Health Science Campus
FINAL APPROVAL OF MASTER THESIS Master of Science in Biomedical Sciences
Potential mismatches in structural and functional organization in the gracile nucleus
Submitted by: Shalini Niranjan
In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences
Examination Committee
Major Advisor: John Wall, Ph.D.
Academic Nicolas Chiaia, Ph.D. Advisory Committee: Richard D. Lane, Ph.D.
Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D.
Date of Defense: July 25, 2008
Potential mismatches in structural and functional
organization in the gracile nucleus
Shalini Niranjan
Neurosciences and Neurological Disorders
University of Toledo, Health science campus
2008
Acknowledgements
I would like to thank Dr. John Wall, my major advisor, who has supported me all through my research and thesis, with his valuable knowledge and suggestions and by his unending support, while at the same time allowing me room to work in my own way. He has challenged and encouraged my thinking throughout the academic program which has immensely helped me in writing this thesis. My master’s degree and this thesis, too, would not have been completed if not for his patience and continued guidance.
I would also like to thank Dr. Richard D. Lane and Dr. Nicolas L. Chiaia who have guided me through the dissertation process, giving me many invaluable suggestions, and serving on my graduate committee.
I would like to thank Xin Wang for his friendship. He has helped me a lot both with his suggestions and his technical expertise.
Finally I would like to thank all my friends and family, especially my mom and dad for cheering me through the whole program and my husband for his support and my two baby girls for their patience and giving me time to work on my thesis.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS………………………………………………………...……..ii
TABLE OF CONTENTS……………………………………………………………….iii
INTRODUCTION…………………………………………………………………….....1
Background for thesis……………………………………………………….…….1
Focus of thesis ………………………………………………………………….....5
LITERATURE REVIEW…………………………………………………………….…7
How the GN contributes to the somatosensory system……………………….…..7
Features of GN structural and functional organization………………………..…..8
A. A main input to the GN is from primary sensory neurons that
process tactile inputs from the skin……..…………………………….8
B. What is know about the spatial organization of ascending axon
terminations in the GN…………………………………………………9
a) GN organization of dorsal column inputs from Golgi
material ...……………………………………….……..9
b) GN organization of primary neurons terminations
from labeling of inputs from dorsal root ganglia or nerve
locations ………………………….…………………10
c) GN organization of primary neurons terminations
from labeling of inputs from specific body
locations………………...…………………………….12
C. GN neurons …………...…………………………………………..…13 iii
D. GN connectivity ...…………………………………………………...14
E. GN functional RFs……………………………………………………16
Literature Summary……………………………………………….………….....18
DESIGN………………………………………………………………………………. .20
MANUSCRIPT……………………………………………………… …………...…. .23
DISCUSSION ………………………………………………...………………………...63
Present results……………………………………………………………………63
How the present results supplement understanding of primary
input organization in the GN…...………………………...………………66
How the present results supplement current understanding of GN
functional RFs on the toes……………………………...………………...70
Some broader implications of the findings of this thesis………………………...72
A. Possible mismatches in GN structure and function and
a potential explanation………………………………………………..72
B. Some features of cortical and GN processing may be similar………..74
C. Potential contribution to plasticity of GN RFs……………………...... 75
BIBLIOGRAPHY………………………………………………………………………77
ABSTRACT……………………………………………………………………………..91
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Introduction
Background for thesis
The somatosensory system conveys tactile and pain sensation from our body. Transmission of touch information from the body is initiated by peripheral primary sensory neurons that project axons from endings near the body surface to the central nervous system, including a large ascending central projection to the dorsal column nuclei (DCN), within the brainstem (Willis & Coggeshall, 2004; Tracey, 2004).
Peripheral sensory axons from the toes on the foot, for example, enter the spinal cord and ascend to the brainstem where they terminate and make excitatory synaptic connections on postsynaptic neurons in the gracile nucleus (GN) subdivision of the DCN. GN neurons, in turn, send ascending projection axons to the thalamus where connections through a second set of excitatory synapses is made onto postsynaptic thalamic neurons.
Thalamocortical axons of thalamic neurons project to primary somatosensory cortex where subsequent synapses are made onto cortical neurons. In this way, tactile stimulation of a toe on the foot leads to excitation of GN and thalamic neurons which, in turn, initiate cortical activation that contributes to feelings of touch.
Individual somatosensory neurons commonly have an excitatory tactile receptive field (RF). This is an area of skin within which tactile stimuli must be located to cause excitation of the neuron. Thus, individual GN, thalamic, and cortical somatosensory neurons usually have a delimited RF that encompasses the body location where touch can activate that neuron. It is generally thought that our ability to localize touch stimuli to specific body parts like a toe results, in part, because stimulation of RFs 1
on different parts of the body activates restricted clusters of central neurons.
Understanding how ascending projections and related postsynaptic neurons are
structurally organized to process information from different locations on the body is an
important part of understanding normal somatosensory functional organization.
The detailed synaptic arrangements of ascending axonal inputs onto neurons in the GN, thalamus, and cortex remain only partly understood. This is due to uncertainties about the diverse arrangements of connections that are possible. For example, at each level it is possible that terminations from only a few or, alternatively, a large number of ascending axonal projections may converge onto an individual postsynaptic neuron. It is also possible that an individual projection axon may send
divergent terminations to only a few or to a large number of postsynaptic neurons.
Moreover, at each synaptic level the excitatory connections of ascending axonal
projections onto postsynaptic neurons are supplemented by further connections of
inhibitory neurons which inhibit excitatory convergent and divergent connections of input
projection axons. Given these and other connection possibilities, it is difficult to know
how structural convergence of excitatory input terminations versus inhibitory connections
and other factors shape the functional RF properties of a postsynaptic neuron at any
particular time.
Some insight into this issue has come from studies of RF plasticity in the
adult somatosensory cortex. Cortical neurons that normally have RFs on a particular part
of the body have been studied after peripheral inputs from their RFs were lost as a result
of a range of manipulations including injury of nerves, loss of a part of the body (and its
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nerves) due to amputation or local anesthetic block of inputs. The results show that
cortical neurons that lose their normal inputs and RFs often acquire new RFs in other parts of the body that remain normally innervated. These findings show that cortical neurons can ‘reorganize’ by becoming responsive to new inputs and to shifted RFs on new locations on the body. Cortical RF reorganization has been seen in adult animals
and humans and, thus appears to reflect an important concept of organization. (Jones,
2000; Florence, 2002; Kaas, 2002; Calford, 2002; Wall et al., 2002).
The above RF reorganization of cortical neurons indicates adjustments can
occur in adult ascending connections at some level(s) of the system between the body and cortex. Cortical studies initially focused on RF reorganization after chronic input disruption because it was thought that once developmental changes were complete, adult
somatosensory connections were highly resistant to modification and would require long
times to change. However, much subsequent work has shown that initial cortical
reorganization can occur quite rapidly (Calford, 2002; Wall et al., 2002). For example,
after injury of nerves to the hands of adult monkeys, cortical RFs and maps of noninjured
tactile inputs from the hand undergo changes in organization within minutes to hours.
Similarly, after disruption of peripheral inputs by cutaneous injections of local anesthetics
like lidocaine, RFs of cortical neurons rapidly enlarge within minutes (Byrne and
Calford, 1991; Calford and Tweedale, 1991; Faggin et al., 1997; Krupa et al., 1999;
Nicolelis et al., 1993; Panetsos et al., 1995; Shin et al., 1995).
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The rapidity of these initial cortical RF changes suggested a broader range of inputs was available to activate a cortical neuron than was normally seen at a particular time, and that some of these available inputs were normally masked (Florence, 2002;
Calford, 2002; Wall et al., 2002). Loss of peripheral inputs, then, led to conditions that rapidly unmasked available inputs.
Two further lines of evidence suggested that masked inputs were at least partly derived from highly convergent/divergent thalamocortical axon projections onto cortical neurons. First, rapid cortical RF reorganization, like that seen within the initial minutes after disruptions of peripheral inputs can also be produced if local cortical inhibition was disrupted by blocking inhibitory effects of cortical interneuron connections with GABA receptor antagonists (Alloway & Burton, 1991;Dykes et al., 1984;
Chowdhury & Rasmusson, 2002). These findings suggested that local cortical inhibition normally decreases RF size by tonically inhibiting functional expression of some subset of structurally existent and broadly convergent/divergent thalamocortical inputs. Second, structural studies, which traced thalamocortical axon arbors as they traveled to synapse on cortical neurons, indicated that individual thalamocortical axons can project across large distances of cortex, and that these distances were broader than cortical distances that contained neurons with RFs on a single body part (Garraghty et al., 1989; Arnold et al.,2001; Rausell & Jones, 1995; Rausell et al. 1998; Garraghty et al. 1990; Churchill et al. 2004; for reviews see Snow and Wilson, 1991). This suggested that thalamocortical axons provide inputs to cortical neurons that have RFs that normally do not reflect all available inputs. 4
Focus of thesis
The above cortical findings point to a simple but important concept about
structural organization of ascending presynaptic inputs and associated RF locations of postsynaptic neurons: i.e., the normal RF location of a neuron at a particular time reflects only a part of a larger range of locations that is possible from available overlapping convergent (and divergent) input axons onto that neuron. This concept has important implications for understanding how postsynaptic functional organization arises from presynaptic structural organization. For example, structurally broad sets of overlapping inputs that are only partially expressed at a particular time could give postsynaptic neurons greater functional adaptability than organization based on narrow inputs.
Cortex is commonly thought to be the most complex part of the nervous system, and adaptation and modifiability of connections is an important intrinsic cortical property. It is possible that this concept may apply to cortex but be less pertinent in other central structures where organization may be less complex or based on different concepts.
For example, presynaptic input organization in subcortical circuits may reflect concepts that support accurate information transmission rather than adaptability, and may be more narrowly defined than presynaptic cortical organization.
Working from the above perspectives, the question addressed in this thesis is: does the concept that RF location reflects only part of an available overlapping convergence (and divergence) of ascending input axons also apply to connections at an 5 earlier level of central touch pathways, and more specifically, at the brainstem level in the GN? To examine this question, this thesis tested whether GN terminations of peripheral primary sensory projections from different locations on the body of adult rats overlap and are organized in convergent fashion or, alternatively, whether these terminations are more discretely organized in nonoverlapping, nonconverging ways. To do this, I studied GN terminations of primary neuron inputs from hindpaw toes, first, because each toe is physically distinct and offers a body location that can be distinguished from other body locations and, second, because high densities of primary neuron inputs originate from the toes. Locations on one or more hindpaw toes were injected with horseradish peroxidase conjugates to permit labeling and reconstruction of
GN terminations of sensory axons from specific toes. Degrees of overlap of input terminations from different toes were assessed from these reconstructions. In addition, patterns of GN terminations from different toes were compared to GN toe RFs. This permitted examination of whether overlap of structural inputs from different toes matched or was different than the numbers of toes that are usually included in normal functional RFs. This approach could also complement other recent studies, including inhibition block studies, which deal with GN RFs but not from the present perspective of
RF relationships to structural inputs (see discussion).
