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Enhanced Synaptic Inhibition Disrupts the Efferent Code of Cerebellar Purkinje Neurons in Leaner Cav2.1 Ca2+ Channel Mutant Mice

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Abstract

Cerebellar Purkinje cells (PCs) encode afferent information in the rate and temporal structure of their spike trains. Both spontaneous firing in these neurons and its modulation by synaptic inputs depend on Ca2+ current carried by Cav2.1 (P/Q) type channels. Previous studies have described how loss-of-function Cav2.1 mutations affect intrinsic excitability and excitatory transmission in PCs. This study examines the effects of the leaner mutation on fast GABAergic transmission and its modulation of spontaneous firing in PCs. The leaner mutation enhances spontaneous synaptic inhibition of PCs, leading to transitory reductions in PC firing rate and increased spike rate variability. Enhanced inhibition is paralleled by an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) measured under voltage clamp. These differences are abolished by tetrodotoxin, implicating effects of the mutation on spike-induced GABA release. Elevated sIPSC frequency in leaner PCs is not accompanied by increased mean firing rate in molecular layer interneurons, but IPSCs evoked in PCs by direct stimulation of these neurons exhibit larger amplitude, slower decay rate, and a higher burst probability compared to wild-type PCs. Ca2+ release from internal stores appears to be required for enhanced inhibition since differences in sIPSC frequency and amplitude in leaner and wild-type PCs are abolished by thapsigargin, an ER Ca2+ pump inhibitor. These findings represent the first account of the functional consequences of a loss-of-function P/Q channel mutation on PC firing properties through altered GABAergic transmission. Gain in synaptic inhibition shown here would compromise the fidelity of information coding in these neurons and may contribute to impaired cerebellar function resulting from loss-of function mutations in the CaV2.1 channel gene.

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Abbreviations

PC:

Purkinje cell

BC:

Basket cell

IPSC:

Inhibitory postsynaptic current

sIPSC:

Spontaneous IPSC

eIPSC:

Evoked IPSC

mIPSC:

Miniature IPSC

TTX:

Tetrodotoxin

SERCA:

Sarcoplasmic/endoplasmic reticulum Ca/ATPase

ISI:

Inter-spike interval

IEI:

Inter-event interval

CV:

Coefficient of variation

CICR:

Calcium-induced calcium release

[Ca2+]i :

Cytosolic free calcium concentration

WT:

Wild type

tgla :

Leaner

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Acknowledgments

David Friel would like to thank Dr. Roberto F. Galán for discussions regarding sIPSC burst statistics. This work was supported by a grant from NIH/NINDS (NS 33514) to DDF.

Conflict of interest

The authors report no conflicts of interest regarding the contents of this manuscript.

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  1. Saak V. Ovsepian

    Present address: Dublin City University, ICNT, X Block, Room X241, Science Building, Dublin 9, Republic of Ireland

Authors and Affiliations

  1. Department of Neurosciences, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH, 44106, USA

    Saak V. Ovsepian & David D. Friel

  2. Department of Neurosciences, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH, 44106-4975, USA

    David D. Friel

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  1. Saak V. Ovsepian
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Correspondence to David D. Friel.

Electronic Supplementary Material

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Supplementary Fig. 1

Comparison between tonic firing in PCs under whole cell and cell-attached recording conditions. a, b Representative examples of spontaneous tonic firing activity recorded in wild-type (a) and leaner (b) PCs under cell-attached (top) and whole cell (bottom) recording conditions. Note the pronounced irregular spiking of leaner PCs under both experimental conditions, in contrast to the more regular firing of WT PCs. c, d Comparison between measurements of firing rate and CV in WT and leaner PCs obtained under cell-attached (CA, WT: n = 24; leaner: n = 13) and whole cell (WC, wild type: n = 18; leaner: n = 15) recording conditions. The similar values for firing rate and CV obtained under cell-attached and whole cell conditions supports the use of both experimental approaches for evaluating PC spike activity in slices. e, f A typical cell-attached recording of spontaneous spiking from WT PC and after the transition to whole cell recording mode (black arrow), during which firing rate and CV remained approximately constant. f Collected results from three WT PCs in which recordings we made as in e (JPEG 623 kb)

Supplementary Fig. 2

Detection and analysis of sIPSCs. a Representative current recording from a leaner PC under voltage clamp at −75 mV in the presence of kynurenic acid (5 mM) illustrating spontaneous IPSCs. Dashed line indicates baseline holding current. b Negative derivative of the current trace in a. Dashed line indicates zero and dotted line shows the threshold used for identifying synaptic currents. The threshold was defined as 2.5 times the standard deviation of −dI/dt during a manually defined time interval that lacked obvious synaptic currents (see horizontal bar above trace in a and corresponding values of −dI/dt in b). c Normalized distribution of −dI/dt during the event-free period illustrated in a. Mean value differs from zero due to the small non-zero current slope during the event-free time interval in a. d Normalized distribution of inter-event intervals from the same cell. This distribution could be approximated by a single exponential function, consistent with a homogeneous Poisson process. Mean sIPSC frequency in this cell was 50.2 Hz. Sample interval, 50 μs (JPEG 278 kb)

Supplementary Fig. 3

Variability of firing rate and CV measurements in PCs and molecular layer interneurons. a Distribution of firing rate (top) and interspike interval CV (bottom) under whole cell current-clamp conditions in WT and leaner Purkinje neurons before (left) and after (right) exposure to kynurenic acid (5 mM) and picrotoxin (200 μM) to block spontaneous synaptic currents. Measurements were from 19 WT and 16 leaner PCs (pre-block) and 18 WT and 12 leaner cells (post-block). b Distribution of firing rate (top) and CV (bottom) in molecular layer interneurons under cell-attached recording conditions. Measurements were from 13 WT to 7 leaner basket neurons. In each panel, horizontal bars indicate mean values (JPEG 381 kb)

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Ovsepian, S.V., Friel, D.D. Enhanced Synaptic Inhibition Disrupts the Efferent Code of Cerebellar Purkinje Neurons in Leaner Cav2.1 Ca2+ Channel Mutant Mice. Cerebellum 11, 666–680 (2012). https://doi.org/10.1007/s12311-010-0210-9

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