ELECTROPHYSIOLOGICAL CHARACTERIZATION OF CEREBELLAR PURKINJE CELLS FROM THE PCP2-L7- DEFICIENT MICE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Emilia Maria Iscru
*****
The Ohio State University June 2008
Dissertation Committee:
Dr. Mike Xi Zhu, Adviser Approved by
Dr. John D. Oberdick
Dr. Georgia A. Bishop Advisor, Biophysics Program
Dr. Jackie D. Wood
ABSTRACT
An increasing body of knowledge suggests that the cerebellum performs
functions outside its traditionally attributed role in motor coordination. The cerebellum is
considered to mediate diverse non-traditional functions ranging from motor learning
[DeZeeuw, 1998], emotion [Sacchetti, 2004], to cognition [Kim, 1994; Allen et al.,
1997]. Another non-traditional view is that some functions of the cerebellum may be
primarily sensory rather than motor, or they concentrate on optimizing motor functions
required for sensation [Gao, et al., 1996; Hartmann and Bower, 2001]. However, so far it
has been difficult to investigate these non-traditional functions due to the lack of good
animal models in which these presumed functions can be explored in isolation without
the confusing interference of motor coordination defects.
This project explores the possible non-traditional roles of cerebellum using a
mouse model ablated of the gene for a Purkinje cell-specific protein called Pcp2-L7
[Oberdick et al., 1988; Luo and Denker, 1999; Zhang et al., 2002; Willard et al., 2006].
Because Pcp2-L7 contains GoLoco domains and has been implicated in the functional
regulation of P/Q-type Ca2+ channels [Kinoshita-Kawada et al., 2004] we focussed on the
ii electrophysiological properties of the cerebellar Purkinje cells of the wild type and L7
knockout mice.
The GoLoco domain acts both as a binding domain and as a guanine nucleotide
dissociation inhibitor (GDI) selective for Gα subunits of heterotrimeric G proteins
[Natochin et al., 2001; Willard et al., 2006; Kimple et al., 2001]. Much is known about the biochemistry of these proteins and their effects on mechanisms of G-protein signalling, but the specific downstream effectors and functions in vivo are only just beginning to emerge.
The L7 protein has the additional novelty that it is highly expressed cerebellar
Purkinje cells and retinal bipolar neurons [Oberdick et al., 1990; Berrebi et al., 1991].
Inactivation of Pcp2-L7 in mice was reported to have no apparent phenotype as determined using behavioral, anatomical, and ultrastructural analysis [Mohn et al., 1997;
Vassileva et al., 1997]. Despite the lack of a reported phenotype in the L7 mutant mice, previous work in Xenopus oocyte expression system suggests that one downstream effector of L7 modulation of G-protein signalling is likely the P-type Ca2+ channel which
is the primary voltage-dependent Ca2+ channel expressed in Purkinje cells [Kinoshita-
Kawada et al., 2004]. As Ca2+ plays a major role in Purkinje cell function and plasticity,
and P-type channels are widely held to play an important computational role in these
cells, the phenotype of L7 null mutant mice has been re-examined. These recent results
suggest subtle but significant anatomical, behavioural and electrophysiological changes
in the absence of L7 protein. Based on these observations we propose that the Purkinje
cells and L7 serve a sensory damping function that also acts to limit or delay changes in
motor performance over time.
iii The entire project is a collaboration between Dr. John Oberdick and Dr. Mike
Zhu’s laboratories. My focus in this project is the electrophysiological characterization of the wild type and mutant cerebellar Purkinje neurons.
iv
Dedicated to Professor Puiu Balan, my Physics teacher from sixth-grade until grad school
v
ACKNOWLEDGMENTS
I am profoundly grateful to all those who have offered me their support, advice,
encouragement and patience in this accomplishment. While I will only acknowledge a
few of you here, for those I fail to mention, please forgive the omission and take heart that without everyone who has been there for me over the last few years I could never
have made it to this day.
First, I would like to thank Dr. Mike Zhu, for his great mentoring and leading by
example, for his kindness and support, which gave me the strength and courage to follow
my dreams in spite of the short term hardship.
I also want to thank Dr. John Oberdick for providing me with this project and for
being patient and understanding, as well as for his generous supply and genotyping of
animals that made this project possible.
vi I am forever grateful for the generosity and the precious guidance of Dr. Georgia
Bishop. Special thanks also go to Dr. Kamran Khodakhah, for his useful consultancy in this project.
I also want to thank Ms.Lorry Kapple and Mr. Mike Shade for their valuable technical support. I deeply appreciate the support of my colleagues Jin-Bin Tian, Rui
Xiao, Craig Colton, XueMei Hao and Dina Zhu.
I cannot be thankful enough to my family for all their endurance and love they offer me over these years and to all of my friends.
vii
VITA
August 22, 1974……..Born - Rm. Vilcea, Romania
1993 - 1997 …………Bachelor of Science, Physics, University of Bucharest, Romania
1997 – 1999………….Master of Science, Biophysics, University of Bucharest, Romania
2002 – present………..PhD candidate, Biophysics Program, The Ohio State University
PUBLICATIONS AND ABSTRACTS
1. Iscru, E.M., Serinagaoglu, Y., Schilling, K., Tian, J. Bowers-Kidder , S.L., Morgan, J.I., DeVries, A.C., Nelson, R.I., Zhu, M.X., and Oberdick, J. (2008) Sensorimotor enhancements in mouse mutants lacking the Purkinje cell-specific G i/o modulator, Pcp2(L7).
2. Colton, C.K., Hu, H.Z., Iscru, E.M., Tang, J., Wang, C., Wood, J.D., and Zhu, M.X. (2005) Acid activation of TRPV3 channel expressed in HEK 293 cells. Biophys. J. 88, 113A, Part 2
3. Serinagaoglu, Y., Iscru, E.M., Tian, J., Bishop, G.A., Morgan, J.I., Zhu, M.X., and Oberdick, J. (2005) Sensorimotor behavioral changes and alteration of Purkinje cell firing patterns in Pcp2(L7) null mutant mice. Program No. 986.2. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience.
4. Hu, H.Z., Colton, C.K., Iscru, E., Tang, J., Wang, C., Wood, J.D., and Zhu, M.X. (2005) Acidic pH activates transient receptor potential vanilloid 3 channel (TRPV3) when expressed in HEK 293 cells. Gastroenterology 128 (4): A362-A362 Suppl. 2
FIELDS OF STUDY
Major Field: Biophysics/Electrophysiology viii
TABLE OF CONTENTS
Page Abstract...... ii Dedication...... v Acknowledgments……………………………………………………………………….vi Vita……………………………………………………………………………………...viii List of Tables…………………………………………………………………………….ix List of Figures………………………………………………………………………… …x Abbreviations………………………………………………………………………...... xii Chapters: 1. Introduction 1.1. Cerebellum- anatomy and functions……………………………….……...1 1.2. L7 protein- expression and function ……………………………………...6 1.3. Overview of calcium signaling- voltage gated calcium channels……………………………………………………8 1.4. Role of P/Q-type channels in cerebellar function………………………..13 1.5. Purkinje cells- mechanisms underlying different firing patterns…………………………………………………..17
2. Methods
2.1. Mice…………………………………………………………………...... 29 2.2. Tissue preparation…………………………………………………. ……30 2.3. Data acquisition-Experimental set-up……………………………………30 2.4. Electrophysiology………………………………………………………..33
ix 2.4.1. Extracellular recordings………………………………………….33 2.4.2. Complex spikes recordings………………………………………33 2.4.3. Whole cell patch clamp………………………………………….34 2.5. Statistical analysis of data………………………………………………..36
3. Results
3.1. Spontaneous firing patterns of Purkinje cells
in cerebellar slices of wild type and L7 knockout mice………………….41
3.1.1. Tonic firing………………………………………………45 3.1.2. Phasic firing-trimodal pattern……………………………47 3.2. Gender-genotype interaction of firing behavior
between Purkinje cells of L7 knockout and wild type mice……………..50
3.2.1. Tonic firing- gender*genotype interaction………………50 3.2.2. Burst firing parameters in the trimodal
pattern- gender*genotype interaction……………………51
3.3. The effect of age on the tonic firing properties from the wild type and L7 knockout mice………………………………55 3.4. Complex spikes analysis for wild type and L7 knockout mice…………………………………………………..57 3.5. Whole-cell recording of the Purkinje cells in
cerebella slices from wild type and L7 knockout mice………………….58
4. Discussion and future studies………………………………………….96
5. Conclusions…………………………………………………………….103
List of References……………………………………………………………………...105
x
LIST OF TABLES
Table Page
1. Neurotransmission in the cerebellar cortex……………………………………27
2. VGCC-summary……………………………………………………………….28
3. Electrophysiology solutions- extracellular solutions…………………………..38
4. Electrophysiology solutions- internal solutions………………………………39
5. Cutting solution………………………………………………………………..40
6. Summary of the number of mice and number of cells
per genotype and gender in bursting analysis………………………………….95
xi
LIST OF FIGURES
Figure Page
1. Stuctural organization of the cerebellum………………………………………22
2. Cerebellar circuitry……………………………………………………………23
3. Cerebellar cortex………………………………………………………………24
4. Graphic representation of the VGCC………………………………………….25
5. Proposed mechanism of regulation of the P/Q-type channels by L7…………26
6. Diversity of firing pattern in PC………………………………………………62
7. Firing pattern of Purkinje cells in wild type and L7-/-………………………..63
8. Firing pattern distribution……………………………………………………..64
9. Tonic firing- initial set of data ………………………………………………..66
10. Tonic firing-all data……………………………………………………………68
11. Tonic firing frequency in different external conditions……………………….69
12. Bursting firing- all data………………………………………………………..71
13. Tonic firing analysis by genotype and gender…………………………………72
14. Bursting analysis by genotype and gender…………………………………….73
15. Cumulative plots for the bursting parameters…………………………………75
16. Cumulative plots for the bursting parameters- alternative representation…….76
xii 17. Spikes/ second- genotype*gender interaction…………………………………78
18. Mean interspike interval……...………………………………………………..80
19. Mean frequency………………………………………………………………..82
20. Burst duration………………………………………………………………….84
21. Interburst interval……………...………………………………………………86
22. The effect of age on tonic firing..……………………………………………..87
23. Complex spikes………………………………………………………………..88
24. Distribution histogram for the complex spikes………………………………..89
25. Whole cell recordings from white mice; current injection recordings………..90
26. Representative recordings for L7 knockout mice
in whole cell current clamp …………………………………………………..92
27. Representative recordings for wild type in whole cell current clamp ………..93
28. Action potential waveform for wild type and L7 knockout…………………..94
xiii
ABREVIATIONS
AP: action potential
AHP: afterhyperpolarization
BC: Basket cells
BK: big conductance calcium activated potassium channels
CF: Climbing fibre
CIRC: Calcium –induced calcium release
ER: endoplasmic reticulum
GDI: guanine nucleotide dissociation inhibitor
GDP: guanine triposphate
GTP: guanine triposphate
GEF: guanine nucleotide dissociation facilitator
GC: Golgi cells
GPCR: G protein coupled receptor
IPS: ipsapirone
IP3: inositol triphosphate
LTD: long term depression
NE: norepinephrine
xiv NMDA: N-methyl-D- aspartic acid
PF: Parallel fibre
PC: Purkinje cells
Pcp2: Purkinje cell protein 2
SC: Stellate cells
SK: small conductance calcium activated potassium channels
VGCC: voltage gated calcium channels
xv
CHAPTER 1
INTRODUCTION
1.1. Cerebellum-anatomy and function
Cerebellum is a major component of the central nervous system (CNS) and it is
responsible for motor coordination, fine control of movement and balance. While
cerebellum does not initiate movement, it receives sensory, motor, perceptual and
cognitive information from all parts of the CNS and it transmits the information to the
motor system. The cerebellum has a central region called vermis (mainly responsible for
somatosensory functions), flanked by paired lateral hemispheres (with role in motor
coordination and limb movement) and floculi (responsible for balance and equilibrium)
[Nolte, 1999].
Cerebellum has a lobulated structure with 10 distinct lobules [Altman and Bayer,
1978]. At the microscopic level, the cerebellar cortex consists of three layers: molecular layer, Purkinje cell layer and granule cell layer (Figure 1). In the molecular layer there are stellate and basket cells (also known as interneurons), the huge dendritic tree of the
1 Purkinje neurons, the axons of the granule cells (parallel fibers) and the axons of the inferior olive neurons (climbing fibers). The Purkinje cell layer consists of Purkinje neuron cell bodies, which are pear-like shaped cells and are the largest cerebellar neurons
(~20 µm in diameter). The granule cell layer consists of granule cells, unipolar brush cells, interneurons and Golgi cells.
Purkinje cells are the only output of the cerebellar cortex. The way these neurons
receive inhibitory/excitatory input from the neighboring cells and how they integrate this
input and then transmit an inhibitory signal is presented below. Cerebellum receives
afferent input via climbing fibers and mossy fibers.
Climbing fibers are the axons of the neurons in the inferior olive. They go to all
parts of the cerebellum and relay information from the spinal cord, red nucleus and
cerebellar motor cortex to the Purkinje cells. They also send a collateral excitatory input
to the deep cerebellar nuclei [O’Leary, et al., 1970; Altman and Bayer, 1978]. As the
name suggests, they “climb” up, wrap around the Purkinje cell body and form excitatory
synapses all over the dendrites (see Figure 2). Although one Purkinje cell receives input
form only one climbing fiber, the synaptic connection between the climbing fiber and the
Purkinje cell is one of the most powerful in the nervous system. A single action potential
in a climbing fiber is able to elicit a burst of 3 or 4 action potentials of different
amplitudes in the targeted Purkinje cell, called a complex spike. Purkinje cells exhibit
complex spikes at a rate of about 1 per second.
Mossy fibers are the axons of the neurons in the brain stem. They also originate
from pontine nuclei and spinal cord. Their fibers spread information from the cerebral
motor cortex and body to cerebellar granule cells, unipolar brush cells, and also send
2 collateral innervations to the deep cerebellar nuclei neurons [Chan-Palay, et al., 1977;
Oschlowka and Vijayan, 1980; Altman and Bayer, 1978; Morin, et al., 2001].
Unlike climbing fibers, mossy fibers do not make synaptic contacts with the
Purkinje cells; instead they branch in the white matter and exert excitatory input on granule cells. The name mossy fibers was given by the unique synapses formed by their projections, namely the mossy fiber rosettes (brunches of mossy fiber axons twisting through the granule cell layer with synaptic contacts that have the appearance of a knot).
One mossy fiber can have up to 50 rosettes, so the mossy fiber signal is highly divergent.
The axons of the granule cells, also called parallel fibers, run into the molecular layer of the cerebellar cortex, perpendicular to the dendritic tree of the Purkinje neurons. Each parallel fiber establishes excitatory synapses with dendritic spines of numerous Purkinje cells. However, the synaptic effect of a single parallel fiber is very week, and therefore it takes many mossy fibers to fire rapidly and simultaneously cause excitation of many granule cells, which in turn excites enough spines of Purkinje cells, in order to elicit an action potential. This is called simple spike and it can be fired spontaneously at a rate of
50-100/second.