The following Literature Review outlines salient properties of organization in the rat GN that served as a base of understanding for the present GN work.
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Literature Review
How the GN contributes to the somatosensory system
The GN is a major brainstem processing nucleus in the somatosensory
system that, together with the cuneate nucleus (CN), makes up the dorsal column nuclei
(DCN) (Willis and Coggeshall, 2004; Tracey, 2004). The GN nucleus is located dorsally
in the medulla, is close to the midline and medial to the adjacent CN, and is elongated in
the rostrocaudal direction. In rats, it occupies about 39% of the volume of the DCN and
does not appear to have clear cytoarchitectonic subdivisions (Bermejo et al., 2003).
The DCN form the brainstem processing component of the dorsal column-
medial lemniscal system, a large ascending projection system from the body to cortex
(Tracey, 2004). This system processes touch and proprioception information, and the GN subdivision of the DCN is recognized as the main processing complex for touch and proprioception signals from skin areas on the foot, hindlimb, and lower body (Kaas,
2004). There is further evidence that the GN also contributes to processing of pain information (Willis & Westlund, 2004).
It is of interest to know whether concepts of processing that apply at one level of this system, e.g., cortex, apply at other levels, e.g. GN. Present understanding of major structural and functional features of the GN that are pertinent to the question that is addressed in this thesis is outlined next. This comes from studies of the GN as well as the adjacent CN.
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Features of GN structural and functional organization
A. A main input to the GN is from primary sensory neurons
that process tactile inputs from the skin
Neurons in the GN receive a major part of their inputs from primary
sensory neurons. The distal axons of these primary sensory neurons end in the body and,
via the actions of receptors and voltage-gated ion channels, transduce mechanical and
other stimuli into receptor potentials which, in turn, are converted to action potentials that
are transmitted up the primary neuron’s distal and central axons. The central axons of
primary sensory neurons to the GN enter the spinal cord at thoracic, lumbar, or sacral
levels and ascend in the ipsilateral gracile fascicle of the spinal dorsal column to
terminate and make excitatory synapses on ipsilateral GN neurons (Willis & Coggeshall,
2004).
Each primary sensory neuron has a RF area or volume on the body within
which stimuli must fall to activate that primary neuron. For primary neuron inputs to the
GN, these RFs are commonly on lower body skin locations including, for example,
hindpaw toes. Thus, tactile stimuli activate primary neurons with RFs on a stimulated
skin area which, in turn, activate GN neurons on which activated primary neurons
terminate and synapse. As a consequence, most GN neurons have excitatory tactile RFs
on the skin (Nord, 1967; Berkley & Hubscher, 1995; Panetsos, 1995; Kondo et al., 2002;
Costa-Garcia & Nunez, 2004; Malmierca & Nuñez, 2004). Thus, by nature of its main
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structural input connections, a major function of the GN is to process primary neuron
inputs from toe and other lower body skin areas.
B. What is known about the spatial organization of ascending
axon terminations in the GN?
A main focus of the present thesis is on how primary neuron terminals
from individual hindpaw toes are distributed in the GN. Of particular interest is whether
ascending input terminations from individual hindpaw toes overlap or, alternatively, occupy nonoverlapping GN locations.
As discussed next, present information about the organization of ascending input terminations in the GN come from: a) Golgi staining of dorsal column inputs, b) labeling of primary neuron inputs from dorsal root ganglion or peripheral nerve locations, and c) labeling of primary neuron inputs from specific skin locations.
a) GN organization of dorsal column inputs from Golgi material
Early studies suggested that Golgi-stained dorsal column axons that
entered the GN had extensively overlapping terminations (Valverde, 1966; Odutola,
1977). Terminal arborizations of individual and small groups of dorsal column axons
formed intertwining glomeruli which surrounded dendrites of GN neurons. Adjacent
glomeruli appeared to form a continuous and relatively uniform plexus of terminations.
Axons that enter the GN from the dorsal column can potentially originate from the
ascending axons of primary sensory neurons from lower body locations or, alternatively,
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from ascending axons that originate from central spinal neurons. Thus, early Golgi
studies suggested that GN terminations of ascending dorsal column axons contribute to
glomeruli that appear to be part of a continuous input termination plexus. However, these
studies do not distinguish terminations of axons that are from primary neurons versus
spinal neurons, and do not indicate how primary neuron terminations from specific
locations on the body, like the toes, are organized in the GN.
b) GN organization of primary neuron terminations from labeling of
inputs from dorsal root ganglion or nerve locations
With development of axonal transport methods for labeling primary
sensory neuron axons and their central terminations, a large number of studies focused on
organization of GN terminations of primary neuron inputs that were labeled at dorsal root ganglion or peripheral nerve locations (Leong & Tan, 1987; Ding et al., 1999; Hwang et al., 2001; Novikov, 2001; Robertson & Grant, 1985; LaMotte et al., 1991; Ueyama et al.,
1994; Tong et al., 1999; Shehab et al., 2003). In these studies, horseradish peroxidase conjugates were injected into dorsal root ganglia, which contain cell bodies of primary neurons, or into peripheral nerves, which contain distal axons of primary neurons. The label was taken up, transported, and deposited in the central terminations of primary neurons that originated in the labeled structure. The results show that, regardless of the injection site, virtually all labeled terminations were in the GN ipsilateral to injections.
GN terminations of inputs from different dorsal root ganglia, including lumbar ganglia
3-5, which contain cell bodies of primary neurons from the hindlimb, hindpaw, and toes,
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were organized as rostrocaudally continuous columns; in addition, columns of terminations from different ganglia overlapped to substantial extents (Rivero-Melian and
Arvidsson, 1992; Prats-Galino et al., 1999; Puigdellivol-Sanchez et al., 2000). Similarly,
GN terminations of inputs from different peripheral nerves, including the sciatic and saphenous nerves, which contain primary neuron axons from the toes, hindpaw, and hindlimb, were also organized as rostrocaudally continuous columns that overlapped to large extents (LaMotte et al., 1991; Ueyama et al., 1994).
These studies clearly advanced understanding beyond the findings of the early Golgi studies by selectively identifying GN terminations that were associated with ascending primary neuron inputs. In addition, they identified GN termination locations of primary neurons associated with specific dorsal root ganglia and peripheral nerves, and established that terminations from different ganglia or nerves overlapped. From the perspective of the present thesis, however, these studies are limited by the fact that ganglia and nerves contain primary neurons with peripheral endings originating in broad body locations; thus, these studies do not have the spatial resolution to determine whether
GN terminations from a delineated body location, like an individual toe, overlap or are separate from terminations from a second delineated body location, like another toe.
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c) GN organization of primary neuron terminations from labeling of
inputs from specific body locations
In contrast to the above extensive work involving labeling of primary
neuron inputs from ganglia and nerves, there has been little work on GN termination patterns of primary neuron inputs from specific body locations (Maslany et al., 1991;
Maslany et al., 1992). In this work, HRP conjugates were cutaneously injected into toe or hindpaw locations. Terminations from toes were in dorsal locations of the ipsilateral
GN that appeared separated from locations occupied by hindpaw and hindlimb terminations. The results further suggested that toe inputs appeared to be organized in a complex pattern (Maslany et al.,1991) . Specifically, it was shown that rostro-caudal columns of terminations from toes 1 and 4 appeared to reversibly and repeatedly interchange in their relative medial and lateral positions at different rostrocaudal GN locations. Whether medial and lateral positioning of other toe termination columns similarly changed with respect to each other, or whether there was interchanging of termination columns of toes 1 and 4 versus columns of other toes, remained unclear. It also remained unclear whether interchanging termination locations of toes 1 and 4 involved overlapping of terminations from these or other toes. There have been no further studies of GN termination patterns of toe inputs, or inputs from other specific body locations, since this early report. Thus, there presently is no picture of how central terminations of primary neuron inputs from different hindpaw toes are organized in the rat GN and particularly, whether termination areas of different toes overlap or are distinct.
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C. GN neurons
The DCN, including the GN, contain two type of neurons, projection
neurons and interneurons (Snow and Wilson, 1991; Tracey, 2004) ). These neurons
have the following properties.
Projection neurons have long main axons that project signals from the GN
to other central structures. The main axons of projection neurons commonly also branch within the GN to give rise to collateral axons which terminate and synapse within the
GN. Projection neurons are glutamatergic and the main and collateral axons provide excitatory inputs to neurons they synapse on. The cell bodies of projection neurons can have large dendritic trees that can extend up to a few hundred microns from the cell body
(Kemplay & Webster, 1989; Weinberg et al., 1990; Lue et al., 2000; Bermejo et al.,
2003). Projection neurons to thalamic levels of the brain appear to make up a large percentage (up to 80%) of DCN projection neurons in rats (Bermejo et al., 2003).
In contrast to projection neurons, interneurons have short axons which terminate locally within the GN. DCN interneurons, including GN interneurons, all appear to be inhibitory. Immunohistochemical analyses indicate interneurons can be
GABAergic, glycinergic, or combined GABA- and glycinergic (Lue et al., 1993; Lue et al., 1996; Lue et al., 2000; Popratiloff et al., 1996a). Interneuronal cell bodies and dendrites are generally smaller than cell bodies and dendritic trees of projection neurons.
Interneurons are believed to make up a smaller percentage (up to about 10-25%) of DCN neurons than projection neurons (Bermejo et al., 2003).
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D. GN connectivity
Previous work has provided a partial understanding of the synaptic
connections of primary neurons, projection neurons, and interneurons (Willis &
Coggeshall, 2004). This connectivity includes the following main features (Fig.1).
Primary neuron inputs to the GN largely come from large diameter, myelinated afferents that transmit touch and proprioception signals. A further small fraction of primary neuron inputs appears to come from small diameter afferents.
