythmic bursts of spikes) in a regular or periodic khion, Thus, the periodicity of the neurond discharge should be similar to the mean Gring rate. This was always the case for analyzed trials which revealed a periodic discharge pattern: the hpency of the periodic discharge closely 93 matched the mean firing rate ofthe cell forthat trial. This is evidenced in the tail pinch theta analysis presented here: the frequency revealed by the Lomb periodogram (19.5 Hz), and the figrate for this neuron during this trid (21 spikes/sec) are almost a 1: 1 ratio. This phenomenon is important as it firrther codkm one aspect in which these basal ganglia neurons are not related to the hippocampal field state. For example, the fact that a neuron has a higher mean fhquency of periodicity in its discharge pattern during theta states as compared to the LIA state has nothing to do with the neuron's periodic discharge behavior and everythmg to do with the mean rate of finng of the neuron. For a non-bursting periodic neuron, one should not be concerned with comparing mean tiequencies of periodicity (provided by the Lomb periodogram) because these values are dependant on the mean firing rate of a cell (by means of which, in this case, the neuron has been classified as theta-related or not). It is the relative percentage of periodic trials during each field state (for each individual neuron) hmwhich one might infer a relationship between a neuron's periodic discharge behavior and the hippocampal field state (this type of analysis will be presented below). The top panel of Figure 9A shows the dynamic celi behavior of a representative theta ON non-periodic cell (located in the GP) during a transition from hippocampal LIA to tail pinch induced theta An increase in cell discharge rate is readily apparent. The three bottom panels display representative squired trials from which data were obtained and used to classify the neuron. Again, a lower cell discharge rate during the representative LIA trid (0.5 spikes/sec) as compared to the theta trials can be seen This neuron had a mean firing rate of 0.3 +/- 0.3 spikes/sec over all LIA trials. For the trials presented for this neuron, it appears to have a lower discharge rate duriag spontaneous 94 theta (13.1 sprkeslsec) than during tail pinch theta (18.1 splkes/sec). However, upon @on of all trials for each state, the cell discharge rates for these field states were considered equivalent since there was overlap between the error bars constructed around the mean discharge rate over dl spontaneous theta trials (10.3 +/- 4.3 spikesisec) and the error bars constructed around the mean discharge rate over all tail pinch theta trials (12.0 +I- 7.8 spikeslsec). Although the cell tiring rate is quite high, the lack of @odic discharge pattern for this neuron is not difficult to perceive in the presented trials. The analyses used to clamthis theta ON non-periodic neuron are presented in Figure 9B. In the FFT analyses, sizeable peaks are seen for all the mals. However, upon comparing the relative promineme, number, and position of the peaks in each FFT, LIA and theta trials are easily distinguishable. The LIA trial depicted here had a principal Eequency component of 1.6 Hi. The apparent diffkrence in fiequwcy between the spontaneous theta trial and the tad pinch theta trial presented in Part A is confirmed in the bottom two FFTs of Part B. The sjmntaneous theta shown has a main frequency of 3.4 HZ the tail pinch theta has a faster 4.2 Hz. These F?Ts suggest that both signals are somewhat corrupt, but not to a degree such that their distinction fiom LIA is in confusion. The mean principal fresueucy over all spontaneous theta triaIs for this neuron was 3.4 +/- 0.0 Hz. The mean frequency over all tad pinch induced theta trials was 4.0 +I- 0.3 fEz RepmWive analyses suggesting that this neuron did not discharge in a periodic manner are displayed in the second and third coIlmtns of Part B. None of the AC histograms preseated suggest any periodicity- This is amfirmed by the lack of any simcant peaks in the @ve Lomb periodograms. As all trials analyzed for this 95 Figure 9A. Top Panel. Dynamic behavior of a theta ON non-periodic cell recorded 60m the GP during the transition Erom LIA to tail pinch induced theta. Bottom Punels. Representative segments of cell discharge during LIA, spontaneous theta, and tail pinch theta hippocampal field states used to classlfjr this cell as theta ON non-periodic. Respective field and unit amplitude scales apply to each segment. Figure 9B. Corresponding analyses used to distinguish hippocampal field states and classifL this cell as non-periodic. The FFT analysis for each respective hippocampal field data segment in Part A is shown in the first column. The AC histogram and accompanying Lomb periodogram for each respective segment of cell discharge activity in Part A are shown in the second and third columns. The last column depicts the CCFs constructed for the respectwe unit and field activity shown in Part A 98 neuron followed this pattern, the neuron was classified as non-periodic. It shouid be mentioned that the auto-correlation and subsequent spectral analyses for the LIA trials of this neuron are not dependable as the neuron discharged at a firing rate of 0.5 spikes/sec during this state. As mentioned in the Methods section for this experiment, Kaneoke and Vitek's software analyzed 500 msec portions of the acqrred data. Therefore, relevant data for neurons with mean firing rates below 2 spikeslsec might not be detected by the analysis package. However, it is improbable that any periodic infoxmation represented by the discharges of a neuron with a potential periodicity in its firing pattern lower than 2 Hz could be considered relevant to the 2.5-7 Hz theta signal. The CCF produced for the presented LIA trial is flat. Again, this probably has to do with the neuron's very low firing rate during this mal. As the CCF is constructed by shifting the unit signal +I- 1 sec about the peak positivity of the field signal, discharges for neurons with mean fhgrates equal to or less than 0.5 spikedsec might fail to be detected, The CCF obtained for the spontaneous theta trial shows very little oscillation. The CCF produced for the tail pinch theta trial is one of the few that suggested a moderate correlation between the unit discharge aud the theta wave, with a spike preference for the positive phase of the wave. The dynarmc cell behavior of a repmmtative theta OFF periodic cell (located in the CPu) during a transition fiom hipjwampal LIA to tail pinch induced theta is presented in the top panel of Figure IOk A drastic reduction in the number of cell discharges upon the evocation of tail pinch theta can be seen. The three bottom paneIs display representative acquired trials fiom which data were obtained and used to classifL the neuron. A higher cell discharge rate during the representative LIA trial (26.8 99 spikedsec) as compared to the theta trials is clear. This neuron had a mean frring rate of 27.0 +I- 0.5 spikeslsec over all LLA triaIs. The neuron appeared to decrease its discharge rate with increasing theta frequencies. That is, in the presented trials, the cell had a higher discharge rate during spontaneous theta (14.6 spikedsec) than during tail pinch theta (6.0 spikes/sec). However, upon considering all trials for each state, the cell discharge rates for these field states were considered equivalent since there was overlap between the error bars constructed around the mean discharge rate over all spontaneous theta trials (17.7 +/- 6.2 spikes/sec) and the emr bars constructed around the mean discharge rate over all tail pinch theta trials (9.0 +/- 3.0 spikeslsec). For this neuron. analyses revealed that during the presented LIA state, unit firing was periodic. This is difficult to see in the presented trace of Figure I OA as the mean firing rate is so high. but is apparent upon consideration of the accompanying analyses presented in Figure 10B. In the FFT analyses, sizeable peaks are seen ford the trials. Yet. LIA and theta trials are distinguishable. The LLA uial depicted here had a principal fiequency component of 1.9 Hz. This peak was large, but not as prominent as the theta peaks: as is characteristic for the FFT of an LIA signal, a number of peaks are visible. The apparent difference in fiequency between the spontaneous theta trial and tail pinch theta trial presented in Part A is confirmed in the bottom two FFTs of Part B. The spontaneous theta shown has a main fiequency of 3.6 Hz, the tail pinch theta has a main fiequency of 4.2 Hz. The mean principal field hcpency over all spontaneous theta trials for this neuron was 3.2 +I- 0.8 Hz The mean fkquency over all tail pinch induced theta trials was 4.2 +I- 0.0 Hz. 100 Figm 10A. Top Panel. Dynamic behavior of a theta OFF periodic cell recorded fiom the CPu during the transition hmLXA to tail pinch induced theta Bottom Panels. Repmentative segments of cell discharge during LIA, spontaneous theta, and tail pinch theta hippocampal field states used to class* this cell as theta OFF periodic. Respective field and unit amplitude scales apply to each segment. Figure 10B. Corresponding analyses used to distinguish hippocampal field states and classlfjl this cell as periodic. The FFT analysis for each respective hippocampal field data segment in Part A is shown in the first column. The AC histogram and accompanying Lomb periodogram for each respective segment of cell discharge activity in Part A are shown in the second and third columns. This cell was classified as periodic as the Lomb periodogram for the LIA segment shown in Part A contained a significant peak. The last column depicts the CCFs constructed for the respective unit and field activity shown in Part A. 103 The representative analyses that suggest that this neuron discharged in a periodic mmer during the presented LIA trial are displayed in the respective second and third columns of Part B. An underlying periodicity in the occurrence of the peaks, so clear in the AC histogram, is confirmed by the significant peak (p=0.00014) in the Lomb periodogram. Thus, for this trial, the neuron was considered to discharge in a periodic manner, and as such, was classified as periodic. The principal fiequency of the periodic activity in this LIA spike train was 28.7 Hz, quite disparate fiom the principal fiequency of the simultaneously recorded theta wave, but similar to the frequency of firing for this trial (26.8 spikedsec). Although the spontaneous theta AC histogram appeared to approach a periodic signal, it produced no significant spectral peak, nor did the AC histogram for the tail pinch theta trial. The oscillations in the CCFs produced for the LIA and spontaneous theta trials are insignificant. The CCF produced for the tail pinch theta trial suggests a moderate correIation between the unit discharge and the theta wave, with a spike discharge preference for the negative phase of the wave. The top panel of Figure I 1A shows the dynamic cell behavior of a representative theta OFF non-periodic cell (located in the SN) during a transition fiom hippocampal LLA to tail pinch induced theta. A decrease in cell discharge rate can be perceived The three bottom panels dispIay representative acquired trials hmwhich data were obtained and used to classify the neuron. Again, a higher celI discharge rate during the representative LIA trial (38.3 spikedsec) as compared to the theta trials is apparent. This neuron had a mean firing rate of 37.2 +/- 2.3 spikdsec over all LIA trials. There seems to be little difference between the firing rate of this neuron during simultaneously 104 recorded spontaneous theta (26 spikes/sec) and the firing rate of this neuron during simultaneously recorded tail pinch theta (29.3 spikes/sec). The cell discharge rates for these field states were considered equivalent since there was overlap between the error bars constructed around the mean discharge rate over all spontaneous theta trials (27.8 +I- 1.6 spikeslsec) and the error bars constructed around the mean discharge rate over all tail pinch theta trials (28.4 +/- 1.8 spikedsec). Without consulting the accompanying analyses (Figure 1IB), it is difficult to assess whether this neuron had a non-periodic firing pattern. The prominence of the peaks in the FFTs for the theta triab relative to the peak for the LIA trial suggests the differences between these signals. The LIA trial depicted here had a principal fkquency component of 1.6 Hz This peak was large, but not as prominent as the theta peaks, as it was not entirely distinct. The apparent difference in fkquency between the spontaneous theta trial and tail pinch theta td presented in Part A is codinned by the two FITSshown in the lower section of Part B. The spontaneous theta shown has a relatively low main frequency of 2.6 Hz, while tail pinch theta occurred at a frequency of 3.4 Hz While the FFT for the tail pinch theta suggests that this signal was pure, the FFT for the spontaneous theta trial is less clear. Quite a sigdicant peak can be seen in this FFT at a lower fkquency than the principal hquency. This indicates that the signal presented may be representative of a nearly completed transition between LIA and spontaneous theta. The mean principal kquency over all spontaneous theta trials for this neuron was 2.5 +/- 0.1 Hz. The mean fiqyency over all tad pinch induced theta trials was 3.2 +/- 0.3 Hi. 10s Figure 1IA Top Panel. Dynamic behavim of a theta OFF non-periodic cell recorded from the SN during the transition from LIA to tail pinch induced theta Bonom Panels. Representative segments of cell discharge during LLA, spontaneous theta, and tail pinch theta hippocampal field states used to classify this cell as theta OFF non-periodic. Respective field and unit amplitude scales apply to each segment. Figure 1 13. Conesponding analyses used to distinguish hippocampal field states and classitjl this cell as non-periodic. The FFT analysis for each respective hippocampd field data segment in Part A is shown in the first column. The AC histogram and accompanying Lomb periodogram for each respective segment of cell discharge activity in Part A are shown in the second and third columns. The last column depicts the CCFs constructed for the respective unit and field activity shown in Part A. 1sec LIA t Tail pinch theta - - - 1 SBC 1- 1 S8C LIA Spontaneous Tail pinch theta theta 108 Representative analyses showing that this mmon did not discharge in a periodic manner are displayed in the second and third columns of Part B. Neither of the AC histograms for the LIA and spontaneous theta state suggest any periodicity. This is confirmed by the lack of any si@cant peaks in the respective Lomb periodograms. The AC histogram for the tail pinch state seems to approach a periodic signal, but is not significantly oscillatory as shown by the respective Lomb periodogram. As no trials analyzed for this neuron produced Lamb periodograms with significant peaks, the neuron was classified as non-periodic. The oscillations in the CCFs produced for the presented LIA and spontaneous theta trials are insignificant, The CCF produced for the tail pinch theta trial suggests a moderate correlation between the unit discharge and the theta wave, with a spike preference for the positive phase of the wave. These analyses hinted at one other manner in which it is possible to Wersub classifj. any cells within the four groups of theta-related neurons whose discharge properties were recorded during both the hippocampal theta field states. Any theta ONIOFF periodiclnon-periodic neuron codd be fiirther sub-classified depending on its relative discharge rate during spontaneous and tail pinch theta states. For example, a theta ON neuron could have a higher mean discharge rate (by at least 4 SEs) during tail pinch induced theta (higher frequency) as compared to during spontaneous theta (lower fkquency). This cell might be considered to be in a different group of cells than a theta ON neuron that had an equivalent or lower mean discharge rate during tail pinch induced theta as compared to during spontaneous Wa. ConverseIy, a theta OFF neuron codd have a lower mean discharge rate (by at least 4 SEs) during tail pinch induced theta 109 (higher fkquency) as compared to during spontaneous theCa (lower fkquency), This cell might be considered to be in a different group of cells than a theta OFF neuron that had an equivalent or higher mean discharge rate during tail pinch induced theta as compared to during spontaneous theta. Although all of the above examples followed the latter pattern (equivalent discharge rates across different theta states), the former pattern is certainly possible and was present in the data This type of classification has been applied to neurons before (Colom et d., 1987; Ford et al., 1989), but more stringent rdes for distinguishing between these two patterns of unit discharge were used Specifically, electrical stimulation of components of the ascending system was used This provides: 1) a wide range of potential hippocampal theta frequencies; and, 2) precise control of the hippocampal theta frequency. Using these techniques coupled with linear regression analysis, the authors were able to distingwgwshbetween "linear" and "non-linear" theta- related cells. Because it is possible to obtain only a limited range of theta frequencies during the spontaneous and tail pinch field states, this sub-classification scheme is never used in experiments where one does not have the degree of control over the field state that is provided by electrical stimulation. Figure 12A displays typical acquired data for a representative non-related periodic neuron recorded fkom the SN. The top pane1 shows how the discharge rate and pattern were unaffkcted by changes in the hippocampal field state. The three bottom panels display representative aqumd trials from which data were obtained and used to class@ the neuron. The cell discharge rate remains unchanged across the three hippocampal states (8.3 splkedsec discharge rate during the qmmtative LIA trial, 9.6 +I- 1.6 spikedsec mean discharge rate over all LIA trials; 8.8 spikedsec discharge rate during the 110 representative spontaneous theta trial, 8.9 +/- 0.2 spikes/sec mean discharge rate over all spontaneous theta trials, 9 sphdsec discharge rate during the representative tail pinch trial, 8.2 +I- 1.0 spikes/sec mean discharge rate over all tail pinch theta trials). The periodic nature of this neuron's discharge pattern is qute apparent in all three presented Accompanying analyses for this non-related periodic neuron are shown in Figure 12B. In the FFT analyses, sizeable peaks are seen for all the trials. However, LIA and theta trials are distinguishable based on the number and position of discernible peaks. The LIA trial depicted here had a principal fhquency component of 1.8 Hz. The apparent difference iu frequency between the spontaneous theta trial and tail pinch theta trial presented in Part A is confirmed in the bottom two FFTs of Part B. The spontaneous theta shown has a main fiequency of 3.4 Hz, the tail pinch theta has a main fiequency of 4.2 Hz The peak for both FFTs is evident. The mean principal fiesuency over all spontaneous theta trials for this neuron was 3.4 +/- 0.0 Hz The mean fkequency over all tail pinch induced theta trials was 4.0 +/- 0.2 Hz. The representative analyses that suggest that this neuron discharged in a periodic manner are displayed in the second and third coturnas of Part B. An underlying periodicity in the occurrence of the peaks in the AC histograms for all trials is confirmed by significant peaks (p4.00074 for the LLA trial, p4.00016 for the spontaneous theta trial, and p4.O 18 for tk tail pinch trial) in the respective Lomb periodograms. Thus, this neuron was classified as periodic. The principal fieqwncy of the periodic activity in the LIA spike train was 7.6 Hz, quite disparate from ?he principal fiqmcy of the simultaneously recorded LIA, but simiIar to the kquency of £iring for this ma1 (8.3 111 Figure 12A Top Panel. Dynamic behavior of a non-related periodic cell recorded fiom the SN during the transition from LIA to taiI pinch induced theta. Bottom Pds. Representative segments of cell discharge during LIA, spontaneous theta, and tail piach theta hippocampal field states used to class* this cell as non-related periodic. Respective field and unit amplitude scales apply to each segment. Figure 128. Corresponding analyses used to distinguish hippocampal field states and classify this cell as periodic. The FFT analysis for each respective hippocampal field data segment in Part A is shown in the first column. The AC histogram and accompanying Lomb pendogram for each respective segment of cell discharge activity in Part A are shown in the second and third columns. This cell was classified as periodic as the Lomb periodograms for all data segments shown in Part A contained significant peaks. The last column depicts the CCFs constructed for the respective unit and field activity shown in Part A. 0-5mV 1 1111- 11111111111 111111111111 111 11111111111111i1111111111111111111111111111111IIIIIIIIU 1sec LIA Tall pinch theta - - - lsec t sec 1S8C LIA Spontaneous Tail pinch theta theta 114 spikedsec). The principal fiequency of the periodic activity in the spontaneous theta spike train was 8.1 Hz, not a good match for the principal fiequency of the simultaneously recorded theta, but a good match to the frequency of firing for this trial (8.8 spikeslsec). The principal eequency of the periodic activity in the tail pinch theta spike train was 9.1 Hz, quite distant from the principal kequency of the simultaneously recorded theta, but similar to the fiequency of firing for this trial (8.2 spikeslsec). The low frequencies of periodicity account for the relative sloppiness of the peaks in the AC histograms. Simply, fewer peaks are resolved in the same time window. None of the CCFs for the represented trials show significant correlations. Figure 13A displays typical acquired data for a representative non-related non- periodic neuron recorded fiom the GP. The top panel shows how the discharge rate and pattern were unaffected by changes in the hippocampal field state. The three bottom panels display representative acquired trials from which data were obtained and used to classify the neuron. Although the signal fiom the unit recording electrode showed some slight amplitude variation (due to animal movement) the cell discharge rate remains unchanged across the three hippocampal states. This neuron discharged at a rate of 1.9 spikesfsec during the representative LIA mai with a mean discharge rate of 1.5 +/- 0.4 spikeslsec mean discharge rate over all LZA trials. It fired at a rate of I -7 spikes/sec during the spontaneous theta trial shown with a mean rate of 2-5 +/- 0.9 spikes/sec over all spontaneous theta trials. Finally, this neuron discharged at a rate of 1.4 spikes/sec during the representative tail pinch trial and 1.5 +/- 1.6 spikes/sec over all tail pinch theta triaIs. The non-periodic nature of this neuron's discharge pattern is quite apparent in all three presented signals. 