The single unit innervation of climbing fibers to Purkinje cell is developmentally regulated. In early development, one Purkinje cell receives synaptic input from multiple climbing fibers. It has been intensively studied the phenomenon of regression of the multiple climbing fiber innervation of Purkinje cells [Kakizawa et al, 2000; Hashimoto and Kano, 2003; Scelfo and Strata, 2005]. The remodeling and regression processes of multiple transient contacts between climbing fibers and Purkinje cells, which result in the usual monoinnervation of Purkinje cells is a fundamental characteristic of the
3 olivocerebellar development [Sugihara, 2006]. Electrophysiological recordings revealed that functional synapses between climbing fibers and Purkinje cells appear at postnatal day 3 (P3) in the rat. The majority of Purkinje cells are multi-innervated from P3 to P7.
Then there is a rapid decrease at P10 and eventually at P17, all Purkinje cells are single innervated [Crepel et al., 1976; Crepel et al., 1981; Mariani and Changeux, 1981a;
Sotelo, 2004]. Parallel fiber-Purkinje cell synapses appear first at P7 in rats. The later development of the parallel fibers-Purkinje cell synapses is highly correlated with the time course of the multiple climbing fiber regression. If the parallel fiber are deleted, degenerated or impaired in their function the multiple climbing fiber innervation of
Purkinje cells will persist [Sotelo, 2004; Lohof et al., 2005]. In summary, climbing fiber input on Purkinje cells is direct, very strong and it elicits complex spikes (1 per second), while mossy fiber input is indirect, occurring via granule cells and parallel fibers and it causes simple spike firing at a constant frequency ( 50-100 per second).
In the cerebellar cortex there exist also stellate cells and basket cells, which are inhibitory to Purkinje neurons [Altman and Bayer, 1997; Voogd and Glickstein, 1967].
The activity of Purkinje neurons is the result of the integrated inhibitory and excitatory inputs. Purkinje cell output is inhibitory to the granule cell layer interneurons, Golgi cells
[Eccles, et al., 1967], which are also inhibitory to granule cells; this creates a negative feedback loop on Purkinje cell excitability. On the other hand, the unipolar brush cells are excitatory to granule cells, which in turn provide a positive feedback loop on Purkinje cell excitability [Dino, et al., 2000]. Cerebellar Purkinje neurons are the only output of the cerebellar cortex and they are inhibitory to the deep cerebellar nuclei and vestibular nuclei. In the traditional view, the functional role of the cerebellum is to dictate the
4 posture of the body and the ability to execute movements. However, cerebellum also
performs functions outside its traditionally attributed role in motor coordination. It is
considered that cerebellum is able to mediate non-traditional functions from motor
learning and emotion to cognition and it has been speculated that cerebellum may be
primarily sensory rather than motor [DeZeeuw et al., 1998; Sacchetti et al., 2004; Gao et
al., 1996; Hartmann and Bower, 2001; Kim et al., 1994; Allen et al., 1997]
The output from the cerebellum is the result of the integrated information at the
Purkinje cell level. Table 1 summarizes the neurotransmission associated with cerebellar
innervations. Figure 2 illustrates the cerebellar circuitry as well as the complex spikes and
simple spike evoked firing, while a three-dimensional diagram of the cerebellar cortex,
highlighting different layers as well as the appearance of various cell types and fibers is
showed in Figure 3.
In terms of cerebellar development, the deep cerebellar nuclei are the ones that
develop first, followed by Purkinje cells and Golgi neurons [Altman and Bayer, 1978;
Altman and Bayer, 1985a; Altman and Bayer, 1985b]. In mice, prenatal day 13 (E13), when the deep cerebellar nuclear neurons and Purkinje cells stop dividing [Miale and
Sidman, 1961], the external granule cell layer is formed [Goldwitz and Hamre, 1998].
Unipolar brush cell interneurons develop around E14 and they complete their
differentiation and maturation by P20 [Abbott and Jacobowitz, 1995; Morin, et al., 2001].
During early postnatal development in the mouse, molecular layer interneurons (stellate
and basket cells) are generated [Zhang and Goldman, 1996]. Basket cells migrate into the
molecular layer at P6 – P7 and complete their differentiation from P10 to P14, while
stellate cells move into the molecular layer at P8 – 11 and complete differentiation by
5 P20 [Zhang and Goldman, 1996; Collin, et al., 2005]. Dendrites of Purkinje cells develop
beginning at P3 as apical swellings, which then take the appearance of main stem
dendrites by P10 –P12 [Altman and Bayer, 1997]. Branching of the main stem dendrite
starts immediately and the Purkinje cell dendritic arbor is further elaborated and
developed from P12 – P20 [Altman and Bayer, 1997]. During this time, Purkinje cells form synapses at dendritic spines with granule cell parallel fibers [Larramendi and
Victor, 1967]. Most of the development and elaboration of the dendritic arbor is
dependent on the synaptic contacts between granule cells and Purkinje cells [Schrenk, et al., 2002; Adcock, et al., 2004]. Also during this time, Purkinje cells limit synaptic contacts between its dendritic tree and climbing fibers to achieve a 1:1 association between Purkinje cells and inferior olive neurons [Mason and Gregory, 1984; Mason, et al., 1990]. At P20 cerebellar Purkinje cells are developed into their distinctive shape with complex two dimensional dendritic ramifications [Altman and Bayer, 1997].
1.2. PCP2-L7 protein- cerebellum specific expression
Purkinje cell protein Pcp2, also called L7 is specifically expressed in cerebellar
Purkinje cells and retina bipolar neurons. Despite the widely used of L7 gene promoter in driving transgene expression in mouse cerebellar Purkinje neurons, the functional role of
L7 protein was not known for a long time. Recently it was found that L7 belongs to a novel family of proteins that include the mammalian regulator of g protein signaling
(RGS)-12 and its Drosophila homologue, Loco. These proteins all contain one or several
6 19-amino acids homologue motifs designated as GoLoco domains. These domains bind
to Gαi and/or Gαo subunit of heterotrimeric G proteins.
Initially, functional characterization of the L7 protein suggested that L7 might
work as a guanine nucleotide exchange factor (GEF) for the pertussis toxin (PTX)-
sensitive G proteins Gi/o. However, all later studies indicated that L7 should rather work
as a guanine dissociation inhibitor (GDI), like all other GoLoco domain proteins [Luo
and Denker, 1999; Siderovski et al., 1999]. GoLoco domains are found in a number of
proteins such as RGS12, RGS14, activator of G protein signaling 3 (AGS3), as well as
L7/Pcp-2. Unlike most of the GoLoco proteins that interact with all Gαi, L7 binds only to
Gαo. It is believed that GoLoco domains are involved in releasing Gβγ from heterotrimeric G proteins, independent of activation of G protein-coupled receptors.
Therefore, there is a continued activation of the Gβγ pathway without the activation of
Gαi/o.
The generation of L7 knockout mice provided excellent animal models to study
the physiological function of L7, although these animals appeared normal in gross
anatomy and simple standard motor behavior [Mohn et al., 1997; Vassileva et al., 1997].
Purkinje cells are a suitable model to investigate the role of L7 because they express
voltage–gated calcium channels that are regulated by Gi/o proteins via physical
interactions and furthermore, L7 is almost exclusively expressed in Purkinje cells. In a
series of studies using real-time RT-PCR to examine RNA prepared from wild-type rat dorsal root ganglia, cochlea and other tissues it was found that although L7 is present in
some other tissues, the level is extremely low, being about 2000-fold less than in retina
7 and about 10,000-fold less than in cerebellum [Serinagaoglu and Oberdick, unpublished
results] .
1.3. Overview of calcium signaling- voltage-gated calcium channels in
Purkinje cells
Calcium signaling is a very complex process implicated in many cellular
functions. For instance, calcium acts as a second messenger responsible for modulating neuronal excitability, neurotransmitter release, neurite outgrowth, synaptogenesis,
activity dependent gene expression, cell death and survival [Pietrobon, 2002].
Extracellular concentration of calcium is about 1 mM, while inside the cell the calcium is
divided between the cytosol and storage compartments such as endoplasmic reticulum
(ER). At rest intracellular concentration of calcium is about 100 nM. It can rise to 1 µM
during calcium signaling events. Calcium concentration gradient between inside and outside of the cell is very critical for the proper function of the neurons. Voltage-gated and ligand-gated calcium permeable channels, calcium-binding proteins, calcium transporters and exchangers, store-operated calcium channels have to function properly for calcium signaling [Bootman, et al., 2001]. For instance, as a result of depolarization of neuronal cell membrane, sodium channels will open and allow sodium to move into the cell. This will activate and open voltage-gated calcium channels (VGCC), leading to calcium influx. Following depolarization, voltage-gated potassium channels will open allowing potassium to exit the cell, which repolarizes the cell membrane, causing the
VGCC to close [Koester and Siegelbaum, 2000].
8
Also, there are ligand-gated channels that are involved in synaptic transmission. For instance the N-methyl-D-aspartic acid (NMDA) receptor will be activated upon binding
to its neurotransmitter, causing sodium and calcium influx [Kandel and Siegelbaum,
2000]. Calcium-binding proteins contribute to intracellular calcium buffering. They are
also involved in second messenger signaling pathways. Some of the most important
calcium-binding proteins in adult mouse cerebellum are: calbindin (abundant in Purkinje
cells and also present in Golgi cells), calmodulin (in Purkinje cells and also in granule
cells, basket and stellate cells), parvalbumin (present in Purkinje and more abundant in
basket and stellate cells) and calretinin (abundant in granule cells) [Bastinelli, 2003].
Calcium influx can be further modulated by another process called calcium-induced
calcium release (CICR). Inositol trisphosphate (IP3) receptors and ryanodine receptors
are two ligand-gated calcium channels on the ER and they are mainly responsible for
CICR [Beridge, et al.; Ashby and Tepikin, 2001]. When activated, the receptors will
trigger calcium release from the ER. Neurons use a series of mechanisms to re-establish
the cytosolic calcium concentration to the resting levels. Ca2+-ATPases at the plasma
membrane will remove excess intracellular calcium while the sarco-endopasmic
reticulum Ca2+-ATPases pump cytosolic Ca2+ back into the ER [ Beridge, et al., 2000;
Thayer, et al., 2002].
VGCCs are calcium channels that are activated by membrane depolarization and
they allow Ca2+ to enter the cell [Catterall, 2000]. They have a multisubunit structure,
consisting of a pore-forming subunit (α1) and secondary β, γ, and α2δ subunits, all of which work together to modulate the biophysical properties of the pore-forming subunit
9 [Westenbroek, et al., 1995; Koester and Siegelbaum, 2000]. The pore-forming subunit is composed of four homologous domains flanked by cytoplasmic amino and carboxyl ends.
Each of the four domains consists of six transmembrane spanning α-helices (indicated by
cylindrical segments in figure 4.). One of the segments (the 4th) spanning the membrane
carries positively charged amino acid residues (see "+" symbols) that form voltage
sensors. The extracellular loop between segments five and six of each domain forms the
ion selectivity filter. The Ca2+ channel β subunits are cytoplasmic proteins closely
associated with the α1 subunit [Richards et al, 2004]. The α2δ subunit is translated as a
single protein, which is post-translationally cleaved into α2 and δ subunits, which are
linked via a disulfide bond [Arikkath and Campbell, 2003; Klugbauer et al, 2003]. The δ-
subunit has a single transmembrane region, which is attached to the extra cellular δ 2
protein. The γ-subunit consists of four transmembrane spanning helices also with
cytoplasmic NH2 and COOH termini. The activity of the channel is not only influenced
by the auxiliary subunits, but also by phosphorylation [Li, et al., 2005], Ca2+ and calmodulin binding (Lee, et al., 1999), and G proteins, such in the case of P/Q channels and N and R type channels. The family of VGCCs includes high voltage-activated channels N-, R-, P/Q- and L-types and low voltage activated channels, T-type. P/Q-type channels are sensitive to ω-agatoxin, L-type channels to dihydropyridine, N-type channels to ω-conotoxin and R-type channels are resistant to all these inhibitors. A summary of the voltage-gated Ca2+channels is presented in Table 2.
Calcium influx/efflux mechanisms are very important for the proper function of
neurons, as calcium signaling regulates functions as complex as neurotransmission, cell
proliferation, death and survival. P/Q type calcium channels are of major interest for this
10 project because they mediate about 90% of calcium current in the cerebellar Purkinje
cells. The P-type channel is a high-threshold, slowly inactivating channel which was first
described in Purkinje cells [Llinas et al, 1992; Mintz et al, 1992]. Although the P/Q-type
channels present in most of the other types of cells are typically involved in synaptic
transmission (presynaptic neurotransmitter release), the P-type channel in Purkinje
neurons seems to have a computational role for the overall output of these cells [Genet
and Delord, 2002]. It is believed that these particular channels are responsible for the
large dendritic Ca2+ spikes and the slow plateau potentials in the some detected by current
clamp recordings in Purkinje cells in cerebellar slices [Womack and Khodakhah, 2000].
The dendritic Ca2+ influx is mediated by the Ca2+ channels has a crucial role in the
development of long term depression (LTD) at the following two synapses: parallel fiber-
Purkinje cell and climbing fiber-Purkinje cell.
Previously it has been shown that the P/Q-type Ca2+ channels may be a primary
downstream effector of L7 modulation of Gi/o proteins [Kinoshita-Kawada et al., 2004].
Using the Xenopus oocytes expression system, it was shown that the relative expression
levels of L7 proteins as compared to the Gi/o proteins strongly influence the degree of G
protein-induced inhibition of P/Q-type Ca2+ channels. At low concentrations, L7
enhanced whereas at high concentrations it reduced the inhibition of the Ca2+ channel. A schematic representation of the proposed mechanism by which L7 modulates Ca2+ channels via Gi/o proteins is shown in Figure 5.
The concentration-dependent dual regulation of P/Q-type channels by L7 can be explained by the fact that both Gαi/o and Gβγ are involved in the inhibition of the Ca2+ channels. By binding to Gαi/o, the low concentration of L7 causes Gβγ release of and
11 hence facilitated the Gβγ-induced channel inhibition. However, as the L7 concentration
increases the availability of Gαi/o subunits becomes more limited, leading to a reduction
of inhibition mediated by Gα, assuming there is a more pronounced inhibition by Gαi/o than Gβγ and the latter is easily saturable. The enhancing effect of the high concentration of L7 should reflect a disinhibition of the channel from Gαi/o through competitive binding as well as the inhibition of GDP dissociation from Gα subunits.
There are two isoforms of L7, L7A and L7B, containing one and two GoLoco
domains, respectively. The presence of the two isoforms is well conserved across species.
The mRNAs from both forms were localized in the distal dendrites of Purkinje neurons
[Zhang at al, 2002]. As a result of the mRNA migration through dendrites and use-
dependent protein synthesis, there are different levels of expression of L7 proteins in
different parts of the dendrites.
