ABSTRACT
The well established anatomy of the cerebellar cortex has led to suggestions that cerebellar molecular layer interneurons laterally inhibit Purkinje cells. In support of the anatomical predictions, on-beam excitation and off-beam inhibition of Purkinje cells have been shown to occur when the surface of the cerebellum is electrically excited. Patchy excitation of Purkinje cells with flanking inhibition of sagittally oriented Purkinje cells have also been demonstrated following peripheral stimulation in vivo. To extend these observations, we mapped the functional connectivity between granule cells, molecular layer interneurons, and Purkinje cells in rats. Patches of granule cells were asynchronously activated by photostimulation to mimic their excitation by a mossy fiber as it occurs in vivo. We found with remarkable consistency that, in the sagittal orientation, granule cells elicit a stereotypic set of responses. Granule cells immediately underneath a Purkinje cell provide pure excitation. Granule cells positioned 340-400 µm laterally provided pure inhibition, consistent with the lateral inhibition proposed earlier. The net effect of exciting granule cells in between these two extremes was to provide a systematic change in the response of Purkinje cells, from net excitation to net inhibition moving laterally from the Purkinje cell. In contrast to the sagittal orientation, in the coronal orientation the organization of Purkinje cell responses with granule cell activation was remarkably different. Independent of the location of granule cells, within the 480 µm lateral distance examined, molecular layer interneurons reduced the strength of granule cell inputs to Purkinje cells to a comparable extent.
Subject(s)
Cerebellar Cortex/cytology , Interneurons/physiology , Purkinje Cells/physiology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Action Potentials/physiology , Age Factors , Animals , Animals, Newborn , Central Nervous System Stimulants/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Electric Stimulation/methods , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , GABA Antagonists/pharmacology , In Vitro Techniques , Myasthenic Syndromes, Congenital/drug therapy , Nerve Net/drug effects , Nerve Net/physiology , Neural Inhibition/drug effects , Patch-Clamp Techniques/methods , Phosphinic Acids/pharmacology , Photic Stimulation/methods , Photolysis , Picrotoxin/pharmacology , Propanolamines/pharmacology , Pyridazines/pharmacology , Rats , Rats, WistarABSTRACT
At the center of the computational cerebellar circuitry are Purkinje cells, which integrate synaptic inputs from >150,000 granule cell inputs. Traditional theories of cerebellar function assume that all granule cell inputs are comparable. However, it has recently been suggested that the two anatomically distinct granule cell inputs, ascending and parallel fiber, have different functional roles. By systematically examining the efficacy of patches of granule cells with photostimulation, we found no differences in the efficacy of the two inputs in driving the activity of, or in producing postsynaptic currents in, Purkinje cells in cerebellar slices in vitro. We also found that the activity of Purkinje cells was significantly increased upon stimulation of lateral granule cells in vivo. Moreover, when we estimated parallel fiber and ascending apparent unitary EPSC amplitudes using photostimulation in cerebellar slices in vitro, we found them to be indistinguishable. These results are inconsistent with differential functional roles for these two inputs. Instead, our data support theories of cerebellar computation that consider granule cell inputs to be functionally comparable.
Subject(s)
Afferent Pathways/physiology , Cerebellum/cytology , Nerve Fibers/physiology , Purkinje Cells/physiology , Afferent Pathways/drug effects , Age Factors , Animals , Animals, Newborn , Brain Mapping , Excitatory Amino Acid Agonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , GABA Antagonists/pharmacology , Glutamates/pharmacology , In Vitro Techniques , Membrane Potentials/drug effects , Membrane Potentials/physiology , Nerve Fibers/drug effects , Patch-Clamp Techniques/methods , Phosphinic Acids/pharmacology , Picrotoxin/pharmacology , Propanolamines/pharmacology , Purkinje Cells/cytology , Rats , Rats, WistarABSTRACT
Fast synaptic inhibition in the mammalian central nervous system is mediated primarily via activation of the gamma-aminobutyric acid type A receptor (GABAA-R). Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state. This transition, known as gating, is thought to be associated with a sequence of conformational changes originating at the agonist-binding site, ultimately resulting in opening of the channel. Using site-directed mutagenesis and several different GABAA-R agonists, we identified a number of highly conserved charged residues in the GABAA-R beta2 subunit that appear to be involved in receptor activation. We then used charge reversal double mutants and disulfide trapping to investigate the interactions between these flexible loops within the beta2 subunit. The results suggest that interactions between an acidic residue in loop 7 (Asp146) and a basic residue in pre-transmembrane domain-1 (Lys215) are involved in coupling agonist binding to channel gating.