115 Accompanying analyses for this non-related non-periodic neuron are shown in Figure 13B. The peaks for the FFT analyses are not very prominent as the signal is of low arnpiitude (see Part A). However, LIA and theta trials are distinguishable as a specific prominent peak was difficult to discern in the FIT for the LIA trial, but not in the FFTs for the theta trials. The LIA trial depicted here had a principal Erequency component of 1.6 Hz. Although difficult to differentiate as a result of the amplitude of the signal, the spontaneous theta trial and tail pinch theta trial presented in Part A have different hquencies as confirmed in the bottom two FFTs of Part B. The spontaneous theta shown has a principal fiequency of 3.4 Hz, the tail pinch theta shown has a principal frequency of 4.2 Hz The main peak for both FFTs may be differentiated, yet there appear to be some lower frequency sinusoids in the spontaneous theta sigal. The mean principal fiequency over all spontaneous theta trials for ttus neuron was 3.3 +I- 0.1 Hz. The mean frequency over all tail pinch induced theta trials was 4.5 +/- 0.3 Hz Considering the low tiring rates of this neuron in all three hippocampal field states, none of the AC histograms or Lomb periodogram analyses shown in Figure B are dependable. Nevertheless, at such low firing rates, it is doubtfid that any periodicity that could potentially underlie this neurons £kingpattern is significant in the context of hippocampal th- The CCF for the LIA trial presented here shows ao mss-correlation between the cell discharge pattern and the field activity. The CCF for the spontaneous theta trial shows an osciuation too weak to be significant. Fiiy, whde one half of the CCF presented for the tail pinch theta trial shows a very weak oscillation, the other half is completely flat. Again, considering the low firing rate of this unit during the presented 116 Figure 13A Top Pmel. Dynamic behavior of a non-related non-periodic cell recorded £?om the GP during the transition fiom LIA to tail pinch induced theta. Bottom Panels. Repmeatative segments of cell discharge during U, spontaneous theta, and tail pinch theta hippocampal field states used to classify this cell as non-related non-periodic. Respective field and mit amplitude scales apply to each segment. Figure 13B. Corresponding analyses used to distinguish hippocampal field states and classify this cell as non-periodic. The FFT analysis for each respective hippocampal field data segment in Part A is shown in the first column. The AC histogram and accompanying Lomb periodogram for each respective segment of cell discharge activity in Part A are shown in the second and third columns. The last column depicts the CCFs constructed for the respective unit and field activity shown in Part A. - 1s UA + T~IIpinch theta 0 0 - lsec 1- 1- Tall pinch LIA Spontaneous theta theta 119 tail pinch trial (1.4 spikedsec), it is entirely possible that unit discharges during the trial were extraneous to the +I- 1 second lag window about the peak positivity of the theta signal. Representative field and unit traces for the sensory activated non-periodic neuron in the CPu are presented in Figure 14A As seen in the top panel showing the cell behavior during the transition between the tail pinch and spontaneous theta states, and in the bottom three panels showing the cell behavior during each field state, this neuron only had an elevated firing rate during tail pinch conditions, and not during spontaneous theta or LIA. The representative trials chosen indicate that the neuron did not fire at all during the LIA and spontaneous theta states, and at a reasonable rate (5 spikdsec) during the representative tail pinch condition, despite there being no difference in the theta Frequencies for the spontaneous and tail pinch trid shown. Indeed, this neuron never fired at dl during the LIA state and had an equivalent mean firing rate of 1.3 +/- 2.1 spikedsec over all the spontaneous theta trials, yet maintained a mean tiring rate of 4.9 +/- 0.2 spikedsec over all the tail pinch trials recorded The FFTs displayed in Figure 14B confirm that the spontaneous theta and tail pinch theta depicted in Part A have equivalent frequencies (5.2 Hz for representative trials, 4.9 +/- 0.3 Ht and 5.3 +/- 0.3 Hz means over all trials -vely). The LIA field state was confbned with a pseudo-prominent peak at 2.1 Hz None of the subsequent anaiyses were performed for the presented LIA and spontaneous theta trials as no spikes could be analyzed The neuron was classified as non-pexiodic as no trials produced a signrficant peak in the Lomb periodogram. The CCF on the bottom row of Figure B 120 Figure 14A Top Panel. Dynamic behavior of a sensory activated non-periodic cell recorded fiom the CPu during the transition fiom tail pinch induced to spontaneous theta Bottom Panels. Representative segments of cell discharge during LIA, spontaneous theta, and tail pinch theta hippocampal field states used to class@ this cell as sensory activated non-periodic, ns neuron was generally silent during the LIA and spontaneous theta field states. Figure 14B. Corresponding analyses used to distinguish hippocampal field states and classifL this cell as non-periodic. The FFT analysis for each respective hippocampal field data segment in Part A is shown in the first column. The AC histogram and accompanying Lomb periodogram for the tail pinch theta segment of cell discharge activity in Part A is shown in the second and third columns. The last column depicts the CCF constructed for the unit and field activity of the tail pinch theta data segment shown in Part A - 1 sec Tail pinch t Spontaneous theta theta No cell No cell discharge discharge LIA Spontaneous Tail pinch theta theta 123 shows that dwing the presented tail pinch theta trial the neuron preferentially discharged in a weakly correlated manner during the negative phase of the theta wave. Figure 15A displays typical acquired data for the sensory inactivated periodic neuron located in the CPu. The top panel shows how the discharge rate of the neuron increased during subsequent spontaneous theta following a period of tail pinch theta. The hebottom panels display representative acquired trials fiom which data were obtained and used to classifL this neuron. Although it is difficult to delineate because of the high firing rate of this neuron during all three hippocampal field states, for the trials shown, this neuron fired at similar rates during LIA (25 spikedsec) and spontaneous theta (27.7 spikedsec), and at a somewhat depressed rate during tail pinch theta (22.3 spikeslsec) even though the two theta trials have similar frequencies. On average, this neuron fied at a rate of 24.4 +/- 0.7 spikeslsec during LIA, 28.0 +I- 0.3 during spontaneous theta, and 21 -9 +I- 0.8 spikedsec during tail pinch theta. Again, because of these high firing rates, the periodic nature of this neuron's discharge pattern is not qite apparent in these panels, but is revealed in the accompanying analyses shown in Figure 15B. In the FET analyses, the LIA and theta trials are easily distinguishable because the LIA trial produced an FFT with no single prominent peak, but a pair of depressed peaks. The peak frequency for this LIA trial was 2.9 Hz Indeed, the two theta signals were composed primarily of a 3.6 Hz sinmid, evidenced by prominent peaks at this frequency for the FFTs for both trials. The mean principal frequency over all spontaneous theta trials for this neuron was 3.9 +I- 0-3 Hz, equivalent to the mean fkquency over all tail pinch induced theta trials of 4.0 +/- 0.3 Hz 124 Figure 15A Top Panel. Dynamic behavior ofthe sensory inactivated periodic cell recorded from the CPu during the transition fiom tail pinch induced to spontaneous theta Bonom Panels. Representative segments of cell discharge during LIA, spontaneous theta, and tail pinch theta hipcampal field states used to class@ this cell as sensory inactivated periodic. Respective field and unit amplitude scales apply to each segment. Figure 15B. Corresponding analyses used to distinguish hippocampal field states and classifj. this cell as periodic. The FFT analysis for each respective hippocampal field data segment in Part A is shown in the first column, The AC histogram and accompanying Lomb periodogram for each respective segment of cell discharge activity in Part A are shown in the second and third columns. This cell was classified as periodic as the Lomb periodograrns for all data segments shown in Part A contained significant peaks. The last column depicts the CCFs constructed for the respective unit and field activity shown in Part A I 1sec Tail pinch t Spontaneous theta theta - - - lsec ISBC 1sec LIA Spontaneous Tail pinch theta theta 127 The representative andyses suggesting that this neuron discharged in a periodic manner are displayed in the second and third columns ofpart B. Clear periodicity in the occurrence of the peaks in the AC histograms for all trials is confhed by significant peaks (p4.014 for the LIA trial, p4.0003 1 for the spontaneous theta trial, and p=0.039 for the tail pinch trial) in the respective Lomb periodograms. Thus, this neuron was classified as periodic. The principal fkquenq of the periodic activity in the LIA spike train was 22.4 Hz, not a good match for the principal frequency of the simultaneously recorded LIA, but a good match to the fkquency of unit discharge for this trial (25 spikdsec). The principal Erequency of the periodic activity in the spontaneous theta spike train was 27.6 Hz, quite disparate from the principal frequency of the simultaneously recorded LIA, but airnost exactly the frequency of firing for this trial (27.7 spikedsec). The principal fkquency of the periodic activity in the tail pinch theta spike train was 2 1.6 Hz, quite distant from the principal frequency of the simultaneously recorded theta, but similar to the tiequency of firing for this trial (22.3 spikedsec). None of the CCFs for the represented trials contained significant cross- correlations. IU. Distribution By examining Tables 1 to 4, it is possible to assess the regional distri'butions of theta-related and non-related cells recorded The 45 cells recorded fiom the SN were in a nearIy 1 :1 ratio of related neurons to non-related neurons, while the 45 cells recorded hmthe GP were in a 2:l ratio, and the 62 cells recorded fiom the CPu were in a 3:l ratio. Outside these stmtures, almost 7 times more theta-related cells were recorded than non-related cells. Table 1 - Number and discharge properties of substantla nigra neurons in the urethane anaesthetized rat. Theta-related cells change their firing attern and/or rate between hippocampal EEG field states. In the substantla nigra, cells do not cR ange their firing patterns between field states, but do change their rates. Thus, ON cells have an increased rate of firing during hippocampal theta as compared to LIA, while OFF cells have a decreased firing rate. Non-related cells do not change firing rates across hippocampal field states. The bottom portion of the table indicates the distribution of cells with periodic firing patterns. A number of neurons within the substantia nigra discharged with regular, periodic intervals between each spike. Because no neurons discharged in a bursting mode, the periodicity of these intervals was always closely matched to the mean rate of firing for these neurons. Theta-related Non-related 24 21 ON OFF 15 9 Non- Non- Non- Periodic Periodic Periodic Periodic Periodic Periodic ?? , 3 5 4 ., 18 3 Table 3 - Number and discharge properties of caudate/putamen neurons in the urethane anaesthetized rat (see Table 1 for explanation). Some non-related caudate/putamen neurons were further sub-classified as sensory related. A cell was classified as sensory activated if there was no difference in its discharge rate between spontaneously occurring theta and LIA, but there was an increase in the tail pinch condition compared to spontaneously occurring theta and LIA conditions. In these cases, field frequencies during spontaneous1 occurring theta and tail pinch induced theta were similar. Conversely, a neuron was classi! ied as sensory inactivated if there was no difference in its dischar e rate between spontaneously occurring theta and LIA, but there was a decrease in the tail pinc?I condition compared to spontaneously occurring theta and LIA conditions. Again, theta frequencies during spontaneous and tail pinch conditions were similar. lhab-relatad Nondated 47 15 - lrlQ OFF Non-sensory Sensory i activated inactiva ed & I1 7 i 13 1 1 . . Non* Non. Perkdic , Perbdic Periodic ' Periodic Non- Non- Non- Periodic Periodic Periodic Periodic Periodic Periodic 20 16 5 6 5 8 0 1 1 0 - Table 4 - Number and discharge properties of neurons outside basal ganglia in the urethane anaesthetized rat (see Tables 1 and 3 for explanation). The majority of neurons located outside basal ganglia were in ventral aspects of the thalamus. Seven neurons were recorded within the ventrolateral thalamic nucleus, three within the ventromedial thalamic nucleus, two within the ventral posterolateral thalamic nucleus, two within the ventral posterornedial thalamic nucleus, and one within the anteroventral thalamic nucleus. Two neurons were also recorded within the reticular thalamic nucleus. One neuron was recorded In the ventral part of the medial geniculate nucleus, another in the medial part of the medial geniculate nucleus. A pair of neurons were recorded within the medial lemniscus, one was found within the deep mesencephalic nucleus, and one within the fundus striati, Thefwd8W l4owalatd 20 3 i c 'ON OFF Non-sensory Sensory Sensory , . f* activated inactivated I8 2. 2 1 0 Mil- Non- '. Perkdlc P&&' * Periodic . Periodic " ,. Non- Non- Non- b. ' Periodic Periodic Periodic Periodic Periodic Periodic 2 16 I f 1 1 0 1 0 0 r 132 Scnrtiny of the anatomical IocaIjzation figures alongside Tables 1-4 reveals no strict regional differences in the distriiution of theta-related and non-related cells, although the ratios suggest that there may be more theta-related neurons in the CPu. There were other evident distributions however. Particularly, neurons located in the SNR tended to have much higher firing rates across all hippocampal field states than neurons recorded elsewhere, including the SNC. This phenomenon helped guide and localize electrode placement in the SN. Furthermore, a higher proportion of periodic cells (either theta-related or non-related) were recorded from the SN. Another distribution existed which deals with periodicity and may indirectly be related to the hippocampal field state. As a high number of theta-related cells recorded in the basal ganglia had spike trains which contained periodic neuronal discharge, a usell indicator of theta-relatedness for each neuron might be the proportion of trials in the LIA and theta field states which were classified as periodic (had a significant peak in the Lomb periodogram). As seen in Figure Id, these proportions were sometimes dependent on where the neuron was located and on how its discharge profile was classified It should be restated that only one analyzed trial per neuron had to produce a sigdicant peak in the Lomb periodograrn for that neuron to be classified as periodic. For SN neurons classified as periodic there appears to be a slight trend for theta-related cells (both ON and OFF cells) to have higher proportions of both LIA and theta trials be considered periodic than non-related cells recorded fiom the same structure. Thus, for SN neurons, there may be some positive interaction between their "degree of periodicity" (number of periodic trials) and their relatedness to the hippocampal field state. For periodic GP cells, there is a strong interaction between these factors. Periodic theta-related cells (only theta ON cells as 133 Figure 16. Interactions between degree of periodicity and theta-relatedness for periodic neurons recorded hmthe basal ganglia For each periodic cell recorded fiom the basal ganglia, the proportion of LIA trials considered periodic was plotted against the proportion of theta trials considered periodic. (Spontaneous and tail pinch theta trials were collapsed as no diffiefences in the distribution of periodic trials were observed between these two groups). Separate diagrams were constructed for each structure examined, and the distributions were delineated along the cell type dimension (theta ON, theta OFF, non-related). Clustering and possible interactions are apparent for theta- related cells recorded from the SN and GP, but not for cells recorded fiom the CPu. Overlap was reduced among points by adding and subtracting small random numbers to percentage values. 135 there were no periodic theta OFF cells recorded from the GP) had no LIA trials which contained periodicity. OnIy non-related cek in the GP showed periodic LIA and theta trials. This implies that periodic theta ON cells in the GP were only periodic during theta states, and not during LIA states. No clear reIationship between the theta-relatedness of a cell and its "degree of periodicity" could be identified for the periodic CPu neurons. Discussion-Experiment One The data recorded fkom the 175 neurons suggest that there is a relationship between cellular activity in the basal ganglia and the hippocampus. Sixty-nine percent of all cells recorded exhibited discharge properties whch were related to the hippocampal field state. Discharge profiles were classified using a scheme which was based on Colom and Bland's (1987) but adjusted so that comparisons could be drawn with the basal ganglia literature. Theta-related cells were categorized as ON or OFF depending on changes in their firing rate related to hippocampal field activity. These cells were also classified according to their fbing pattern. In 1987, Colom and Bland proposed that two subtypes of theta ON and theta OFF neurons could be distinguished based on their firing patterns: 1) phasic, which discharge in bursts with a consistent phase relation to each slow wave theta cycle, and 2) tonic, which have regular or irregular discharges with no consistent relation to the phase of the hippocam@ them Using Kaneoke and Vitek's (1996) method for burst detection, none of the cells recorded in this study were considered to discharge in bursts during LIA or theta Thus, no neurons were cIdedas phasic ON or phasic OFF according to Coiom and Bland's scheme- The neurons recorded here would be classified as tonic ON and tonic OFF. In this study, using the Lomb periodogram to identie significant periodicityin the discharge pattern of a cell, a distinction was made between the regular (periodic) and irregular (non-periodic) single- spike firing patterns of tonic neurons. This additional periodic/non-periodic classification is comparable with schemes used to distinguish units recorded fiom the basal ganglia (Boraud et al., 1998; Magdl et d., 2000). It should be noted that periodic neurons do not constitute another category of theta-related cells. Indeed, cells classified as non-related could also be classified as periodic: the intervals between spikes for these cells remained fairly uniform and regular across all hippocampal field states as their firing rates did not change. Neurons with single-spike and bursting discharge patterns, which may or may not be periodic have been found throughout the basal ganglia However, these reports are oflea inconsistent as they appear to depend on the species studied, anaesthetic regime used (if any), and analysis employed to class@ a spike train as periodic, bursting, or both. As the statistical analysis of neuronal firing pattems proves to be a powem tool in the neurophysiological identification of fUnctionaI cell populations, a number of studies over the past 30 years have characterized discharge properties of neostriasal neurons. These studies generally emphasized the same observations: the low firing rates of neurons in this structure and the rhytiuxuc bursting of these neurons under light anaesthesia (for a review see Wilson, 1993). Initially, chloral hydrate or pentobarbital was used as an auaesWic in these prepamtiom, but it was eventually found that the spontaneous activity in animals 137 anaesthetized with urethane most closely resembles that of awake animals (Wilson, 1993). Thus, much of the most recent and significant work in this field has been performed in urethane anaesthetized rats. Although the same model was used for this investigation, the data presented here are not in agreement with the general £indings of burst activity for CPu neurons. This discrepancy can probably be attri'buted to the method used to identtfy burst periods and rhythmic firing. Kaneoke and Vitek's method of using discharge density for burst detection was first introduced in 1996 and has since then been used to identifL burst periods in spike trains for neurons recorded in the pallidal complex (Boraud et al., 1998; Magill et al., 2000; Duque et al., 2000) and the STN (Magill et al., 2000), but not the neostn'atum. In his 1993 review, Wilson noted that the cells of the CPu are generally classified as either tonic or phasic, but that there is little consensus on specific criteria used to place a neuron within one of these categories. This lack of agreec-upon criteria may account for the large discrepancy in the relative proportions of these two types of cells reported in the literature (about 40% tonic by Kimura et al. in 1990, about 8% tonic by Alexander and DeLong in 1985). Wilson (1993) states that the clearest method which can be used to identify burst periods in a spike train is the IS1 histogram. However, the interval histograms of neostriatal neurons generally do not plainly show the bimodal distniution (one peak for intraburst intervals, one peak for interburst intervals) characteristic of bursting cells. This is because the intervals between bursts for CPu neurons are long and inconsistent as compared to the interspike intends within burst periods, and as such make less of a contribution in the IS1 histogram. In 1990, Kimura et al. descrii evidently bimodal IS1 histograms, but for a subset of what they classified as tonically 138 firing cells (which by most definitions should have a unimadal IS1 histogram). In 1991, Aldridge and Gilman used the Legendy and Salcman (1985) surprise method for burst detection. The population of cells analyzed was distriiuted continuously along most of the parameters measured, malo'ng it diflicult to distinguish categorical types of Evidently, it is quite difficult to design a set of quantitative criteria with which to standardize the identification of cell discharge patterns and, in addition, relate these identifications to previous classifications used for hippocampal theta-related cells. Kaneoke and Vitek's burst detection algorithm was used in conjunction with IS1 histograms as it is believed that this combination provides the most objective method to identi@and quantifL burst periods in a spike train. When applied to data from neurons within nuclei of the ascending hippocampal synchronizing system, these techniques provide classifications which are in agreement with Colom and Bland's (1987) scheme. That is, neurons previously classified as phasic are revealed to have burst periods in their spike trains. In addition, the Lomb periodogram analysis provided by Kaneoke and Vitek's software is useful for identimg and providing significance levels for underlying periodicities in single-spike and bursting data This type of analysis ha. never been applied to CPu neurons, even though AC histograms obtained fiom these cells have previously suggested rhythmic bursting (Wilson, 1993). Thus, although burst periods have been reported for CPu spike trains in the past, they are not reported here. This contrariety is due to differences in the methods used to identify burst periods. As a post-hoc precaution, hardcopy output of CPu spike trains was 139 , - visually scmmmd: no clear burst penpen&could be identified. Also, penodic single- spike bebavior was reported here but has not been previously demonstrated, although it has been suggested that this is a discharge property which could be observed in the giant aspiny interneurons of the striatum (Wilson, 1993). While the data presented here for the CPu neurons investigated does not correspond well with data provided by previous studies, the data presented here for the GP neurons investigated does. The cell discharge profiles of GP neurons recorded in this experiment are similar to those recorded in vitro and in vivo tiom the rat. In 1985, Nakarishi et al. recorded spontaneous unit discharges fiom GP neurons in rat slice preparations. Firing patterns were classified as either regular or irregular, as revealed by IS1 histogram analysis. Interestingly, neurons classified as regular had a higher mean discharge rate than those classified as ineguIar. This observation is relevant to the interesting distribution presented in Figure 16. The population of theta ON neurons recorded from the GP in this study which were classified as periodic only exhibited periodic behavior during theta states. By definition, theta ON neurons discharge at higher rates during theta triais. For these neurons, a jump in firing rate resulted in a shift to periodic firing. Thus, it is hypothesized that periodicity in these neurons is pethaps not theta-related, but simply firing rate related. Indeed, for these theta ON neurons, the transition fiom LIA to theta conditions emailed an increase in discharge rate from low levels, which were similar to those reported by Nakanishi et at. for their irregular neurons (approximately 0 to 10 spikedsec), to high levels which matched those reported by this group for their regular neurons (above 15 spikes/sec). In addition, the non-related GP cell which was periodic during both LiA and theta states (as seen in Figure 16) had a very 140 elevated (above 40 spikdsec) mean discharge rate across all field states. However, this hypothesis is not supported by the pair of non-related GP neurons in Figure 16, which only discharged periodically during LIA; one of these ceIls discharged at high levels during all hippocampal field states, the other fired at low levels during all field states. Furthermore, a number of GP neurons (both theta- and non-related) discharged at high rates but did not show periodic discharges. Thus, at this time, it is concluded that periodic 6ring in GP spike trains is not related simply to degree of theta-relatedness or firing rate, but perhaps to an interaction of these factors with other variables such as morphology and location. Napier and Peterson (1983) recorded fiom the GP of rats anaesthetized with pentobarbital or chloral hydrate. They observed three types of hngpattern under both anaesthetic regimes: regular firing, irregular firing, and some short burst activity. However, identification of burst periods was based on purely subjective criteria and it is dubious whether these burst wouId be identified as such using more stringent, objective methods. The data most comparable to that presented here were reported by Magi11 et d. in 2000. Amongst other recordings, this group recorded unit discharges of GP neurons and coincident cortical EEG in rats anaesthetized with either ketamine or urethane. Using Kaneoke and Vitek's burst detection methods, this group demonstrated that, under ketamine anaesthesia, GP units exhiiit bunting, regular, and irregular figpatterns. Ninety-one percent of GP neurons recorded in this state produced AC histograms which had a sigdicant oscillation (as revealed by Lomb periodogram) in them. The kquemy of this oscillation was closely matched to the comment slow wave oscillation recorded L4 I fiom the fiontal cortex (appmrdmateIy 1.5 Hz). In addition, bursting GP unit activity could be divided into "miniature burs&'' (one to four spikes) that were phase-locked to 7- 14 Hz spindle sequences superimposed on the slow wave activity. Spontaneous or sensory evoked desynchronization of the cortical EEG resuited in a loss of bursting activity of GP neurons paired with an increase in discharge rate (6/30 neurons tested) or a decrease in discharge rate (8D0 neurons tested). Cortical and hippocampal EEG patterns are generally thought to be inversely related, such that cortical synchrony is associated with hippocampal desynchrony (WA)and cortical desynchrony is associated with hippocampal synchrony (theta) (Bland and Oddie, 1998; Sainsbury, 1998). Thus, if one inverts the results discussed above, it would appear that, in ketamine anaesthetized rats, GP neurons cycle between a rhythmically bursting discharge pattern during LIA and an irregular pattern during theta. Furthennore, small populations of cells display theta ON and theta OFF firing rate profiles. Importantly, different results were found for the urethane recordings. Under those conditions, ail GP neurons recorded discharged single spikes in a regular manner @=IS). The periodicity of these discharges was revealed (by Lomb periodograrn) to be similar to the mean rate of firing for these celis, as should be the case for regular, single-spiking neurons. There were no changes in the presence of periodic discharges as the cortical EEG cycled from synchronized to desynchronized patterns. However, in seven instances, a hindpaw pinch was associated with a signifTcant increase in £iring rates (equivaIent to the theta ON cells reported here). En six other animals, the transition from synchronized to desynchronized cortical EEG patterns was not associated with any signiscant changes 142 in fking rate. In one case, hindpaw pinches fhiled to desynchronize the cortical field activity. The data presented here are in agreement with the results presented by Magdl et al. (2000) for urethane anaesthetized rats. The only disqcyis between the relative proportions of periodic and non-periodic neurons recorded from the GP. At this point, it is unclear why Magdl et al. did not obsereve any GP neurons which discharged in an irregular fashion. This discrepancy can not be attn'buted to species, anaesthetic regime, or analysis used as these were equivalent across the studies. Perhaps the differences are due to sample size (45 cells examined here, 15 cells examined by Magill's group). If Magill et al. had recorded &om more neurons under these conditions, it is suggested that they might aIso have observed irregularly firing neurons and, in addition, neurons that decreased their firing rates during cortical desynchronization (as was the case for their ketamine anaesthetized rats). Evidently, a population of GP neurons can be recorded fiom urethane anaesthetized rats which display periodic discharge properties. Furthermore, the discharge rates of these cells are related to changes in cortical and hippocampal field activity patterns. Whether two distinct populations of cortical and hippocampal EEG related neurons exist, or whether these properties interact within a single population of neurons, remains to be determined Magdl et d. (2000) proposed that the loss of burst Eiring in GP neuron spike trains in the urethane preparations, as compared to the ketamine preprations, could be accounted for by the behavior of STN cells (which bey also recorded, both individually and simultanmusly with GP neurons), As mentioned in the Introduction, the STN 143 projects massively to both the EP and the GP. Furthermore, although the GP receives some projections fiom the cortex, it has been shown tfmt it is the STN that mediates the excitatory responses of GP neurons to brief electrical stimulation of the cortex Ergo, the burst firing of GP neurons observed in their ketamine preparations is probably a consequence of periodic excitation of the STN and subsequent feed-forward input to the GP (Magill et al. review a body of literature which provides support for this idea). Magill et al. found that the bursting activity of STN neurons was less intense under the urethane regime as compared to the ketamine regime. They suggest that the weaker STN activity under urethane is insufficient to relay oscillatory activity (proposed to come fiom the cortex) to the GP and drive burst firing. In theory, this might also apply to oscillatory activity (theta) sent fiom the hippocampus and relayed through the ne~~aturn, explaining the lack of burst discharge behavior in the GP neurons examined here. Wilson et d.(1977) did much of the pioneering work on SN unit behavior in awake, locally anaesthetized, immobilized rats. This seminal research suggested that neurons throughout the SN could be classified as having either irregular or bursting discharge pattern. Within this classification, two patterns were apparent 1) irregular cells undergo a "regul~tion"process with increases in firing rate, and 2) while bursting cells can be recorded in both the SNC and !WR, they are about twice as common in the latter. In addition, some of these bursts may occur periodically at low frequencies. The group suggested that these bursting cells were probabIy interneurons and, apparently, this has not been contested Since this report, a number of studies have been conducted to elucidate the firing patterns of SNC and SNR neurons. 144 In 1984, Grace and Bunney reviewed a body of literature which suggested that the dopaminergic cells of the SNC have "...&ah of spikes which discharge at steady but inegular kmals... ", in gallmine-paralyzed or chloral hydrate mesthetized rats. Furthermore, a Iess typical burst discharg pattern can be recorded fiom some cells of the SNC. In 1993, Hajos and Greenfield showed that the majority of SNC neurons recorded from the guinea-pig slice preparation fired spontaneously in a very regular fashion without bursting activity. A minority of cells discharged in a "phasic", bursting manner. Less data suggest that neurons of the SNR fire in a single-spike periodic manner, although the investigations that do suggest this are those that best approximate normal physiological conditions. In addition to the report fiom Wilson et al. (1977) that SNR cells fire more regularly at high firing rates in localIy anaesthetized, awake, immobile rats, Gulley et al. (1999) described periodic cells recorded fiom the SNR of freely moving rats. Again, the other pattern of firing reported for SNR neurons is a burst pattern, first suggested by Wilson et al. in 1977. This report was corroborated by Villa and Bajo Lorenzana in 1997 for rats anasthetiid with equithesin or ketamine. hterestingly, in accordance with the report for STN neurons (Magdl et al., 2000), ketamine increased the tendency of SNR units to fire in bursts. These reports of bursting SNR unit activity are contradicted by studies that found only tonically active neurons in the SNR of rats anaesthetized with chloraI hydrate (Rohlfs et al., 1997) and freely moving rats (GulIey et al., 1999). Again, the manner in *. which a neuron's discharge pattem is classified must be closely satmmd as it depends on a number of variables, inc1uding species ded,anaesthetic regime used (ifany), and analytical technique used 145 There appears to be a paucity of studies in which SN unit activity is recorded hrn udme anaesthetized rats. As mentioned above, Wilson (1993) found that, after experimenting with a number of anaesthetics, the spontaneous activity of neostriatal neurons recorded from animals anaesthetized with urethane most closely resembles that of awake animals. Thus, it is cautiously suggested that the methodology used in this study is most comparable to studies in which rats were freely moving. Unfortunately, results from these studies are not consistent: there is little consensus on burst firing in the SN of unanaesthetized animals. While Wilson et al. (1977) report burst Gnag in the SN, and particularly the SNR, of locally anaethetized, awake, immobile rats, Gulley et al. (1999) do not report burst firing patterns in the SNR of freely moving rats. In this study, using the combination of Kaneoke and Vitek's burst detection method and IS1 histogram analysis, no burst activity was reported for neurons located in the SN. In past work, the classification of SN neuronat activity as bursting was based on AC histogram aaaiysis, a technique which is potentially fraught with errors (Kaneoke and Vitek, 1996). Although Ruskin et al. (1999) have used the Lomb periodogram techniq~ to analyze firing rate oscillations for SN neurons, no other group has yet used the discharge density histogram analysis to search for burst properties in SN spike trains. In accordance with previous data fiom freely moving, in vivo, and in vitto experiments, a large proportion of neurons recorded from the SNC and SNR in this study were considered to have discharge pattern which contained sections of periodicity. As can be seen for the SN neurons in Figure 16, a number of neurons were highly periodic: they had high percentages of periodic LIA and theta trials. Based on the "regularhtion" that SN neurons have been observed to undergo at high 6ring rates (Wilson et al., 1977), 146 it would be hypothesized that those cells clustered near the top right of the SN panel in Figure 16 should have higher mean discharge rates than other SN neurons. Interestingly, this may be the case for theta ON neurons, but not for theta OFF or non-related neurons. The SN theta ON neurons which had high proportions of LIA and/or theta trials which were analyzed to be periodic fiad correspondingly high firing rates (all above 15 spikes/sec, usually around 25 spikedsec). Accordingly, the three SN theta ON neurons which were not considered to be periodic had mean firing rates well below 15 spikdsec for ail field states. For the SN theta OFF and non-related neurons, there appears to be no cIw relationship between degree of peridcrty and firing rates. It is suggested that the degree of periodicity or regularity in the spike train of a neuron recorded frorn the SN is probably not related solely to discharge rate or theta-relatedness, but to a third variable which might also account for why theta ON cells do show "reguIarizationn at high f3ng rates. It is unlikely that this third vmiable is Iocation within the SN, as the positions of periodic neurons were scattered throughout this structure, as seen in Figure 7. While it remains debatable whether the discharge properties of neurons recorded from the basal ganglia in this study are similar to the discharge properties of basal ganglia neurons recorded in previous studies, the propties of the individual neurons that were classified as theta-related in the present study correspond to those described for neurons in "Iower" ascending hippocampd synchronizing nuclei such as the RPO and PPT (Hmada et d., 1999) and the superior colliculus (Nabme et aI., 1999). Proportions of theta-reIated cells reported for the pon~eregion (59%) and superior colliculus (81%) are comparable to the number reported here for tbe basal gangIia (69%). As is the case in this study, only tonic theta ON (80% of all theta-related I47 cells) and tonic theta OFF (20% of all theta-reIated cells) neurons were identified in the pontine region (90% ON, 10% OFF) and the superior colliculus (8 1% ON, 19% OFF). In urethane anaesthetized rats, the first appearance of bursting phasic cells in the ascending synchronizing system is at the level of the diencephalon, specifically the SUM and MM (for reviews see Bland and Oddie, 1998; Kirk, 1998). This important division suggests that, in terms of functional significance, the theta-related cells of the basal ganglia are more comparable to those of the RPO, PPT, and superior colliculus than to those of "higher" ascending hippocampal synchronizing structures. However, a pair of features potentially set the theta-related neurons of the basal ganglia apart fiom those of the pons and superior colliculus. First, the periodic discharge nature found for a high proportion of basal ganglia cells sets them apart fiom other "Iowei' ascending system structures. Regular discharge patterns have been described for neurons within the hippocampus (Colom and Bland, 1987) and the MS(Ford et al., 1989), but not for other nuclei related to, or considered part of, the ascending hippocampal synchronizing pathways, including the PPT, RPO, and superior collicuIus. The periodic nature of some neuronal discharges was noted in the two papers listed above, but neither attempted to objectively quantify this pattern. Again, how this periodic £iringpattern actualy relates to the hippocampal field activity (if at all) has yet to be determined. The periodic theta ON and theta OFF neurons recorded in this study did not show any preferential frequency of discharge. These cells had a wide range of firing rates: there was no clustering of single-spike periodic activity around theta frequencies. In addition, only a low number of these neurons had discharges which were cross-correlated 148 with the theta wave, and there was no consensus as to what portion of the wave these regular cells preferentially discharged at. No cross-correlation analysis was performed in the study of theta-related cell activity in the superior collicdus (Natsume et al., 1999), but crosscorrelation analysis revealed a low percentage of neurons in the pontine region had discharges which were weakly conelated with the slow theta wave (Hanada et al., 1999). These neurons appeared to preferentially discharge during the negative phase of theta recorded fiom the dentate gyrus. Because such a low number of weakly cross- correlated neurons were found, this group did not attach much significance to these findings. The second feature of theta-related basal ganglia neurons which delineates them fiom pontine and superior colliculus theta-related neurons is the relative position of these structures vis-bvis the ascending brainstem hippocampal synchronizing system. While the relative positions of the pons region and the superior colliculus are known, the relative position of the basal ganglia remains to be made clear. Evidence derived fiom ekctricai and chemical stimulation studies, paired with anterograde and retrograde tracer studies, has Myindicated that the pons region is in fact the origin of the ascending brainstem hippaampal synchronizing system (for a review see Bland, 2000). While the exact position of the superior collicdus relative to nuclei of the ascending pathway remains unknown, it is likely that this structure plays a role in integrating sensory information from a number of mdties, and projects this information to the hippocampus via the connections between its intermediate, theta-related cell rich layer, and the PH and PPT (Natsume et al., t 999). Whether the theta-related cells of the basal 149 ganglia are part of the ascending system which synchronizes hippocampal EEG, or part of an output pathway from the hippocampus to other areas (or both), remains to be seen In reference to the distriiutions of theta-related cells witbin the basal ganglia (Tables 1 to 3), the data presented here suggests that the CPu contains a higher relative proportion of theta-related cells than the SN and GP. Differences in the relative proportions of theta- and non-related ceHs in nuclei throughout the hippocampal synchronizing system have never been properly addressed in the literature. It is very tentatively suggested that the finding of more theta-related cells in the CPu potentially supports the hypothesis that the basal ganglia receives output from the hlppocampus, as anatomical data suggest that the most likely destination for thls hippocampal output is the ventromedial section of the CPu (Heimer et al., 1995). That is, the CPu is the most "theta-relatednstructure as it probably has the most direct interaction with the hippocampus. It is preswned that this "theta-rehtes' information, if transmitted at all, is projected on to relay (GP, SNC) and output (SNR)stmtmes in a less than one-to-one manner, accounting for the higher number of theta-related cells at the input side of the basal ganglia As the hippocampal projection to the CPu is said to terminate mainly in ventromedial regions, this idea wouId be supported by demonstrations that more theta- related cells are found in this area of the CPu as compared to other areas. As seen in Figure 5, the majority of CPu neurons recorded in this study were medially located; there is a lack of more lateralIy positioned recording sites and thus no comparison can be made. The low number of theta OFF neurons relative to theta ON neurons recorded in the CPu, GP, and SN is consistent with findings tkom other studies of the ascending 150 system (for a review see Bland and Colom, 1993). The possibility that theta OFF neurons, at least in the septum and hippocampus, are Likely small interneurons, has already been alluded to (for a review see Bland, 2000; Chapman and Lacaille, 1999). It might be proposed then, that although theta ON and theta OFF cells exist in equal numbers, lower numbers of theta OFF cells are generally reported simply because there is a lower probability of placing a recording electrode near one. Yet, this cannot be the case for theta OFF neurons in the -*aturn, since interneurons in this structure are larger (large aspiny interneurons) than other neurons (Heimer et al., 1995). Thus, if it can be genedized that theta OFF cells are always interneurons, it should be easier to record them tiom the CPu. Theta OFF neurons are probably not strictly interneurons in the GP or SNR either, as few interneurons are found in these structures, and aII cells are generally long and fusiform; yet theta OFF cells were found in these structures. Small nondopaminergic intemurons have been reported for the SNC of rats (FalIon and Loughlin, 1995). However, histological data (see Figure 7) do not support the hypothesis of theta OFF cells preferentially located in the SNC. The most parsimonious explanation for why, in this study, fewer theta OFF cells were recorded as compared to theta ON cells, is simply because theta Off cells make up a smaller population. Why this is the case in the basal ganglia, and seemingly throughout the ascending hippocampal synchro&g system, is unknown. The distinction between non-sensory and sensory non-related neurons is one that has only recently appeared in the literatu~(Hanada et d,1999). Because these cells modulate their firing rates during tail pinch induced theta but not during spontaneously occttrring theta, it has ken suggested that these cells code the occurrence of sensory 151 stimulation rather than the occurrex~ceof hippocampal theta. Very few sensory related neurons were recorded fiom the basal ganglia This is not surprising because, even though this structure may play a role in the initiation and maintenance of internally generated movements, it likely does not pIay a significant role in the initiation of stimulus-triggered movements, and is not a sensory processing region of the brain per se (Cote and Crutcher, 1991; &Long, 2000). It might be argued that the differential discharge rates of the theta-related cells reported here may represent general changes in the animal's state that may not be directly related to theta, but rather to genera1 excitability or "arousal". However, given that the sensory stimulus used here specificalIy influenced hippocampal theta activity and revealed corresponding changes in basal ganglia theta-related cell activity, it is equally likely that theta-related cell discharge is either a causal reflection of, or causally reflected in, simultaneously occurring theta field activity. In addition, the fact that non-sensory non-related cells did not cbange their discharge rates across the LIA, spontaneous theta, and tail pinch theta field states is not supportive of general excitation. The distinction between theta ON cells that increase their firing rates during both spontaneous and tail pinch induced theta relative to LIA, and sensory activated cells that only increase their discharge rates during the tad pinch state (and vice versa for theta OFF and sensory inactivated cells), provides fbther suppart that changes in theta-related cell activity are not a by-product of "arousaln. Of the 175 neurons recorded in this study, 23 were located in areas outside the basal ganglia The majority of these cells (15) were recorded fiom ventral aspects of the thalamus. 