Hence, one way to study the functional role of L7 proteins, which are mainly
expressed in Purkinje cells dendrites in an activity-dependent manner, is by looking at the
electrophysiological characteristics of these neurons. Through differential regulation of
the availability of the two arms of Gi/o protein, i.e. Gαi/o and Gβγ, the L7 protein is
believed to regulate the activity of P-type channels, which represent the majority of
calcium channels in cerebellar Purkinje cells and play a computational role in signal
processing. Furthermore, these calcium channels are critical determinants for the firing
pattern of the Purkinje neurons, which in part is achieved via the consequential activation
of the calcium-activated potassium channels. The possible mechanism by which P-type
channels regulate firing behaviors of Purkinje cells was proposed by Edgrton and
Reinhart (2003). They suggest that Ca2+ entry through Purkinje cells P-type channels
12 activates small-conductance Ca2+-activated K+ (SK) current and large-conductance Ca2+-
activated K+ (BK) current, both being involved in modulating the firing pattern. They also
proposed that SK plays a role in determining the intrinsic firing frequency, while BK
currents are regulators of the action potential shape and are they are involved in the
climbing fiber response. Given that L7 modulates P/Q-type channel activity in a concentration-dependent manner Kinoshita-Kawada et al, 2004], the lack of L7 expression in the L7 knockout mice is expected to alter the firing pattern and/or frequency of the cerebellar Purkinje cells.
1.4. Role of P/Q-type channels in cerebellar function
P/Q-type VGCCs are highly expressed in the dentate gyrus and CA fields of the
hippocampus, the cerebellar cortex, pontine nucleus, olfactory bulb and cerebral cortex
layers II and VI. Moderate levels are also found in the striatum, hypothalamus, substantia
nigra, red nucleus, lateral reticular nucleus and inferior olive [Stea, et al., 1994].
Although N-, L-, and R-type channels are also present in the cerebellum, the big majority
of VGCCs in the cerebellum is represented by P/Q-type VGCCs [Westenbroek, et
al.,1995]. These channels are found predominantly all over the dendritic arbor and at the
cell body of Purkinje cells, and to a lesser extent in cerebellar granule cells
[Westenbroek, et al., 1995]. In addition, molecular layer interneurons express P/Q-type
VGCCs at their presynaptic terminals which form synaptic contacts with Purkinje cells
[Westenbroek, et al., 1995].
13 The pore-forming subunit of P/Q-type VGCCs is Ca v2.1 or α1A. Mutations of
this particular gene are found to be responsible for several inherited neurological
disorders, including in human familial hemiplegic migraine (FHM), episodic ataxia type
2 (EA-2) and spinocerebellar ataxia type 6 (SCA-6) [Ophoff, et al., 1996; Klockgether and Evert, 1998; Ducros, et al., 1999; Pietrobon, 2002]. FHM is associated with 12
different mutations in the α1A gene that cause migraine accompanied by intermittent
weakness or paralysis [Ophoff, et al., 1996; Klockgether and Evert, 1998; Pietrobon,
2002]. These mutations are found in different domains in the voltage sensor, P-loop and
in transmembrane segments five and six [Pietrobon, 2002]. About half of the FHM
mutations cause a slowly progressive cerebellar ataxia and atrophy [Ducros, et al., 1999].
The most common mutation for FHM with progressive cerebellar atrophy is T666M in
the P-loop of domain II of the α1A subunit [Pietrobon, 2002]. EA-2 has been linked to 15
different mutations in the α1A gene. These mutations interrupt the open reading frame
and cause truncation, intron inclusion or exon skipping of the α1A subunit [Ophoff, et al.,
1996; Klockgether and Evert, 1998; Pietrobon, 2002] resulting in episodes of ataxia that
may be progressive and cerebellar atrophy localized to the anterior vermis [Klockgether
and Evert, 1998; Pietrobon, 2002]. SCA-6 is caused by polyglutamine repeats in the
carboxy-terminus of the α1A subunit that result in a slowly progressive cerebellar ataxia
with cerebellar atrophy and a severe loss of cerebellar Purkinje cells and granule cells
[Pietrobon, 2002].
There are several mutations of the α1A gene found in mice in which the
phenotype or clinical signs closely resemble those in humans. Therefore, they have been
used as animal models to investigate the cellular and molecular consequences of α1A
14 mutations in FHM, EA-2 and SCA-6 [Fletcher, et al., 1996]. Naturally occurring mouse
mutations include leaner, tottering, rocker, Nagoya rolling, and possibly pogo [Hyun, et
al., 2001; Zwingman, et al., 2001]. A genetic knockout of the α1A gene (P/Q null) has
also been created [Jun, et al., 1999].
The leaner mouse (tgla or Cacna1atg-la) has a nucleotide substitution at an intronexon junction causing an aberrant splicing event in the carboxyl tail that results in a
severe cerebellar ataxia with a loss of both granule cells and Purkinje cells [Fletcher, et
al., 1996]. The tottering (tg or Cacna1atg) mouse mutation is an amino acid substitution
(leucine for proline) in the P-loop of domain II of the α1A protein [Fletcher, et al., 1996;
Ducros, et al., 1999; Pietrobon, 2002] and is characterized by an intermittent dyskinesia
and a mild to moderate cerebellar ataxia without a significant loss of cerebellar neurons
[Green and Sidman, 1962; Isaacs and Abbott, 1992; Klionsky, 2005]. The rocker (tgrkr or
Cacna1arkr) mutation resulted from an amino acid substitution (lysine for threonine) in
the P-loop of domain III and the phenotype is also characterized by a mild to moderate
cerebellar ataxia without a loss of cerebellar neurons [Zwingman, et al., 2001]. The
Nagoya rolling (tgrol or Cacnalatg-rol) mouse mutation occurred from the replacement of
a glycine with arginine in the voltage sensor of domain III [Oda, 1981] and the phenotype
has a moderate to severe ataxia with a minor loss of granule cells [Suh, et al., 2002]. The
α1A knockout or null mouse (Cacna1aFctm1) resembles the leaner mouse and its
phenotype includes a severe cerebellar ataxia with a loss of cerebellar granule cells and
Purkinje cells [Jun, et al., 1999; Fletcher, et al., 2001].
15 All these mutant mice are characterized by very dramatic functional alterations compared to the wild type. They have strong motor coordination impairments and the complexity of their defects, as well as the wide spread expression of P/Q channels in multiple brain regions, make it difficult to associate the mutation and hence the P/Q-type channels activity changes with the function of the cerebellum itself. Furthermore, the strong defect in motor coordination displayed by these mutants occludes the possibility of accessing the role of cerebellar P/Q-type channels in sensorymotor gating.
By contrast, the mutant mice used for this project are an excellent model to investigate the role of cerebellum, especially Purkinje cells and L7 protein in motor function and sensorymotor gating, as well as to investigate the P/Q-type channel regulation. These mice do not have a direct mutation of the P/Q-type channels, but they are designed not to express the Pcp2-L7 protein, which is normally expressed only in cerebellar Purkinje neurons. This protein is known to modulate the activity of P/Q-type channels in a concentration dependent manner. Although initial studies did not show any anatomical difference, later examinations revealed subtle cerebellar hypoplasia, thinner molecular layer and a reduced size of Purkinje cell soma. Behaviorally, the mutants are characterized by functional enhancements related to sensorymotor learning including: improved rate of gross motor learning, increased maximal gross motor performance after training, and increased rate of acquisition in tone-conditioned fear as well as increased acoustic and cutaneous sensory responsiveness [Oberdick, Serinagaoglu, Nelson, unpublished results].
16 1.5. Purkinje cells - mechanisms that regulate different firing patterns
The traditional view of cerebellar function is to coordinate movement and to maintain balance. Cerebellum has a complex architecture based on repeats of an anatomical well defined circuit, whose most important neurons are the Purkinje cells.
These cells are the only output of the cerebellar cortex and it is believed that they encode timing signals in their firing rate [Ito, 1984]. A good understanding of the cerebellum circuitry requires a detailed study of the intrinsic firing and pattern activity of Purkinje cells, and the regulatory factors that influence this activity.
Cerebellar Purkinje cells have the intrinsic ability to fire action potentials (AP) in
the absence of external stimulation. Some Purkinje cells display a complex pattern of
activity that includes a tonic firing phase, followed by a bursting phase, and then a silent
period. Basically, the rate of tonic firing increases until the cell starts a bursting mode of
activity, where few spikes are separate by short (10-20 ms) pauses. The bursting phase is then followed by total silence, which lasts about 10-20 s. This has been recognized as the trimodal pattern of activity, originally defined by Womack and Khodakhah [2002] on
Purkinje neurons in acutely prepared cerebellar slices. This pattern may repeat for hours, but can be very different from one cell to another. The trimodal pattern appears to require the maturation of the dendrites, especially the increase in the dendritic arborisation, as in young animals (less than P16) cells fire only in a tonic mode, while in adult animals higher percentage of trimodal firing cells is detected [Womack and Khodakhah, 2002].
Electrophysiological experiments have been applied to study the spontaneous firing patterns of Purkinje cells in dissociated cultures, cerebellum slices, or in vivo
17 preparations, using extracellular as well as intracellular recordings. Firing behaviors of
the Purkinje cells have been studied very early on in guinea pig cerebella slices, where
intracellular recordings showed a pattern of alternating bursting and silent periods [Llinas
and Sugimori, 1980]. Whole-cell recording in slices of rat cerebellum revealed a similar
pattern of activity for Purkinje cells [Jaeger and Bower, 1999]. The extracellular
recording, however, is the approach that causes minimum damage to the cell.
Factors that may or may not influence the cyclic firing of the Purkinje cells are listed below. For instance, firing frequency does not strongly influence whether a cell can or cannot go into the trimodal pattern. Second, the firing rate is temperature sensitive.
The recordings are normally performed at 34°C, but if the temperature drops 3-7°C, the cell may stop firing. Third, synaptic input strongly affects the firing patterns. Although the trimodal pattern of activity can be observed in the presence of fast synaptic input in some cells, with the inhibition of the fast synaptic input, the trimodal pattern appears even more regular [Womack and Khodakhah, 2002]. In order to eliminate the fast
synaptic transmission, excitatory and inhibitory inputs can be pharmacologically blocked
(glutamate receptors and GABAA receptors are blocked by kynurenic acid and picrotoxin, respectively) [Womack and Khodakhah, 2002, 2004]. In our experiments we found that the presence of synaptic blockers significantly reduces the firing rate (Figure 9). Fourth, although cerebellum receives inputs from noradrenergic, serotonergic, histaminergic and cholinergic fibers, it has been shown that the cyclic activity of Purkinje cells is not driven by the diffused release of these neurotransmitters. Finally, even though in many cells with the cyclic activity, the hyperpolarizing current (Ih) is responsible for the rhythmical
18 behavior, the trimodal pattern in Purkinje cells it is not driven by Ih current [Womack and
Khodakhah, 2002].
Ion channels involved in shaping the action potential and firing behavior of
Purkinje cells have been actually evaluated. In many neurons the voltage-gated Ca2+ channels are shown to be involved in their oscillating activity [Llinas, 1988; Huguenard,
1996; McCormick and Bal, 1997]. P/Q-type voltage gated Ca2+ channels account for
more than 95% of the Ca2+ channel activity in Purkinje cells. When these channels are
blocked, the spontaneous activity of the Purkinje cell is inhibited. The loss of the inward
calcium current could be one reason; however, if the effect of the calcium entry is to
hyperpolarize the membrane by activating calcium-activated potassium channels, the lack
of calcium channels activity would result in persistent membrane depolarization, making
the cell inactive. Mathematic modeling has showed that small changes in the P-type
channel activity can dramatically modify the firing pattern of Purkinje cells [Womack
and Khodakhah, 2002]. In fact, among all the studied channels (see below), only the P/Q
type channels are absolutely required for sustaining regular spontaneous bursting.
It has been shown that calcium-activated potassium channels are critical
regulators of the spontaneous firing of Purkinje cells [Womack and Khodakhah, 2002].
These include SK and BK. Originally it was considered that the decrease in the
expression of SK channels, which in rat Purkinje cells appears within the first few weeks
of life, makes SK channels have an insignificant role in regulating the excitability of the
adult rat Purkinje neurons [Cingolani et al, 2002]. Later study showed that there is no age
dependent difference in the way SK channels regulate the firing rate of Purkinje neurons,
19 but rather a distinction between the functional roles of SK in soma as compared to dendrites [Womack and Khodakhah, 2002].
The functional role of somatic SK channels is thought to prevent the cell from firing by producing a pronounced afterhyperpolarization (AHP). At short AHP a faster repolarization is allowed and the threshold is reached earlier for the discharge of a subsequent sodium spike. The functional role of SK in dendrites is rather related to regulation of dendritic excitability when calcium is elevated, like for example in the case of climbing fiber stimulation. Also, the dendritic SK channels activity does not modulate the tonic firing rate, but the length of the trimodal pattern. The shortening of the trimodal cycle when dendritic SK channels are blocked suggests that the dendrites can easily initiate calcium spikes under these conditions [Womack and Khodakhah, 2002]. A typical burst is composed of 2 to 30 action potential spikes. The burst has a stereotypical waveform. It develops after an increase in firing rate during the tonic phase of the trimodal pattern. The final few spikes in each burst are closer to each other and with smaller amplitude. The ionic mechanism of the spontaneous burst firing has been studied also in dissociated Purkinje neurons [Swensen and Been, 2003]. Pharmacological studies
[Womack and Khodakhah, 2002] showed that SK channels have a major contribution in controlling the firing rate during the burst, and the interburst interval, but have little or no effect on burst termination. On the other hand, BK channels mainly modulate burst duration and interburst interval. The spontaneous bursting develops with less hyperpolarization, suggesting the involvement of the T-type, i.e. the low voltage- activated Ca2+ channels. In fact, T-type calcium channels also contribute to the interspike and interburst intervals [Womack and Khodakhah, 2004]. Clearly, the bursting firing
20 phenomenon is the result of the interplay between somatic and dendritic contributions: tonic firing is mainly somatic sodium-dependent, while the burst firing is terminated by dendritic calcium spikes. It is believed that in Purkinje cells the P/Q-type channels are responsible for burst termination by generating dendritic calcium spikes, which result in the inactivation of sodium channels and hence cell silent for a short period of time
[Womack and Khodakhah, 2004].
In in vivo, studies Purkinje cells display an alternating pattern of activity consisting of tonic firing with sporadic silence intervals and single bursts, associated to climbing fibers input [Rubia and Hennemann, 1997; Ebner and Bloedel, 1981]. However, a regular trimodal pattern of activity is not observed in vivo, where all the synaptic input is present. This fact raises an intriguing question: what is the physiological significance of the trimodal firing pattern? Most likely, the trimodal pattern of activity is maintained and regulated by rhythmic changes in the excitability of the cells. In vivo, there are factors, such as second messengers that may alter the pacing of the excitability adjustments of the cells and if, for instance, the ion channel whose activity regulates the firing becomes phosphorylated as a result of messenger signaling, then it is probable the trimodal pattern could be masked or disrupted.
21 A B GL ML
PL
CD
RE
Figure 1: Structural organization of cerebellum.
(A) Sagittal section of the cerebellar vermis area. (B) Closer image of the lobule reveals the molecular layer (ML), the granule cell layer (GL) and the Purkinje cell layer (PL).