Primary neuron inputs have glutaminergic excitatory synapses on GN projection neurons and interneurons. Inhibitory interneurons synapse on other inhibitory interneurons,
projection neurons, and terminations of primary neurons to mediate postsynaptic and
presynaptic inhibition (Fig.1). Projection neurons receive inputs from primary neurons
and interneurons and, via their axon collaterals, activate interneurons and projection
neurons. Primary neuron inputs comprise a major fraction, i.e. about 15-25%, of DCN
inputs. Other smaller input contributions come from central structures including cortex
(Chimelli et al., 1994; Desbois et al., 1999), spinal cord, (Cliffer and Willis 1994; De
Biasi et al. 1995; French et al. 2002), cerebellum (Asanuma et al., 1983), and other brainstem nuclei ( Weinberg & Rustioni, 1990). From this connectivity, activation of primary neuron inputs from the body can produce a number of effects on GN neurons including: (a) direct excitation of GN interneurons and projection neurons, (b) indirect
inhibition of interneurons, projection neurons, and primary neuron axon terminations through connections on inhibitory interneurons, and (c) more complex GN disinhibition
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through serial inhibitory interneuron connections (Fig.1). These effects of primary
neuron activation are, in turn, presumably integrated with effects of other inputs.
Figure 1. Primary sensory neuron inputs to GN neurons and GN synaptic connectivity
Primary sensory neurons (S) from the body have excitatory synapses on GN projection neurons (P) and on inhibitory interneurons (I). P axon collaterals have excitatory synapses on I and P (not shown) neurons. I neurons have inhibitory synapses on I and P neurons postsynaptically, and on S neuron terminations presynaptically. GN synaptic activity is dependent on inputs from S neurons and from inputs from central structures. Activation of S neurons can: (a) directly activate I and P neurons, (b) indirectly inhibit I and P neurons and S neuron terminations via I neuron activation, and (c) mediate more complex disinhibition through serial I neuron connections.
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With regard to output connections, GN and DCN projection neurons
transmit information to a number of central nuclei. A major projection ascends from the
GN to thalamic nuclei, particularly the ventroposterior lateral nucleus (VPL) via the medial lemniscus, to form the brainstem tract component of the dorsal column medial lemniscal system. VPL neurons, in turn, project to cortex; consequently, the GN serves as the first level nucleus in the dorsal column medial lemniscal system for processing of
ascending signals to cortex. Other central structures receiving direct GN and DCN projections include spinal cord, cerebellum, brainstem nuclei, and diencephalic nuclei
(Kemplay et al., 1989; Di Biasi et al., 1994; Bermejo et al., 2003).
E. GN functional RFs
The focus of this thesis was on how terminations of primary neuron inputs
from the toes are spatially distributed in the GN. This was done with an eye toward also
examining how structural organization of input terminations from different toes
contributes to GN neuron functional RFs on the toes. Of specific interest was how many
toes are usually included in normally-sized GN RFs on the toes.
It is known that a major percentage of GN neurons have an excitatory
tactile RF on the skin of the toes, hindpaw, hindlimb, or some other lower body location
(Nord, 1967; Berkley & Hubscher, 1995; Panetsos, 1995; Kondo et al., 2002; Costa-
Garcia & Nunez, 2004; Malmierca & Nuñez, 2004). In addition, beside a skin RF, many
GN neurons can have further RF components in visceral locations including, for example,
anal and vaginal locations ( Al-Chaer ED et al., 1996; Hubscher & Berkley, 1994). Most
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data that pertain to normal sizes of GN RFs on the hindpaw indicate RF size in terms of
areas of skin, but do not indicate size with respect to numbers of toes (McComas, 1963;
Suzuki & Dickenson, 2002; Miki et al., 1998). A small number of GN RFs that include
toes have been illustrated anecdotally in figures of reports on other issues (Nord et al.,
1967; Berkley et al., 1995; Kondo et al., 2002; Costa-Garcia et al., 2004; Wang & Wall,
2005; 2006; Panetsos et al., 1995; Pettit & Schwark, 1993 ). These RFs range from a part
of one toe to up to 5 toes and appear to emerge from summation of smaller RF’s of
primary neurons (Lynn & Carpenter, 1982; Leem et al., 1993; Reinke & Dinse, 1996;
Shortland & Woolf, 1993; Shortland et al., 1989; Sanders & Zimmermann, 1986).
However, the few GN RFs shown in different studies do not provide indications of the
usual number of toes that are included in GN RFs. Other than these observations that
numbers of toes in GN toe RFs can vary to some degree, there is little understanding of
typical degrees of convergence of inputs from different toes.
Consistent with the above findings that primary neuron inputs provide the
major structural input to the GN, disruptions of primary neuron inputs from specific skin
locations, including the toes, causes loss of parts of GN RFs that are innervated by the
disrupted inputs (Pettit & Schwark, 1993; Panetsos et al. 1997; Schwark et al., 1999;
Wang & Wall, 2005; 2006). In contrast, GN RFs, including RFs on the toes, continue to
be generated after disruption of GN inputs from central structures like cortex and
cerebellum (Pettit & Schwark, 1993; Wang & Wall, 2005; 2006). This suggests GN
functional RFs depend to a large degree on primary neurons.
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Literature Summary
The above literature review points out the following salient points about
what is and is not known about the GN, and about relationships between GN distributions
of primary neuron terminations from hindpaw toes, and GN RFs on these toes.
First, the GN is a major brainstem processing nucleus for information that
reaches cortex via the dorsal column medial lemniscal system. As part of a major cortical
input system, it is of interest to know whether concepts of processing that apply to cortex,
also apply to the GN. This issue has received little attention and is poorly understood.
Second, although cortex and other central structures provide inputs to the
GN, primary neuron inputs and, more specifically, large diameter primary neuron inputs
that process touch and proprioception signals, provide the major structural input to the
GN.
Third, axons from the dorsal column terminate in glomeruli which appear
to be organized in a plexus of terminations in the GN. Axons from dorsal root ganglia or
nerves terminate in rostrocaudal columns, and termination columns associated with
different ganglia or nerves overlap. Limited evidence suggests GN terminations of
primary neurons from toes versus hindpaw and hindlimb locations occupy different GN
locations. GN terminations of primary neurons from toes have been reported to
interchange relative locations at different rostrocaudal levels of the GN. Whether this interchanging involves overlap or some other complex arrangements of termination areas
of inputs from different toes remains unclear.
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Fourth, primary neurons activate GN inhibitory interneurons and projection neurons. Through the activity of inhibitory interneurons, primary neuron inputs can be inhibited at postsynaptic locations on inhibitory interneurons and projection neurons, and at presynaptic locations on primary neuron axon terminations. Thus, inhibition within the GN may attenuate or cancel excitatory effects of some primary neuron terminations.
Fifth, a major percentage of GN neurons have tactile RFs on the toes, hindlimb, or lower body. These RFs are dependent to a large degree on activity of large diameter primary neuron inputs. Available data do not indicate the number of toes that typically contribute to GN toe RFs.
The above findings provide the foundation for examining the thesis question of whether structural inputs from different toes overlap or are distinct, and whether structural termination patterns of inputs from the toes match or differ from the numbers of toes that are usually included in functional RFs.
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Design
This thesis examined whether a concept of cortical processing, i.e. that
excitatory RFs reflect functional expression of only part of an available overlapping
larger structural pool of ascending inputs, also applies to the GN.
The design was simple. The main part of the study examined whether GN
termination fields of primary neuron inputs from individual hindpaw toes overlap in a convergent fashion or, alternatively, are discretely organized in a nonoverlapping fashion.
The degree of overlap of structural inputs from different toes was then compared with the number of toes that are typically included in functional RFs on the toes. Inputs from toes were studied because toes are physically distinct (as compared to other continuous skin surfaces) and have higher densities of tactile inputs than more proximal body locations.
Toe RFs that were used for comparison with termination patterns were derived from our recent studies in which normal rat GN tactile RFs were assessed prior to experimental
manipulations (Wang and Wall, 2005; 2006).
To carry out this design, GN termination patterns of inputs from the toes
were reconstructed following toe microinjections of BHRP (cholera toxin subunit B
conjugated to horseradish peroxidase) or a mixture of BHRP and WGA-HRP (wheat germ agglutinin conjugated to horseradish peroxidase). The label was taken up and
transported by distal endings of primary neurons in the injection site. BHRP was used as the primary label because BHRP and the cholera toxin B subunit have been shown to be taken up and transported mainly by large diameter primary neurons that provide the main
20 input to the GN, and that provide activation for GN tactile RFs on the toes (LaMotte et al., 1991; Grant, 1993; Tong et al., 1999; Maslany et al., 1991).
Previous studies suggest that BHRP provides a more comprehensive estimate of total extents and densities of terminations than other labels (e.g., WGA-HRP, free HRP). Use of BHRP was, thus, consistent with the goal of identifying total terminal fields of inputs from individual toes and potential overlap of fields of different toes.
Electron microscopic studies indicate that label is located in terminals of sensory inputs after cholera toxin B subunit injections in hindlimb nerves (Hwang et al., 2001), and other studies have reported that label is mainly in sensory input terminals in the dorsal horn after BHRP or cholera toxin B subunit injection of hindlimb nerves or skin locations
(LaMotte et al., 1991; Levinsson et al., 2002; Maslany et al., 1992; Takahashi et al.,
2003). Previous reports also indicate BHRP that is taken up by primary neurons is not trans-synaptically transported (Robertson & Grant, 1985; LaMotte et al., 1991; Rivero-
Melian and Arvidsson, 1992). Consistent with previous observations (Maslany et al
1991; Rivero-Melian et al., 1992; Ueyama et al., 1994; Novikov, 2001), labeled axons were virtually all contained in the ipsilateral GN. This permitted microinjections of different toes in the two hindpaws, and cross-side comparisons of termination fields of different combinations of toes.
GN tactile RFs were defined using cutaneous tactile stimulation with hand-held probes and single or multiple unit GN responses in normal rats that were anesthetized with ketamine (80-90 mg/kg, im) and acepromazine (1.7 mg/kg, im). RF
21 locations on the toes were charted on scaled figures of the hindpaw, and counts were made of the numbers of toes per RF.
These structural and functional data provided analyses of degrees of structural overlap of GN terminations from different toes, and degrees of functional expression of toe inputs. These analyses were used to test whether structural overlap or non overlap matched or did not match functional expression. Further details about procedures are given in the following manuscript that summarizes the findings.
22
Manuscript
Title: Spatial relationships of structural and functional inputs to the brainstem gracile nucleus and potential functional underexpression of inputs
Shalini Niranjan, Xin Wang, and John T. Wall
Department of Neurosciences University of Toledo Medical Center Toledo, OH 43614-2598
Keywords: rat gracile nucleus, primary afferents, dorsal column nuclei, receptive fields, transganglionic transport
We gratefully acknowledge support of National Institutes of Health Grant HD 39791.