152 The ventral thalamic nuclei have a number of roles, but because these nuclei are a major source of Wamic efferents to the basal ganglia, and the major thalamic target of afferents from the basal ganglia, it is to be expected that they are important in motor control (Amaral, 2000). Because of this connectivity, and because of their hctional significance, it should not be surprising that theta-related cells couId be recorded fiom these structures. As stated in the Introduction, theta-related cells have been recorded born other thalamic nuclei, inctuding the cend median nucleus (Bland et al., 1995). The only thing particdarIy different or notable about these theta-related cells which were recorded outside of the basal ganglia was their seemingly high numbers. A very high proportion of these neurons were classified as theta-related. This skewed distribution is entirely due to sampling bias. That is, for the vast majority of these investigations, recording locations were unknown during the course of the experiment, and were only known upon histological verification. Because the position of the unit recording eIectrode could ody be estimated during the course of these uncommon experiments, only those cells which displayed evident theta-related behavior were recorded. This accounts for the high number of theta-reiated cells examined outside the basd ganglia In summary, by relating basal ganglia extracellular unit activity to the hippocampal EEG, it has been demonstrated that lines of communication exist between these structures. The discharge profiles of the neurons recorded could be classified using a slightly modified version of CoIom and Bland's (1987) scheme. Two distinct populations (ON and OFF) of theta-reIated neurons were recorded from the SN, GP, and 153 CPy such that the basal gmgha can be included within a wider group of cortical and subcortical struchms which contain neurons that have discharge properties related to the field activity recorded fiom the hippocampus. For many of these structures, the functional connection between their theta-related cells and the hippocampus has been established using electrical and chemical mapping techniques. Experiments two and three will attempt to establish a similar co~ectionfor theta-related cells of the bd gan@ia Methods-Experiment Two L Objectives Theta has been shown to be elicited by electrical stimuIation of the ascending brainstem hippocampal synchronizing pathways. At subcortical levels (PWSUM, RPO), relatively high frequencies (100 Hz) must be delivered in order to consistently elicit theta At the level of the MS, "theta driving" fkquencies (3-12 Hz) are applied (Vertes, 1981; Oddie et al., 1994; Ball and Gray, 1971; Scarlett and Bland, 1997). The use of electrical stimulation has two advantages: I) precise control over the production of theta per se; and 2) precise control over the fhpency of the induced theta One posst'ble disadvantage of using electrical stimulation in terms of localization is that axom of passage may be activated in addition to the cells in the nucleus being stimulatd Stimulation effects can be blocked by microinfusing the local maesthetic procaine hydrochloride into ascending system nuclei Using this drug, it has been shown that the effects of electrical stimulation of the RPO can be blocked at the level of the PH-SUM (Oddie et al., 1994), and at the level ofthe MS (Kirk et al,, 1996). In turn, the effects of 154 electrical stimulation of the PH-SUMregion are reversibly blocked by microinfusion of procaine into the MS (Smythe et aL, 199 1). The objective of this experiment was to examine whether electrical stimulation of basal ganglia structures has any effect on the hippocampal field activity. An attempt was made to characterize this possible effect: 1) are effective stimulation paratneters more like those used subcortically or more Iike those used at the level of the MS? and 2) might any stimulation effects be blocked by chemical inactivation (procaine) of other basal ganglia structures or by nuclei within the ascending system? Results should help define through which route(s) hippocampal fieId activity and basal ganglia cell activity interact. 11. Subjects Twenty-eight male Long Evans rats weighing between 125 and 150 g served as subjects. The animals were maintained and experiments were timed as previously outlined for experiment one. IIL Surgery Surgical preparation followed a protocol similar to that used for experiment one. Coordinates for the indifferent electrode, jeweler's screw, and the hippocampal reference electrode remained the same- In addition to these* depending on the investigation, coordinates (described in experiment one) for one basal ganglia structure (either the SN,GP, or CPu) and the MS (0.5 mm AP, 0 mm L) were marked on the brain, and holes (respective diameters maintained fiom experiment one) were drilled In a limited number of investigations, holes were Medfor the basal ganglia nuclei ipsiIateral to the reference electrode hoIe. 155 In a second group of investigations for experiment two, coordinates for a pair of basal ganglia structures (SN and GP, GP and SN, CPu and SN, CPu and GP) were marked and holes were drilled No holes were Wed for the MS in this group. In these cases, basal ganglia holes were always ipsilateral to one another and contralateral to tbe refereace electrode position. Following placement and fastening (with dental acrylic) of the hippocampal field recording electrode, in the first group of investigations described above, an infusion cannula was lowered at a rate of 10 @set (using the micromanipulator) into the MS to a depth of 5.5 mm DV. This cannula consisted of a 1 inch length of 30 gauge hypodermic needle (beveled end descending isto the brain), and contained either 10 or 20% procaine hydrochloride to be infUsed in the MS. lnfusrons were performed using a Ward Apparatus Idision pump (mode1 22). One 10 pL HamiltodGas tight syringe (#701) was connected via PE 30 intramedic tubing to the cannula. The 10 @ syringe was placed into the pump and the flow rate was set to 0.5 flmin. Upon reaching its destimtiog this cannula was attached to the jeweler's screw with dental acrylic. At this point the animal was allowed to wait without perturbon for about one half hour so that the hippocampal sigoal could stabilize. FoUowing this, a bipolar stimdating electrode was Iowered at a rate of 10 @set (using the micr~m~ptdator)into one of the basal ganglia structur~~to a depth range of 7.1 to 8.4 mm DV for the SN, 4.8 to 7.5 mrn DV for the GP, and 3.2 to 7.6 mm DV for the CPu A diagram of this experimental setup can be seen in Figure 17. 156 Figure 17. Diagrammatical representation of the placement of the recording electrode, stimulating electrode, and micro~ioncannula used for the first set of investigations described for experiment two. Hippocampal field activity was recorded itom the dentate gyrus. A stimulating electrode was placed into one of the three basal ganglia structures examined and a microinfusion cannula was lowered into the MS so that the effect basal gangIia stimulation had on hippocampal field activity could be observed before, during, and after chemical inactivation of the MS. 158 The bipolar stimulating electrode was constructed by twisting two 2 '/t inch tengths of 250 pm insulated stainless steel wires together. Each end of wire was clamped to a male subminiature connector, which facilitated attachment to a Grass 4678 stimulation isolation unit (SIU) and a Grass CC UIA constaut current unit A Grass S44 stimulator provided biphasic square wave output. The stimulator was connected to the STU in order to reduce stimulation artSact in the recording. Its output then passed through to the constant current unit, to change its voltage output to a current intensity (which was varied by altering the voltage setting on the S44). For each nucleus, the final depth of the stimulating electrode was selected when threshold levels of electrical stimulation produced low frequency theta (2.5-3.5 Hz). initially, an attempt was made to induce theta with 1 msec duration pulses at 5 Hz and 0.1 rnA. Ifthis did not induce theta, current intensity was raised through a range up to 0.6 mk If none of these settings induced any theta, the frequency of the pulses was raised to 10 Hz, the current intensity was reset to 0.1 mA, and the whole process repeated If no stimulation effects occurred at these settings, fiequency was raised to 25 and the whole process was repeated Finally, if no theta was induced at these settings, a final attempt was made at a much higher fiequency setting (100 Hz) with shoder pulse durations (0.1 msec). Again stimulation intensity started at 0.1 mA and, if necesuy, was raised up to 0.6 mA. For the first investigation, this whole protocol was performed at varying depths within the SN, GP, and CPu Once an optimal electrode depth had been assessed, this depth was used for subsequent investigations. Optiinal driving was demonstrated when theta of amplitude and fiequency (scnmnized on-line) comparable to tail pinch induced theta was obtained Also, behavioral signs such as viirissae 159 movement, increased rate of respiration, and pupil dilation were evident only during optimal driving and served as indicators of good stimulating electrode placement For the second group of investigations for experiment two, after the reference electrode was placed and secured, the aforementioned infusion cannula was not lowered into the MS, but into the relay or output basal ganglia nucIei examined The cannula was Iowered at a rate of 10Crm/sec (using the micromanipulator) to a dew of 7.5 mm DV in the SN or 6.15 mm DV in the GP. These depths were chosen as they represent the so- &led "middle of the middle" for each nucleus, so that the diffused drug had maximal effect within the nucleus and minimal effect in surrounding ganglia. Mer fastening this cannda to the jeweler's screw with dental acryIic and letting the animal rest for one half hour, a bipolar stirnufating electrode (equivalent to those used in the first group) was lowered into a different basal ganglia nucleus to depth ranges described above. For stimulating eIectrodes placed in the SN, infusion cannulas were positioned in the GP and vice versa. For stimulating electrodes placed in the CPy infUsion cannuIas were lowered into either the SN or the GP (one per investigation). Again, a11 stimulating eIectrodes and infusion caundas were ipsilaterd to one another and contralateral to the hippocamp$ reference electrode. This experimental setup can be seen in Figure 18. The protocol for omtionof stimulating electrode position and stimulation parameters was equivalent to the one descni above. Both of these surgicaI prepar&ons and subsequent data acquisitions took between 3 and 5 hours. After placement of all mrding electrodes, idision cannulas, and stimulating electrodes, urethane anaesthetic IeveIs were maintained such that spontaneous cycling between hippocampal LIA and theta field states occurred. Figure 18. Diagrammaticaf representation of the placement of the recording electrode, stimulating electrode, and microinfusion caunda used for the second set of investigations descrii for experiment two. Hippocampal field activity was recorded kom the dentate gym. A stimulating eiectrode was placed into one of the three basal ganglia structures examined while a microinfWion cannula was lowered into a different basal ganglia structure. The effect GP stimulation had on hippocampal field activity was observed before, during, and after chemical inactivation of the SN,and vice-versa Within an investigation, stimulation of the CPu was observed before, during, and after chemical inactivation of either the GP or the SN. 162 At the conclusion of a limited number of investigations, a volume of Pontamine Sky Blue (matching the volume of procaine used in the investigation) was infused through the cannula for subsequent localization and analysis of drug diffusion patterns. After all data had been acquired and this final protocol had been performed, animals were perfbed and brains were removed and stored as descnied in experiment one. IV. Data Acquisition All field activity recorded by the hippocampal reference electrode was amplified, filtered, displayed, and recorded with the same apparatus and in the same manner as descn'bed for experiment one. As no unit recordings were made for experiment two, these channels were turned off except during reference electrode placement. Once optimal "theta driving" was obtained the hippocampal field state was recorded "pre-drug"as it spontaneously cycled between LIA and theta, during tail pinch induced theta, and during electrical stimulation induced theta. Once an adequate number of baseline field states had been recorded, the procaine hydrochloride was infUsed (into the MS or a basal ganglia structure, depending on the investigation). At a flow rate of 0.5 Wmin, an initial dose of Z pL (for 20% procaine) or 2 pJ., (for 10% procaine) was given, and the animal was allowed to sit without disruption for 4-5 minutes. At this time, the effectiveness of the drug was tested via tail pinches and electrical stimulation. If during either of these manipulations no theta was produced, another series of recordings was made of the animal's hippocampal EEG during LIA, tail pinches, and electrical stimulation. Electrical stimulation induced theta was considered abolished once stimulation intensities equal to or greater than those used in the pre-drug state (up to 100 163 Hz,0. I msec, 0.6 mA) were no longer effective. If no change in the effectiveness of tail pinches or eI&cal stimulation to elicit theta was observed, another 0.5 pL of procaine was infused, the animal was dowed to sit for 2-3 minutes, and another assessment of theta inducement was perfofmed This process was repeated until both tail pinch and stimulation induced theta were abolished, or up to a minimum of 3 pL of procake had been infused Once hippocampal theta was abolished and a "drug staten data series was recorded, the animal was allowed to sit as the drug wore off such that spontaneous theta reappeared, or for up to 2 W hours. At this point, "post-drug" recordings of LIA, spontaoeous theta, tail pinch induced theta, and stimulation induced theta were gathered, after which data acquisition was considered complete. If the maximal infusion of drug had no effect on taii pinch or stimulation induced theta, a series of "post infusion" recordings were made, after which data acquisition was considered completed As was the case for experiment one, hardcopy output of dl data was produced and scrutinized off-line, and data segments were selected for subsequent analysis. In order to analyze the effects of electrical stimulation of the bad ganglia on hippocampal field states, the stimulation parameters and depths of stimulation electrodes were noted. Furthennore, at lease two segments of acquired data for each 6eId state during the baseline condition were selected to be and@ The concentration and volume of procaine hydrochloride used was noted, and at least two segments of acquired data during "drug staten LM,tail pinch and electrical stimulation conditions were selected to be analyzed Finally, the amount of time required for the return of spontaneous theta was noted, and at least two stretches of acquired data for each "postdrug" field state were 164 selected for subsequent analysis. Each selected data segment was at least 4 seconds long and, for each drug condition, the chronological order of selected segments was random. All selected segments of hippocampal field data were digitized in the same manner as described for experiment one. V. Data Analysis For each data segment acquired, the hippocampal field state was characterized and classified in the same manner as descnifor experiment one. An attempt was made to differentiate the principal frequencies of LIA, spontaneous theta, tail pinch induced theta, and stimulation induced theta for each drug state using methods descrii in experiment one. Finally, electrode and cannula placement was analyzed histologicaily as in experiment one. Stimulation electrode and infusion cannula positions were assessed by visually identifjlng their tracks, and procaine spread was assessed by identitjing blue dots (Pontamine Sky Blue). Results-Experiment Two L SN stimulation EIectrical stimulation of the SN was able to elicit hippocampal field activity for 8 stimulating electrodes placed in thehemisphere contralateral to the hippocampal recording site, and 1 stimulating electrode placed in the same hemisphere as the hippocampal recording site. Initial investigations revealed robust theta activation at electrode placements throughout the SN. Thus, on subsequent trials, optimal stimuIating 165 electrode placement was considered to be the so-called "middle of the middle" of the SN,at a depth 7.1 mm DV. Histological adyses enabled verification of SN stimulating electrode placement. All electrodes were located within the SN. One stimulating electrode tip was located within the SNC,six were located within the "middle of the middle" of the SNR (including the ipsilatrally placed electrode), and two were located in the extreme Iatdportion of the SNR Two stimulatingelectrodes (both in the optimal stimulating site) effectively elicited robust hippocampal theta with 1 msec, 25 Hz stimulation trains at a current intensity of 0.6 mA. For the other seven electrodes, 0.1 msec, 100 Hz stimulation trains at current intensities between 0.5 and 0.6 rnA elicited regular, robust theta. All stimdation induced theta was easily distinguishable from LIA as, within an investigation, it had similar amplitude and frequency ranges as the spontaneous and tail pinch indud theta also recorded. In all four cases tested, all stimulation effects were blocked by micoinfbion of small volumes of procaine hydrochloride into the MS. Application of procaine to the MS also disrupted atl spontaneous and tail pinch theta. Representative data can be seen in Figure 19. In the top panel, before any drug was infused, typical hippocampd theta was elicited by Imsec long, I00 Hz pulses delivered at an intensity of 0.55 mk Similar theta occurred spontaneously and upon tail pinches but is not shown. Five minutes after the microinfusion of 2.5 pL of 10% procaine into the MS, neither tail pinches or equivalent levels of electrical stimulation were able to elicit hippocampal theta (middle panels). As seen in the bottom panel, following a recovery period of 1 hour, these same intensities of 166 Figure 19. Representative hippocampal field activity recordings in response to electrical stimulation of the SN. Before the microinfusion of procaine into the MS, SN electrical stimulation elicited robust theta (top panel). AAer the application of drug, neither tail pinches (second panel) nor SN electrical stimulation (third panel) were able to elicit theta. Stimulation effects were re-apparent once the drug had worn off (bottom panet). Amplitude and time scales apply to all panels. Hoilow bars in this and subsequent Figures represent duration of manipulation. stimulation were again able to produce theta. Spontaneous theta and tail pinch induced theta (not shown) had also recovered after this period This pattern of drug effect was seen in all four cases tested In one of these cases, stimulation was delivered for 1 msec, at 25 Hz, at a cment intensity of 0.6 mA. In the other three cases, 100 Hz pulses that were 0.1 msec long were used, at intensities of 0.5, 0.55 (depicted here), and 0.6 mA (ipsilateral electrode placement). In the three investigations where 10% procaine was used, volumes ranging from 2.5 to 3.5 pi, were necessary to abolish all hippocampal theta For these investigations, all drug effects had worn off after one hour. In the fourth case, only 1 JLof 20% procaine was required to abolish ail theta. In this case, drug effects were persistent for only 30 minutes. Cannula placement was assessed histologically for a11 four investigations. All cannulas were located within the MS. In two cases, equivalent volumes (to those infitsed into the MS during the investigation) of Pontamine Sky Blue were infirsed in the cannulas once data acquisition was complete. In both cases, the blue stain was focused within the MS. In four investigations, SN stimulating effects were shown to persist upon microinfusion of procaine hydrochloride into the GP. Mimkhsion of procaine into the GP also had no effect on the occunence of spontaneous and tail pinch induced theta. Representative data are presented in Figure 20. As has been discussed above, regular theta was elicited by electrical stimulation of the SN(top panel). In this case, stimulation was delivered in 100 Hz pulses, 0.1 msec in length, at au intensity of 0.6 mk The regular occunence of spontaneous and tail pinch induced theta has not been depicted in the "predug" state. Five minutes after the application of up to 4 of 100/o ptocaine 169 Figure 20. Representative hippocampal field activity recordings in response to electrical stimulation of the SN. Before the microinfusion of procaine into the GP, SN electrical stimuiation elicited robust theta (top pel). The application of drug had no effect on tail pinch (middle panel) and SN electrical stimulation (bottom panel) elicited theta. Amplitude and time scales apply to all panels. 171 into the GP, spontaneous theta (not shown), taiI pinch induced theta (middle panel), and stimulation induced theta (bottom panel) remained This pattern of no drug effect was seen in all four cases investigated Stimulation parameters for all cases were 0.1 msec, I00 Hz puIses delivered at intensities of 0.5 (one stimulating electrode located in the SNC), 0.55, and 0.6 (depicted here) mk In aIl investigations, either 3 (one case) or 4 pL (other three cases) of 10% procaine was microinfused into the GP. Cannula placement was assessed histobgically for all four investigations. Three cannulas were located in the "middle of the middle" of the GP, one cannula was located in the medial GP. In two cases, equivalent voIumes (as those used during the experiment) of Poutaxnine Sky Blue were microinhsed into the respective cannulas upon completion of data acquisition. In both of these infirsions, the blue dot was nicety localized to the GP. II. GP stimulation Electrical stimulation of the GP was able to eIicit hippocampal theta field activity for 6 stimulating electrodes placed in the hemisphere contralateral to the hippocampal recording site, and 1 stimulating electrode pIaced in the same hemisphere as the hippocampal field recording electrode. One implanted stimulating electrode was not able to elicit hippocampal theta, even at stimulation intensities of 0.6 mA delivered in 100 Hz pulses. Initial investigations revealed robust theta activation at electrode placements throughout the upper two-thirds of the GP, but not at depths beyond 6.8 mm DV. Thus, on subsequent trials, optimal stimulating electrode placement was considered to be the "middle of the middlen of the GP, at a depth 6.15 turn DV. Histological anaIyses 172 provided verification of GP stimulating electrode placements. All electrodes but one were located within the GP. One stimulating electrode tip was located near the border between the GP and the basal nucleus of Meynert, and six were located within the "middle of the middle" of the GP (induding the ipsilatrally placed electrode). The stimulating electrode which could not elicit theta was found to be located in the ventral posteromedial thalamic nucleus (VPM). No stimulating sit& were able to elicit robust hippocampal theta with low frequencies (25 Hz and lower) of stimulation However, at current intensities between 0.3 and 0.6 mA, with 0.1 msec, 100 Hz pulses, GP stimulation always elicited consistent theta. All stimulation induced theta was easily distinguishable from LIA as, within an experiment, it had similar amplitude and frequency ranges as the spontaneous and tail pinch induced theta also recorded, In all three cases tested, stimulation effects were blocked by microinfbion of small volumes of procaine hydrochloride into the MS. Application of procaine to the MS also disrupted all spontaneous and tail pinch theta Representative data can be seen in Figure 2 1. In the top panel, before any drug was infused, typical hippocampal theta was elicited by lmsec long, 100 Hz pulses delivered at an intensity of 0.4 mA. In this case, stimulation was delivered ipsllateraly to the hippocampal field recording site. Similar theta occurred spontaneously and with tail pinches but is not shown. As can be seen in the middle panels of Figure 21, five mirmtes after the microinfusion of 3 pL, of 10% procaine into the MS, neither taiI pinches nor elevated (0.5 mA) levels of electrid stimulation were able to elicit hippocampal theta As seen in the bottom panel, following a recovery period of 45 minutes, these same intensities of stimulation were again able to 173 Figure 21. Representative hippocampal field activity fecordings in response to electrical stimulation of the GP. Before the microinfirsion of procaine into the MS. GP electricalstimulation elicited robust theta (top panel). After the application of drug, neither tail pinches (second panel) nor GP electrid stimulation (third pel)were able to elicit theta Stimulation effects were re-apparent once the drug had worn off (tattom panel). Amplitude and time scales apply to all paneis, 175 produce theta, though not at equivalent "pre-dmg" amplitudes. Spontaneous theta and tail pinch induced theta (not shown) had also recovered after this period. This pattern of drug effect was seen in all three cases tested. Stimulation intensities of 0.3 (stimulating electrode located near the basal nucleus of Meynert), 0.4 (shown here), and 0.5 mA were required to elicit theta in these investigations. In the investigation where 10% procaine was used (depicted here), 3 pL. of procaine were necessary to abolish ail hippocampal theta. For this investigation, all drug effects had worn off after 45 minutes. In the other two studies, only 0.5 to 1 pL of 20% procaine was required to abolish all theta In