Images provided by Dr. Georgia Bishop, (C) Hoffman modulation contrast (HMC) image of a Purkinje neuron patched by a recording electrode (RE). (D) Image of Purkinje neurons with intact processes
22
Figure 2: Cerebellum circuitry and examples of simple spikes and complex spikes obtained by extracellular field recordings.
The solid lines indicate excitatory input and the dashed lines indicate the inhibitory input.
PC- Purkinje cell, GC-granule cell, CF- climbing fiber, PF- parallel fiber, DCN- deep
cerebellar nuclei, PN- Pontine Nuclei, MF- Mossy fiber, IO-inferior olive, BC- Basket
cell, SC-stellate cell, RE- recording electrode, SE- stimulation electrode, SS- simple
spikes, CS- complex spikes
23
Figure 3: Three-dimensional view of cerebellar cortex (adaptation from Broedel and Bracha, [1998]) The diagram reveals the organization and appearances of the Purkinje cell dendrites and how they interact with climbing fibers, interneurons (basket, Golgi, stellate cells), as well as mossy fiber system and parallel fibers.
24
NH2 A α1 γ S-S α2-δ
+ + + + + + + + + + + + + + + +
COOH NH2 COOH
COOH NH2 α2 NH2 B δ γ α1 COOH β 2+ β Ca
Figure 4: Schematic representation of molecular composition of the high voltage- activated calcium channels. A typical channel has a complex structure consisting of the main pore-forming subunit α1
plus the auxiliary subunits β, γ, α2-δ. (adopted from Khosravani and Zamponi, [2006] with modifications) A). Layouts of membrane topology of each subunit. B). Putative three dimensional structure of the channel.
25
P/Q type VGCC
α1A
Gβγ Gβγ
Gαo
G protein cycle GTP αi *
βγ ∗ H2O
Pi α GDP α GDP α GTPβγ i i i L7
L7 GTP GDP αβγ GDP i αi βγ Receptor
Figure 5: Proposed mechanism of regulation of P/Q-type Ca2+ channels by L7
(adopted from Konishita-Kawada, 2004).
The gray arrows represent how low concentration of L7 interferes with G-protein cycle: it binds to Gαi/o and causes Gβγ release and facilitates the Gβγ-induced channels inhibition
(yellow arrow).The black cycle represents interference of high concentration of L7 of the
G-protein cycle: when in high concentration, L7 binds to Gαi/o, which limits the supply of the subunit leading to a reduction of inhibition mediated by Gα .
26 Type of Synapse Neurotransmitter synapse
Climbing fiber-Purkinje cells CRF, aspartate Excitatory
Climbing fiber-deep cerebellar nuclei CRF Excitatory
Mossy fiber-granule cells glutamate, acethylcholine Excitatory
Mossy fiber-unipolar brush cells glutamate Excitatory
Unipolar brush cells-granule cells glutamate Excitatory
Granule cells-Purkinje cells glutamate Excitatory
Granule cells-stellate cells glutamate Excitatory
Granule cells- basket cells glutamate Excitatory
Stellate cells-Purkinje cells GABA Inhibitory
Basket cells-Purkinje cells GABA Inhibitory
Golgi cells- granule cells GABA, glycine Inhibitory
Purkinje cells- Golgi cells GABA Inhibitory
Purkinje cells-deep cerebellar nuclei GABA Inhibitory
Table 1: Neurotransmission in cerebellar cortex
27 Ca2+ Current Primary Primary channel type Location Function Cav1.1 L skeletal muscle initiate contraction, calcium homeostasis, gene regulation
Cav1.2 L cardiac muscle initiate contraction, endocrine cells initiate hormone secretion neurons gene expression
Cav1.3 L endocrine cells initiate hormone secretion neurons gene expression
Cav1.4 L retina neurotransmitter release
Cav2.1 P/Q nerve terminals neurotransmitter release nerve dendrites Ca2+ transients
Cav2.2 N nerve terminals neurotransmitter release nerve dendrites Ca2+ transients
Cav2.3 R nerve soma Ca2+ dependent action potentials nerve terminals neurotransmitter release nerve dendrites Ca2+ transients
Cav3.1 T cardiac muscle control repetitive firing pattern skeletal muscle control repetitive firing pattern neurons control repetitive firing pattern
Cav3.2 T cardiac muscle control repetitive firing pattern neurons control repetitive firing pattern
Cav3.3 T neurons control repetitive firing pattern
Table 2: Voltage gated calcium channels- summary
28
CHAPTER 2
METHODS
2.1. Mice
L7 homozygous breeders were obtained from St. Jude Childrens Research
Hospital [Vassileva, et al., 1997]. L7 heterozygotes (L7+/-) were crossed in each
generation to commercially obtained stock C57BL/6NTac mice (Taconic), and the L7+/- pups identified by PCR of tail biopsy. All animals were obtained from crosses between
L7+/- parents that were 8-10 generations in C57BL6. For the electrophysiological
recordings all animals were 2-4 months old at the time of recording. All experiments on
animals were conducted in compliance with the guidelines for animal research described in “PHS Policy On Humane Care and Use Of Laboratory Animals” and the PHS “Guide for the Care and Use of Laboratory Animals” from the U.S. Dept. of Health and Human
Services.
29 2.2. Tissue preparation
Animals were quickly decapitated and the cerebellum was extracted and was paced in ice cold aCSF, in the presence of 5% CO2–95% O2. Anesthetics were not used in order to eliminate the possibility of any ataxic effect the alcohol-based anesthetics on the subjects and consequentially on the cerebellum output. Sagittal slices of 250-300 μm thick were prepared from the vermis of the cerebellum with a vibratome and incubated at
34-36°C in the recording solution for one hr before being stored at the room temperature
(22-24°C) until use. The incubation chamber is custom-made, it has a grid that holds the slices and the oxygen is provided through a vertical tube making the incubating solution to circulate in vertical plane, which not only holds the slices on the grid (the direction of the flow has to be top to bottom), but also it assure o good oxygenation of the tissue, as compared to having the slices sitting on the bottom of a beaker. Preparation of the slices usually took about 15 minutes and the slicing solution was ice-cold in order to assure a good quality slicing and also a good survival rate of the neurons.
2.3. Data recordings and experimental set-ups
Extracellular and whole cell recordings on cerebellar Purkinje cells in slice were performed using two experimental set-ups (A and B) as well as slightly different solutions for each of them. Both set-ups and the experimental conditions are described below.
30 Set-up A was equipped with a Zbicz Top tissue slice chamber (Harvard Apparatus
Co.). A Tektronix 5A26Dual Differential amplifier was used and the signal was
visualized on a 5110 oscilloscope display. The audio monitor (Grass AM8) also facilitated a good isolation of the cell and the optimization of the signal and the temperature was maintained around 34-35ºC and monitored with a TC-102 temperature controller. The data was recorded with Spike2 software. Set-up B was more complex and had an EXT-10C (npi) amplifier for extracellular recordings and a Heka EPC10 amplifier for the whole cell recordings. Both of these were assisted by PatchMaster software. The visualization of the slices was possible with a Nikon Eclipse E600FN microscope, equipped with an RT KE/SE Spot camera (Diagnostic Inc.). The experimental set-up includes also an LPBF-01GX (npi) and a HumBug (Quest Scientific) filters, which make easier to isolate the signal. In addition we have a biological audio monitor BAM-10
(ALA Scientific Instruments) which allows us to distinguish the cells by the specific firing tone they have. The climbing fiber activation was carried out with an isolation stimulator, IsoStim01M (npi). The stimulation was triggered externally through a specific recording protocol. The slices were taken with a NVSL manual advance vibroslicer
(WPI), which was equipped with one-time use stainless steel blades or a Sapphire blade.
The electrodes were pulled using a Narishige PC-10 vertical puller in one step pulling.
The recordings duration was 1 to 15 minutes. Frequency of the recording was 20 kHz.
Data presented in sections 3.1.1, 3.1.2, 3.2 and 3.3 was recorded on set-up A. In
these experiments, slices were placed on a Zbicz Top tissue slice chamber (Harvard
Apparatus Co.) and continuously perfused with a warm (34°C) artificial cerebrospinal fluid (Table 3) bubbled with 5% CO2–95% O2. Slices were visualized with an inverted
31 stereo microscope which although allowed a good identification of the Purkinje cells location, did not make possible the detection of individual cells. No synaptic blockers were used for these experiments in order to mimic as close as possible the physiological conditions.
The complex spikes recordings (data presented in section 3.4) and the whole cell recordings (data presented in section 3.5) were performed on set up B. In these experiments, the slices were placed in an RC-27L perfusion chamber (Harvard
Apparatus) mounted on the stage of a Nikon E600-FN upright microscope and continuously perfused with a modified aCSF solution (Table 3) supplemented with 5 mM kynurenic acid and 100 μM picrotoxin, bubbled with 5% CO2–95% O2. Kynurenic acid, a broad-spectrum ionotropic glutamate receptor antagonist [Stone, 1993], and picrotoxin, a GABAA receptor antagonist [Yoon, et al., 1993], were used in order to block the fast synaptic input, so that only spontaneous activity was recorded. The solution was heated to 34-35°C with an SC-20 in-line solution heater (Harvard Apparatus).
However, there is no difference (in firing frequency and firing pattern) between recordings from the two experimental set-ups when performed in similar conditions of external solution and temperature.
32 2.4. Electrophysiology
2.4.1. Extracellular recordings
Extracellular field potentials were recorded using glass electrodes with tip
resistance of about 3-6 MΩ filled with 4 mM NaCl or plain ACSF without the synaptic
blockers. Sampling rate was 20 kHz. For this set of experiments, slices were placed in a
Zbicz Top tissue slice chamber, maintained at 34-35ºC on top of a piece of optical tissue and submerged in aCSF for the entire duration of experiment. The chamber was partially covered during the recording, so that the slices were kept in a moist, oxygenated environment and cells were alive for up to 6-8 hours. After the cell was identified and isolated to a good signal to noise ratio, the spontaneous activity was recorded for 1-10 minutes.
2.4.2. Complex spikes recordings
This set of measurements require a good identification of the cell body; therefore
RC-27L perfusion chamber and the immersion microscope were used. For complex spike
measurement, kynurenic acid was omitted, picrotoxin was reduced to 20 μM, and the
temperature was maintained at 32°C. Climbing fibers were stimulated using a low
resistance (0.1 MΩ) bipolar electrode (MicroProbe Inc) placed in the granule cell layer
within 50 μM from the soma of the Purkinje cell of which the extracellular filed
potentials were continuously monitored while 200 μs square pulses of 0.1-5 V constant
33 voltages were applied at a frequency of 1 Hz. The procedure involves two electrodes, one
for recording and one for the stimulation of the climbing fiber. After the recording
electrode was positioned and good signal was obtained from the cell, the second electrode was placed as mentioned above and then the stimulation was triggered. The climbing fibers are not actually visible and the stimulation, even if it was not very strong, could activate granule cells and generate a lot of excitation around the cell. In order to prevent that very small (0.1-0.2 V) stimulation was applied first, and then it was increased until the complex spikes were obtained. The occurrence of complex spikes was “all or none”; therefore, a small adjustment of the stimulation to below the threshold can eliminate the complex spikes. However, the threshold values are influenced by the position of the stimulating electrode as well as the condition of the cell.
2.4.3. Whole cell measurements
Whole cell recordings of the spontaneous activity of Purkinje cells were
performed at room temperature using a 3-5 MΩ glass electrodes filled with a potassium
based internal solution (Table 4) These experiments were performed using cerebellar
slices placed in the RC-27L perfusion chamber. The immersion microscope was used to identify the cells. Sometimes, the targeted cell was cleaned using a blunt patch pipette with a tip diameter of 2-3 µm. The pipette was inserted into the bath and filled with some bath solution by capillary action. The cleaning pipette was brought close to the slice surface and positive pressure was applied to produce a flow of solution, which would break up the tissue over the cell of interest. Then light suction was used to remove the
34 debris covering the cell. To make the whole cell patch the recording electrode is placed in
the solution, positioned just above the targeted cell, and then moved toward the cells
under the visualization of the immersion objective. Typically, the resistance would increase as the electrode came closer to the surface of the cell. At this point the positive pressure that was applied to the recording electrode would form a dimple on the cell membrane. The pressure was removed and a small suction was applied to help form a gigaohm seal, which occurred either immediately or in a few minutes. Sometimes, a
positive hold potential of 65 mV was applied to help the seal formation. After the
formation of the giga-seal further gentle suction will rupture the membrane and allow the establishment of the whole cell configuration. The Purkinje cells are about 20 µm in diameter, and they have a huge dendritic three, therefore, the cells cannot be voltage- clamped due to the space clamp issue. Current clamp recordings will allow visualization of the action potentials elicited spontaneously or under current stimulation by the
Purkinje cells.
The animals older than 17 days develop a perineuronal net of proteoglycans. This
net impedes the access of the patching electrode to the cell membrane; therefore a special
cutting solution would be used for mice older than 17 days, to help remove the neuronal
net before the patching. The cutting solution is made fresh or the night before the
experiment (in this case the calcium and glucose are added the day of the experiment.
Cutting solution replaces sodium with choline and calcium with magnesium. The
osmolarity of the solution is around 300mOsm and the pH is adjusted to 7.3-7.4.
35 2.5. Data analysis
Due to a high diversity of the firing pattern of Purkinje cells, a trustworthy
analysis requires a huge number of recordings and consequentially accurate statistical
analysis. The extracellular recording method allowed us to accumulate a big volume of
data. For the mean values comparison, either t test (in the case of the two-group
comparison) or one way Anova test (in the case of three-group analysis) were used in
Origin or Excel.
Firing pattern of Purkinje cells, especially the bursting phase, is very complex and
difficult to analyze. As the mean differences are rather subtle, a multifactorial ANOVA is
required to verify the interaction of two or more factors (gender and genotype interaction)
that will show significant difference in bursting behavior of the mutant, as compared to
the wild type.
Distribution histograms and cumulative probability plots are also suitable way to
analyze a huge volume of data, where the means are not obviously different. The way
data is distributed as compared to the mean, will give information about the variance of
certain parameters, the overlapping range of the data. Kolmogorov –Smirnov two-sample
test was used to test for the significant difference between two data samples. The statistic
analysis compares cumulative distributions, in the sense that large differences between
cumulative samples distributions indicates that data is not drown from the same
distribution. Chi-Square test was used to examine if the difference in data distribution is
significant. This analysis is a non-parametric statistics method, which does not require normality of data, but it does have a series of restrictions, such as: no more than 20% of
36 the cells can have a frequency < 5, frequency 0 is not acceptable and data should not be subjected to other preliminary analysis.
Data were and analyzed with the Spike2 program, (Cambridge Electronic
Design). A series of scripts were used to characterize the continuous and the bursting firing of the neurons. The software generates parameters such as instantaneous and average firing frequency based on the raw data, as well as parameters describing the bursting behavior of the neurons: interburst interval, burst duration, number of events per burst. The statistical analysis (one way Anova, multifactor ANOVA and t-test) as well as the graphs were made using Excel, Origin or Statistica. Count data (e.g. CS spikelet number) were analyzed using a generalized linear model (GLM) with a logarithmic link and a quasipoisson model [GLM(qp)] to account for overdispersion [McCullagh and
Nelder, 1989; see also, Zeileis et al: Regression Models for Count Data in R; available at http://cran.r-roject.org/doc/vignettes/pscl/countreg.pdf]. For the statistical analysis of the bursting properties by gender and genotype Kolmogorov–Smirnov two-sample test was performed in Statistica, in order to prove that samples come form the same distribution.
