Pharmacological evidence for L‐aspartate as the neurotransmitter of cerebellar climbing fibres in the guinea‐pig.

Climbing fibre responses (c.f.r.s) evoked by white matter stimulation and the depolarizations induced by iontophoretically applied L‐glutamate and L‐aspartate were recorded intracellularly from the proximal dendrites of Purkinje cells in in vitro slice preparations of the guinea‐pig cerebellum. Short pulses of L‐glutamate and L‐aspartate dose‐dependently depolarized the Purkinje cell dendrite. Even small doses of these amino acids reduced the input resistance. The maximum decrease in input resistance induced by L‐glutamate was 36% and that by L‐aspartate was 38%. Intracellular injection of Cs+ allowed Purkinje cell dendrites to be depolarized to a range of ‐15 to +30 mV. The mean reversal potential for the c.f.r. (Ec) was found to be +10.2 mV (n = 4). The mean reversal potentials obtained for L‐glutamate (Eg) and for L‐aspartate (Ea) were +7.3 mV (n = 7) and +5.6 mV (n = 7) respectively. When external Na+ concentration was reduced, Ec, Ea and Eg were linearly and similarly shifted in the negative direction, indicating that all these reversal potentials are determined primarily by a Na+ conductance. The effects of the glutamate antagonists 2‐amino‐5‐phosphonovaleric acid (APV), gamma‐D‐glutamylglycine (gamma‐DGG), N‐methyl‐DL‐aspartic acid (NMDLA) and glutamic acid diethylester (GDEE) were compared as to the responses to L‐glutamate and L‐aspartate and Ca2+‐activated focal climbing fibre responses (c.f.c.f.r.s) in order to investigate the receptor type at the synapses formed by the climbing fibres with Purkinje cell dendrites. The order of antagonistic potency to the c.f.c.f.r. was : APV (mean percentage blockade = 99%) greater than gamma‐DGG (87%) greater than NMDLA (71%) greater than GDEE (28%). The order of antagonistic potency to the response to L‐aspartate was: gamma‐DGG (69%) greater than APV (66%) greater than NMDLA (60%) greater than GDEE (31%), and that to the response to L‐glutamate was: GDEE (63%) greater than NMDLA (22%) greater than gamma‐GDD (15%) greater than APV (14%). APV was found to be the most effective anatagonist of the c.f.c.f.r. Its action was reversible, selective for L‐aspartate‐induced depolarization and had no effect on the responses to L‐glutamate. NMDLA, which has no activity as an agonist, was a greater suppressant of the responses to L‐aspartate than those to L‐glutamate. These electrophysiological and pharmacological findings suggest that the receptor for the transmitter at the synapses formed by climbing fibres with Purkinje cell dendrites is of the L‐aspartate‐preferring type, and are thus consistent with the bio‐and histochemical findings that L‐aspartate may be the endogenous transmitter at this synapse.

[1]  T. Sears,et al.  Effect of glutamate, aspartate and related derivatives on cerebellar Purkinje cell dendrites in the rat: an in vitro study , 1982, The Journal of physiology.

[2]  R Llinás,et al.  Interaction experiments on the responses evoked in Purkinje cells by climbing fibres , 1966, The Journal of physiology.

[3]  J. T. Hackett,et al.  Glutamate and synaptic depolarization of Purkinje cells evoked by parallel fibers and by climbing fibers , 1979, Brain Research.

[4]  K. Okamoto,et al.  Pharmacological characterization of postsynaptic receptors for excitatory amino acids in Purkinje cell dendrites in the guinea pig cerebellum. , 1985, Journal of pharmacobio-dynamics.

[5]  J. Watkins,et al.  Selective antagonism of amino acid‐induced and synaptic excitation in the cat spinal cord. , 1979, The Journal of physiology.

[6]  R. Llinás,et al.  Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices. , 1980, The Journal of physiology.

[7]  G. Collingridge,et al.  The antagonism of amino acid‐induced excitations of rat hippocampal CA1 neurones in vitro. , 1983, The Journal of physiology.

[8]  A. I. Shapovalov,et al.  Mechanisms of post‐synaptic excitation in amphibian motoneurones. , 1978, The Journal of physiology.