these cases, drug effects were persistent for ody 30 to 40 minutes. Cannula placement was confiied to be within the MS for dl three experiments. GP stimulating effects persisted upon microinfUsion of procaine hydrochloride into the SN, tested in three investigations. Microinhion of procaine into the SN also had no effect on the occurrence of spontaneous and tail pinch induced theta. Representative data are presented in Figure 22. As has been discussed above, reguIar theta was elicited by electrical stimulation of the GP (top panel). In this case, stimulation was delivered in 100 Hz pulses, 0.1 msec in length, at an intensity of 0.5 mA. The regular occurrence of spontaneous and tail pinch induced theta has not been shown in the "pre-drug state. Five minutes after the application of up to 3 pL of 10% procaine into the SN, spontaneous theta (not shown), tail pinch induced theta (middle panel), and stimulation induced theta (bottom panel) remained. This pattern of no drug effect was seen in all three cases investigated. Stimulation parameters for all cases were 0.1 msec, 100 Hz pulses delivered at intensities of 0.5 (one 176 Figure 22. Representative hippocampal field activity reardings in response to electrical stimulation of the GP. Before the microidusion of procaine into the SN, GP electrical stimulation elicited robust theta (top pel). The application of drug had no effect on tail pinch (middle panel) and GP electrical stimulation (bottom panel) elicited theta, Amplitude and time scales apply to all pels. 178 depicted here) and 0.6 mA. In aIl investigations, either 3 (two cases) or 4 pL (other case) of 10% procaine was microinfirsed into the SN. Cannula placement was assessed histologically for only two investigations. Both cannulas were located in the "middle ofthe middle" of the SN. In one of these cases, an equivalent volume (as that used duriag the experiment) of Pontamine Sky Blue was microinfiwd through the cannula after data acquisition was complete. The resulting blue dot was focused high in the SNR, with stain also seen in the SNC. III. CPn stimulation Hippocampal theta field activity was elicited by electrical stimulation of the CPu for 10 electrodes all placed contralaterally to the hippocampal reference electrode. Initial investigations revealed theta activation at electrode placements throughout the CPu. Thus, on subsequent trials, an attempt was made to lower electrodes into the "middle of the middlen of the CPu, to a depth of 5.1 nun DV. Histological dysesconfirmed that all stimulating electrodes were located within the CPu. The majority of electrode tips were located at the optimal theta driving position, while one electrode tip was located in the medial CPu, quite near the wall of the laterai ventricle. No stimulation electrodes were able to evoke theta at low frequencies of stimulation However, at current intensities of 0.3 to 0.6 mA, with 0.1 rnsec long puIses, I00 Hz of stimulation always elicited regular, robust theta. This stimulation theta was easily distinguishable tiom LIA as, within each investigation, it had amplitude and frequency ranges similar to those for the spontaneous and tail pinch induced theta recorded 179 In all three cases tested, all stimulation effects were blocked by microinfusion of small volumes of procaine hydrocholride into the MS. Application of this drug also abolished all spontaneous and tail pinch theta. R-tive data are shown in Figure 23. As seen in the top panel before any drug was infused, typical theta was elicited by 100 Hz, 0.1 msec long, 0.35 mA stimulation In the two middle panels, it is clear that, five minutes after infusion of 1 pL. of 20% procaine into the MS, neither tail pinches nor "pre-drug" equivalent levels of electrical stimulation were able to elicit hippocampal theta. Following 40 minutes of recovery, spontaneous and tail pinch induced theta were again seen in the hippocampal field (not shown), and equivalent levels of electrical stimulation were again abIe to produce theta This pattern of drug effect was observed in all three investigations. Stimulation intensities of 0.35 (depicted here), 0.55, and 0.6 mA were required to elicit theta in these experiments. In the one experiment where 10% procaine was used, 2& were necessary for disruption of the hippocampal theta fieIds. in this case, drug effects had worn off after 70 minutes. In the other two investigations where 20% procaine was used, 1 to 1.5j.L of procaine were necessary. For one of these investigations, spontaneous theta reappeared in the hippocam@ field 20 minutes after the application of procaine. In the other case (depicted here), 40 minutes were requked for recovery. Cannula placement within the MS was confinned histologically for all three experiments. The effect of micro~onof procaine into the SN on CPu stimulation induced hippocampal theta was tested in three investigations. In two cases, a relatively large (as compared to idhion into the MS) volume of drug was required to abolish stimulation 180 Figure 23. Representative hippocampal field activity recordings in response to electricai stimulation of the CPu. Before the microinhsion of procaine into the MS, CPu electrical stimulation elicited robust theta (top panel). After the application of drug, neither tail pinches (second panel) nor CPu electrical stimulation (third panel) were able to elicit theta. Stimulation effects were re-apparent once the drug had worn off (bottom panel). Amplitude and time scales apply to all panels. 182 effects. Spontaneous theta and tail pinch induced theta were only abolished in one of these investigations. In the third investigation, spontaneous theta, tail pinch induced theta, and stimulation induced theta remained unperturbed at high levels of procaine. The most interesting data are presented in Figure 24. As has been described above, regular theta was elicited by electrical stimulation of the CPu (top panel). In this case, stimulation was delivered in 100 Hz pdses of 0.1 msec duration at an intensity of 0.35 rnA. The regular occurrence of spontaneous and tail pinch induced theta have not been shown in the "pre-drug" state. In the investigation presented in Figure 24, five minutes after the microinhion of 2 pL of 20% procaine into the SN, spontaneous theta (not shown), tail pinch theta (second panel from the top), and stimulation theta (third panel tiom the top) could still be seen in the hippocampal field recording. However. five minutes after the infusion of an additional 2 pL (4 @ total) of the same procaine, stimuIation theta was abolished (second panel from the bottom) while spontaneous theta (not shown) and tail pinch theta (third panel fkom the bottom) remained relatively intact. Theta could not be induced by electrical stimuIation nearly doubled in intensity (0.6 rnA) after this treatment. Finally, after 20 minutes of recovery, theta could be elicited by electrical stimulation of the CPu at "predrug" levels (0.35 mA). In the other two procedures that investigated the effect of SN procaine on CPu theta stimulation, 10% procaine was used, and produced mixed results. In one case, after 3 pL of drug infbed into the SN, spontaneous theta was no longer observed, tail pinch theta could no longer be induced, and levels of electrical stimulation (100 Hz, 0.1 msec, 0.5 mA) which previously had elicited theta could no longer do so. Even after 90 minutes, no theta couId be evoked by either tail pinches or electrical stimulation. In the 183 Figure 24. Representative hippocampal field activity recordings in response to electrical stimulation of the CPu. Before the microinfUsion of procaine into the SN, CPu electrical stimulation elicited robust theta (top panel). The application of a small volume of drug had no effect on tail pinch (second panel) adCPu electrical stimulation (third panel) elicited theta. However, a larger volume ofdrug disrupted theta produced by electrical stimulation of the CPu (second panel fiom the bottom) while tail pinch induced theta (third panel fiom the bottom) persisted. Stimulation effects were re-apparent once the drug had worn off (bottom pel). Amplitude and time scales apply to all panels. 185 second case, 4 pL of drug microinfused into the SN had no effect on spontaneous theta, tail pinch induced theta, or theta induced by electrical stimulation of 100 Hz, 0.1 msec in duration, and 0.3 mA in intensity. In all three cases infusion cannula tracks were 1- to be within the SNR For the investigation depicted in Figure 24, an equal volume (4 pL) of Pontamine Sky Blue was ihedthrough the cannula in the SN once data acquisition was complete. The blue dot obtained was focused within the extreme lateral and posterior SNR, and some stain appeared to have diffused into peripheral areas (including the deep Ch). The effect of microinfusion of procaine into the GP on the ability of CPu stimulation to elicit hippocampal theta was tested in three investigations. All stimulation effects were abolished by the microinbion of a small volume of drug into the GP. However, in only one case were spontaneous theta and tail pinch theta also abolished Representative data are presented in Figure 25. As has been shown above, typical theta was elicited by stimulation of the CPu (top panel). For this investigation, stimulation was delivered in 100 Hz pulses of 0.1 msec duration at an intensity of 0.3 olA The regular occurrence of spontaneous and tail pinch induced theta has not been shown in the "pre- drug" state. For the experiment presented here, five minutes after the infusion of I pL of 200h procaine in the GP, spontaneous theta (not shown) and tail pinch induced theta (second panel from the top) remained. However, stimulation levels twice (0.6 mA) those previously used to evoke theta in the "predrug" state were no longer able to do so. StimuIation theta could be evoked at origiaal levels after 30 minutes of fecovery. This pattern was observed in one of the two other investigations. In this case, while 0.1 msec, 100 0.3 mA stimulation codd elicit theta before the microhfhsion of 186 Figure 25. Representative hippocampal field activity recordings in response to electrical stimulation ofthe CPu Before the mimidhion of procaine into the GP, CPu electrical stimulation elicited robust theta (top panel). After the application of drug, theta produced by electrical 5-timulation of the CPu (third panel) was disrupted while tail pinch induced theta (second panel) persisted Stimulation effects were re-apparent once the drug had worn off (bottom panel). Amplitude and time scales apply to all panels. 188 procaine, Ievels of stimulation up to 0.6 mA no longer had the same effect after hfbion of 1.5 pL of 20% procaine into the GP. Again, spontaneous theta and tail pinch theta survived this drug treatment The ability to elicit hippocampal theta through CPu stimulation at "pre&gn levels recovered after 40 minutes. In the other investigation of this type, spontaneous, tail pinch, and CPu stimulation theta were all disrupted five minutes after the microinfusion of 1.5 pL of 20% procaine into the GP. "Pre-drug" stimulation parameters of 100 Hz, 0.1 msec, 0.3 mA were only able to elicit theta after 40 minutes of recovery, at which point both spontaneous theta and tail pinch theta were also observed For the two experiments in which spontaneous md tail pinch induced theta remained intact after drug infusion, cannulas were localized withthe "middle of the middle" of the GP. For the investigation where these field states were disrupted, the infbsion cannuIa was localized in the media1 posterior GP. Equivalent volumes (to those of procaine used during the experiment) of Pontamine Sky Blue were infused through the canndas after data acquisition for dl three investigations. In all cases, the focus of the bIue dot was in the GP, but stain had diffused into the bordering CPu in all three cases, and was quite diffuse in the last investigation described. In this case, the blue dot appeared to be spread into a major portion of the CPu, and into the vend aspects of the thalamus. Discussion-Experiment Two Electrical current applied ldyto brain tissue induces membrane potential depolarizatious in neurons within the vicinity of the stimulation This induction of 189 summed neuronal activity has been used at various points throughout the ascending pathway to elicit hippocampal theta (for reviews see Bland and Oddie, 1998; Bland, 2000). The principal finding of this experiment was that electrical stimulation of the SN, GP, and CPu evokes hippocampal theta. There appears to be no difference in this effect if stimulated nuclei are contra- or ipsilateral to the hippocampus. These findings corroborate work performed by Sabatino et al. in 1985, 1986, and 1989. The major difference between the data presented here and the data presented in the above papers is that, in the latter, lower frequencies of current were required to elicit hippocampal theta: electrical pulses deIivered at, on average, 30 Hz were able to elicit theta. In some cases it appears that stimulation as low as 5 Hz was able to elicit theta (Sabatino et al., 1989). These electrical pulses were delivered at intensities between 0.1 and 0.6 mA, and for a duration of 1 msec. in the present study, theta could only be elicited using equivalently low stimulation frequencies in two of nine cases for stimulating electrodes positioned in the SN, and for no experiments in which the GP or CPu were stimulated. The stimuIation parameters for these two investigations were 1 msec long pulses, delivered at a minimum kquency of at least 25 Hz, with output intensities at the maximum 0.6 mA. Two differences between the present study and the Italian studies might account for this discrepancy in stimulation kquencies. First, there was a preparation difference. The present study was performed in urethane anaesthetized rats, whereas Sabatino st al. used encephale isole halothane anaethetkd cats. Second, in the studies performed by Sabatino et al., prior to measuring any stimulation effects, epileptiform activity was induced in these cats via an intravenous injection of penicillin. Because of the extensive 190 circuitry of the hippocampus, small areas of activation within a restricted site produce enhanced activation of the entire structure. The drug-induced activation of the hippocampi formations of these cats probably facilitated the synchronization of widespread neuronal activity through basal ganglia electrical stimulation. The stimulation parameters used to eIicit theta in the present study group thk bd ganglia with "lower" ascending system nuclei, where high frequency (100 Hz) stimulation is required to synchronize the hippocampal EEG, and separately tiom the MS, where each pulse of stimulation induces a corresponding wave in the hippocampa1 EEG. This is corroborated by the fact that basal ganglia stimulation effects were blocked by chemical inactivation of the MS (see Figures 19,2 1, and 23). Both caudal diencephalon (Smythe et d., 1991) and pontine electrical stirnuIation (Kirk et al., 1996), which synchronizes the hipgocampal field activity, can be revem'bly biocked by microinfusing procaine into the MS. In 1985, Sabatino et al. demonstrated that stimulation of the CPu was no longer effive in eliciting hippocampal theta after the MS was lesioned electrically. Similar results were demonstrated for SNC stimulation in 1985. The present study extends these findings, in that GP and SNR electrical stimulation which elicited robust theta was also blocked by MS inactivation. One other manner in which the present study attempted to develop beyond previous work was through mapping the route via which stimulation effects travel through the basal ganglia nuclei. As expected, theta induced through stimulation of a basal ganglia output stnrcture (the SNR) could not be blocked by chemical kctivation of a relay structure [the GP) (see Figure 20). 191 hteresthgty, the theta evoked by stimulation of a relay strum (the GP) couId not be blocked through procaine inactivation of an output structure (the SNR) (see Figure 22). Histology suggests that, in addition to the SNR being blocked (the focus of the Pontamine Sky Blue stain), the SNC may also have been blocked. Thus, it also appears as though GP stimulation effects could not be blocked through inactivation of the other relay structure of the basal ganglia (the SNC). These results may not be entirely unexpected as, in the rat, information travelling through the indirect pathway of the basal ganglia (wherein is included the GP) can exit the basal ganglia through two structures, the EP and SML It may be necessary to inactivate both output structures in order to see a disruption of this stimulation effect. If synchronizing effects do indeed travel f?om the GP to the hippocampus through normal basal ganglia pathways (and not by circumventing the other relay and output nuclei), it appears as though this output is not equally split between the SNR and EP. Tbat is, chemical blockade of the SNR did not reduce the amplitude or frequency of the induced theta by half Although this is not supported anatomically, perhaps hippocampal synchronizing information generated or passing through the GP is projected solely to the EP, and thence to the PPT or lateral habenda The data gathered here appear to suggest that CPu stimulation elicited theta can be blocked through inactivation of both relay (GP) and output (SNR)nuclei. However, after considemion of the data derived fiom the histological verification of drug focus and di&rsion, it appears this may not be the case. The volumes of procaine used to block CFu stimulation effects appeared to be too large, as equivalent volumes of Pontamine Sky Blue were seen to also stain stlrrounding nuclei, including the CPu For the 192 experiment in which procaine was iafUsed into the SN depicted in Figute 24, it is evident that small volumes of procaine had no effect on CPu stimulation induced theta, but that large volumes disrupted this effect (spontaneous and tail pinch induced theta remained intact). However, as revealed by an equivalent volume of Pontamine Sky Blue microinfised post-hoc, this procaine had ditlked beyond the borders of the SNR, and into the dorso-posterior region of the CPu. Ergo, the cells in some parts of this structure could not be made to discharge as they were chemicdly silenced It may be that widespread Chactivation is necessary for synchronization of the hippocampal fieId activity, and that this widespread activation was blocked in some areas by diffused procaine. In one other case, an equivalent volume of a lower concentration of procaine probably also diffused into the CPu, but had little effect due to its low potency at such a concentration. The results fiom the investigation where a lower volume of lower concentration procaine was used remain codking. The drug was infkd into the SNR as evidenced by histological identification of the cannula track, but the spread of the drug was never verified, although it can be hypothesized that the procaine remained in the SNR as a lower volume of drug was used (3 pL as opposed to 4 a).In this investigation, all theta (spontaneous, tail pinch induced, and CPu stimulation induced) was abolished and never recovered These results are probably not a reflection of any of the manipulations performed, but of a third variable which would have led to the degradation of all hippocampal theta independently. Indeed, this animal had been experiencing breathing problems throughout the prepation. Independent complications might also account for why theta never recovered to fidl amplitude in the investigation pictured in Figure 2 1, 193 where MS procaine was shown to reversibly block theta evoked by stimulation of the Pontamine Sky Blue analysis revealed similar drug *ion patterns for the GP blocking experiments: drug appears to have spread from a focus in the GP into surrounding nuclei. For two experiments, procaine spread out of the GP and into the directly adjacent CPu. It is proposed that theta evoked by CPu stimulation was dis~pted in these investigations not because of inactivation of the GP relay nucleus, but because drug had spread into the site of stimulation itself For the third of these experiments, Pontamine Sky Blue analysis revealed that drug had spread fiom the GP, into the CPu, and into a ventral territory of the thalamus. Again, it is proposed that theta evoked by CPu stimulation was disrupted in this investigation not because of inactivation of the GP relay nucleus, but because drug had spread into the site of stimulation itself. However, for this experiment, both spontaneous and tail pinch induced theta were also abolished It is postulated that this is not because of some external variable which would have permanently disrupted dl theta independently of drug action, but because some aspect of the ascending hippocampal synchronizing system was also blocked by the: widely diffused drug. This is corroborated by the fact that spontaneous and tail pinch induced theta returned, alongside theta induced through CPu stimulation, &r the drug had worn off Yet, post-hoc stain analysis did not reveal that any nucleus of the ascending system was affected by the drug however, some stain was observed in thalamic nuclei. It is tentatively suggested here then, that some nuclei of the thalamus (prtkularly those ventrally located and implicated with motor control) may play a role in relaying ascending sensory information from brainstem regions to the septohippocampal system 194 This has been suggested in the litemre before (Fortin, 1999, but no work has been performed to assess the role of the thaIamus in the ascending hippocampal synchronizing pathway. As striataI projection fibers follow both indirect and direct routes to the output nuclei of the basal ganglia, it appears that, if synchronizing effects do indeed travel fkom the CPu to the hippocampus through normal basal ganglia pathways (and not by circumventing relay and output nuclei entirely), chemical inactivation of only one relay nucleus or one output nucleus is not sufl'icient to block these effects. It may be necessary to inactivate both the SNR and the EP (and possibly also the GP) at the same time in order to disrupt this synchronization, which might then ascend to the hippocampus via the PPT or the Iaterai habenula In summary, it appears that the SN, GP, and CPu comprise nuclei which can project ascending synchronizing information to the hippocampus. The manner in which this occurs is similar to that observed for "lower" ascending system nuclei rather than that observed for the MS. The route@)through which these basal ganglia nuclei exert these ascending synchronizing effects may or may not be independent of one another and, in turn, may or may not be independent of the ascending brainstem hippocampal synchronizing pathway. However, all of these routes must converge at some level below the MS as all effects are blocked by MS inactivation. Methods-Experiment Three L Objectives The objective of this experiment was to mermap the established relationship between hippocampal theta and the basal ganglia structures examined As has been previously discussed, there is very strong agreement that the synchronizing influences of the ascending pathway on the hippocampus are mediated by the MS (for reviews see Bland and Colom, 1993; Bland, 2000). A number of studies, including experiment two discussed above, have examined the effects that focal anesthetics (particularly procaine hydrochloride) injected into this sbucture have had on hippocampal field activity and the ability to induce theta through activation of the ascending system (Lawson and Bland, 199 1b; Srnythe et al., 199 1; Oddie et al., 1994; Kirk et al., 1996). An interesting extension of these studies is the investigation of what occurs to hippocampal theta-related cells (throughout the ascending pathway) upon application of these anaesthetics to the MS. In the Smythe et al. study (1991), it was shown that the dynamic relationships theta- related cells within the hippocampus had with the hippocampal field activity were disrupted upon the application of procaine to the MS. That is, the discharge activity of theta ON neurons was reduced to almost zero while theta OFF neurons continued to discharge at rates that were not significandy different from the pre-procaine levels accompanying LIA, until the a-e of spontaneous theta