Chi-Square test of independence was used to compare the cumulative probability plots.
For data analysis every cell was considered individually for tonic firing. Originally, for bursting analysis a number of about 100 bursts were considered for each cell. However, some data presented here shows the average per individual cell, in order to eliminate the possibility of an artificially increased sample number (cumulative plots, for instance).
37
extracellular solutions
aCSF modified aCSF (mM) (mM) NaCl 124 124 KCl 5 2.5
K2HPO4 1.3 1.3
NaHCO3 28 28
CaCl2 2.5 2.5
MgSO4 1.2 1.2 glucose 20 20 adjusted to pH 7.4 with 1 N HCl
Table 3: Electrophysiology solution: aCSF plain and modified
38
internal solutions
whole cell Extracellular recording recordings (mM) (mM) NaCl 4
KCH3SO3 110 NaCl 10
CaCl2 0.2 MgCl 0.2 EGTA 0.5 HEPES 10 adjusted to pH 7.4 with 1 N HCl
Table 4: Electrophysiology solutions: internal electrode solutions for whole cell and extracellular recordings
39
Molarity Chemical (mM)
Choline Cl 110
NaHCO3 25 KCl 3
MgSO4*7 H2O 7 NaH2PO4 1.5 Add the following on the day of use: d-Glucose 10
CaCl2*2 H2O 0.5
Table 5: Cutting solution
40
CHAPTER 3
RESULTS
3.1. Spontaneous firing patterns of Purkinje cells cerebellar slices of wild
type and L7 knockout mice
In order to study the physiological significance of Pcp2-L7 in motor learning and
sensory gating, the L7 knockout mice were obtained from Dr. James Morgan in St. Jude
Children's Research Hospital and backcrossed to C57/BL6 background for more than 10
generations. Littermate L7 knockout (L7-/-), heterozygote (L7+/-), and the wild type (L7+/+)
mice were subjected to a series of behavioral tests. These tests were performed by Dr.
Oberdick’s group, in collaboration with Drs. Nelson and DeVries. Surprisingly, they revealed significant improvement and maximal performance in the rate of gross motor learning in the accelerated rotarod tests, in the rate of acquisition for the tone-conditioned
41 fear, as well as increased acoustic and cutaneous sensory responsiveness [Iscru et al.
2008, unpublished results].
The behavioral changes found in the L7 knockout mice are more sensory rather than motor, suggesting that L7 proteins may be more involved in sensory gating than in motor coordination aspect of the cerebellar function. If these effects are attributable to alterations in Purkinje cells, we might expect to see changes in the firing properties of these cells. Therefore, I studied the spontaneous firing patterns of Purkinje cells in cerebellar slices from wild types, L7+/-, and L7-/- mice (2-4 months old) using the
extracellular recording technique.
This method was chosen because it was anticipated that just like the anatomical
and behavioral changes, the effect of the mutation on the firing properties may also be
subtle and therefore requires a large sampling size of each genotype in order to reveal the
change, if any. Although Purkinje cells fire spontaneously in the absence of any synaptic
input [Raman and Bean, 1999; Wormack and Khodakhah, 2002], synaptic transmission is
not disrupted in behavioral studies. Thus, firing properties need to be acquired both in the
absence and presence of synaptic blockers. The extracellular recording technique allows
quick analyses of many cells without disrupting the cellular contents.
The spontaneous firing patterns of Purkinje neurons, although very diverse from
cell to cell and from animal to animal, follow the trend of either tonic or phasic firing
patterns (Figure 6). The tonic pattern firing can last spontaneously for hours and the frequency is very uniform (Figure 6A). The firing rate differs from cell to cell and is influenced by temperature, K+ concentration of aCFS and the presence of the fast synaptic
blockers. On average, the firing rate is about 50 % lower in the presence than in the
42 absence of the synaptic blockers (see figure 9). The phasic firing can be rather irregular
(Figure 6B) or it follows a uniform trimodal firing pattern as exemplified in figures 6C
and 7. As described in the Introduction, section 1.5, the trimodal firing pattern consists of
cycles of tonic firing, regular bursting and silent periods, which repeat in a regular
fashion. Within each cycle, there is a relatively long period of tonic firing which then
transits to more than 100 short bursts which are characterized by parameters such as burst
length, the number of spikes per burst, the interburst interval and the interspike interval
(see later bursting description). These bursts are followed by a long period of silence
before the cell start to fire tonically again. During the recording a tonic firing cell could
convert into firing, which typically occurred after an obvious and steady increase in the
firing rate (Figure 6D).
The firing patterns of Purkinje cells in acute cerebellar slices were studied using
L7+/+, L7+/-, and L7-/- mice. Initially, the recordings were performed at 34C using plain
aCSF solution (Table 3) and in the absence of synaptic blockers. More than 100 Purkinje cells in cerebellar slices were recorded for each genotype (192 L7+/+, n=14 mice; 134
L7+/-, n= 14 mice; and 172 L7-/-, n=14 mice).Originally, we tried to include all recordings
for an unbiased analysis. However, this posted problems for some cells because the noise
levels were high in some recordings and the firing frequencies for a few cells were
extremely low. Those became outliers in the entire data sets. In addition, the microscope
for the original setup I used for recordings does not have a high enough magnification for
visually identifying Purkinje cells from the cerebellar slice. Firing cells were identified
based on audio signal transmitted through the amplifier. Occasionally, spikes from
neighboring cells could be interviewed within the same recording and it is not always
43 possible to separate them during the post-aquisitional analysis. These recordings were also eliminated in the later analysis. Furthermore, there is not a clear distinction between irregular phasic firing and trimodal firing, because of the variability among different cycles of the trimodal pattern. However, because the burst firing behavior is thought to be regulated by VGCCs, especially the P-type channels, and therefore could be altered in the
L7-/- cells, we have identified and analyzed all cells that possess reasonably good trimodal firing pattern.
Collectively, among the 192 wild type cells recorded, 102 were non-bursting cells, while the rest 72 cells were phasic. Out of the 72 phasic firing ones, 8 cells showed regular trimodal patterns. For L7+/-, there were 90 non-bursting and 44 phasic cells, of which 20 cells displayed regular trimodal patterns. The recordings from the L7 knockout mice were 130 non-bursting cells and 42 phasic cells; among which, 12 cells had regular trimodal patterns. When compared based on mean values obtained for each animal, the distribution between tonic and phasic firing cells among the three genotypes is not significantly different, although L7-/- cells showed somewhat less percentage of phasic firing cells (Figure 8). For all genotypes, more than 60%of the cells (63% for L7+/+, n=14 mice; 67% for L7 +/-, n=14 mice; 75% for L7-/-, n=14 mice) displayed continuous firing at the time of the recording. The remaining cells showed phasic firing pattern. In
Figure 7 are shown two typical sample recordings for wild type and L7 knockout. Closer analysis of the firing properties is required in order to establish changes in the firing timing, which might be caused by altered ion channels regulation mechanism in the absence of L7.
44 3.1.1. Tonic firing
For data analysis, firing frequency is the main parameter used to characterize the
property of tonic firing. This is presented as the number of spikes per second and the
calculation was automated in the Spike2 program. The program also gives the possibility
of an additional filtering of the data, by setting a proper threshold value. This way large
noise can be eliminated. Furthermore, the program provides a possibility to separate
signals that came from two different cells, in case that firing from a neighboring cell was
included in the recording. When this happens, most times the signals can be distinguished
based on the amplitude. If a cell fires tonically the amplitude of the spikes does not change dramatically. Therefore, by setting up the right threshold, the high amplitude spikes can be separated from the lower amplitude spikes and individually analyzed.
In an initial data set we analyzed 60, 101 and 41 tonic firing cells from 3 L7+/+,
8L7+/- and 4L7-/- animals, respectively (Figure 9). Note: because of the more stringent criteria and better refined methods used in later analysis, some of the cells were not included in the final data set. Nonetheless, it was noticed that although the mean firing frequency did not differ significantly, the distribution of the firing rate seems to be narrower for the knockout than for the wild type. This is consistent with the hypothesis that L7 increase the dynamic range of P-type VGCC regulation by G proteins and thereby allowing more diversity of firing behaviors of the Purkinje cells. In the absence of L7 the
Ca2+ channel activity maybe is more uniform and hence the firing frequency might also be
more uniform. This encouraged us to include more cells for the analysis. However, because of the more stringent criteria applied to the later analysis many cells from the
45 initial data set were excluded. Therefore, the final set of the all tonic firing data (non-
bursting) (120 L7+/+, 90 L7+/-, and 130 L7-/-) revealed not only no significant difference
among the mean firing rate, but also no noticeable difference in the distribution histograms between L7 knockout and the wild type cells (Figure 10).
On the other hand, it might be possible that the differences between L7 knockout
and the wild type were masked by fast synaptic transmission. To examine this possibility,
the recordings were repeated in the presence of kynurenic acid and picrotoxin to block the
glutamatergic and GABAergic transmissions, respectively. For this experiments, the
recordings were performed using a new set up, equipped with a 10X objective with an
infrared camera that allow us to visually identify the Purkinje cells in the slice. The uncertainty of capturing signals from multiple cells is minimized. In addition the aCSF solution was modified (Table 3) to contain 2.5 mM instead of 5 mM K+. However, except
for an overall reduction of the firing rate as compared to in the absence of synaptic blockers (about 125 spikes/second without synaptic blockers and around 50 spikes/second with one or both synaptic blockers) , there is again no significant difference between L7-/-
and the wild types (Figure 11). It is likely that the higher K+ of the initial aCSF caused
slightly depolarization of the membrane potential that accelerated the firing. However, the
blockade of the fast synaptic transmission is also expected to reduce the firing rate.
Furthermore, the experiments with picrotoxin were performed at 32C instead of 34C in
order to accommodate the conditions for observing complex spikes (see section 3.5. of the
Results). However, under these conditions there is still no significant difference between
L7 knockout and wild type in firing rate for tonically firing cells.
46 3.1.2. Phasic firing- trimodal pattern
As mentioned above, 25 to 37 % of the recorded cells displayed phasic firing
patterns that can be either irregular or regular trimodal. The irregular firing is difficult to
analyze and compare. However, the trimodal firing can be meaningfully analyzed using
the Spike2 program. Repetitive patterns were searched automatically and summarized.
The bursting phase of the trimodal firing pattern was specifically isolated and analyzed
using a custom made script, which automatically calculated a set of parameters including
the number of spikes per burst, interspike interval, burst duration, interburst interval, as
well as the frequency within the burst, which is the reciprocal of the interspike interval.
The first step in the analysis is to set a series of threshold values, which have to be carefully chosen. This will define the accuracy of the later analysis. For instance, the values for maximal burst length and the maximal interval between two consecutive spikes will determine weather a gap between two consecutive spikes will be treated as interspike interval or as interburst interval.
For all cells the number of spikes per burst is on the average between 15 and 20.
The correctness of this value depends on the threshold set for the burst duration, so that
all the events in a burst are included. A wrong threshold value could lead to the lost of the
last few spikes. However, the number of spikes per burst (and the burst duration as well)
is not constant within the bursting phase of a single trimodal firing cycle. Typically, the
bursting phase starts with longer bursts, having 10-20 spikes/ burst, and then the burst
becomes shorter and shorter (sometimes with just 2-3 spikes per burst) at the end of the bursting phase. The interspike interval is the time interval between two consecutive
47 spikes within a burst and is usually in the range of 3 to 4 ms, on the average. The
interspike interval varies within a burst. It normally starts higher at the beginning and then is reduced by more then 50% at the end of the burst. The few last spikes at the end of the burst, often referred to as calcium spikes, are closer and smaller in amplitude. The burst duration is defined as the time between the first and the last spike of a burst and it is on
the average about 60 ms. The interburst interval is the silent period between the last spike
of a preceding burst and the first spikes of the subsequent burst. It is typically about 5
times larger, on the average, than interspike interval. The firing frequency is calculated as
the number of spikes/second. For burst firing typically is much higher (more than 200
spikes/second) than that during the tonic firing phase of the trimodal pattern for the same
cell and it is, on average, higher than the frequency of tonically firing cells.
The bursting analysis (Figure 12) for all trimodal firing patterns (8 L7+/+, n = 6 mice; 20 L7+/-, n = 12 mice; 12 L7-/-, n = 9 mice) revealed subtle but significant
differences in bursting parameters between L7 knockout and wild type Purkinje cells for
burst duration, spikes/burst, interspike and interburst intervals. Figure 12A shows means
± SEM. In order to reflect the diversity of the data, about 100 bursts were extracted for
each cell. Assuming that burst is independent from each other we pooled all bursts together. Therefore, 784 bursts for wild type, 1529 bursts for L7+/- and 1321 bursts for
L7-/- mice were used to make distribution histograms of the bursting parameters (burst
duration, spike/burst, mean interspike and interburst intervals) (Figure 12B). The firing
rate was not included, to avoid the redundancy of the data, as the frequency is just the
reverse of the interspike interval.
48 Although the mean values for interburst (21 ± 0.2 ms for L7+/+ vs. 24 ± 0.4 ms for
L7-/-, p < 0.05), interspike interval (3.6 ± 2.1E-07 ms for L7+/+ vs. 3.8 ± 4.06E-07 ms L7-/-,
p < 0.05) and burst duration (64.3 ± 0.9E-03 ms for L7+/+ vs. 59.7 ± E-03 ms L7-/-, p <
0.05) suggest significant differences between wild type and L7 knockout when analyzed with one-way ANOVA, the differences are very small. The apparent significance might result from the large number of dataset, which, however were from limited number of cells and animals. The distribution histograms do not support the original hypothesis of data distribution being narrower in the absence than in the presence of L7. The result for the heterozygote are difficult to interpret, as both the mean values and the distribution histograms seem to be outside of the range delimited by the wild type and L7 knockout cells. Given the complexity of the firing behavior in bursting phases, it would be difficult to propose a valid firing model for wild type as compared to the knockout, but it is interesting to note the tendency for each genotype. Overall, the results suggest a bursting model in which the L7 knockout Purkinje cells have longer interburst and interspike intervals, therefore a slower burst firing rate than the wild type cells. We conclude that the loss of L7 does not significantly alter the spontaneous firing pattern and the firing frequency of tonic firing Purkinje cells. Subtle changes may exist in burst firing properties during the trimodal firing cycle. However, the data maybe influenced by the relative small number of cells that showed regular trimodal pattern. Nonetheless, the gender of the animal appears to have a strong influence on firing properties, especially the burst properties (see next). This sexual dimorphism could obscure the differences among genotypes.
49 3.2. Gender-genotype interaction of firing behavior between Purkinje cells of
L7 knockout and wild type mice
When data from the male and female mice are grouped together, differences
observed in firing properties of L7 knockout as compared to the wild type, although
significant in some cases (see figure 12), are very subtle. In order to examine whether
gender is a contributing factor, we separated the data based on the sex of the animals. The mean values for firing frequency and also for bursting parameters as well as cumulative
probability plots were used to compare the males and females from L7+/+, L7+/-, L7-/-.