23
Abstract
The gracile nucleus (GN) is a major brainstem processing center for somatosensory inputs. GN neurons in rats often have excitatory tactile receptive fields
(RFs) on hindpaw toes, but there is little consensus on how the locations and sizes of these RFs emerge from GN substrates. To study this issue, the present experiments assessed relationships between spatial distributions of GN terminations of afferents from the toes, and sizes of GN tactile RFs on toes. Subcutaneous injections and transganglionic transport of tracers were used to reconstruct the distributions of GN termination areas of inputs from the toes, and neural recordings were used to define RFs on the toes. Overlap in termination areas of inputs from different toes was assessed and compared to the numbers of toes that contributed to toe RFs. GN terminations from each toe were distributed in a continuous rostrocaudal column in the GN. There was intermixing of termination columns of at least 3-4 toes across dorsal GN areas which, in cytochrome oxidase stained sections, appeared uniformly stained. Although a small percentage of toe RFs had inputs from up to 4-5 toes, on average RFs involved 2 toes, and most were on 1-2 toes. These results raise the possibility that, to some degree, toe
RFs in the rat GN emerge from sensory afferent substrates that have wider capacities than may be functionally expressed at a particular time.
24
Introduction
Afferent fibers from the hindpaws of rats project to the brainstem where
they terminate and make excitatory synaptic connections on GN neurons (Willis and
Coggeshall, 2004). GN neurons commonly have tactile receptive fields (RFs) on
hindpaw skin which, when stimulated by touch, cause neuronal activation. The locations
and sizes of these RFs, to some degree, reflect available direct input connections;
consequently, the structural distribution of primary afferent terminations from different
hindpaw locations is one factor in generating spatial properties of GN RFs. In addition,
GN input connections are influenced by intrinsic GN mechanisms, including inhibition
mechanisms, that affect GN processing and RF size (Broman, 1994; Willis and
Coggeshall, 2004; Wang and Wall, 2006). How spatial properties of excitatory tactile
RFs emerge from distributions of afferent terminations, GN processing mechanisms, and
other factors remains conjectural.
To study this issue, the present experiments evaluated relationships between the distributions of GN terminations of primary afferents from hindpaw toes and
GN excitatory tactile RFs on hindpaw toes. Tracers were injected into the hindpaw toes
of normal adult rats to permit labeling and reconstruction of the spatial distributions of
GN terminations of toe afferents. Overlaps in termination areas of different toes were
assessed and compared to results from a sample of GN toe RFs that estimated
percentages of RFs involving different numbers of toes. In addition, cytochrome oxidase
staining was used to test for subdivisions in GN areas with toe terminations.
25
Materials and Methods
All experiments were done in accordance with protocols that were approved by
the institutional animal care and use committee. Adult female Sprague Dawley rats were
used.
Toe injections
Animals were anesthetized with ketamine ( 80-90 mg/kg, im) and xylazine (8-12
mg/kg, im), and one or more toes on one or both hindpaws were injected subcutaneously
with cholera toxin subunit B conjugated to horseradish peroxidase (BHRP, 1%) or a mixture of BHRP plus wheat germ agglutinin conjugated to horseradish peroxidase
(BHRP, 1% and WGA-HRP, 14%). BHRP was used as the main tracer because it
effectively labels terminations of large diameter sensory inputs in normal rats (Robertson
and Grant, 1985; LaMotte et al., 1991; Tong et al., 1999; Shehab et al., 2003), and an aim
was to label large diameter sensory neurons that contribute to GN tactile RFs.
Microinjections were made from a microsyringe in one shot (≤8 µl) into the distal
phalanx. This was done to focus delivery in distal parts of toes that are expected to have
high innervation by large diameter cutaneous inputs, and to avoid spread beyond the
injected toe. Due to possible loss of label from uptake by blood, leakage from the
injection site, and other factors, it is difficult to stipulate exact amounts of label that were
transported. A total of 59 toes were injected in 20 rats.
26
Tissue processing
Following a post-injection time of 3 days, animals were given an overdose
of anesthesia and transcardially perfused with heparinated saline, 2% paraformaldehyde
in 0.1 M phosphate buffer, and 2% paraformaldehyde in 0.1 M phosphate buffer with
10% sucrose. The brainstem was removed, placed in the latter solution, and refrigerated
overnight.
The next day, a block of medulla spanning the entire rostrocaudal length of the GN was serially cut into 50 µm transverse sections. Each section was sequentially
placed in an individual well in 0.1 M phosphate buffer. A 5 or 7 in 10 series of these
sections was reacted in successive stages using tetramethylbenzidine (TMB) as a
chromogen (Florence et al., 1994). Given the interest in visualizing total termination
areas, sequential TBM reaction stages were monitored under microscopic view and
repeated to a point where emerging areas of GN labeling progressed no further, and
before background labeling became apparent in the GN and neighboring cuneate nucleus.
The remaining alternate 5 or 3 in 10 series of sections was processed with cytochrome
oxidase (CO) methods (Xu and Wall, 1999; Wang and Wall, 2006). The TMB series and
the CO series were separately mounted in sequence on slides and coverslipped.
In attempts to further document injection sites, in early experiments (n=4)
hindpaws were degloved by surgically dissecting all toe and other hindpaw skin from
underlying hindpaw tissues as one dorsal, and one plantar, skin piece. Dorsal and plantar
pieces, and the underlying hindpaw, were reacted separately using the above TMB
27 procedures. These attempts proved unreliable due to reactions with residual blood in paw tissues.
Reconstructions of GN termination areas
GN label typically appeared as collections of fine particles that were interpreted to involve terminal label (see Discussion). Transported label was clearly distinguishable from label associated with blood vessels or background. Labeled soma were not seen. GN areas containing labeled terminations were systematically reconstructed from transverse sections using dark field illumination and a drawing tube.
In agreement with previous findings (Rivero-Melian and Arvidsson, 1992; Ueyama et al.,
1994; Novikov, 2001), the present procedures resulted in labeling in only the ipsilateral
GN; consequently, it was possible to do cross-side comparisons following injections of toes on the two sides. Cross-side comparisons were done by first defining the left and right GN borders on individual sections. The GN borders on one side were then rotated around the midline axis and matched to the mirror image GN borders on the opposite side. Following matching of borders, reconstructions of labeled termination areas and lines indicating perimeters of labeled termination areas on each side were added at their respective locations within their GN borders. Cross-side matching was done entirely on the basis of GN borders and without reference to labeled terminations. Thus, cross-side analyses were “blinded” to the locations of the termination areas that were compared.
Digital images of TMB and CO sections were taken with a digital camera on a Nikon microscope. Images were collected using Spot basic software that was
28
interfaced with Adobe Photoshop programs. Images were adjusted using only brightness and contrast controls.
Toe receptive fields
Excitatory tactile RFs on hindpaw toes were used as indicators of suprathreshold functional convergence of toe inputs in the GN. A sample of 78 toe RFs
(39 single and 39 multi unit) was derived from studies in this lab in which normal GN tactile RFs were defined prior to subsequent experimental manipulations (Wang and
Wall, 2005; 2006). The sample reflected the range and distribution of sizes of normal toe
RFs defined with our procedures.
Recording, stimulation, and RF definition procedures have been previously described (Wang and Wall, 2005; 2006). Briefly, extracellular single unit
(window discriminated, ≥3:1 signal:noise) or multi unit GN activity was monitored as toe and hindpaw skin areas were tactilely stimulated with hand-held probes or brushes in anesthetized adult rats (ketamine, 80-90 mg/kg, im; acepromazine, 1.7 mg/kg, im). A RF was defined as the total area of skin that elicited neuronal discharges to cutaneous stimulation, and was judged to reflect a maximal excitatory tactile RF. For purposes of the present study, the sizes of GN RFs on toes were characterized in terms of the number of toes that were included in each RF.
29
Results
GN terminations of toe inputs
Injections were made into all five toes of a hindpaw to estimate the total
GN distribution of inputs from all toes. Injections of all 5 toes resulted in label in the ipsilateral, but not contralateral GN (e.g., Fig 1A). Across rostrocaudal levels, the composite termination area for inputs from all toes was roughly centered at mid- mediolateral locations in the more dorsal half to two-thirds of the ipsilateral GN (e.g. Fig.
1A-C). Terminations were continuously and densely distributed within this termination area across sections and extended as a continuous column along the rostrocaudal axis of the nucleus (e.g. Fig. 1B,C).
Injections of an individual toe were used to study terminations of inputs from different toes. After injection of any toe, associated terminations were centered around mid-mediolateral GN locations and, with sparing of label along the dorsal edge of the GN, were located in the dorsal half to two-thirds of the GN (e.g., Figs. 2,3). With
some variations in density, terminations were continuously distributed mediolaterally
and dorsoventrally across sections, and extended as a continuous column along the
rostrocaudal axis of the nucleus (e.g. Figs. 2,3).
A pattern of continuous terminations also occurred following injections of
nonadjacent toes on the same hindpaw. For example, injections of nonadjacent toes 3+5 or 2+4 resulted in largely continuous mediolateral and dorsoventral distributions of
labeled terminations, which extended as a continuous rostrocaudal column (e.g., Fig. 4).
A similar largely continuous pattern of terminations was produced after injections of toes
30
1+5, even though these toes were separated by the remaining three intervening toes 2-4
(e.g., Fig. 4).
Taken together, the above results suggested that the termination areas of
all five toes, any single toe, or nonadjacent toes were distributed in largely continuous
fashion in mediolateral and dorsoventral directions in the ipsilateral GN. Across
rostrocaudal levels, the termination areas of individual toes or combinations of toes
consistently formed continuous elongated columns in the GN.
Spatial relationships of termination columns of different toes
Further analyses were directed at assessing whether termination columns of inputs from different toes overlap. Three lines of evidence suggest that terminations of a given toe overlapped terminations of the other toes to substantial extents.
First, the termination column of each toe spanned similar locations and extents of the dorsal GN, with little apparent somatotopic shifting of termination areas of different toes. These results held when termination columns of different toes were compared across cases, or across sides in the same case. For example, the column of terminations from toe 1 occupied dorsal GN locations that appeared to coextend to some extent with termination columns of toes 2, 3, or 4 from other cases (e.g., compare Fig. 3A and B-D). Similarly, across cases, the columns of terminations from toes 1, 2, 3, or 4, and the termination column of toe 5 occupied overlapping locations (e.g., compare Fig. 3
A-D and E). Analogous statements applied when termination columns were compared across sides within the same case, as indicated for example by cross-side comparisons of
31
the termination columns of toes 2 and 4 (Fig. 3B and D), and the most separated toes 1
and 5 (Fig. 3E).