once the maximal eff'ects of the drug had worn off. It is interesting to compare these results with those hmthe Kirk et al. study (1996). In this study, it was shown that that the dynamic relationships theta-related cells within the PH and SUM had with the hippoampal field activity were hardly disrupted upon the application of procaine to the MS. This should not come as a 1% surprise as these nuclei are considered to be below the MS in the ascending hippocampal synchronizing systems. Yet, theta-related ceUs within the MM were disrupted by this drug treatment, The authors concluded that the MM in fact receives descending information from the hippocampus. Based on the evidence provided from experiment two, it would appear that application of procaine to the MS should have little effect on the firing repertoires of theta-related cells in the basal ganglia. The major finding fiom this experiment was that the basal ganglia interact with hippoampal theta by sending ascending information to the hippocampus through the MS. Thus, it would be hypothesized that a theta ON cell in the CPu, for example, would still be activated during a taiI pinch even though hippocampal theta was abolished through procaine inactivation of the MS. However, some of the anatomical evidence presented in the Introduction suggests that the hippocampus sends descending efferent5 to basal gangiia structures- Thus, the main goal of experiment three was to test the hypothesis that procaine inactivation of hippocampal theta would have no effect on the dynamic cell discharge patterns of theta-related cells in the SN, GP, and CPu. This experiment should provide a better picture of how neurons within these structures communicate with the hippocampus, and should provide some indication of the basaI ganglia's putative position relative to the ascending hippocampal synchronidng system. IL Subjects Data were obtained from 35 male Long Evans rats weighing between 125 and 197 150 g. These animals were already inciuded in the count for experiment one as they also provided cell classification data The animals were maintained and experiments were timed as previously outlined for experiment one. III, Surgery Surgical preparation followed a protocol simiIar to that used for experiments one and two. In the same fashion as was descnid for the first group of investigations performed for experiment two, after the indifferent electrode, jeweler's screw, and hippocampal reference electrode were secured, an infusion cannula was lowered into the MS and glued to the screw with dental acrylic. However, for one control animal, the infusion cannula was only lowered to a depth of 2.5 mm DV. From this point in the investigation, throughout all data acquisition, urethane anaesthesia levels were maintained such that spontaneous cycling between hippocampal LIA and theta fieId states occurred. Once a neuron had been isolated and enough data had been gathered to characterize it (as described in experiment one), a procaine infusion protocol was performed (as descnkd in experiment two). For another control animal, equivalent volumes of saline solution were infhed instead of procaine. This surgical preparation and subseqwnt data acquisition took from 3 to 8 hours. Recording locations for gIass electrodes were stained using the protocol descnid in experiment one, and an attempt was made to subsequently localize a limited number of inkion cannulas and drug spread, using techniques outlined in experiment two. 198 IV. Data Acquisition The materids and methods required to acquire &?a for ceI1 classification have been previously outlined in experiment one. Indeed, going into neuronal classification here is redundant as these neurons were already included in the data set for experiment one. Once it was felt that enough data had been coIIected to construct a baseline "pre- drug" neuronal profile for a cell, the procaine infhsion protocol descnid in experiment two was commenced. The same volumes of drug were idused (depending on what concentration of procaine was used) at the same rates. As no hippocampal theta was induced via electrical stimulation for this experiment, infbsions were stopped only after tail pinches no longer induced hippocampal theta If, following the infusion of up to a minimum of 3 @ of procaine hippocampal theta still persisted, one final series of recordings ("post infusion")or each field state was made, and data acquisition was terminated. Once hippocampal theta could no longer be induced by tail pinch, enough data were collected in order to class* a cell's discharge rate and pattern in the "drug state". That is, hippocampal field and lmit discharge recordings were made during LIA and during tail pinches while the drug was active. Once these data had been recorded, the animal was permitted to recover unperturbed until the reapprance of spontaneous theta or for up to 2 % hours, dl the while keeping close attention to the condition of the unit recording, and adjusting the glass electrode position to compensate for any tissue movements during this sometimes protracted recovery period. Once recovery was complete, "'postdrug"recordings of the unit during the three hippocampal states were obtained, after which time data acquisition was considered complete. I99 Representative data segments for cell classification during the "predrug" and "postdrug" states were selected in the same manner as descnid for experiment one. Data segments for analysis of field and cell behavior during the "drug state" were selected in a similar manner, except no representative selections could be made for the spontaneous theta state as it did not exist. Each selected data segment was at least 4 seconds long and, for each state, the chnologicaI order of selected segments was random. Selected acquired data segments were digitized as described in experiment one. V. Data Analysis For each data segment, the hippocampal field state was characterized and distinguished as described for experiment one. Field states were classified for the "pre- drug" data, the "drug state" data, and the "post-drug" data The unit discharge rates and patterns for each neuron were characterized as described for experiment one. Representative examples of the classification of these neurons were described in the results section of experiment one. Of importance here is the classification of these cells for the "drug state" and "post-drug" state, and how these compare to one another and to their "predmgn classification. Scrutiny of the data from experiment three led to the post hoc decision that, for the purposes of this experiment, the classification scheme being used might be too stringent, and could lead to the ignoring of trends in the data that are, in fact, important Thus, all changes in king rate andlor pattern will be reported Finally, glass electrode placement was analyzed as in experiment one, and infusion cannula positions and drug spread were analyzed as described in experiment two. Results-Experiment Three L Bistoiogy The effect that microinfusion of procaine hydrochloride into the MS had on cell discharge profiles was tested for neurons at 25 recording sites. Of these, twelve blue dots were localized histologicalIy in the CPu and the positions of four unstained sites were determined to be in this structure fiom histologically located tracks and electrode depths. Three blue dots were localized histologically in the SNR. Finally, three blue dots were localized histologically in the ventrolateral thalamic nucleus (VL), one in the ventromedial thalamic nucleus (VM), one in the reticular thalamic nucleus (Rt), and one in the medial I-scus (d). For dl investigations, infusion cannulas were histologically located in the MS (except for one position control investigation where the cannula was located just above the corpus callosum, in the cingulate cortex). For those limited initial investigations where equivalent volumes of Pontamine Sky Blue were infused into the MS after data acquisiton, blue dots were found to be focused within the MS, with little to no stain spread outside of this structure (again, except for the position control investigation where stain was focused on the border between the hntal and cingulate cortex). All neuronal recording sites were previously reported in the Results section of experiment one, and all stained sites were represented in Figures 5,6, and 7. IL Control experiments As was reported in the Results section of experiment two, the microiafusion of small volumes of procaine hydrochIoride into the MS has a profound effect on the 201 recorded field activity of the hippocampus, such that the regular occurrence of spontaneous theta and taiI pinch induced theta are no longer observed This in turn may or may not have affected the discharge properties of simultaneously recorded neurons, depending on how they were related to the hippocampal field activity before the drug had abolished theta It should be mentioned that all neurons reported here (and in subsequent sections) have been previously included amongst those classified in experiment one. Only the andyses provided by data acquired in the "pre-drug" state were used for that classification. In both control experiments, microinhion had no effect on the spontaneous and tail pinch induced theta recorded ftom the hippocampus. In one controI experiment, 4 of saline was microinfused into the MS. while a unit was simultaneously recorded from the CPu. Before ttre microinfirsion, this ceU could be classified as theta OFF non-periodic. This classification held throughout the experiment That is, cell discharge rates were d&ed by this treatment: no change in basal rate was observed, and no change in the relative difference in discharge rate between LIA and tail pinch trials was apparent. Also, this treatment had no effect on the nompe~odicfiring pattern of this &I. In the other control experiment, 4 pL of procaine was microinfused into the fiontal/cingutate cortex, while a unit was simultaneousIy recorded hmthe CPL Before the microhhiooq this cell was classified as theta ON non-periodic. After the mim~ion,using +/- 2 SF%, this cell could still be classified as theta ON. However, the basal firing rate of this neuron was slightly depressed across aII field states following 202 drug treatment. Furthermore, tbis ceU was less responsive to sensory stimulation following the microinlision of procaine. That is, although the cell increased its discharge rate during tail pinches in the "drug state", tbis increase in rate, while large enough for the ceU to be classified as theta ON using stringent criteria, was not as large an increase as in the "pre-drug" state. MS suppression had no effect on the firing pattern of this cell. IIL Effects of procaine suppression of MS on discharge properties of CPu neurons The effect of procaine suppression of MS on ceU discharge profiles was tested for one non-related non-periodic CPu neuron. In this investigation, five minutes after the rnicroinfhsion of 3 pL, of 10% procaine into the MS, all spontaneous theta was abolished Furthennore, tail pinches which were previously successll at eliciting hippocampal theta were no longer effective. Theta disruption had no effect on the neuron's discharge profile, and it could still be categorized as non-related in the "drug state". This cell had a very low discharge rate, close to 0 spikes/sec, throughout the whole experiment For this investigation, at 50 minutes post-ieion, the neuron was lost before any recovery of spontaneous and tail pinch theta Thus, no "post-drug" unit data were collected Procaine abolishment of hippocampal theta had profound effects on a population of theta-related cells of the CPu. The effect of procaine iafusion into the MS was tested for eleven theta ON (six periodic, five non-periodic) neurons located in the CPu. In all cases, the occurrence of spontaneous theta was no longer observed approximately five minutes after the MS microinfusion of 10% procaine at volumes ranging from I to 5 pL and of 20% procaine at volumes ranging fiom 1 to 1.5 pL. Also at this point, no theta was elicited by vigorous tail pinches. In five of these cases, cell discharge rates were depressed to 0 spikes/sec for the ciura!ion of MS suppression. This level of firing was 203 equivalent to the level of figassociated with LIA in the "pre-drug" state for three of these cells. In one cell, firing was depressed to 0 spikes/sec in the "drug statep fiom a mean basal level of 1.9 spikdsec during LIA in the "pre-drug" state. For the last of these cells, the mean tiring rate during LIA in the "predrug" state had been 0.4 spikedsec. In four of these five experiments, partial recovery was observed before the cell was lost. That is, for two of these investigations in which 10% procaine was used, at least one trial of tail pinch induced theta could be recorded 1 to 2 H hours after the initial infusion of the drug. For the other two of these investigations, "partial recovery theta" was recorded 35 minutes after the microidision of 20% procaine into the MS. This "partial recovery theta" was either very low in amplitude, corrupted in the frequency domain, or inconsistently elicited within a pinch, but was still considered to have more activity in the theta-band ftequencies compared to LIA, as assessed by FFT analysis. During these episodes of weakly induced tail pinch theta, these neurons all increased their firing rates relative to the ongoing LIA, however, these pinches were still not as effective as they had been previously. That is, the relative increase in discharge rate was not as large for these cells as it had been in the "pre-drclg" state. In the experiments in which basal 6ring levels were depressed, the mean firing rate during LIA trials had recovered to "predmg" levels. In one of these five experiments, robust spontaneous theta was again apparent in the hippocampat field recording 30 minutes after the microinfusion of I pL, of 20% procaine into the MS. Although long segments of spontaneous theta were apparent in this "post-drug" state, only one LIA trial and one tail pinch theta trial were ace 204 This cell continued firing at 0 spikedsec duriag the LIA state, as it had for the whole experiment. During spontaneous theta "post-drug" trials, the cell discharge rate was highly variable, but the mean rate was observed to lie between the mean discharge rate associated with spontaneous theta and the mean discharge rate associated with tail pinch theta in the "pre-drugyycondition. The one tail pinch theta trial recorded in the "post- drugn condition was slightly depressed as compared to "predrug" trials. Representative data for dl drug conditions are shown for the cell in Figure 26. For one theta ON neuron recorded from the CPu, while cell discharge was not reduced to 0 spikedsec after drug treatment, basal fking rates were depressed, and tail pinches could no longer effectively increase cell tiring. Using the same criteria emptoyed to class@ this cell as theta ON before the microinfusion of 2.5 jL of 10% procaine, this cell could be classified as non-related in the "drug staten. In fact, during the period of maximal effect of the drug, the mean cell discharge rate for this neuron across dl tail pinch trials was slightly lower then the mean discharge rate during LIA trials. Again, all firing levels were depressed after microinfUsion. Although the animal was allowed to sit unperturbed for 80 minutes, only very weak theta could be induced by tail pinches before the ceU was lost. During this "partial recovery" period of the experiment, the basal Ievel of firing had returned to "pre-drug" levels, but the theta ON relationship had not recovered That is, this neuron continued to fire at a slightly lower rate during the one tail pinch trial recorded at this time, as compared to LIA trials recorded fiom the same period For the remaining five theta ON neurons recorded Ecom the CPu, the microinfusion of procaine into the MS had no effect on the relative firing rate 205 Figure 26. Repesmtative responses to tail pinches before, during, and after chemical inactivation ofthe MS for a theta ON non-periodic neuron recorded hmthe CPu Five minutes after the microhfision of a small volume of procaine into the MS, all spontaneous theta and theta induced by a tail pinch was abolished. This was associated with a depression of ali cell firing to 0 spikedsec. Following the recovery of theta, this cell responded to tail pinches at discharge levels that were very slightly depressed as compared to the "predrug"condition. 207 relationships between LIA and tail pinch tn'als, For these neurons, approximately five minutes after the disruption of theta via MS procaine, mean discharge rates across alI LIA trials remained sigruficdy loww tima mean discharge rates across all tail pinch trials. Amongst these cells, there was a wide range of absolute differences between mean LIA and tail pinch firing rates in the "drug staten. Indeed, adopting the scheme used to classifi these cells as theta ON pre-procaine, one of these neurons could be classified as non-related during the period of maximal effect of the drug. However, for this cell in the "drug staten, the firing rate during every segment of acquired tail pinch data was higher than the firing rate during every segment of acquired LIA data There was quite a bit of variability within these trials however, and the mean increase in discharge rate was only slightly higher. In the "predmgn condition, the mean cell firing rate increased by 10 spikedsec during the transition from LIA to tail pinch theta. In the "drug state", this mean increase was only 2 spikeslsec. It should also be mentioned that, for this investigation, all cell discharge across all states was depressed following the microinfusion of a volume of 2.5 pL of 10% procaine. Basal f~ngrates had ody partially recovered after 45 minutes, at which point robust spontaneous and tail pinch theta was observed Furthermore, as seen in Figure 27, while the increase in cell discharge rate associated with tail pinch theta in the "post-drug" state (a mean increase of 7 spikdsec) more closely matched the haease seen in the "pre&gn state (a mean increase of 10 spikes/sec), it still had not Myrecovered Using the scheme which bad previously classified these cells as theta ON, the four remaining neurons could still be classilied as such in the "drug staten. However, for all four of these cells, the relative increase in mean discharge rate observed during tail 208 Figure 27. Representative responses to tail pinches before, dduring, and after chemical inactivation of the MS for a theta ON periodic neuron recorded from the CPu Five minutes after the microinfbsion of a small volume of procaine into the MS, all spontaneous theta and theta induced by a tail pinch was abolished This was associated with a depression in the basal firing rate, and a decrease in the discharge rate of the cell in response to tail pinches. Following the recovery of theta, this cell responded to tail pinches at discharge levels that were still slightly depressed as compared to the "pre- drug" condition. Furthermore, basal firing rates had only partially recovered at this time. 2 10 pinches in the "drug state" was smaller than that observed in the "predrug" condition. In addition, for two of these cells, all cell discharge was reduced after the microinfusion of procaine. For the other two neurons, LIA discharge rates remained highly comparable across all drug conditions. In one of these investigations the neuron was lost before any recovery of hippocampal theta. In the other three cases, 11l recovery of robust spontaneous theta was observed 20,40, and 45 minutes after the microinfusion of 1 pL. of 20% procaine into the MS. In all three experiments, mean cell firing rate was higher during "post-dmgn tail pinch induced theta trials than during "post-drug" LIA trials. These neurons remained theta ON in all three "pre-drugn, "drug state", and "post-drug" portions of the investigation, However, for one of these neurons, basal levels remained depressed in this "postdrug" state, and the gap between mean discharge rate during LLA and tail pinch theta, although it had increased, still did not approach "predrug" levels. The other two cells were those in which no baseline firing drop occurred, and fidl recovery of theta was mirrored by 111 recovery of tail pinch effects. Sample data for one of these cells are presented in Figure 28. There appeared to be no consistent effect on the discharge patterns of investigated theta ON CPu neurons. Although all previously classified non-periodic neurons remained non-periodic throughout and after drug treatment, and all periodic neurons continued to have at least one periodic trial throughout and after drug treatment, the relative percentages of trials considered periodic for this latter group changed 21 1 Figure 28. Representative responses to tail pinches before, during, and after chemical inactivation of the MS for a theta ON non-periodic neuron recorded from the CPu Five minutes after the microinfirsion of a small volume of procaine into the MS, all spontaneous theta and theta induced by a tail pinch was abolished. This was associated with a decrease in the discharge rate of the cell in response to tail pinches. Following the recovery of theta, this cell responded to tail pinches at discharge levels that were equivalent to those observed in the "pre-drug" condition. 213 The effect procaine microinfused into the MS had on CPu cell discharge properties was tested for two theta OFF cells (one periodic, one non-periodic). For both investigations, spontaneous and tail pinch induced theta was abolished five minutes after the microinfusion of 3pL of 100/o procaine into the MS. In both cases, at the time of maximal drug effect, mean cell discharge rate across all LIA trials was considered equivalent to mean cell discharge rate across dl tail pinch trials. Thus, these "pre-drug" theta OFF neurons could be classified as non-related in the "drug state". However, the changes in discharge rate which dowed for tbis non-related "drug state" classification were different for each cell, and this is possl%ly more relevant. For one of these cells, the basal rate of firing during hippocampal desynchrony changed after microidision That is, in the "drug staten, sensory stimulation still inhliited ceU firing to pre-procaine levels, but the baseline LIA firing rate had decreased from "pre-drug" levels so that it could be considered equal to the mean tail pinch firing rate in the "drug staten. Only partial recovery was observed for this cell after 80 minutes. For the one tail pinch induced theta trial recorded before the cell was lost, cell discharge rate was lower than the mean cell discharge rate calculated over LIA tn'als recorded from the same period. However, the LIA mean thing rate at this time was still considerably depressed as compared to "predmg7' IeveIs. For the other CPu theta OFF cell recorded in these conditions, there was no change in basal firing rate. In the "drug st&"' sensory stimulation was plainly no longer able to depress cell discharge rate: the mean firing rate over all LIA trials and all tail pinch trials at this time were nearly identical, and very similar to the mean LIA firing rate 214 in the "pre-drug" state. Although fidl theta recovery was observed in this experiment after 40 minutes, and although, using the "postdrug"data collected, this neuron could again be classified as theta OFF, the depression of figrate induced by sensory stimulation in the "post-drug" state was not as robust as it was in the "pre-drug" state. That is, the mean cell firing rate over all tail pinch trials in the recovered condition was still higher than the mean cell firing rate over all tail pinch trials in the pre-procaine condition As with the theta ON periodic neurons, this one theta OFF periodic neuron remained periodic throughout the whole investigation, although the relative proportions of LIA and theta trials considered periodic changed IV. Effects of procaine suppression of MS on discharge properties of SNR neurons The effect microinhion of procaine into the MS had on cell discharge profiles was tested for three theta ON neurons located in the SNR In all three cases, the occurrence of spontaneous and tail pinch induced hippocampal theta was abolished five minutes after the microinfusion of 1 p.L of 20% procaine into the MS. During the "drug state", the mean discharge rate for each neuron across all LIA trials was considered equivalent to the mean discharge rate for each neuron across all tail pinch trials. AIl three cells could be classified as non-related during the period of maximal effect of the dntg Yet, closer scrutiny beyond this classification reveals some interesting results. For two of these cells, the mean firing rate over all tail pinch trials was higher than the mean firing rate over all LlA trials in the "drug staten. Although this increase in firing rate was relatively small (allowing for the non-related classification), it was consistent For one of these cells, all cell discharge was depressed as compared to "pre- 215 drug" levels. While the first celi was lost before the reappearance of theta, this depressed cell produced one trial of robust tail pinch induced theta before it was lost 40 minutes after the infusion of procaine. Data segments for this neuron during the "pre- drug", "drug state", and "post drug" situations are shown in Figure 29. As might be iaferred fiom the bottom panel, the cell had a higher discharge rate during this one "post drug" tail pinch ma1 as compared to the mean discharge rate calculated over LLA trials acquired around the same time. Furthermore, the drop in baseline firing rate, which occurred upon the infirsion of procaine, was no longer apparent once the drug lost its efficacy. Interestingly, this periodic theta ON neuron which, in the "pre-drug" condition, had 100% of LIA trials considered periodic and 100% of theta trials considered periodic had, in the "drug statencondition, only 25% of LIA trials considered periodic and only 66% of theta trials considered periodic. All the "post-drug* condition LIA trials for this cell were considered periodic, as was the one tail pinch theta trial. For the third theta ON cell recorded fiom the SN in these conditions, cell discharge rates were plainly equivalent across all states during the period of maximal suppression of the MS. Tail pinches could no longer increase the firing rate of this celI above baseline "predrugn and "drug state" LIA levels. Unfortunately, this cell was lost before any recovery of theta field activity- V. Effects of procaine suppression of MS on discharge properties of neurons outside of basal ganglia The effect MS procaine had on cell discharge profiles was assessed for one neuron recorded fiom the ml, and five neurons recorded hmregions of the thalamus. 