Also, multifactor ANOVA was used, along with graphic representation (box plots) in order to highlight differences in firing properties among the genotypes, and between genders.
3.2.1. Tonic firing- gender*genotype interaction
The genotype*gender interaction was performed for tonic firing. Figure 13A shows the average firing rate for males and females, of the three genotypes of all cells that included both continuously tonic firing cells and phasic firing cells. For the later, the firing frequency was obtained from the tonic firing period. Table 7 shows the number of animals for every genotype and gender, along with the number of cells recorded having tonic or phasic firing pattern. When the entire tonic firing is considered (continuously tonic firing cells and tonic firing phase of phasic firing cells), there is no significant difference in the average values among all the genotypes for both males and females,
50 even though the male L7 knockout has a lower frequency, than L7+/+and L7+/-. However,
when the continuously firing cells are considered separately from the phasic firing cells,
it becomes obvious that the average firing rate in the tonic phase of the phasic firing cells
for the L7-/- male is significantly lower (p < 0.05) than for the wild type male ( Figure
13B). At the same time, the corresponding firing rate in females doesn’t seem to be
affected by the absence of L7 protein, as the firing frequency of the knockout females
does not differ significantly from the firing frequency of the wild type females. Also, the
firing rate for the heterozygote females is significantly higher than for the wild type and
knockout. In fact, the firing behavior of heterozygote females is difficult to interpret
because all parameters analyzed for this genotype do not fall in between wild type and
knockout as expected if modulation of the firing activity of Purkinje cells is proportional
with the amount of L7 protein. On the other hand, for continuously firing cells, the
average firing rate appears to be very uniform among all genotypes and both genders
(Figure 13C).
3.2.2. Burst firing parameters in the trimodal pattern- gender*genotype
interaction
Bursting properties in trimodal firing cells were also analyzed for males and
females separately (3, 10, 5 cells from 3, 7, 4 male mice of L7+/+, L7+/-, L7-/-, respectively; 5, 10, 7 cells from 3, 5, 5 female mice of L7+/+, L7+/-, L7-/-, respectively)
(Figure 14). The number of spikes/burst for L7 knockout male is significantly lower than
for wild type male (p < 0.05) and knockout female (p < 0.05). In addition, for both the
51 wild type and L7 knockout, but not for the heterozygote, the spikes/burst numbers are significantly different between males and females. However, the changes are in different directions. For wild type the number of spikes/ burst is lower in females, but it is higher for L7-/-. At the same time, the interspike interval for L7-/- male is significantly longer, than for the wild type male and for the L7 knockout female (p < 0.05). This suggests an overall slower firing rate for Purkinje cells in the L7 absence in the male, but not in the female mice. Interburst interval is another parameter that shows sexual dimorphism. The interburst interval for L7 knockout males is significantly longer (p < 0.05) than for the wild type males and the L7 knockout females. In fact, this is the parameter that is the most dramatically changed in the absence of L7 in males, but not in females. Also, if we look at the gender differences in each genotype, it is noticeable that the wild types are not affected by gender as much as the L7 knockout mice. However, the heterozygote are least affected by gender. This suggest that in the absence of L7 the differences between males and females become more pronounced than in the presence of this protein in terms of the burst firing behavior of the Purkinje cells. Also, if we look at the interburst interval for the wild type and the L7 knockout, it is surprising to observe that, on average, the interval is shorter for male than for female in wild type, but this is the opposite in the L7 knockout. This also appears to be true for spikes per burst and interspike interval although the differences are less dramatic. It is interesting to note that the firing properties for heterozygote female are outside the range defined by the wild types and the
L7 knockouts. By contrast, both tonic and burst firing parameters for heterozygote males do fit in between the wild type and L7 knockout males, although some parameters are more similar to the wild types (such as tonic firing frequency, spikes /burst) and some
52 other are more similar to the knockouts (such as interburst interval). In summary, the
bursting phase of the trimodal pattern showed clear sex- or genotype-dependent changes
in several burst parameters including mean interburst interval, mean interspike interval,
and the number of spikes per burst.
An interesting way to analyze these differences is to look at the cumulative
probability plots, which give a good representation of how data are distributed. Figure 15
shows the cumulative probability plots for burst firing parameters (spikes/burst,
interspike and interburst intervals) for wild type and L7 knockout males and females.
Cumulative plots were done in Origin software and at least 100 bursts were included for
each cell. The frequency probability data was first obtained for individual cells and then
they were averaged. Thus, the final plots represent the mean cumulative probability of the
corresponding number of cells for each gender and genotype (3, 10, 5 cells from 3, 7, 4
male mice of L7+/+, L7+/-, L7-/-, respectively; 5, 10, 7 cells from 3, 5, 5 female mice of
L7+/+, L7+/-, L7-/-, respectively). From the cumulative probability plots in figure 15 it is
obvious that there is a more pronounced gender difference for the L7 knockout than for
wild type mice, in terms of how data are distributed, for all parameters. For instance, the interburst interval varies in similar ranges for all the knockout mice; however 80% of the
cells from the L7 knockout females have interburst interval < 30 ms, while just 40% of
the L7 knockout male have the interburst interval within that range. The distribution
profile for the interburst interval is very different between the wild types and the L7
knockouts. From the plots (Figure15, bottom plots) the sigmoid shape of the distribution
is evident for the wild types, which can be correlated to less variation among the data.
53 Therefore, 80% of the wild type cells have the interburst intervals being < 30ms, while just 50 % of the L7 knockout cells can fit in this range.
Figure 16 shows the same data separated by gender but grouped by the genotypes.
There is significant difference in distribution of the data between wild type and L7 knockout males for all burst firing parameters. Also, it is obvious that the heterozygote females have unusual distributions, as the mean interspike and the interburst intervals look the most different from both wild type and knockout. Chi-square test has been used to examine the statistically significant differences between the distributions. For statistical analysis one way ANOVA was applied using Excel and the p-values are shown in table 6. The results of the statistical tests for cumulative probability plots indicate that the two distributions emerged from different populations.
Another representation of the data is shown in figures 17-21. The analysis was performed using Statistica, with the multifactor ANOVA test, as well as post-hoc analysis. The box graphs give an estimation of the average value for each parameter, as well as the standard deviations. If there is a significant interaction between gender and genotype the lines connecting wild type values and L7 knockout values will intersect.
Figure 17 shows the spikes/ burst parameter analyzed for changes dictated by the gender*genotype interaction, for the wild type and the L7 knockout mice (3 and 5 cells from 3 and 4 male mice of L7+/+, L7-/-, respectively; 5 and 7 cells from 3 and 5 female mice of L7+/+, L7-/-, respectively). The box plot reveals large variations for all genotypes and genders, significant difference between males and females in the L7 knockout (p <
0.005) but no gender*genotype interaction. The interspike interval box plot is showed in
Figure 18. There is significant difference between genders within the same genotype,
54 although L7 knockout mice are more obviously affected by gender than the wild types.
Also, there is gender*genotype interaction. The similar result is given in figure 19 for the frequency. The box plot is just the reverse of the previous one for the interspike interval.
Analysis of the burst duration is shown in figure 20. Although there is no significant difference between genders within the same genotype, there seems to be gender*genotype interaction for this parameter. Figure 21 shows the box plot for interburst interval, and it reveals large variations of this parameter in both genotypes, but no gender*genotype interaction.
3.3. The effect of age on the tonic firing properties for Purkinje cells of wild
type and L7 knockout mice
All the results analyzed so far, are from animals of 2 to 4 months of age. However, at the beginning of this project some older mice (6 months of age) were also examined by extracellular recording. After the initial analysis of all data, the effect of age on Purkinje cell firing patterns was investigated. Although the Purkinje cells are fully developed by
P20, it seems that there are age–dependent differences in terms of their firing activities.
We compared the initial set of data, recorded from animals of up to 4 months of age (60
L7+/+, n= 3 mice; 101 L7+/-, n= 8 mice; 41 L7-/-, n= 4 mice) with a set of data recorded from 6 month-old mice (69 L7+/+, n= 3 mice; 30 L7+/-, n= 1 mouse; 52 L7-/-, n= 3 mice).
The firing rates in the continuously firing cells were measured and the means ± SEM are shown in figure 22. The results indicate a higher firing frequency for older animals, with not significant difference among the genotypes. When compared with all the data
55 obtained from the 2-4 month-old mice (Figure 10), the differences between the two age
groups are even larger. However, since the data for older mice were obtained from a small
number of cells and at the early phase of the project, it is more reliable to compare them
with the initial set of recordings. The number of cells is comparable at least for the wild
type and the L7 knockout mice. Our analysis of tonic firing in different age groups,
suggested that in order to observe potential differences between wild type and L7
knockout, similar age subjects should be used. This analysis convinced us to use younger
animals (between 2 and 4 months of age) for the extracellular recordings.
Age is actually a limiting factor in designing our experiments. Because the L7
protein synthesis is activity dependent, and it may play a role in motor learning, the ideal
situation would be to examine the electrophysiological properties of the animals that had been subjected to some behavioral tests. By doing so, the animals could easily reach the age of more than 2 months, which, on the other hand, are difficult to record by whole cell patch clamping.
3.4. Complex spikes in Purkinje cells from wild type and L7 knockout mice
Previously it has been shown that the P/Q-type Ca2+ channels may be a primary
downstream effector of L7 modulation of Gi/o proteins [Kinoshita-Kawada et al., 2004].
In Purkinje cells, voltage-gated Ca2+ channels are major contributors of the slow
components of complex spike waveform evoked by the stimulation of climbing fibers
[Hansel and Linden, 2000, Weber et al., 2003]. We reasoned that altered modulation of
P-type channel activity due to the lack of L7 might change the complex spike waveform.
56 Figure 23 shows examples of extracellular recording of Purkinje cell complex spikes evoked by climbing fiber simulation in a wild type and an L7-/- cell. Picrotoxin was
included in the aCSF to block inhibitory synaptic input and it did not reveal any significant difference in firing frequency between the two genotypes before climbing fiber stimulation (Figure 11, picrotoxin only cells). However, the number of small spikelets, which is indicative of Ca2+ action potentials and hence the Ca2+ channel activity, in the complex spike is significantly reduced from 3.32 ± 0.10 in the wild types to 2.62 ± 0.08 in the L7-/- (p < 0.05, n = 34 wild type, n = 42 knockout cells). As shown
by the histograms in figure 24, most L7-/- cells have 2 to 3 spikelets whereas the wild
types have more cells with 4 to 5 spikelets. Related to this is the duration of the complex
spikes, which is changed from 8.18 ± 0.50 ms in the wild type to 6.08 ± 0.40 in mutants
(p < 0.05). On the other hand, the duration of the pause after the spikes is not significantly changed due to the lack of L7 (57.34±4.7 ms for L7+/+ vs. 49.15±3.8 ms for
L7-/-, p>0.05). The reduced number of spikelets in L7-/- Purkinje cells is indicative of
alterations in voltage-gated Ca2+ channel activities and consistent with the hypothesis that
L7 contributes to the firing properties and synaptic transmission of Purkinje cells through
modulation of P-type channels.
57 3.5 Whole-cell recording of the Purkinje cells in cerebella slices from wild
type and L7 knockout mice
One alternative to the extracellular recording investigation is to make whole-cell gigaseal patch on the Purkinje cells and directly record the spontaneous and evoked action potentials from these neurons. The Purkinje cell not only have a large size soma
(~20 µm in diameter) but also a very elaborated dendritic tree, making it difficult to voltage clamp the entire cell because of the space clamp problem. On the other hand, current clamp is feasible because it is unaffected by the size and cell shape. Under the current clamp conditions, Purkinje cells fire action potentials spontaneously without injection of any current. The recorded action potential waveforms reveal many detailed characteristics that are indicative of the activities of Na+, Ca2+ and K+ channels [Womack and Khodakhah K., 2003, 2004]. These include the resting membrane potential, the threshold potential for action potential, the rate of depolarization and repolarization, as well as the size and duration of afterhyperpolarization. For example, the resting potential is mainly set by the action of K+ channels, the depolarization is due to activation of voltage-gated Na+ channels; the afterhyperpolarization is related to the activity of Ca2+- activated K+ channels. The resting membrane potential of Purkinje cells is typically in the range of –50 to –60 mV [Womack and Khodakhah, 2004]. The half width of the action potential, defined as the width of the action potential at half the amplitude, is a useful parameter that describes the relative duration of depolarization and repolarization phases and is typically in the range of hundreds of µs [Womack and Khodakhah, 2004; Bean,
2007]. However, it is greatly affected by temperature. The procedure for whole-cell
58 recording of Purkinje cells in mouse cerebellar slices is described in detail in the
“Methods” section. The whole-cell data have an advantage of being high resolution, i e.
having very low noise. Nonetheless, the method is also more technically challenging and therefore less number of cells can be recorded. In addition, slices prepared from order animals (> 20 days) are difficult to record because of the development of perineuronal network, which interferes with the access of recording pipette. Furthermore, dialysis of cellular content through the patch pipette is a potential problem, which for most studies, however, is insignificant.
In order to master the whole-cell recording technique and to test the new
recording station, I started with the wild type ICR(CD-1) mice. Mice of either sex from
14 to 25 days were used. The recordings were performed at the room temperature (22-
24oC) because it allows the cells to be maintained healthier for longer time. However,
the action potential waveform is significantly broader at the room temperature than at
32ºC, as compared to the values reported in the literature [Wormack and Khodakhah,
2003, 2004; Bean, 2007]. A total of 20 cells from the 16 mice were successfully recorded
under these conditions. As shown in figure 25 the action potential is very uniform during tonic firing and is detected without injection of any current (spontaneous). However, injection of negative current reduced the firing frequency and at certain levels the cell
stopped firing. On the other hand, injection of positive current enhanced the firing
frequency in a dose dependent manner. These changes are entirely expected according to
the current understanding of how activation potentials are generated. Once the method
was established, I also recorded 3 cells from 2 wild type C57Bl/6 mice under the same
condition. The results were similar to that from the ICR (CD-1) mice.
59 To compare the action potential waveforms between Purkinje cells of the wild type and the L7 knockout mice, the recording was performed at 32ºC A total of 6 cells from 3 wild type and 14 cells from 4 L7-/- mice were successfully recorded. The results obtained from the whole-cell patching resemble the diversity of the firing properties observed with the extracellular recording. Figures 26 and 27 show representative whole- cell recordings from the wild type and from the L7 knockout mice, respectively. It is obvious that all firing patterns observed with the extracellular recording: tonic and trimodal firing, as well as irregular patterns, are all detected in the whole-cell records, demonstrating that the extracellular recording method is a reliable and efficient way to investigate Purkinje cell firing patterns. Its utility is especially valuable for this project given the technical difficulties related to making the whole cell patches and the limited age constrains on the animals.