Second, injections of two nonadjacent toes on the same hindpaw resulted
in a continuous column of labeled terminations (Fig. 4). Given that the combined
terminations of all five toes also occupied a continuous area (e.g. Fig. 1), continuous
termination areas of nonadjacent toes suggest that terminations of inputs from the
injected toes likely overlapped termination areas of inputs from other (e.g., intervening)
toes that were not injected. The continuous termination area seen when the most distant
toes 1+5 were injected (e.g. Fig. 4A), suggests terminations of toes 1 and/or 5 cross areas
of terminations of intervening toes 2-4, thus reflecting overlap of terminations from at
least 3-4 toes. Further consistent with this possibility, cross-side comparisons of the
continuous termination areas seen, for example, after injection of nonadjacent toes 3+5
on one side and toes 2+4 on the other side, also suggested there was substantial overlap in the termination areas of at least 3-4 toes (Fig. 4C).
Finally, other analyses indicated that the column of terminations that was associated with any individual toe occupied substantial extents of the column of terminations associated with combined injections of either the most separated toes 1+5, or all five toes. For example, in different cases the continuous column of terminations that was associated with combined injection of the most separated toes 1+5 on one side colocalized to major degrees with the columns of terminations that were associated with toe 5 (Fig. 5A), toe 3 (Fig. 5B), or toe 4 (Fig. 5C) on the opposite side. Similarly, the column of terminations associated with injection of one toe on one side occupied
32
substantial extents of the column of terminations seen after injections of all five toes on
the other side. For example, across rostrocaudal levels, the termination column of toe 5
inputs spanned major central fractions of the termination column for all toes on the
opposite side, leaving relatively narrow margins for terminations from the remaining 4
toes (e.g., Fig. 5D).
The above results suggest that inputs from each toe terminated in a
continuous rostrocaudally elongated column in dorsal GN locations, and that there was
consistent and substantial overlap in the columns of terminations from at least 3-4, and
most likely all 5, toes. From this it appears that the GN contains areas where terminations
from each toe may intermix to substantial extents with terminations from most or all
other toes.
GN structure from CO stained sections
In CO stained sections, caudal levels of the GN contain densely stained neuropil areas that are disrupted by lightly stained bundles of fibers which are continuous with the gracile fascicle (e.g. Fig. 6A,B, asterisks). At successively more rostral levels, fiber bundles occupy reduced areas and densely stained areas occupy increased space as the main part of the nucleus first enlarges and then recedes (e.g., Fig. 6B-E). No consistent subdivisions were seen in the densely stained areas across the caudorostral axis
(e.g., Fig. 6A-E). On adjacent CO and TMB sections, the termination columns of inputs from the toes avoided fiber areas with light CO staining, and were localized in densely stained areas. Termination areas of toes were located in dorsal GN locations that had
33
relatively uniform CO staining (e.g., compare Figs. 6 and 3). These areas did not contain recognizable CO patches or other signs of subdivision, even though patches were consistently seen in the neighboring cuneate nucleus on the same sections. These results suggested that extents of the GN that contained terminations from different toes colocalize with GN areas with relatively uniform, dense CO staining.
GN excitatory receptive fields on the toes
Excitatory RFs on the toes were studied to assess functional convergence
of toe inputs in the GN. A sample of 78 toe RFs (39 single and 39 multi unit) was used to
analyze numbers of toes in RFs. Given the observed overlap of terminations of inputs
from the toes, RFs involving at least 3-4 toes might be expected to be common. In this
direction, GN RFs could involve excitatory inputs from up to 5 toes (e.g., Fig. 7).
However, further results suggest that most RFs did not reflect this expectation, including
that: (a) substantial percentages of toe receptive fields were restricted to one toe (single
unit = 46%; multi unit = 28%), (b) the majority of fields involved 1 or 2 toes (single unit
= 85%; multi unit = 56%), (c) few toe receptive fields involved 4 or 5 toes (single unit =
0%; multi unit = 11%), and (d) the overall mean number of toes per RF was 2 (single unit
= 1.7, multi unit = 2.3). These results suggest that although GN terminations of each toe
appeared to commonly intermix with terminations from most or all other toes, most RFs
did not functionally reflect this intermixing. These findings raise the possibility of
common mismatching in functional and structural organization where numbers of toes in
functional RFs underexpress existing structural overlaps in terminations of toe afferents.
34
Discussion
1. Present results
The present study suggests three main results. First, GN termination areas of toe inputs were organized as continuous rostrocaudal columns, and the column of terminations associated with a given toe overlapped the columns of other toes to large extents. As a consequence, there appeared to be intermixing of terminations from 3-4 or all toes along the GN rostrocaudal axis. Second, as assessed with CO staining, the termination areas of the toes avoided fiber areas with light CO staining, and were located in densely stained dorsal GN locations that did not appear to be subdivided. Finally, a small percentage of GN excitatory tactile RFs functionally reflected inputs from up to 4-5 toes, however, toe RFs averaged 2 toes, and the majority were on 1-2 toes. Taken together, these findings raise the possibility that there is some structural-functional mismatching in the form of common overlapping of afferent terminations from 3 or more toes, but common functional expression of inputs from 1-2 toes.
There is little understanding of how structural distributions of toe afferent terminations and functional RFs of toe inputs are related in the rat GN. The present results supplement current understanding in the following ways.
2. Peripheral sensory inputs to the rat GN
Extensive GN studies have been done in rats on sensory input projections from nerves (Leong and Tan, 1987; LaMotte et al., 1991; Ueyama et al., 1994; Shortland et al., 1998; Ding et al., 1999; Tong et al., 1999; Hwang et al., 2001; Shehab et al., 2003),
35
and dorsal root ganglia (Robertson and Grant, 1985; Rivero-Melian and Arvidsson, 1992;
Novikov, 2001). Although these studies clearly define GN termination patterns of
primary sensory inputs from the injected nerves or ganglia, they do not distinguish
termination patterns of toe inputs.
GN termination patterns of sensory inputs from hindpaw toes have been
examined in one previous study in which label was injected into toes (Maslany et al.,
1991). Results from that study, which were also based on transport of HRP conjugates,
resemble the present results in that terminations from an individual toe were distributed
as a rostrocaudal column in the ipsilateral dorsal GN. The present findings and this study
further agree that GN terminations from different toes are not distributed in a simple somatotopically shifted pattern. In this respect, the previous study suggested that the
termination columns of toes 1 and 4 reversibly and repeatedly interchanged their medial
and lateral positions with respect to each other across the rostrocaudal GN axis. How the
columns of these and other toes were related as this mediolateral interchanging occurred,
remained unclear; however, it seems possible that with mediolateral interchanging, the
termination columns of toes 1 and 4 may have overlapped the termination columns of
intervening toes. In the previous study, injections were designed to label limited inputs
from small areas on the plantar side of a toe (Maslany et al., 1991). In the present study,
the distal injections presumably involved most distal plantar and dorsal inputs of a toe;
consequently, termination areas of toes 1 and 4 in the present study appeared larger and
overlapping across the rostrocaudal axis. The present study also tested input pattern
relationships that weren’t analyzed in the previous report, including termination patterns
36
after combined injections of different multiple toes on a hindpaw, and termination
patterns of individual toes versus combinations of toes. These additional analyses provide
new evidence for substantial overlap in termination columns of different toes.
Overlap in GN terminations of toe afferents resembles the overlap in other terminations in the rat GN. For example, substantial overlap is seen in GN termination areas of afferents from the sciatic and saphenous nerves (LaMotte et al., 1991; Ueyama et al., 1994). Overlap is also seen in the GN termination areas of afferents from different lumbar dorsal root ganglia, with substantial overlap in termination areas of lumbar ganglia 3-5 which contain afferents from the hindpaw (Rivero-Melian and Arvidsson,
1992).
The present results are based on injections of BHRP or a combination of
BHRP and WGA-HRP. In normal rats, the cholera toxin B subunit and BHRP are taken up and transported mainly by large diameter sensory inputs (Robertson and Grant, 1985;
LaMotte et al., 1991; Tong et al., 1999; Shehab et al., 2003). A main component of large diameter inputs from the toes originates from cutaneous tactile inputs. Consistent with the present results, previous studies suggest hindlimb nerve inputs predominantly terminate in the ipsilateral GN, with no or only a few fibers projecting to the contralateral
GN (Rivero-Melian and Arvidsson, 1992; Ueyama et al., 1994; Novikov, 2001).
Previous work suggests that BHRP also provides a more complete estimate of total extents and densities of GN terminations than WGA-HRP (LaMotte et al., 1991; Maslany et al., 1991).
37
In the present studies, label was located in GN areas that appeared
uniformly stained in CO sections, avoided GN fascicles, and was not seen in soma. This
suggests that GN label mainly reflected terminal or preterminal, rather than axonal or
soma, labeling. This is consistent with electron microscopic findings of label in GN terminals of sensory afferents after cholera toxin B subunit injections in hindlimb nerves
(Hwang et al., 2001), with findings that label is mainly in sensory afferent terminals in the dorsal horn after BHRP or cholera toxin B subunit injection of hindlimb nerves or skin sites (LaMotte et al., 1991; Levinsson et al., 2002), and with findings that BHRP uptake by primary neurons is not trans-synaptically transported (Robertson and Grant,
1985; LaMotte et al., 1991; Rivero-Melian and Arvidsson, 1992).
Other pertinent findings come from Golgi studies of the rat GN. Terminal fields of Golgi stained dorsal column fibers have been reported to overlap extensively
(Odutola, 1977) and to be organized in a largely continuous fashion (Valverde, 1966).
Terminal arborizations of individual and small groups of dorsal column inputs have been described as intertwining to form glomeruli, within which dendrites of neurons receive axo-dendritic synapses. Glomeruli that occupy adjacent locations also fused to form a relatively uniform dense plexus of terminal arbors (Valverde, 1966). Although dorsal column inputs include spinal as well as primary afferents, the terminal labeling and CO staining patterns in the present studies could be consistent with this organization if primary afferents from different toes are organized in an overlapping plexus.
Postsynaptically, dendritic fields of GN neurons, including fields of projection neurons, appear to also extensively overlap (Valverde, 1966; Odutola, 1977).
38
We suggest that label in the present study is associated with afferents from injected toes, rather than with nonspecific uptake or transport by other inputs.