216 Figure 29. Representative responses to tail pinches before, during and after chemical inactivation of the MS for a theta ON periodic neuron recorded hmthe SNR. Five minutes after the microinhion of a small voIume of procaine into the MS, all spontaneous theta and theta induced by a tail pinch was abolished This was associated with a depression in the basal figrate, and a decrease in the discharge rate of the cell in response to tail pinches. FoUowing the recovery of theta, this cell responded to tail pinches at discharge levels that were equivalent to those observed in the "pre-drug" condition. Furthermore, basal firing rates had Myrecovered at this time. 218 For the neuron recorded hmthe ml, all simultaneously recorded spontaueous and tail pinch induced theta was abolished after tbe microinfusion of 3 pL of 20% procaine hydrochohide into the MS. Although this neuron was classified as a theta ON periodic neuron using "pre-drug" data, it could be classified as a non-related cell using data acquired while the hippocampus was under the maximal effect of the procaine. That is, mean cell discharge rates for this neuron were considered equivalent for LIA and tail pinch trials once the drug had been infUsed However, closer inspection of mean discharge rates during this treatment reveals that this cell consistently fired at a higher rate during tail pinches than during LIA trials in the "drug state". This increase in discharge rate was simply more variable and less profound in this condition. Although no spontaneous theta was recorded, tail pinch induced theta was recorded folIowing one hour of recovery. Using this "post-drug" data, the cell could be again classified as theta ON. It should be mentioned, however, that all cell discharge rates for this neuron were radically depressed in the "postdrug" state, and that the increases in discharge rate associated with tail pinches in this state were also not as substantial as in the "pre-drug" state. Interestingly, this neuron remained periodic throughout the whole investigation. The proportion of trials considered periodic for this cell remained the same (100%) for dl hippocampal field states across all drug conditions. For one of the neurons in the VL, 10% procaine was micro~edinto the MS; for the other two neurons, 20% procaine was used. For the former, 6 pL were necessary to abolish spontaneous theta and theta that could be previously induced with a tail pinch For the latter, 1 and 1.5 pL of drug were required AII three of these neurons were 2 19 classified as theta ON non-periodic before the rnicroinfUsion of procaine; these classifications held after the microinfusion, However, as was the case for a number of theta ON cells descriiabove, the effect of tail pinch in the "drug state" was not as manifest as it was in the "predrugn state. In the most extreme case, while one cell increased its firing rate in response to sensory stimulation by 20 spikeslsec in the "pre- drug" condition, this cell only increased its firing rate by 3 spikeslsec after drug had been microinhed The neuron depicted in Figure 30 was the only VL neuron that was not lost prior to any reappearance of spontaneous theta or before tail pinch theta codd again be induced. For this cell, tail pinch induced theta was apparent after 30 minutes. As can be seen, the same pattern of lower cell discharge rate in the LIA state was maintained in the "post-drug" condition. However, although discharge rates associated with sensory stimulation were definitely higher at this point than during the period of maximal effect of procaine, tail pinches still could not induce the higher tiring rates associated with sensory stimulation in the "predrug" condition. The neuron recorded from the VM which was tested under these conditions was classified as theta ON non-periodic in the "pre-drug" state. For this investigation, five minutes after the microinfusion of 1.5 pL of 20% procaine into the MS, spontaneous theta was no longer apparent and tail pinch theta could no longer be elicited During the "drug state", this neuron maintained its theta ON status, as the mean cell discharge rate over all LIA trials was significantly lower than the mean cell discharge rate over all tail pinch trids. Yet, like the cells descri'bed above, tail pinches were not as effective during the period of maximal suppression ofthe MS. While the mean mgrate for this cell increased fiom 0.5 to 21 spikedsec: in the "predrugn state, it only inadfiom 0.9 to 220 Figure 30. Representative responses to mil pinches before, during, and after chemical inactivation of the MS for a theta ON aon-periodic neuron recorded fiom the VL. Five minutes after the rnicro~onof a small volume of procaine into the MS, all spontaneous theta and theta induced by a taiI pinch was abolished. This was associated with a decrease in the discharge rate of the cell in response to tail pinches. Following the recovery of theta, this cell responded to tail pinches at discharge levels that were still slightly depressed as compared to those observed in the "pre-drug" condition 222 15 spikes/sec in the "drug state". This neuron was allow& to sit without perturbation for 70 minutes before it was lost and any spontaneous or tail pinch theta trials could be acquired Finally, the effect of MS procaine on cell discharge properties was tested on one theta OFF periodic neuron located in the Rt. In this case, only 1 pL of 20% procaine was needed to abolish all spontaneous and tail pinch theta. For this cell, once theta was abolished, discharge rate over all LIA trials remained higher than the mean discharge rate over all tail pinch trials. Thus, this cell could be classified as theta OFF during the 'drug state". However, as if showing the inverse of results seen for the theta ON cells recorded from the thalamus, tail pinches did not inhiit cetl firing to the same degree after procaine had been microinfused into the MS. No spontaneous theta or tail pinch induced theta could be recorded before this neuron was lost 70 minutes after the initial infusion of procaine. For this cell, there was seemingly no theta-related pattern to the occurrence of periodic trials in the "pre-drug" stak or the "drug state". Discussion-Experiment Three The criteria previousIy used in hsthesis to classify basal ganglia neurons along the rate dimension in relation to hippocampal field activity leads to conservative judgements. This reduces the number of cells incorrectly classified as theta-dated The disadvantage of this scheme is that subtle changes in the dynamics of a neuron's firing rate, which may be related to activity in the hippocampus, run the risk of not being perceived Procaine suppression of the MS produced subtle effects on the theta-related 223 discharge rates of basal ganglia cells. Thus, discussion of these results based on the classification scheme used in experh.net one would be inaccurate and oversimplified. Chemical inactivation of the MS has previously been used to indicate how the theta-related cell behavior of other ascending system structures is dependent on the integriq of hippocampal electrical activity. Because the hippocampal formation is a large structure, it can not easily be reversibly inactivated It is assumed that precise chemical suppression of the MS produces functionally equivalent inactivation as hippacampal theta generation is dependent on the intactness of septohippocampal neurons (for a hewsee Bland, 2000). The results presented here will be discussed with this assumpon in mind Neurons were recorded fiom the VL, VM, and Rt The data derived fiom these cells indicate that their changes in discharge rate associated with theta activity are, on the whole, independent of the septohippocampal system. As the theta ON cells in the ventral thalamus continued to increase their discharge rates during sensory stimulation after the MS was inactivated, it can be suggested that these cells receive ascending sensory information which simultaneously acts to synchronize the hippocampal field activity. These findings support the suggestions made in the Discussions for experiments one and two that the ventral thalamus may be a relay point in the ascending theta synchronizing system. As evidenced by the behavior of the theta OFF neuron recorded fiom the Rt, other thalamic nuclei may also be involved in this ascending pathway. However, chemical inactivation of the MS was not entirely without consequence for these cells. Indeed, for all ofthese neurons, after procaine microinfusion, sensory stimulation did not increase (or &crease) the discharge rates of these cells to the same 224 degree as it had in the "pre-drug" condition. This suggests that, although these cds receive their primary theta-related input from an ascending source, integrity of the MS appears to be necessary for a certain level of robustness in the theta-related responses of these cells. (A similar set of interactions was descn'bed between the septohippocampal system and the posterior diencephalic region by Kirk et al. in 1996). However, these effects may be mediated through regions of the MS which are not implicated in theta systems. For those investigations in the thaIamus in which spontaneous and tail pinch hippocampal theta activity could be recorded following a period of recovery from the drug, the full effectiveness of sensory stimulation was often not observed to be coincident with this recovgr. In the majority of cases, in the "post-drug" state, while tail pinches induced very regular hippocam@ theta, the accompanying increase in cell discharge rate for theta ON cells in the thalamus (opposite effect for the theta OFF cell) was not as large as it had been in the "pre-drug" condition. It should be stated here that no objective measures were taken to fully compare the theta observed in the "pre-drug" and "post- drug" situations. In 1995, Jakab and Leranth reviewed a body of work which suggested that the MS (particularly the diagonal band of Broca) contxiiutes a small direct projection to the VM and Rt (as well as to the VP). At this point, it is unclear whether the additional tonic contribution the MS makes to the robustness of the theta-related celluIar response of the ventral thalamus is mediated through this direct connection, or through a circuitous route which entails input hmthe MS to the hippocampal formation and thence to the thalamus. Anatomically, the most parsimonious route is the former. It is possible that, while the portion of the MS which projects to the ventral thalamus had not fully 225 recovered from procaine (preventing thalamic theta-related ceUs brnresponding to tail pinches completely), the septohippcampal projection had fully recovered (such that theta could be observed in the dentate gyrus). It is also possible, however, that MS control over ventral thalamic theta-related cells is dependent on an intact theta system, and that the theta recorded in the "post-drug" state in these experiments was actually not comparable to the theta recorded in the "pre-drug" state, resulting in a weaker of theta-related information to the thalamus. Similar effects were seen for a group of striatal theta ON cells, for which the anatomical evidence suggesting descending input from the hippocampal formation is stronger. For a population of theta ON cells recorded from the CPu, MS completeness was not requisite for the observation of tail pinch related increases in firing rates. Thls supports the argument made in experiment two that a population of cells in the CPu participate in an ascending system which transmits synchronidng sensory information to the hippocampus. However, as it was observed that sensory stimulation was not as effective during the period of maximal suppression of the MS, it can also be argued that this structure contn'butes or relays a level of tonic excitatory activity which serves to increase the response of striatal theta ON neurons to sensory stimulation. Although it might be argued that the MS, through its light projections to the SNC and SNR (Jakab and Lmth, 1995), could influence the CPu through a descending input, it seems more likely that the influence of this structure over the CPu is tranmitted via the hippocampal formation, as both the EC and subiculum have been shown to provide significant inputs to this area (for a review see Heimer et al., 1995)- Full recovery of the hippocampal theta state was observed in a number of these experiments. In some, this was mirrored by full recovery of tail pinch effects, in others, tail pinch effects in the "postdrug" state were still not as robust as they had been in the "pre-drug" state. Again, it can be argued that theta recovery was not actually "W,as objective comparisons between the power and frequency of theta in the "pre-drug" and "post-drug" conditions were not made. It remains unclear at this point whether the influence the MS has over this population of CPu theta ON cells is dependent or independent of theta systems. It might be argued that the drop in basal firing rate observed for some of these cells is not associated with suppression of the MS at all, but rather a result of the preparation used This is certainly the case for the investigation in which the firing rate of a neuron recorded fiom the ml was gradually depressed through the experiment (and particularly in the "post-drug" period). The animal used for this investigation was having progressive difficulties in breathing. This might have been the case for a few other experiments in which baseline decreases were observed, but certainly can not be the case for all experiments in which this occurred Indeed, in a number of studies, while baseline firing rates were depressed during the maximal suppression of the MS, the recovery of these baseline rates was correlated with the reappearance of hippocarnpaf theta For three of the five Chr theta ON cells included in the population discussed above, baseline firing rates were attenuated following the rnicroinfUsion of procaine. Two of these celIs were recorded "post-drug". At this time, while baseline levels had increased back towards "pre-drug" levels, they had not fully reached this point Interestingly, for both of these cells, the 111 efficacy of sensory stimulation also had not recovered at this pint, while for the two other CPu cells included in the population discussed above (baseline &ing rates had not changed), sensory stimulation was equally effective in the "predmg" and "post-drug" states. This suggests a possible interaction between the physiology behind a decrease in basal 6ring and the physiology behind a decrease in the effectiveness of sensory stimulation This suggestion is supported by the discharge behavior observed for a theta ON cell in the SNR. Following the microinhion of procaine, this cell experienced a baseline depression. Furthermore, this cell also responded less to sensory stimulation during this period. However, during the "postdrug" period, this cell responded to tail pinches to the same degree as in the "pre-drug" period, and its basal level of firing had returned to "predmg" levels. Thus, a population of theta ON cells exist in the CPu and SNR which are dependent on the integrity of the MS to maintain a certain level of spontaneous firing (although, as discharges for these cells were not completely abolished following MS inactivation, these ceils have other inputs which influence their firing). It is postulated that the mechanism by which the MS exerts this effect is highly related to the mechanism by which the MS provides additional excitatory information to these cells regarding the occurrence of hippocampal theta. For another population of theta-related cells in the CPu, ventral thalamus, and SNR (see below), MS inactivation dtsin no basal firing rate disruption, but does result in a less robust response to sensory stimulation. Thus, for these cells, the mechanism by which the MS provides excitatory information during the occmce of hippocampal theta is independent of an MS influence over spontaneous firing levels. Indeed, these cells appear not to be dependent at all on the integrity of the MS for spontaneous firing. It is tentatively suggested here that the delineation of these two populations of MS dependent cells may indicate the underlying routes through which the 228 MS interacts with these populations. That is, one group of celis receives descending input from the MS which is independent of the hippocampal formation Wely the population which is dependent on the MS for the maintenance of basal firing rate), white another group of cells interacts with the MS through its influence over the hippocampal formation A separate population of theta ON ceUs in the basal ganglia did not respond to sensory stimulation when the MS was inactivated Cell discharges were reduced to 0 spikes/sec for five theta ON cells recorded fiom the CPu following the rnicroinfUsion of procaine into the MS. This total suppression of discharges has also been reported for theta ON cells in the hippocampus (Smythe et al., 199 1) and the MM W*rket al., 1996). Both groups referred to the existence of monosynaptic co~ectionsbetween the septa1 complex and the respective structures investigated to explain this total loss of firing That is, as procaine blocked the sodium channels on all septohippocampd neurons such that they could not be depolarized above their discharge threshold, no information was transferred across the single synapse that connects these to hippocampal neurons (Smythe et al., 1991), and to MA4 neurons (Kirk et a]., I9%), resulting in no celI discharges in these respective targets. This explanation does not suffice for the resuits presented here. There is no evidence for a direct co~ectionbetween the CPu and the MS in the literature and, as revealed by drug spread analysis, it is unlikely that procaine *ed fiom the MS focus of injection to other areas which might be monosynapticalIy co~ectedto the CPu Four of these neurons were lost before the reappearance of hippocampal theta which could be considered equivalent to pre-procaine theta This partial recovery was correlated with only partial recovery of the effectiveness of sensory stimulation: tail 229 pinches which evoked this quasi-theta also evoked cell firing rates which were more elevated than those associated with LIA during this period, but this relative increase was still depressed compared to the "predmg" state. For one cell, spontaneous theta which appeared to be equivalent to theta observed pre-procaine was observed "postdrug". Again, no systematic objective measures were used. During spontaneous theta, the discharge rate of this cell had reached levels associated with spontaneous theta activity in the "predrug" state. As seen in Figure 26, during the one tail pinch trial recorded at this time, the cell discharged at a rate which was only very slightly lower than the mean rate during tail pinch theta in the "prednrg" state. These results suggest that the activity of these neurons is solely dependent on the integrity of the MS. The route through which this is mediated is at present unverified, but some pathways are more plausible than others. Because the loss of firing in these cells is highly correlated with desynchronous hippocampal EEG (that is, these cells almost excfusively discharge when theta is occurring), it is suggested that their MS dependency is actually a manifestation of their intimate interaction with the hippocampal formation. The results presented here provide evidence that, for a population of theta ON cells in the CPu, ascending sensory information reaches the MS, is then transmitted to the hippocampus so that it synchronizes field activity there, and is only then transmitted via the EC andlor subiculum to the ievei ofthe CPu. Another theta ON cell recorded hrnthe CPu appeared to have a similar relationship with the septohippwampal system, aIthough this cell was not lllymuted during the ^drug state". Ch-cal hachtion of the MS changed the discharge pf3e of this cell in two ways: the basal rate of firing was depressed, and the cell no longer 23 0 responded to sensory stimulation This cell is grouped with the population of theta ON cells discussed above because sensory stimulation which previously served to synchronize the hippocampal EEG was not -tted to the cell once the MS was inactivated. Whether, at the level of the MS, this information was transferred directly to this cell or whether it was transferred through the hippocampal formation is unknown at this time, although anatomical evidence would indicate the latter. The decrease in the baseline firing rate of this cell appeared to be related to the suppression of the MS, and not to the general condition of the animal. AIthough ody very poor theta could be recorded after 80 minutes, baseline firing rates were back to "pre-drug" levels. Thus, this baseline firing depression appeared to depend on the integrity of the MS, in a fashion that circumvented the hippocampus (basal firing had returned to normal while hippocampal theta had not). Yet, during this recovery period, the effectiveness of sensory stimulation had not recovered This suggests that this cell might have interacted with the MS through two pathways. One (possibly via the MS comection to the SN) which maintains basal levels of firing, and one (possibly via the hippocampal formation) which transmits sensory information relevant to theta. Evidently, more data must be collected before such conclusions are given much credence. It shouId be mentioned that there is potentially a regionai distri'bution within the CPu of theta-related neurons which primarily transmit ascending information, and theta- related neurons which receive descending information. The position of ody two of the five "ascending" cells reported in this study was verified histologically with a Pontamine Sky Blue stain. Both of these cells were located near the ventral border of