The action potential waveforms between the wild type and L7 knockout Purkinje cells are compared (Figure 28). The wild type action potential waveform shown is the mean of 8 cells from 5 mice and the L7 knockout action potential waveform shown is the mean of 7 cells from 4 mice. Up to 300 action potentials were averaged for each cell. The action potential waveform for the wild type and the L7 knockout Purkinje cells are very similar in shape. One parameter to be considered in analyzing the action potential waveform is the width at half amplitude of the maximal depolarization, designated “half width”. The L7 knockout appears to have normal half width (0.18 ± 0.01 ms for the L7 knockout, as compared to 0.17 ± 0.01 ms for the wild type). Interestingly, the resting membrane potential is changed from -56.9 ± 1.3 mV for the wild type cells to -62.2 ± 1.8 mV for the L7 knockout cells. This difference is significant (p < 0.05) as analyzed by
60 Student’s t-test. Although the number of recorded cells is relatively low at the current stage to further validate the significance, it is interesting to note that there may be a tendency for the L7 knockout Purkinje cells to have a more hyperpolarized resting membrane potential. If this is the case, the more negative resting potential of the Purkinje neurons may be correlated with enhanced K+ channel activities, for example, the G protein-gated inwardly rectifying K+ channels [Tabata et al., 2005] in the absence of L7.
61 A. wild type B. L7 knockout
200 µV 20 s
200 µV 1 s
200 µV 30ms
tonic burst tonic burst
Figure 6: Trimodal firing pattern of the Purkinje cells in cerebellar slices of wild type (A) and knockout L7 (B).
The trimodal firing pattern of the Purkinje neurons consists of cycles of continuous firing,
bursting and a long (at least 10 s duration) silent period. The bursts are defined as groups
of spikes (from 2 to 10-15 or even more), which are also delimited by silent periods of
much shorter duration (10-50 ms). The top panel represents the row data. The lower
panels represent expanded regions, as indicated in the figure and by the scale bars on the
right side.
62 A.
B.
C.
D.
Figure 7: Diversity of firing patterns in Purkinje cells
Raw data are in green and firing frequency in pink. A. Tonic firing; B. Irregular phasic firing; C. Regular trimodal firing, D. Transition from tonic, to phasic-trimodal firing 63
100 tonic phasic
50 Firing pattern (% distribution cells) 0 +/+ +/- -/- genotype
Figure 8: Firing pattern distribution of Purkinje cells in the wild type and L7 mutants.
The Purkinje cells display tonic firing, with a relatively constant frequency, or phasic firing, which consists of repeats of silence and firing. Most of the cells (>60%) present a tonic firing at the time of recording, but L7-/- tended to have higher percentage of tonic firing cells. However, upon averaging the % distribution of firing patterns based on the number of animals, the difference is not statistically significant (n = 14 mice for each genotype). Data are means ± SEM.
64
Figure 9: Distribution histograms of the tonic firing frequency for all three genotypes (initial set of data)
A. The distribution histograms suggest a narrower distribution for the L7 knockout cells,
which is consistent with the hypothesis that L7 plays a role in fine tuning the firing. In its
presence, there is room for much more variability, because of the use- dependent L7 protein synthesis at the Purkinje cell dendrites, which leads to more diversity of calcium channels activities. Therefore, the broader distribution was expected for the wild type
cells; B. Average firing rate for tonic firing. Data are means ± SEM. n = 60, 101, 41 cells
for L7+/+, L7+/- and L7-/-, respectively from n = 3, 8, 4 mice of L7+/+, L7+/- and L7-/-, respectively.
65 Tonic firing (initial set of data)
A.
6 +/+ 4 2
0 0 50 100 150 200 250 300
10 +/-
5 Number of cells
0 0 50 100 150 200 250 300
-/- 8 6 4 2 0 0 50 100 150 200 250 300 average firing rate (spikes/sec)
Average firing frequency B. (spikes/second) (initial set of data) (41) 120 (60) (101)
80
40
0 +/+ +/ --/-
Figure 9: Distribution histograms of the tonic firing frequency (initial set of data) 66 Figure 10: Distribution histograms of the tonic firing frequency for all three
genotypes (all set of data)
A. The distribution histograms suggest no difference between the L7 knockout and the
wild type. This contradicts the previous result and suggests no change in distribution and
firing frequency of the tonic firing phase for the L7 knockout. B. Average firing rate for
tonic firing. Data are means ± SEM. n = 120, 90, 130 cells for L7+/+, L7+/- and L7-/-, respectively.
67
A. Tonic firing (all data) 20
15 +/+
10
5
0 0 50 100 150 200 250 300
20 15 +/-
10
5
0 0 50 100 150 200 250 300
20 -/ - 15
10
5
0 0 50 100 150 200 250 300
Average firing frequency (spikes/second) B. (all data)
150 (120) (90) (130)
100
50
0 +/+ +/- -/-
Figure 10: Distribution histograms of the tonic firing frequency (all set of data)
68
+/+ +/- 15 (90) -/- (130) (120)
y
) 10 uenc
q (56) (33) fre
g (34) (42) ikes/second 5 p
s ( Tonic firin Tonic
0 no aCSF+ aCSF+ synaptic picrotoxin picrotoxin+ blockers at 32°C kynurenic at 35°C acid at 35°C
Figure 11: Tonic firing frequency for the three genotypes in different external conditions
Extracellular recordings were performed in different external conditions and in none of them the firing frequency of the L7 knockout was significantly different from that of the wild type. Without synaptic blockers the average firing frequency for wild type and L7 mutants were much higher, which was expected as the synaptic blockers are supposed to obstruct part of the excitatory input received by Purkinje neurons. In addition, the solution contained higher concentration of K+ than those when synaptic blockers were included.
69 Figure 12: Bursting parameters (all data)
Bursting parameters (burst length, spikes/ burst, burst interspike, interburst interval) for
747, 1529, 1321 bursts pooled from 8 L7+/+ (6 mice), 20 L7+/- (12 mice) and 12 L7-/- (9 mice)cells, respectively. A). Means ± SEM p < 0.05 different from wild type; B). The corresponding distribution histograms
70 700 A. B. +/+ burst duration 350 (ms) 0 0 100 200 300 80 700 * +/- 60 350 0 40 0 100 200 300 700 -/- 20 350 0 0 0 100 200 300 +/+ +/- -/-
400 +/+ spikes/burst 200
20 0 0 20 40 60 80 * 400 +/- 15 200
10 0 0 20 40 60 80 400 -/- 5 200
0 +/+ +/- -/- 0 0 20 40 60 80
500 +/+ interspike interval 250 (ms) 5 0 0246 8 500 4 * +/- 250 3 0 02 4 6 8 2 500 -/- 1 250 0 +/+ +/- -/- 0 0246 8
600 +/+ interburst interval 300 (ms) 40 0 0 50 100 150 200 600 +/- 30 * 300
20 0 0 50 100 150 200 600 -/- 10 300 0 +/+ +/- -/- 0 0 50 100 150 200
Figure 12: Bursting parameters (all data)
71
All cells +/+ 200 +/- -/-
d 150
100
50 spikes/secon 0 male female
B. C. Trimodal firing cells Continuously firing cells +/+ 200 +/- 200 +/+ -/- +/- 150 150 -/-
100 100
50 spikes/second 50 spikes/second 0 0 male female male female
Figure 13: Genotype*gender interaction for tonic firing
A). Mean firing frequency for all cells, including continuously firing cells and phasic firing cells. The firing frequency does not seem to be different among the genotypes.
Although the knockout male appears to have a slower firing rate the difference was not significant; B). Mean firing frequency for the tonic phase of phasic firing cells. Note the significant lower firing of the L7 knockout male than wild type male, but the difference in females is not statistically significant; C). Mean firing frequency is not different for the continuously firing cells between L7 knockout and wild type for either gender. Data are means ± SEM for the number of cells indicated in the Table 6.
72 Figure 14: Genotype*gender interaction for bursting firing
A). The number of spikes/burst for males and females of all genotypes. The knockout male seems to have the lowest number of events per burst (p < 0.05). B). Mean interspike interval for males and females of all genotypes. C). Mean interburst interval for males and females of all genotypes. Both interspike interval and interburst interval are the longest for the knockout male, suggesting a slower firing (p < 0.05). Data are means ±
SEM for the number of cells indicated in the Table 6.
73
A spikes/burst +/+ +/- 20 -/-
15
10
5
0
male female
B interspike interval +/+ (ms) +/- -/- 5 4 3 2 1 0
male female
interburst interval +/+ C (ms) +/- -/- 40
30
20
10
0 male female
Figure 14: Genotype*gender interaction for bursting firing 74
A.
1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 +/+ male 0.4 +/- male 0.4 -/- male +/+ female +/- female -/- female 0.2 0.2 0.2 0.0 0.0 0.0 0102030 0102030 0102030 B. spikes/burst spikes/burst spikes/burst
1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.6 0.6 0.4 +/+ male 0.4 +/- male 0.4 -/- male +/+ female +/- female -/- female 0.2 0.2 0.2
0.0 0.0 0.0 1.5 3.5 5.5 1.5 3.5 5.5 1.5 3.5 5.5 C. interspike interval (ms) interspike interval (ms) interspike interval (ms)
1.0 1.0 1.0
0.8 0.8 0.8
0.6 0.6 0.6
0.4 +/+ male 0.4 +/- male 0.4 -/- male +/+ female +/- female -/- female 0.2 0.2 0.2
0.0 0.0 0.0 0 20 40 60 0204060 0 20 40 60 interburst interval (ms) interburst interval (ms) interburst interval (ms)
Figure 15: Cumulative probability plots for the burst firing parameters
Spikes/ burst (A), interspike interval (B), interburst interval (C) cumulative probability plots, by gender and genotype.
75 1.0 1.0
0.8 0.8
0.6 0.6 +/+ female +/+ male 0.4 +/- female 0.4 +/- male -/- female -/- male 0.2 0.2
0.0 0.0 0 10 20 30 0 10 20 30 spikes/burst spikes/burst 1.0 1.0
0.8 0.8
0.6 0.6
0.4 +/+ female 0.4 +/+ male +/- female +/- male 0.2 -/- female 0.2 -/- male
0.0 0.0 1.5 3.5 5.5 1.5 3.5 5.5 interspike interval (ms) interspike interval (ms) 1.0 1.0 0.8 0.8 0.6 0.6 +/+ male +/+ female 0.4 0.4 +/- male +/- female 0.2 -/- male 0.2 -/- female
0.0 0.0 0 20 40 60 0 20 40 60 interburst (ms) interburst (ms)
Figure 16: Cumulative probability plots for the burst firing parameters (alternative representation to compare genotypes)
76 Figure 17: Spikes/burst analysis- gender*genotype interaction
The spikes /burst analyzed for the gender*genotype interaction reveals significant
differences between males and females for L7 knockout and also show that females have
a significantly higher number of spikes/ burst in the absence of L7 (p-values are indicated in the table)
77
{1} {2} {3} {4} sex genotype 17.584 17.268 18.855 17.200
1 male -/- 0.44981 0.00233 0.62856 2 male +/+ 0.44981 0.00043 0.87069 3 female -/- 0.00233 0.00043 0.00045
4 female +/+ 0.62856 0.87069 0.00045
sex*genotype; LS Means Current effect: F(1, 4367)=5.1429, p=.02339 Effective hypothesis decomposition Vertical bars denote 0.95 confidence intervals 20
19
t
18 spikes/burs
17
male 16 female -/- +/+ genotype
Figure 17: Spikes/burst analysis- gender*genotype interaction
78
Figure 18: Mean interspike interval analysis- gender*genotype interaction
The interspike interval analyzed for gender* genotype interaction reveals significant difference between males and females in the absence of L7. Also the males have longer interspike interval when L7 in not present (p-values are included in the table).
79
sex genotype {1} {2} {3} {4} 4.80 4.09 4.17 4.14
1 male -/- 0.000008 0.000009 0.000022
2 male +/+ 0.000008 0.096649 0.241560
3 female -/- 0.000009 0.096649 0.370019 4 female +/+ 0.000022 0.241560 0.370019
sex*genotype; LS Means Current effect: F(1, 4367)=160.51, p=0.0000 Effective hypothesis decomposition Vertical bars denote 0.95 confidence intervals 5.0
4.5
interspike interval ( ms)
male
4.0 female -/- +/ genotype
Figure 18: Mean interspike interval analysis- gender*genotype interaction
80 Figure 19: Mean frequency analysis- gender*genotype interaction
The mean frequency analyzed for gender*genotype interaction shows differences between males of wild type and L7 knockout and also between males and females of L7 knockout. The p-values are listed in the table. Frequency is the reverse of the interspike interval, as sown also by the graph.
81
{1} {2} {3} {4} sex genotype 219.51 253.22 246.64 252.14
1 male -/- 0.000008 0.000009 0.000022 2 male +/+ 0.000008 0.004684 0.606794 3 female -/- 0.000009 0.004684 0.008459
4 female +/+ 0.000022 0.606794 0.008459
sex*genotype; LS Means Current effect: F(1, 4367)=91.125, p=0.0000 Effective hypothesis decomposition Vertical bars denote 0.95 confidence intervals 260
250
240
230
(spikes/second) frequency 220
male 210 -/- +/+ female genotype
Figure 19: Mean frequency analysis- gender*genotype interaction
82 Figure 20: Burst duration analysis- gender*genotype interaction
The analysis of burst duration for the gender*genotype interaction indicated significant differences between males and females in both wild type and knockout cells. The p- values are listed din the table.
83
sex {1} {2} {3} {4} genotype 83.89 71.62 80.28 73.96
1 male -/- 0.000008 0.086252 0.000028
2 male +/+ 0.000008 0.000135 0.265394
3 female -/- 0.086252 0.000135 0.002741 4 female +/+ 0.000028 0.265394 0.002741
sex*genotype; LS Means Current effect: F(1, 4367)=4.0024, p=.04550 Effective hypothesis decomposition Vertical bars denote 0.95 confidence intervals 90
85
80
75
(ms) duration burst 70
male 65 -/- +/+ female genotype
Figure 20: Burst duration analysis- gender*genotype interaction
84 Figure 21: Interburst interval analysis- gender*genotype interaction
The analysis of interburst interval for the gender*genotype interaction showed that both males and females are significantly affected by the lack of L7. Also, there is significant difference between males and females in L7 knockout. The p-values are listed in the table.
85
sex {1} {2} {3} {4} genotype 36.64 39.50 33.77 38.44
1 male -/- 0.031612 0.011732 0.113920 2 male +/+ 0.031612 0.000010 0.348327
3 female -/- 0.011732 0.000010 0.000139
4 female +/+ 0.113920 0.348327 0.000139
sex*genotype; LS Means Current effect: F(1, 4367)=1.2523, p=.26317 Effective hypothesis decomposition Vertical bars denote 0.95 confidence intervals 45
40
35 interburst interval (ms)
male 30 female -/- +/+ genotype
Figure 21: Interburst interval analysis- gender*genotype interaction
86
average firing frequency –tonic firing (spikes/second) 2- 4months 200 6month (69) (30) (52) (41) (60) (101) 100
0 +/+ +/- -/-
Figure 22: The effect of age on tonic firing of L7+/+, L7+/-, L7 -/- mice
The tonic firing frequency for older mice is significantly higher in the older mice than in
the younger ones, especially in the wild type animals. This difference is much smaller in the L7 knockout.