Nonspecific uptake may occur by peripheral spread of injected label into adjacent toes or other hindpaw or hindlimb locations, or by spread of label from labeled toe axons to adjacent axons in projection systems between the toes and GN. Our attempts to directly assess injection sites, although inconclusive (see Methods), did not indicate spread of label was a problem. Other considerations are also consistent with this. Spread of label across peripheral nerve or dorsal column axons is rarely discussed in transganglionic studies but, when examined, has not been seen (Ding et al., 1999). Primary afferents from the toes project to the GN via the sciatic and saphenous nerves and the dorsal column. These projection systems also contain axons that innervate hindlimb locations that terminate in parts of the GN where inputs from the toes did not terminate in the present study (LaMotte et al., 1991; Maslany et al., 1991; Ueyama et al., 1994). This suggests that spread of label, and particularly spread from afferents of an injected toe to afferents of another toe, would have had to occur without spread to other afferents.
GN overlap of toe terminations appears to be a further reflection of overlapping organization of toe projections at sub-brainstem levels in rats. For example, at peripheral levels, primary sensory neurons innervating the five hindpaw toes are located mainly in lumbar ganglia L3-6, with neurons from each toe occupying two or three ganglia in a coarsely shifted and overlapping manner (Prats-Galino et al., 1999;
Puigdellivol-Sanchez et al., 2000). Individual primary sensory neurons that respond to touch stimulation of the toes usually have a restricted receptive field spanning a small
39
fraction of one toe (Lynn and Carpenter, 1982; Sanders and Zimmermann, 1986;
Shortland et al., 1989; Leem et al., 1993; Shortland and Woolf, 1993; Reinke and Dinse,
1996). Interestingly, a few axons with tactile receptive fields on two toes have also been seen (Lynn and Carpenter, 1982; Woolf, 1987), as have a small number of double- or triple-labeled dorsal root ganglion cells after injection of multiple toes (Puigdellivol-
Sanchez et al., 2000). These findings suggest that some peripheral neurons, including mechanosensory neurons, have “overlapping” toe inputs at the peripheral axon level.
Overlap has also been reported in spinal dorsal horn terminations of toe inputs. Injections of individual toes label terminations in the medial dorsal horn at L3-5 levels in rats and, in both superficial and deeper dorsal horn layers, terminations from adjacent and up to 3-
4 toes can overlap (Molander and Grant, 1985; Maslany et al., 1992; Levinsson et al.,
2002; Takahashi et al., 2003). Injection and labeling of individual sensory fibers from toes, including mechanosensory fibers, suggest that these fibers span long rostrocaudal extents in the medial dorsal horn, and that the termination arbors of individual fibers from one toe are partially offset from, but also overlap with, arbors of fibers from other toes
(Shortland et al., 1989; Shortland and Woolf, 1993). From these findings, overlap of toe terminations in the GN appears to be a property that is shared with hindpaw toe projection systems below the brainstem.
3. CO and other structure in the rat GN
CO stained material from our injection cases suggested that toe termination areas in the dorsal GN were uniformly stained and did not appear to be
40
subdivided. CO staining in other brainstem nuclei in rats reveals distinct, densely stained
patches that colocalize with sensory input termination areas from specific body parts,
including, for example, CO patches that colocalize with main termination areas of each
forepaw toe in the other dorsal column nucleus, the cuneate nucleus (Crockett et al.,
1993b). The present failure to see CO patches in the GN was not attributable to technical
failures in staining because CO dense patches were consistently seen in the neighboring
CN on the same sections where uniform staining was seen in the GN toe area.
The present findings that GN toe termination areas do not involve obvious subdivisions are consistent with other observations from the rat GN. A stereological evaluation briefly noted vertical ‘slabs’ of dense CO staining in the rostrodorsal GN, but indicated staining was not sufficiently prominent or consistent to justify subdivisions
(Bermejo et al., 2003). Another report indicated the dense and uniform CO staining pattern in the GN contrasted with the parcellated pattern of CO dense patches in the CN
(Crockett et al., 1993b). Finally, no distinct dorsal GN subdivisions have been recognized in rat with other methods that reveal presynaptic and/or postsynaptic structural organization, including: Nissl staining (Valverde, 1966; Bermejo et al., 2003), p75NGFR-like immunostaining (Crockett et al., 1993a), neuropeptide immunoreactivity
(Conti et al., 1990; Noguchi et al., 1995; Ma and Bisby, 1999; 2000; Ossipov et al., 2002;
Ma et al., 2003), neurotrophin expression (Li et al., 1999; Ha et al., 2000), synaptic
vesicle associated protein immunostaining (Crockett et al., 1996), substance P receptor
binding (Schwark et al., 1998), GABA and glycine immunostaining (Popratiloff et al.,
1996a), calcium binding protein immunoactivity (Crockett et al., 1996; Magnusson et al.,
41
1996), or retrograde labeling of GN projection neurons from GN projection targets
(Kosinski et al., 1988; Kemplay and Webster, 1989; Magnusson et al., 1996; Li and
Mizuno, 1997; Bermejo et al., 2003; Del Cano et al., 2004; Wree et al., 2005).
4. Comparison of toe RFs with primary afferent organization
The distribution of afferent terminations from the toes presumably affects
GN toe RFs, but how terminal organization contributes to the location and size of these
RFs remains unclear. In the present study, some GN excitatory receptive fields had functionally expressed inputs from up to 4-5 toes, but the majority had inputs from 1-2 toes. Considered with the termination findings, these results raise the possibility that some existing terminations may be functionally underexpressed in toe RFs.
Available data on normal sizes of GN tactile RFs on rat hindpaws are usually expressed as areas of skin (e.g., mm2) and do not distinguish toe areas, or
numbers of toes in RFs (McComas, 1963; Miki et al., 1998; Suzuki and Dickenson, 2002;
Kitagawa et al., 2005). RF data in rats that are pertinent to the present results are limited
to a few toe RFs that are incidentally illustrated in reports on other issues (Nord, 1967;
Berkley and Hubscher, 1995; Panetsos et al., 1995; 1997; Kondo et al., 2002; Costa-
Garcia and Nunez, 2004). Illustrated toe RFs range from part of one toe to up to 5 toes,
but typical number of toes per RF cannot be gauged from the few available examples.
Comparisons of GN toe RFs and toe RFs of primary afferents suggest that
spatial convergence of afferent inputs contributes to GN toe RF size and location, i.e.,
whereas GN RFs commonly involve 1-2 toes, most RFs of primary afferents span a small
42
fraction of one toe (Lynn and Carpenter, 1982; Sanders and Zimmermann, 1986;
Shortland et al., 1989; Leem et al., 1993; Shortland and Woolf, 1993; Reinke and Dinse,
1996). However, unless one assumes that input convergence is the sole determinant of
GN RF organization, it is difficult to use relative sizes of GN and primary afferent RFs to suggest how GN RF size and location are determined.
The present findings raise the possibility that toe RFs may not always reflect all available structural inputs from the toes. Other findings seem consistent with this possibility. Toe RFs in the rat GN have capacities to rapidly enlarge under some conditions. For example, disruption of peripheral input activity by cutaneous injection of the local anesthetic lidocaine can cause immediate enlargements of hindpaw GN RFs, including enlargements involving one or more toes (Panetsos et al., 1995; 1997; Wang
and Wall, 2005). In addition, disruption of local inhibition in the rat GN with the
GABAA receptor antagonist bicuculline methiodide commonly causes immediate RF
enlargements involving larger areas on individual toes and increased numbers of toes
(Wang and Wall, 2006). The lidocaine findings are consistent with an availability of functionally unexpressed cutaneous inputs from toes, and the bicuculline results suggest structural substrates of unexpressed toe inputs are located within the GN itself and are regulated by local inhibition. The present results provide new evidence for the possibility that these substrates may, to some degree, involve overlapping terminations of sensory afferent fibers.
There is recognition of the principle that spatial properties of central RFs can emerge from structural substrates that have wider capacities than are functionally
43 expressed at a particular time. However, identifying substrates where structural and functional convergence can be compared has been difficult. In the dorsal column medial lemniscal projection system to somatosensory thalamus and cortex, thalamic afferent substrates to cortex appear to work by this principle. “Masked” substrates between the skin and GN have been long discussed, but there is little data on relationships between structurally identified afferents, and the size and location of functional RFs. The present findings raise the possibility that this principle may apply to peripheral afferent substrate connections in the GN.
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Figure legends
Figure 1. GN label following combined injections of all 5 toes in one hindpaw and no injection in the other hindpaw. A: Darkfield photomicrograph of GN label after unilateral injection of all 5 toes on the left side, and no injection on the right side. There was a continuous area of label in the indicated area in the left GN, and no label in the right GN (GN borders indicated on each side). B,C: Reconstruction of the caudorostrally
(respective lower to higher numbered sections) continuous column of label that was restricted to the ipsilateral dorsal GN after injection of all 5 toes in two cases. Midline and GN borders are indicated. Dorsal is up in all sections.
Figure 2. Examples of GN label after injection of different individual toes in each hindpaw. An injection was made into toe 5 (T5) on the left side and toe 1 (T1) on the right side. Low power photomics illustrating label on each side at indicated transverse planes. Reconstructions illustrate label across the caudorostral (respective lower to higher numbered sections) axis of the GN. Inputs from each toe were distributed as a
53 continuous caudorostral column spanning similar locations in the dorsal GN. Midline and
GN borders are indicated and dorsal is up in all sections.
Figure 3. Examples of GN label in caudorostral series (respective lower to higher numbered sections) following injections of different individual toes. Reconstructions are for terminations of toe 1 (A), toe 2 (B), toe 3 (C), and toe 4 (D). B and D are from the two sides of one case. E shows cross-side comparisons of areal extents of label for toe 5
(grey area) on one side and toe 1 (black area and dashed lines) on the other side in another case. To facilitate comparisons, the midline is to the right and dorsal is up in all cases.
Figure 4. Examples of GN label following injections of nonadjacent toes on the same hindpaw. A: Reconstructed terminations after injections of toes 1+5. B:
Reconstructed terminations after injections of toes 3+5 on the left side and 2+4 on the right side in one case. C: Cross-side comparison of terminations following injections of toes 3+5 (black area and dashed lines) and 2+4 (grey area) for a series of sections from the case shown in B. Midline and GN borders are indicated and dorsal is up in all sections.
Figure 5. Cross-side comparisons of GN label following injections of combinations of toes on one side, and an individual toe on the other side. A-C: Cross- side comparisons for injections of the most separated toes 1+5 on one side and of toe 5
(A), toe 3 (B), or toe 4 (C), on the other side in 3 cases. D: Cross-side comparison for injections of all 5 toes (1-5) on one side and toe 5 on the other side in another case. To facilitate comparisons, all cases are illustrated with the midline to the right and dorsal up.