the CPa The other three neurons were identified to be at mid to ventral deptbs based on the tapng of 23 1 glass recording electrode tracks. As this method of identifyiog recording sites is not fool-proof, it is possible that these three neurons were in fact located more deeply, near the identified neurons: that is, near the ventral edge of the CPu It is less Likely but also possible that these tbree cells were located even more deeply, such that they were actually outside the CPu The possibiIity that the five CPu neurons recorded in this study, which appear to be components of an ascending pathway to the septohippocampal system, are all located in ventral aspects of the CPu might lead to the proposition that this region of the CPu is more heavily implicated in this ascending pathway than other areas. This suggestion would be premature considering the low number of cells recorded, the uncertainty of their exact location, the fact that electrical stimulation which synchronized the hippocampal EEG was focused in the "middle of the middle" of the CPu, and the fact that the ventral CPu is anatomically identified as the territory most likely to receive descending inputs fiom the hippocampus. As alluded to above, a population of theta ON cells in the SNR appear to receive ascending sensory information which influences their theta-related discharges, while also receiving information fiom the MS concerning the relative robustness of these theta- related responses. As one of these cells also experienced a basal drop of firing after drug administtation, it was argued that this cell interacts with the MS without implicating the hippocampus. For the other SNR cell in this population, no basal drop in firing occurred Thus, this cell would be included amongst the neurons that interact with the MS in a cliff- fishion, possibly through the hippocampal formation. Interestingly, the third SNR theta ON cell recorded under these conditions could be grouped with the other theta ON CPu cells, nameIy those that receive no theta-related 232 ascending input, only descending input fiom the MS. This neuron is similar to the singular CPu theta ON neuron incIuded in this group whose firing rate was not Nly attenuated during the period of maximal effect of procaine. While the third SNR cell no longer responded to sensory shimulation in the "clrug state7', it is slightly different fiom this single CPu cell as no basal drop in its Engrate occurred Although a minor projection fiom the MS directly to the SNR has been demonstrated (Jakab and Leranth, 1995), it is suggested here that the effixts observed for this celI are mediated through a route which involves the hippocampus. Although no spontaneous theta could be recorded "post-drug" before this cell was lost, this hypothesis is warranted as the loss of theta ON cellular response was correlated with the loss of hippocampal theta in this investigation. The hippocampal formation likely projects to the SN via the nucleus accumbens (for a review see Mogensen et al., 1980; Lopes de Silva et al., 1985). Evidently, a larger number of neurons exhibiting these properties must be tecorded before this hypothesis can be fully tested Two theta OFF neurons were recorded fiom the basal ganglia before, during, and after MS suppression. Both of these ceUs were located in the CPu The behavior observed for these neurons was incongruous. Chemical inactivation of the MS appeared to weaken or even destroy the effect sensory stimdation had on these cells. For one cell, sensory stimulation was no longer effective at depressing firing rates fiom LIA baseline levels. For the other cell, sensory stimulation still led to a decrease in cell firing rate in the "drug state7', but the basdine rate associated with LIA had dropped to similar levels. Evidently, more data must be collected before a dear picture can be obtained of how basal ganglia theta OFF cells are functionally reIated to the septohippocampal system, 233 The patterns of cell discharge for theta-related CPu cells appeared to vacillate independently of drug condition AU periodic theta ON and OFF CPu cells remained periodic during and after administration ofprocaine; however, the relative percentages of LIA and theta trials considered periodic changed across drug conditions. Yet, there was no consistent pattern to these changes. This is not unexpected since the periodicity of CPu cells appears to be independent of any theta-related processes. An important manner in which the SNR theta ON neurons differed fiom the CPu theta ON neurons was the interaction between drug condition and periodrc spike trains for the SNR neurons. As was the case for non-periodic cells located in the CPu, those located in the SNR remained non-periodic across all drug conditions. Similarly, the one theta ON periodic cell recorded !?om the SNR in these conditions remained classified as periodic throughout the course of the experiment Interestingly, however, this cell became "less periodic" during the time of maximal suppression of the MS. That is, while in the "pre-drug" and "post-drug" conditions all recorded trials across all hippocampal field states could be considered periodic, fewer were considered periodic in the "dmg state" when the neuron lost its theta-related behavior. This provides some evidence for an interaction between the periodicity and theta-relatedness of SN neurons, an issue which was addressed in more detaiI in the Discussion of experiment one. The finding that the theta-related behavior of a theta ON neuron recorded from the ml was dependent on the MS remaining intact provides reason to investigate theta- related ceuular activity in this strwture. Indeed, the ml is anatomically and probably fimctionaIly Iinked with serotonergic neurons of the brain stem, including the MR (Halliday et al., 1995), the site fiom which ascending hippocampal desynchronizing 234 patterns originate (Vertes, 1981). The ml is also one of the primary pathways that sends sensory information from the brainstem to the thalamus (Arnaral, 2000). In summary,while the maintenance of theta-related cell behavior in the Cpu, SNR, and thalamus during MS suppression corroborates the proposition that these nuclei provide ascending synchronizing input to the hippocampus, the loss of theta-related cell behavior in the CPu and SNR under the same conditions suggests that some basal ganglia nuclei receive descending input fiom the septum or from nuclei that are dependent on its proper hctioning (the hippocampus). These findings are not contradictory as the two possibilities are not mutually exclusive. This descending input may or may not travel through independent routes to reach the CPu and SNR but, hctionally, it provides a connection through which the hippocampus might exert an influence over areas involved in motor control. It is unlikely that this descending information is related to that impinging on the MM. Kirk (1998) has suggested that the MM appears to be involved in a re-entrant loop involving the hippocampus, MM, and anterior thalamus. In such a circuit, the phasic neurons of the MM transmit information about the frequency of ongoing theta back to other structures within the ascending system. The tonic theta-related neurons of the ventral thalamus examined in this study may or may not be implicated in this loop. Based on the data accumuIated in experiments two and three, a putative connectivity model showing how the hippocampus and basal ganglia communicate with each other is presented below. 23 5 General Discussion and Fubre Considerations Despite intensive research, the functional significance of the hippocampus remains enigmatic (Bland, 1986). The EEG patterns indigenous to the hippocampal formation have long been considered to be a reflection of the neural processing going on in these structures. Thus, it seems reasonabIe to assume that the study of its EEG pattern will serve to elucidate at least some aspects of hippoampal function. Measurement of the electrical activity of the hippocampus during different behavioral states has revealed that this structure may be implicated in some fashion with motor behavior. The broad objective of this thesis was to assess whether activity in the hippocampus is related in any way to activity in a brain region known to be involved with movement, namely the basal ganglia. An attempt was made to detect any posshle relationship between the summated electrical activity of the hippocampal formation (as represented in the EEG) and the single cell activity of the basal ganglia Further, an attempt was made to map the pathway(s) through which these possble relationships were established The first major god of this research was completed: unit activity from the CPu, GP, and SN was observed simultaneously with the field activity recorded fiom the hippocampus. Experiment one demonstrated that a large number of neurons recorded fiom these stmctwes had discharge properties which were related to the hippocampltl EEG state. Two populations of celIs existed: those that increased their spontaneous discharge rates during the occmceof synchronous hippocampal field activity (theta), and those that decreased their spontaneous discharge rates during the occurrence of theta These findings rank the badganglia with other regions of the brain which contain populations of neurons with discharge properties that are related to the hippocampal field 236 activity, including the hippocampus (Colom and Bhd, 1987; Colorn et al., 1991; Bland and Colom 1988, 1989; Smythe et al., 1991; Konopacki et al., 1992; Bland et al., 1996), the MS (Ford et al., 1989; Bland et al., 1990,1994; Colom and Bland, 1991), the entorhind cortex (Dickson et aL, 1994, 1995), the cingutate cortex (Colom et al., 1988), the caudal diencephalon (Bland et al., 1995; Kirk et al., 1996), the rostra1 pontine region (Hanada et al., 2999), and the superior colliculus (Natsume et al., 1999). Anatomical data, paired with pharmacological and chemical manipulations, have helped identify the routes through which these relationships are established for a number of these structures. This work has led to the proposition of an ascending brainstem synchronizing pathway: a major source of extrinsic inputs which act to initiate and synchronize the intrinsically oscillatory membrane potentials of pyramidal cells in the hippocampus (for reviews see Bland and Colorn, 1993; Bland and Oddie, 1998: Bland, 2000). This ascending pathway arises fiom the pontine reticular formation, synapses with nuclei in the diencephalic region, and fiom there projects to the region of the medial septum. This region serves as the node of the ascending pathway, sending both cholinergic and GABAergic projections to the hippocampusus The second major goal of this research was also completed: electrical and pharmacological manipulations were used to map the possible routes through which the basal ganglia and the hippocampus interact Experiment two revealed that the basal gangha comprise another source of ascending synchronizing input to the hippocampus. Taken together with resuIts presented by Sabatino et al. in 1985, 1986, and 1989, it appears as though electrical stimulation of the CPu, GP, and SN can act to synchronize hippocampal field activity. The routes through which these effects are md-ated may be 237 independent for each basal ganglia structure and, httrm, may be independent f?om the ascending brainstem synchronizing pathway, but all must converge at some pint below the septa1 complex as all synchronizing effects are blocked by inactivation of this structure. Tbis is an area which merits further research as wiIl be discussed below. Finally, experiment three corroborated that cells in tbe CPu and SNR project ascending information to the hippocampus. In additiou, this expenrnent demonstrated that neurons witbin the Cfu and SNR receive a descending influence from the septum, or fiom mctures which are monosynapticalIy connected to it (most prominently the hippocampus), which can modulate the spontaneous discharges of these neurons in reiation to the hippocampal field state. Paired with the most parsimonius anatomical connections outlined in the Introduction, these findings permit the construction of a model outlining the general manner in which the basal gangIia nuclei examined might interact with the ascending pathway and the hippocampus. This model of functional connectivity is displayed in Figure 3 1, with the basal ganglia nuclei investigated grouped in the darker gray box and the ascending brainstern hippocampal synchronizing stn~ctwesgrouped in the lighter gray box. The electrical stimdation which synchronized the hippocampal EEG likely passed fiom the CPu, GP, and SN along the direct and indirect pathways of the bad ganglia before ascending to the hippocampal formation via the comedon between the SN and the pontine region_ It should be noted, however, that stimulation effects arising hm the CPu, GP, and SNC may be able to bypass the output nuclei via a mute that teaves the basal ganglia through the EP and GP, and impinges on the hippocampal formation via the lateral babenula and the raphe nucleus (Sabatino et ai., 1986, 1987). 238 Figure 3 1. Schematic of the possible routes through which hippocampal theta- related cells of the basal ganglia interact with the ascending brainstem hippocampal synchronizing systems and the hippocampus. The basal ganglia is represented by the dark gray box, the ascending system and the hippocampus by the light gray box Ody the basal ganglia structures examined in this thesis are included, and the connections between these structures have not been shown. Furthermore, possible interactions along a doparninergic pathway, and a lateral habeula-raphe nucleus pathway, have not been shown. 240 (Although the intrinsic connectivity of the basal ganglia has not been depicted for simplicity, the CPu, SNC,and GP can interact without involving the SNR). This pathway has not been included in Figure 3 1, as there has not been much support for it in the literature, and it is likely not the simplest pathway these ascending synchronizing effects could follow. Ascending connections between the basal ganglia and the septum were also suggested by the results of experiment three, in which chemical inactivation of the MS had little effect on the theta-related discharge profiles of a group of neurons in the CPu and SNR. For these theta ON cells, sensory stimulation was still able to elicit increases in their discharge rates once the septum was inactivated, although septa1 influeaces may underlie their basal tiring rates and the robustness of their theta-related responses. Sensory information reaches the basal gangha fiom a number of sources, most importantly the cortex and thalamus. An additional interesting source of sensory information also arises &om the brainstem, specificalIy the PPT. This structure projects to all aspects of the basal ganglia: output (the SNR), relay (the SNC), and input (the CPu, via a thalamostriatal projecton) nuclei. This leads to some perplexing combinations. It appears that, if the basal ganglia does in fact pass the sensory information it receives fiom cortical and Wamic sources on to the hippocampus (through the lateral habenula or the PPT), this information is not necessary for the synchronization of theta. As seen in experiment two, chemical inadvation of the SN or GP had no effect on the occurrence of spontaneous theta, or on the ability to elicit theta with tail pinches. At this point, however, the conclusion that the basal ganglia are unnecessary for hippocampal EEG synchronization is premature: it may be necessary to imtivate all basal gangtia nuclei at 24 1 once (a feat which is at present diflicult) to see auy disniption in the synchronizing effect that relevant sensory stimulus has on the hippocampal field activity. The basal ganglia might not wntriiute ascending synchronizing information to the hippocampus per se, but might simply monitor this information (through its connections with the PIT and the MS) as it climbs the ascending pathway en route to the pyramidal ce1Is of the hippocampus. E'qerhent three revealed that another population of theta-related cells in the basal ganglia exclusively receive descending information from the septohippocampd complex: once the MS was inactivated, theta cells in the CPu and in the SNR no longer responded to sensory stimulation that had previously elicited hippocampal theta. Although some of these cells may receive direct descending information fiom the MS which maintains baseline firing rates, all information which codes a theta-related increase in the firing rates of these cells is most probably transmitted to the basal ganglia through septohippocampal fibers. These transfer excitatory information through the hippocampus proper and out the efferent projections of this structure. The majority of these hippocampal efferent5 likely arise in the subiculum and project directly to the ventromedial striatum, and indirectly through the nucleus accumbens to the SNC (see Figure 3 1). It is therefore suggested that, uuder normal conditions, a population of theta ON cells within the basal ganglia discharge at spontaneous levels (which may or may not be dependent on direct input fiom the MS) until the membrane potential oscillations of pyramidal cells in the hippocampus become synchronized by ascending input (theta state). This accrued synchronized activity is transmitted through hippocampal output fibers such that these basal ganglia theta ON neurons are activated- When 242 septohippocampal neurons are inactivated, hippocampal theta can not occur, and this population of theta ON cells in the basal ganglia continue firing at spontaneous, inactivated levels. The evidence from this thesis suggests that fimctiomally distinct theta-related cells exist in the basal ganglia: those potentially associated with monitoring (and perhaps contributing to) ascending hippocampal synchronizing activity, and those associated with receiving descending signals hrn the hippocampus which may possibly influence motor output. A number of research programs could help further pinpoint these relationships. Work would have to be done both within and outside the basal ganglia in order to further elucidate the influence of this region and/or its dependence on the hippocampus. First, extracellular single-unit activity needs to be recorded From the STN and EP. In urethane anaesthetized rats, Magill et ai. found rhythmically bursting neurons in the STN: perhaps these are the elusive phasic theta-related neurons of the basal ganglia In addition, the neurons of the EP homologue in the cat do not appear to have synchronizing effects on the hippocampal EEG (Sabatino et al., 1986). It is possible that this nucleus is primarily comprised of mostly theta OFF neurons, such that electrical stimulation exerts an inhiiitory effect on hippocampal synchronization. This regional segregation of hippocampal theta-related cell types has been observed at the level of the PH-SUM (Bland et al., 1995; Kirk et al., 1996)- It shodd also be mentioned here that it would be of interest to record and characterize the theta-related ~mnsof the thaIamus. In this study, theta-related cells were found in the ventral tbalam~sand have been reported for the central medial thalamic 243 nucleus. In addition, Kirk (1998) has proposed that the anterior thalamus plays a role in mediating the involvement of theta in learning and memory- Also, although in one experiment electrical stimulation of the VPM did not evoke hippocampal theta, in another investigation it appeared that chemical inactivation of the tbalamus led to the disruption of spontaneous and tail pinch induced theta Furthermore, experiment three demonstrated that a population of cells in the ventral thalamus may receive descending information from the hippocampal formation concerning the robustness of their theta-related responses. Considering its role as a gateway between sensory and motor modalities, it is surprising that there has not been an indepth investigation of the thaiamus in the context of hippoampal synchrony. Further mapping of the intrinsic routes through which the various nuclei of the basal ganglia interact in relation to hippocampa1 theta is needed Verification of the stimulation effects reported here and by Sabatino et al. (1985, 1986, 1989) is necessary, preferably using pharmacological activation, so that the nature of the synaptic chernistq involved is revealed, and so that stimulation effects are limited to local infusion receptor sites and do not include axons of passage. Although technically difficult, it would be useful to record hippocampal theta while inactivating a number of basal ganglia nuclei at one time, so that specific routes of ascending influence can be verified For example, while stimulating the CPu, the hippampa1 field shodd be observed while simultaneously blocking relay (the SNC and GP) and output (the EP and SNR) nuclei. Work of this type could provide clues as to whether or not the input or relay nuclei of the basal ganglia transmit information to the hippocampus via output nuclei, or bypass these structures and transmit information via other routes (through the lateral habenula, for 244 example). Such work could be Werdeveloped with experiments which examine basal ganglia stimulation effects during the inactivation of the habenula andfor the PPT The influence thslr inactivation of MS neurons bas on theta-related cells of the basal ganglia needs to be corroborated and further explicated How do theta-related cells of the GP, EP, and SNC (or their homologues in species other than the rat) react to this manipulation? The hypothesis proposed above, that a certain population of cells in the basal ganglia do not directly contribute, but rather monitor, ascending influences could be verified in a number of ways. One important test would be to specifically inactivate all basal ganglia nuclei and simply observe whether spontaneous or tail pinch induced theta occurs. This work is difficult because of the requirement for very specific foci of inactivation Another technically difficult experiment which might help assess the source of sensory theta-relevant information to the basal ganglia involves the electrical stimulation of certain selective brain regions during chemical inactivation of the MS. For theta-related cells which continue to respond to sensory stimulus after inactivation of the septohippcampal complex, is this response also apparent during stimulation of the PPT? During PH-SUM stimulation? Or during stimulation of the thalmic region? The route through which descending influences arrive from the hippocampus could be elucidated by examining theta-related cells in the basal ganglia during chemical inactivation of subicdar output neurons which project to the ventral stn'atum andlor the nucleus accumbens. Although much literature has suggested this Iatter pathway in particular as a gateway between the hippoampus and motor systems, little work has examined the exact nature of this relationship. Some of the work descrikd above could 245 help delineate whether descending influences impinge on different basal ganglia nuclei independently, or principally on the CPu, and are then simply passed along the direct and indirect pathways. Probably the most important fimue work concerns the transfer of this study into freely moving animals. Initial investigations in freely moving animals could examine whether electn-cal stimulation of basal ganglia structures, in addition to eliciting theta, is also associated with theta-related movements. Electrical stimulation induced theta might either be associated with theta-related behaviors, such as head movements, waIking, limb movements, etc., or could be in fact disassociated from theta-related behaviors, as is the case for septa1 activation (Kramis and Routtenberg, 1977; Kramis and Vanderwolf, 1980). Such work would help provide evidence for or against hippocampal and basal ganglia synergistic influences over motor behaviors. More difficult experiments entail characterizing basal ganglia theta-related cell discharge profiles in freely moving, behaving animals. This work would help clarifjl the discrepancies concerning bursting and/or periodic cellular discharges in the basal ganglia related to hippocampal theta. 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