[9]  Influence of bicuculline and picrotoxin on reversal properties of excitatory synaptic potentials in cerebellar Purkinje cells of the rat , 1979, Brain Research.

[10]  K. Onodera,et al.  Permeability changes produced by L‐glutamate at the excitatory post‐synaptic membrane of the crayfish muscle. , 1976, The Journal of physiology.

[11]  D. Harriman.  CEREBELLAR CORTEX, CYTOLOGY AND ORGANIZATION , 1974 .

[12]  R. Meech,et al.  Potassium activation in Helix aspersa neurones under voltage clamp: a component mediated by calcium influx. , 1975, The Journal of physiology.

[13]  F. Crépel,et al.  Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro , 1983, Brain Research.

[14]  J ECCLES.  Functional Meaning of the Patterns of Synaptic Connections in the Cerebellum , 1965, Perspectives in biology and medicine.

[15]  R. H. Evans,et al.  Excitatory amino acid transmitters. , 1981, Annual review of pharmacology and toxicology.

[16]  H. Lux,et al.  Control of the delayed outward potassium currents in bursting pace‐maker neurones of the snail, Helix pomatia. , 1976, The Journal of physiology.

[17]  K. Okamoto,et al.  Ionic mechanisms of the action of taurine on cerebellar Purkinje cell dendrites in vitro: Intradendritic study , 1983, Brain Research.

[18]  N. Nadi,et al.  Effects of 3‐acetylpyridine on several putative neurotransmitter amino acids in the cerebellum and medulla of the rat 1 , 1977, Journal of neurochemistry.

[19]  C. Cotman,et al.  Evaluation of glutamate as a neurotransmitter of cerebellar parallel fibers , 1978, Neuroscience.

[20]  K Krnjevicacute,et al.  An excitatory amino Acid. , 1982, Science.

[21]  R. Llinás,et al.  Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. , 1980, The Journal of physiology.

[22]  J. Hablitz,et al.  Excitation of hippocampal pyramidal cells by glutamate in the guinea‐pig and rat. , 1982, The Journal of physiology.

[23]  F. Crépel,et al.  Morphological and electrophysiological characteristics of rat cerebellar slices maintained in vitro. , 1981, The Journal of physiology.

[24]  L. Nowak,et al.  Magnesium gates glutamate-activated channels in mouse central neurones , 1984, Nature.

[25]  K. Okamoto,et al.  Climbing and parallel fiber responses recorded intracellularly from Purkinje cell dendrites in Guinea pig cerebellar slices , 1985, Brain Research.

[26]  T. Stone.  GLUTAMATE AS THE NEUROTRANSMITTER OF CEREBELLAR GRANULE CELLS IN THE RAT: ELECTROPHYSIOLOGICAL EVIDENCE , 1979, British journal of pharmacology.

[27]  V. Crunelli,et al.  Blockade of amino acid‐induced depolarizations and inhibition of excitatory post‐synaptic potentials in rat dentate gyrus. , 1983, The Journal of physiology.

[28]  D. Johnston,et al.  Voltage clamp discloses slow inward current in hippocampal burst-firing neurones , 1980, Nature.

[29]  M Cuénod,et al.  Aspartate: possible neurotransmitter in cerebellar climbing fibers. , 1982, Science.

[30]  H. Mclennan,et al.  The actions of the dimethyl and diethyl esters of glutamie acid on glutamate uptake by brain tissue , 1973, Journal of neurochemistry.

[31]  C. Yamamoto,et al.  Selective activation of synapses near the tip of drug-ejecting microelectrode, and effects of antagonists of excitatory amino acids in the hippocampus , 1983, Brain Research.

[32]  H. Mclennan,et al.  The antagonism of amino acid-induced excitation of spinal neurones in the cat , 1979, Brain Research.

[33]  J. Hablitz,et al.  Reversal potential for glutamate responses in hippocampal pyramidal cells , 1981, Neuroscience Letters.

[34]  M. Mayer,et al.  Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones , 1984, Nature.

[35]  R. Dingledine.  N‐methyl aspartate activates voltage‐dependent calcium conductance in rat hippocampal pyramidal cells. , 1983, The Journal of physiology.

[36]  J. Eccles,et al.  The excitatory synaptic action of climbing fibres on the Purkinje cells of the cerebellum , 1966, The Journal of physiology.