54
Figure 6. Caudorostral sequence (respective lower to higher numbers) of
CO stained sections through the GN nucleus. At more caudal levels (A,B), densely stained neuropil areas are interrupted by lightly stained fiber bundles (e.g., indicated by
*). At more rostral levels (C-E), densely stained neuropil areas become more prevalent.
Dorsal GN areas where toe inputs terminated were relatively uniformly stained and did not contain recognizable subdivisions. Medial is right and dorsal is up in all sections.
Figure 7. Examples of GN excitatory tactile RFs involving toes. The shaded area on each hindpaw figure indicates a RF, and the number indicates number of toes in that RF. Lines link parts of a RF that involved plantar and dorsal surfaces.
55
Figure - 1
56
Figure - 2
57
Figure – 3
58
Figure - 4
59
Figure – 5
60
Figure – 6
61
Figure – 7
62
Discussion
Present results
This thesis examined GN structural organization of primary neuron
terminations from the hindpaw toes, and GN excitatory RFs on toes, in an attempt to
understand how distributions of input terminations from different toes and RFs on toes are related. At the beginning of this thesis, the GN termination patterns of primary neuron inputs from toes and the numbers of toes that usually contribute to normal
excitatory toe RFs were not well understood.
The GN is a major player in the dorsal column medial lemniscal system
which, through related thalamocortical projections, is a main input projection system to
primary somatosensory cortex. At cortical levels of this system, there was substantial
evidence that RFs of cortical neurons can commonly reflect incomplete functional expression of available inputs; i.e., there is structural overlap of thalamocortical terminations from a much wider range of body locations than is reflected in the excitatory
RF at a given time (Garraghty et al., 1989; Arnold et al.,2001; Rausell & Jones, 1995;
Rausell et al. 1998; Garraghty et al. 1990; Churchill et al. 2004; for earlier reviews see
Snow and Wilson, 1991). This thesis asked whether this concept might also apply at brainstem levels of the ascending projection system.
No previous attempts had been made to examine GN structural-functional relationships between primary neuron termination patterns and RFs, and different arrangements seem possible. One alternative, for example, is that GN terminations from each toe occupy a discrete GN area that does not overlap the termination areas of other 63
toes and that this structural organization, in turn, is associated with a “matched” GN RF organization where excitatory RFs are mainly restricted to an individual toe (e.g. Fig 2,
RF neuron A). An alternative “matching” possibility is that GN terminations of different toes overlap to some degree and that this is associated with a “matched” GN RF organization where excitatory RFs reflect these overlapping toe inputs (e.g. Fig 2, RFs of
neuron B & C). “Mismatched” structural-functional arrangements also seem possible.
For example, there may be discrete nonoverlapping primary sensory terminations from
individual toes, but excitatory RFs on multiple or most toes may result from excitatory
driving from spinal or other inputs that add to primary sensory inputs (e.g. Fig 2, RF of
neuron D) , An alternative mismatch could result if GN terminations from each toe
overlap termination areas of most or all other toes, but excitatory GN RFs involve only one or two toes (e.g. Fig 2, RFs of E & F). This last possibility could involve, e.g. GN inhibition and would be consistent with how thalamocortical inputs and RFs can be organized in somatosensory cortex.
64
Figure 2. Potential relationships between structural distribution patterns of primary neuron terminations from the toes and functional RFs on the toes.
Left: Structural patterns of termination may “match” functional RF expression. For example, GN terminations from each toe may occupy discrete, non-overlapping GN areas and this structural organization may “match” RF organization where excitatory RFs are restricted to one toe (GN neuron A and its RF). Another possibility is that GN terminations from different toes overlap to some degree and this overlapping is associated with “matched” RFs from multiple toes (GN neurons B and C and their RFs). Right: There may also be “mismatches” in structural and functional organization. For example, GN terminations from each toe may occupy discrete, non-overlapping GN areas but excitatory RFs may be on multiple toes due to contributions that are made by inputs other than primary neuron inputs (GN neuron D and its RF). Another “mismatch” possibility is that GN terminations from each toe overlap terminations of most or all other toes but, due to GN inhibition, RFs involve only one or two toes (GN neurons E and F and their RFs). The present results, taken together with findings on GN inhibition effects on RFs, provide evidence for “mismatches” like that seen in neurons E and F. This evidence does not preclude that some “matches”, e.g., like that seen in neuron B, may also occur.
65
The present results provide evidence that GN terminations of primary neuron
inputs from individual toes were organized as continuous rostrocaudal columns in the
ipsilateral GN, and that columns of different toes overlapped. In assessing the degree of
overlap, results from injections of each individual toe, different combinations of some or
all toes on one side, and different combinations of toes on the two sides in individual
cases, suggest there is substantial overlap in the termination columns of at least 3-4, and
likely all 5, toes. The termination columns of toe inputs were located in dorsal extents of
the GN which, in CO stained sections, were densely stained and did not appear to contain
subdivisions. This pattern of CO staining could also be consistent with an overlapping,
nonparcellated organization of structural inputs from the toes. Finally, a small fraction of
GN excitatory RFs expressed inputs from up to 4-5 toes, however, the mean number of
toes in RFs was 2, and a majority of RFs involved 1-2 toes. Taken together, these results
raise the possibility of functional and structural organization whereby numbers of toes in
excitatory RFs underexpress existing structurally available primary neuron inputs. While
not precluding some “matches” in organization (e.g. Fig 2, RF of neuron B), this is
consistent with “mismatching” in structural-functional organization (e.g. Fig 2, RFs of
neurons E & F).
How the present results supplement understanding of primary input
organization in the GN
Prior to the present work, early Golgi studies indicated that ascending axons from the dorsal column terminate in the GN in glomeruli around GN neuron
66
dendrites, and form an apparently continuous plexus of GN terminations (Valverde,
1966; Odutola, 1977). Although some stained axons were likely from primary neurons,
Golgi staining does not permit distinguishing of axons of primary neurons and spinal neurons, both of which ascend in the dorsal column. Subsequent axonal transport studies identified distributions of GN terminations that were labeled after injections of dorsal root ganglia or peripheral nerves that contain primary neurons from hindquarter and hindlimb areas. These studies clearly distinguished ascending inputs of primary neurons from inputs from spinal neurons, and indicated that there was considerable overlap in the
GN termination areas of primary neurons from different ganglia or nerves (Rivero-
Melian and Arvidsson, 1992; Prats-Galino et al., 1999; Puigdellivol-Sanchez et al.,
2000); however, from injections of these structures it was not possible to resolve how primary inputs from a specific location on the body were distributed, or whether GN termination areas of inputs from different body locations, like different toes, overlap.
One previous study used axonal transport following subcutaneous injections of label into toe and other locations on the rat hindpaw and hindlimb and found
that terminations from toes were organized in rostrocaudal columns in the dorsal GN
(Maslany et al., 1991). In addition, terminations of primary neuron inputs from toes 1
and 4 repeatedly and reversibly interchanged medial and lateral GN positions at different
rostrocaudal levels. It was not clear from these results whether this medial-lateral
interchanging was associated with overlap of GN terminations of these and/or other toe
inputs. Thus, whether or not GN terminations from each toe overlap terminations of
other toes was not resolved.
67
The present study used individual toe injections and a range of combined
toe injections in one or both hindpaws to examine termination patterns of toe inputs in
detail. The findings agree with previous findings regarding GN toe terminations in
suggesting that primary neuron inputs from individual toes occupy rostrocaudally
continuous columns in dorsal GN locations. The present findings further agree with previous results in suggesting that GN terminations from the toes are not arranged in a
simple somatotopic pattern. The present results supplement previous understanding by
suggesting that GN termination columns of primary neuron inputs from individual toes
overlap, and that terminations from most or all toes appear to overlap to significant
degrees.
The CO results further suggest there was no apparent parcellation or subdivision in dorsal GN areas where toe inputs terminated. Consistent with this,
previous studies that have examined rat GN using CO staining (Crockett et al., 1993b), as
well as a range of other pre- and postsynaptic structural stains and markers like Nissl
staining (Valverde, 1966; Bermejo et al., 2003), Golgi staining(Valverde, 1966;Gulley,
1973), p75NGFR-like immunostaining (Crockett et al., 1993a) , neuropeptide
immunoreactivity (Conti et al., 1990; Noguchi et al., 1995; Ma and Bisby, 1999; 2000;
Ma et al., 2003; Ossipov et al., 2002), neurotrophin expression (Li et al., 1999; Ha et al.,
2000) , synaptic vesicle associated protein immunostaining (Crockett et al., 1996) ,
substance P receptor binding (Schwark et al., 1998) , GABA and glycine immunostaining
(Popratiloff et al., 1996a) , calcium binding protein immunoactivity (Crockett et al.,
1996; Magnusson et al., 1996) , or retrograde labeling (Kosinski et al., 1988; Kemplay
68
and Webster, 1989; Magnusson et al., 1996; Li and Mizuno, 1997; Bermejo et al., 2003;
Del Cano et al., 2004; Wree et al., 2005) of GN projection neurons from GN projection
targets , have not observed subdivisions in the dorsal GN. Although all these approaches
are not all specific for presynaptic termination organization, they appear consistent with
the possibility that neuropil in the rat dorsal GN where the toes terminate does not
contain structurally defined subdivisions. These GN findings differ from the distinct
parcellated patterns of CO patches that are associated with input terminations from
different forepaw toes in the CN subdivision of the DCN (Crockett et al., 1993b), and that
were seen in the present study on the same CO sections where the GN was uniformly
stained. Similarly, the present GN findings also differ from the CO patches and
parcellated patterns of input terminations from different vibrissae in some parts of the
adjacent brainstem trigeminal nuclei (Chiaia et al.,1992a; Chiaia et al., 1992b; Jacquin et al., 1993c; Chiaia et al., 1994; Florence & Lakshman, 1995).