87
A. B. 100µV 5ms 1 1 2 3 4 5 2 3
100µV 40ms
Figure 23: Analysis of climbing fiber complex spikes of Purkinje cells
Representative traces of complex spikes detected by extracellular recording for wild type
(A) and L7-/- (B) Purkinje cells with expanded images of the spikelets (inserts). Numbers
indicate how spikelets were counted. Climbing fibers were stimulated with a constant
voltage of 0.2-5 V for 200 µs using a bipolar electrode placed within 50 µm away from
the soma of the Purkinje cell. The number of small spikelets, which is indicative of Ca2+ action potentials and hence the Ca2+ channel activity, in the complex spike is significantly
reduced from 3.32 ± 0.10 in the wild types to 2.62 ± 0.08 in the L7-/- (p < 0.05, n = 34
wild type, n = 42 knockout cells).
88
25 +/+
20 -/-
15 s l cel of
r 10 mbe nu 5
0 234 5 number of spikelets
Figure 24: The number of spikelets within the complex spikes for wild type and L7
knockout.
The number of small spikelets in the complex spike is significantly reduced from 3.32 ±
0.10 in the wild types to 2.62 ± 0.08 in the L7-/- (p < 0.05, n = 34 wild type, n = 42
knockout cells). As shown by the histogram, most L7-/- cells have 2 to 3 spikelets
whereas the wild types have more cells with 4 to 5 spikelets.
89 Figure 25: Representative recordings for wild type mice in whole cell current clamp
A). Spontaneous firing recorded in whole cell current clamp from Purkinje neuron (wild type); B). Tonic firing of action potentials (left), the action potential waveform (right);
C). Burst firing of action potentials; D). Current injection changes the firing frequency
90 A 2 0 -20 -40
B 2 0 -20 1ms 40 -40 -60
C 100 20 0 -20 -40 -60
D current clamp recording
800pA
500pA
300pA
250pA
0 0.1 0.2 Time (s)
Figure 25: Representative recordings for wild type mice in whole cell current clamp
91
20 0 -20 2s -40 -60 -80
1s
(first cycle expanded)
20 0 5ms -20 -40 -60 -80
Figure 26: Representative recordings for wild type mice in whole cell current clamp mode
The wild type cells displayed diverse firing pattern consisting of tonic and phasic firing.
The middle trace shows the first trimodal cycle expanded. The lower traces show tonic and bursting firing
92
20 0 -20 -40 2s -60 -80
100ms
20 0 -20 -40 20ms -60
Figure 27: Representative recordings for L7 knockout mice in whole cell current
clamp mode
The L7 knockout cells displayed diverse firing pattern consisting of tonic and phasic
firing. The middle trace shows one expanded trimodal cycle, as indicated in the figure.
The lower traces show tonic and bursting firing.
93
Action potential waveform 20
0 0 1 2 3 456
-20 +/+ mV -/- -40
-60
-80 ms
Figure 28: Action potential waveform for wild type and L7 knockout Purkinje cells
The action potentials waveform for the wild type (average of 8 cells form 5 mice) and for
the L7 knockout (average of 8 cells from 4 mice) are very similar in shape. There might be a tendency for the L7 knockout cells to have a more hyperpolarized membrane potential.
94
total number number of number tonic phasic genotype of cells and mice by of cells firing firing mice gender by gender
L7+/+ 192 cells 7 males 117 71 46 14 mice 7 females 75 49 26
L7+/- 134 cells 8 males 76 46 30 14 mice 6 females 58 44 14
L7-/- 172 cells 7 males 81 60 21 14 mice 7 females 91 70 21
Table 6: Summary data for the number of cells and mice, males and females, and their firing pattern
95
CHAPTER 4
DISCUSSION AND FUTURE STUDIES
Pcp2-L7 protein is a GoLoco domain-containing protein expressed mainly in cerebellar Purkinje cells. Previous studies in animal models have revealed roles for
GoLoco domain proteins that range from control of asymmetric cell division to electrophysiological properties [Francis et al, 2004; Katanaev and Tomlinson, 2006; Xie and Palmer, 2007]. In addition, in the fruit fly Drosophila the pertussis toxin sensitive Gα protein, now known to be a primary target of GoLoco protein binding, was found to be an essential signaling component controlling certain forms of associative learning mediated by the fly brain mushroom bodies [Ferris et al., 2006]. This dissertation documents the first electrophysiological investigation that compares the firing properties Purkinje neurons in cerebellar slices of the L7 knockout and the C57BL6 wild type mice, and is a part of a large project includes examinations of anatomical and behavioral changes of the mutant animals [Iscru et al., 2008, unpublished results]. In summary, the anatomical analysis showed subtle neural developmental defects characterized by decreased overall
96 size, decreased molecular layer thickness, and increased Purkinje cell density due to a
smaller soma size. The behavioral study suggested behavioral enhancements, including
improved gross motor learning and an increased initial rate of association between a
conditioned stimulus (tone) and an unconditioned stimulus (shock). Overall, it seems like
the L7 knockout mice have increased responsiveness to stimuli. If these effects are
attributable to alterations in Purkinje cells, we expect to see changes in the firing properties of these cells. I studied the spontaneous firing patterns of Purkinje cells in cerebellar slices using the extracellular recording and whole-cell recording techniques.
The discussion here will focus on the findings from the electrophysiological studies.
Purkinje cells have the intrinsic property of spontaneously firing action potentials
in the absence of any synaptic input [Raman and Bean, 1999; Womack and Khodakhah,
2002], Firing properties have been measured in the absence and presence of synaptic
blockers, with extracellular recording technique, which allows a fast analyses of many
cells and is the least invasive electrophysiological recording method. We also performed a
set of patch clamping experiments on Purkinje cells in cerebellar slices, and the results
indicate a high resemblance between the firing behaviors observed with extracellular
recordings and whole-cell intracellular recordings, including tonic and phasic firings. The
later also includes trimodal firing. This validates our choice of using extracellular
recording technique as the main method used for the study, as whole-cell patch clamping
of Purkinje cells is a challenging technique, which limited the rate and efficiency of our
data acquisition. Very often, only one cell was successfully recorded for each animal and
the age of the animals is more restricted than extracellular recordings.
97 The diversity of the spontaneous firing patterns of Purkinje neurons from cell to
cell and from animal to animal, has been previously reported. Among the different firing
patterns, the continuous tonic firing as well as the so called trimodal firing pattern
[Womack and Khodakhah, 2002, 2004], consisting of cycles of tonic firing, regular
bursting and silent periods, which repeat in a regular fashion, can be analyzed and
compared among the genotypes. In analyzing the tonic firing phase of the Purkinje cells,
we found no significant difference in the tonic firing frequency between the wild type and
the L7 knockout mice. However, the burst firing behavior is thought to be regulated by
VGCCs, especially the P-type channels. A closer analysis of the bursting phase in
Purkinje cells revealed subtle, but significant, changes in the bursting properties in the L7
knockout cells. Thus, shorter burst duration and less number of spikes per burst, as well as
longer interspike and interburst intervals in the L7 knockout, support the hypothetic
model of slower burst firing in Purkinje cells of the L7 knockout mice. This model is in a
good agreement with the previous assumption that L7 regulates the activity of P-type
channels [Kinoshita-Kawada et al., 2004]. In its absence there seems to be a reduced
activity of the Ca2+-activated K+ channels, as suggested by the slower activity of the knockout cells, and therefore a fine modulation of the P-type Ca2+ channels. Moreover,
the bursting behavior seems to be sexually dimorphic and the analysis of data by gender
and genotype revealed even more pronounced changes in the absence of L7 protein in the
male knockout. The male animals are the most affected by the loss of L7 and
consequently their Purkinje cells show significantly reduced firing rate in bursting phase.
The whole-cell patch clamp recordings of the spontaneous action potentials in Purkinje
neurons indicate that the L7 knockout cells have more negative resting membrane
98 potential than the wild type cells. In this case, the more hyperpolarized resting potential of
the Purkinje neurons may be correlated with enhanced K+ channel activities, for example,
the G protein-gated inwardly rectifying K+ channels [Tabata et al., 2005], which is
another group of ion channels subject to regulation by Gi/o proteins and potential targets
of GoLoco domain proteins, including L7 [Webb et al., 2005]. The enhanced K+ channel activities mean a less excitability for the L7 knockout Purkinje neurons and consequently a slower firing activity, which agrees with the results from extracellular recording experiments.
The Gi/o coupled 5HT1A serotonin and α2 adrenergic receptors have been previously shown to be involved in the regulation of Purkinje neuron firing frequency via volume transmission mechanism [Darrow et al., 1991; Diverse-Perluissi, 1997; Kerr and
Bishop, 1992]. Activation of these led to changes of the stimulated and spontaneous firing rate of Purkinje neurons [Strahlendorf et al., 1984; Glitsch and Marty, 1999; Liu et al,
2003;]. Volume transmission is an extra-synaptic mechanism driven by weak stimulation applied for a long period of time, as opposed to strong stimulation for a short time in the case of synaptic transmission mechanism. In order to determine weather and which Gi/o- coupled receptors are linked to the L7-mediated modulation of Purkinje cell firing behaviors, a series of pharmacological tests were done on the Purkinje cells of the L7 knockout to examine how they affect the firing behaviors. Consistent with volume transmission hypothesis, the L7-mediated regulation of P/Q-type channels is dependent on the tonic and weak receptor stimulation, rather than the acute and strong stimulation
[Kinoshita-Kawada et al., 2004]. We initiated a series of pharmacological studies that needs further analysis. The α2 adrenergic receptor agonists, clonidine and norepinephrine
99 (NE), were applied by bath incubation at 34ºC for more than 2 hours before cells were recorded, in order to mimic the weak and tonic stimulation, while the serotonin 5-HT 1A
receptor agonist, ipsapirone (IPS), was applied to the recording chamber for 5 minutes
and then washed out, in order to examine the response of Purkinje cells to strong and
acute stimulation. Results are not very conclusive at this time and therefore further investigation in required. Also, pharmacological studies would be helpful to investigate hoe L7 protein modulates the activity of P-type channels. It would also be interesting to test how blocking the P-type channels will affect the bursting behavior in the absence of
L7 protein. Therefore this is an essential step in our future work.
It is difficult to stipulate what is the significance of the electrophysiology
evidence of the Purkinje cells firing behavior for the proposed nontraditional
sensorimotor function of the cerebellum. However, the detailed characterization of tonic and burst firing properties for the L7 knockout mice brings us one step closer to elucidating the physiological function of this protein and its implications in motor
learning and sensory gating carried out by the cerebellum.
The most important piece of evidence regarding the effect of L7 in electrical
activity of Purkinje neurons crucial for cerebellar function was obtained from
measurement of complex spikes. The complex spikes of Purkinje cells evoked via
climbing fiber stimulation were recorded using extracellular recording. The spikelets within the complex spike event are believed to be Ca2+ spikes, and therefore the reduced
number of spikelets that we found in the absence of L7 protein, is a clear indication of
altered P-type calcium channel activity in L7 knockout cerebellar Purkinje cells. Although
it is difficult at this stage to provide a clear mechanism of action for L7 with respect to its
100 sensorimotor function suggested by the behavior data, our analysis of the complex spike
responses of Purkinje cells in L7 mutants opens the door to a possible mechanism.
Climbing fiber signals have been variably proposed to act either as “comparators” of higher command signals (with sensory signals actually generated within the spinal
cord, acting as event, unexpected event, or error detectors of intended versus achieved
movement), as “teachers” based on the ability of climbing fiber activation to modulate the
strength of parallel fiber-Purkinje cell synapses, or as a “timing device” that serves as a
rhythm generator controlling movements [reviewed in Simpson et al., 1996]. All of these models for complex spike function (except perhaps the rhythm model) have as a common feature the detection of a sensory change requiring action. So what does the decreased spikelet number and duration of complex spikes mean? If L7 plays a role in motor learning it will be important to examine long-term depression (LTD) at the granule cell-
Purkinje cell (GC-PC) synapse, which is thought to partially constitute to motor memory trace [DeZeeuw et al., 1998; also see Hansel et al., 2001 for review]. However, the GC-
PC synapse may not be the only relevant synapse in this case. Another form of LTD at the climbing fiber-Purkinje cell (CF-PC) synapse has been reported, which appears both as a depression of CF-evoked dendritic Ca2+ transients as well as of the slow secondary somatic “spikelets” observed in the complex spike waveform [Weber et al., 2003]. While the channel types responsible for the somatic component of the complex spikes are not precisely known, a likely candidate is the P/Q-type Ca2+ channel [Mintz et al., 1992; also
reviewed in Schmolesky et al., 2002]. This form of LTD is observed following tetanic
stimulation of climbing fibers and therefore may be a desensitization process serving a
neuroprotective function rather than a “learning” mechanism per se. One possibility is
101 that the decreased number of Ca2+ spikelets in L7 mutants reflects chronic CF-PC LTD due to hyperexpression of complex spikes. Such a change could be due, for example, to a decreased threshold for complex spike activation, which could also underlie the enhanced sensorimotor behavioral responses we have observed. Alternatively, the complex spike changes in the mutant may convey a better sensory “teaching” signal, and information relevant to time-dependent motor improvement or sensory responsiveness may be encoded in the overall duration of the complex spike, which is reduced in the mutant, or encoded in some way in the number of slow spikelets. The decreased spikelet number is also consistent with increased inhibition of the P/Q-type Ca2+ channel that would be expected in the absence of the uncoupling function of the L7 GoLoco protein for G protein-coupled receptors [Kinoshita-Kawada et al., 2004; Willard et al., 2006]. Further tests will need to be conducted in order to resolve these issues.
102
CHAPTER 5
CONCLUSIONS
This dissertation is a part of a complex project including anatomical, behavioral
and electrophysiological investigation of the L7 knockout mice for possible implications
of the functional role of L7 proteins in the cerebellum. The firing properties of Purkinje
cells in cerebellar slices from mice lacking Pcp2-L7 protein were analyzed in detail for
the first time.
Recently, it has become very common to consider that besides its traditional role
in motor coordination and balance, the cerebellum also mediates non-traditional functions
ranging from motor learning and emotion to cognition. This project explores the possible
non-traditional roles of cerebellum using a mouse model which does not express the
Purkinje cell-specific protein Pcp2-L7. Although the electrophysiological results show
normal firing pattern and frequency of the Purkinje cells in the absence of L7 protein, we
have been able to bring evidence to support the hypothesis that L7 protein is involved in the functional regulation of P/Q-type channels, the primary voltage-dependent Ca2+
channels expressed in Purkinje cells, as well as the regulation of K+ channels in Purkinje
103 cells. The altered number of Ca2+ complex spikes evoked in the L7 knockout Purkinje cells is a clear indication that the L7 protein finely modulates the activity of P/Q-type
Ca2+ channels and it is implicated rather in the sensorimotor damping function of the
cerebellum acting to limit or delay time and sense- dependent changes in motor
performance. The reduced resting membrane potential shown by the L7 knockout
Purkinje cell is indicative of alteration in K+ channel activities that dampen the
excitability of these neurons.
Future work regarding the implication of the L7 protein in different forms of
Purkinje cell LTD will help to elucidate the sensorimotor role of this unique protein, the
Purkinje cells and the cerebellum as a whole.
104
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