The present finding of overlapping organization of toe primary inputs in the GN appears to be a further extension of overlapping organization of primary neuron inputs from toes at pre-brainstem locations. For example, there is evidence that small numbers of primary neurons, including touch input neurons, have distal axons that branch
to end in more than one toe (Lynn and Carpenter, 1982; Woolf, 1987). The cell bodies of
primary neurons that have distal axons that innervate different hindpaw toes appear to be
arranged in a partly overlapping manner in the lumbar ganglia (Puigdellivol-Sanchez et
al., 2000). Injections and transport of label from toes have shown that terminations from
up to 3-4 toes can overlap in the spinal dorsal horn (Maslany et al., 1992; Levinsson et
69 al., 2002; Takahashi et al., 2003). Finally, studies where individual primary sensory fibers were labeled suggest that individual axons span long rostrocaudal extents in the dorsal horn and that terminal arbors of axons from different toes can overlap (Shortland et al., 1989; Shortland & Woolf, 1993). Taken together, these results suggest primary neuron inputs from different toes on the rat hindpaw have features of overlapping organization at peripheral and spinal levels. The present results supplement this understanding by suggesting that primary neuron inputs from different toes have further overlapping features of organization at a higher central level in the GN.
How the present results supplement current understanding of GN functional RFs on the toes
A second interest of this thesis was to identify numbers of toes that are normally included GN excitatory RFs in rats. As the thesis was begun there was little understanding of this issue. There is a longstanding recognition that many GN neurons have an excitatory tactile RF that is located on some hindquarter area, including toe areas.
Previous work on sizes of GN excitatory RFs in rats have expressed RF size in terms of skin area measurements (McComas, 1963; Suzuki & Dickenson, 2002; Miki et al., 1998;
Pettit & Schwark, 1993; Wang & Wall, 2005), but there have been no studies directed at estimating numbers of toes that usually contribute to GN excitatory RFs. Anecdotal illustrations of a limited number of rat GN RFs on toes have been included in reports on other issues (Nord, 1967; Berkley and Hubscher, 1995; Panetsos et al., 1995; 1997;
Kondo et al., 2002; Costa-Garcia and Nunez, 2004). Illustrated RFs include RFs that
70 involve a fraction of one toe to RFs that involve parts of up to 5 toes, thus, suggesting excitatory RFs on the toes can vary in size. Because only a few RFs have been shown, and because available RFs come from different studies, these results do not provide estimates or conclusions about how many toes are included in most GN RFs on the toes.
The sample of excitatory RFs in the present thesis was derived from studies in this lab that defined normal hindpaw RFs from single and multiple unit responses as part of obtaining control data prior to doing subsequent experimental manipulations (Wang & Wall, 2005; 2006). Many of the RFs from these studies involved the toes, and it was these RFs which served as the present sample. A relevant note of interest is that because these RFs were defined as part of other studies, they were defined
“blindly” with respect to their use in this thesis, i.e. to count number of toes included in
RFs. Thus, the numbers of toes per RF in the present sample are not “biased” by a prior recognition of the structural results of this thesis. The resulting sample of GN RFs suggests that a small percentage of RFs can include up to 4-5 toes, but the majority involve 1-2 toes, and the mean number of toes was 2. These findings are consistent with illustrated RFs from previous studies, which suggested RFs could involve different numbers of toes. These results supplement current understanding by indicating that a majority of GN neurons normally appear to be activated by 1-2 toes at any particular time. Additionally, a smaller fraction of GN neurons or, alternatively, some neurons at a particular time, may be activated by inputs from a larger number of toes.
71
Some broader implications of the findings of this thesis
As discussed above, the findings of this thesis supplement current
understanding of how terminations of primary neurons from the toes are normally
structurally organized in the GN, and of the number of toes that normally contribute to
GN functional RFs. Some implications of these results are discussed next.
A. Possible mismatches in GN structure and function and a potential explanation
The present study provides new evidence that terminations of primary
neuron inputs from 3-4 or more toes overlap extensively, but that functional RFs usually reflect inputs from 1-2 toes. This raises the possibility of structural-functional mismatching whereby availability of primary neuron inputs from toes may often exceed normal functional expression of inputs in toe RFs at a particular time.
How can these potential mismatches be explained? GN inhibitory interneurons inhibit projection neurons, other GN inhibitory interneurons, and terminations of primary neuron inputs (Lue et al., 1993; Lue et al,. 1994; Lue et al., 1996;
Aguilar et al., 2002) (Fig.1). Thus, from known structural connectivity, primary neuron
terminations and synapses on GN neurons are inhibited to some degree by pre- and post-
synaptic actions of GN inhibitory interneurons. GN interneurons, in turn, receive inputs
from primary neurons and from central structures including cortex (Lue et al., 1997; Lue
et al., 2001; Willis and Coggeshall 2004; Popratiloff et al., 1996a; Malmierca et al.,
2004). Many GN interneurons are GABAergic, and pharmacological blockade studies of 72
GN inhibition with the GABAA receptor antagonist bicuculline methiodide indicate GN hindpaw RFs, including RFs on the toes, immediately and reversibly enlarge across larger numbers of toes and other hindpaw areas following local microinjection of bicuculline methiodide around recording sites where GN RFs were being recorded (Schwark et al.,
1999; Wang et al., 2006). This suggests that the spatial extents of excitatory RFs of GN neurons are tonically controlled by intrinsic GN inhibition and that decreases in this inhibition can lead to immediate functional expression of inputs from a broader area of the hindpaw, including a larger number of toes. Tonic GN inhibition in turn, may in part be driven by direct peripheral inputs (e.g. tonically active proprioceptive inputs that signal body position and movement) or peripheral inputs that relay in spinal circuits.
Overall, known connectivity and pharmacological evidence suggests that local GN inhibition plays an important role in confining normal spatial extents of GN toe RFs.
Axon terminations of touch primary neurons provide a major source of input to the GN in rats. Primary neurons synapse on and directly activate GN inhibitory interneurons and projection neurons (Willis & Coggeshall 2004). These major, direct connections appear to be a main substrate for generation of GN excitatory RFs on the toes because acute disruption of primary neuron transmission from parts of a toe or hindpaw RF as a consequence of local anesthetic blockade causes immediate loss of parts of the RF innervated by disrupted inputs (Panetsos et al., 1997; Wang & Wall, 2005).
Another direct input to the rat GN originates from cortex but, in contrast to effects of disruptions of peripheral inputs, sizes of GN RFs on the toes and hindpaw remain unchanged before and after acute removal of cortical inputs to the GN (Wang & Wall,
73
2005). These and other findings have led to suggestions that GN excitatory RFs on the toes and hindpaw are dominantly determined by ascending primary neuron projections, rather than descending central projections (Wang & Wall, 2005, 2006).
Taken with the above findings on GN connectivity, the immediate enlargements of GN toe RFs with pharmacological blockades of GN GABA inhibition, and the major excitatory input influence that primary neurons provide to the GN, the present results raise the possibility that tonic GN inhibition of functional expression of some fraction of structurally available toe inputs may normally contribute to mismatches between structurally available, and functionally expressed, toe inputs.
B. Some features of cortical and GN processing may be similar
The GN nucleus is a main brainstem processing center for touch and other inputs within the dorsal column medial lemniscal ascending system. Following GN processing, information in this system is subsequently sent to thalamic neurons in the ventroposterior lateral nucleus which, in turn, sends thalamocortical projections to somatosensory cortex. Previous studies have indicated that thalamocortical arbors can transverse large distances across cortex. When the cortical distances of thalamocortical arbors were compared to cortical spaces in which neurons had functional RFs on a specific body location, like a digit, it appeared that functional RFs normally reflected inputs from more delimited areas of the body than reflected by the expansive thalamocortical arbor organization (Garraghty et al., 1989; Arnold et al.,2001; Rausell &
Jones, 1995; Rausell et al. 1998; Garraghty et al. 1990; Churchill et al. 2004; for reviews
74
see Snow and Wilson 1991). Consistent with this mismatch in thalamocortical input
availability and spatial extent of cortical RFs, pharmacological blockades of local cortical
inhibition lead to immediate enlargements of cortical RFs (Alloway & Burton, 1991;
Dykes et al., 1984; Chowdhury & Rasmusson, 2002). These and other findings have led
to suggestions that spatial extents of normal cortical receptive fields are limited by tonic
cortical inhibition and that disruption of this inhibition causes immediate RF
enlargement, in part, via drive of normally suppressed thalamocortical inputs (Calford,
2002; Snow & Wilson, 1991)
An implication of the present findings is that the GN organization of
broader ascending structural inputs than are expressed in functional RFs appears similar
to cortical organization. In concept, the present findings raise the possibility that GN organization resembles cortical organization in this respect.
C. Potential contribution to plasticity of GN RFs
GN tactile RFs on the hindpaw and toes of adult rats enlarge within
minutes following disruptions of primary neuron inputs by subcutaneous local anesthetic blockades of inputs (Panetsos et al., 1997; Wang & Wall, 2005). Further studies have shown that enlargement of an individual GN RF on the toes or hindpaw following these blockades spatially resembles the enlargement that is produced in the same RF following acute pharmacological blockade of local GN GABA inhibition with bicuculline methiodide (Wang & Wall, 2006). Immediate changes in GN RFs after peripheral anesthetic blockades of inputs are paralleled by similar concurrent changes in cortical
75
RFs (Byrne et al., 1991; Calford et al., 1991; Panetsos et al., 1995). These cortical
changes are not required for GN changes because acute removal of cortical descending
inputs to the GN does not block GN RF changes (Wang & Wall, 2005). These findings have led to proposals that immediate enlargements in GN RFs after acute disruptions of
peripheral inputs may be caused by decreases in local GN inhibition that do not require
descending cortical inputs (Panetsos et al., 1997; Pettit & Schwark, 1993; Schwark et al.,
1999; Wang & Wall, 2005, 2006).
GN RF enlargements after disruptions of peripheral inputs or GN GABA
inhibition occur within minutes and are clearly dependent on existing structural
connections. Thus a final implication of the present findings is that terminal organization
of primary neuron inputs from toes may serve as a structural substrate that contributes to
immediate plasticity of normal toe RFs after these disruptions, and that provides the GN
with an ability to rapidly adapt RFs to input conditions.
76
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Abstract
The gracile nucleus (GN) is a major brainstem processing center for
somatosensory inputs. GN neurons often have functional receptive fields (RFs) on the
toes, but there is little consensus on how these RFs are related to primary sensory inputs from toes. This project compared structural overlap in GN terminations of afferents from different toes, and numbers of toes in GN functional RFs. The results suggest that
structural convergence of afferent terminations from at least 3-4 toes is common; in
contrast, although some GN RFs reflected functional inputs from up to 4-5 toes, most
RFs reflected inputs from 1-2 toes. These findings raise the possibility that structural
convergence of sensory afferents from the toes is broader than the afferent convergence
that is usually reflected in functional RFs. This type of potential mismatch has been
previously described in somatosensory cortex and may partly explain rapid GN RF
changes after disruptions of afferents.
91