Induction of long-term depression in cerebellar Purkinje cells requires a rapidly turned over protein.

Evidence is presented indicating that the induction of long-term depression (LTD) in Purkinje cells (PCs) requires a rapidly turned over protein(s) during a critical time period within 15 min after the onset of LTD-inducing stimulation and that synthesis of this protein is maintained by mRNAs supplied via transcription. LTD was induced in granule cell axon (GA)-to-PC synapses by stimulation of these synapses at 1 Hz for 5 min in conjunction with the climbing fibers (CFs) forming synapses on the same PCs and represented by a persistent reduction in the GA-induced excitatory postsynaptic potentials (EPSPs). Not only a prolonged but also a brief (5 min) pulse application of translational inhibitors (anisomycin, puromycin, or cycloheximide) effectively blocked the LTD induction. Pulses applied during the period from 30 min before to 10 min after the onset of conjunctive stimulation blocked the LTD induction, but those applied 15 min after were ineffective. The three translational inhibitors blocked the LTD induction similarly, suggesting that the effect is due to their common action of inhibiting protein synthesis. Infusion of a mRNA cap analogue (7-methyl GTP) into PCs also blocked LTD induction, ensuring that the postsynaptic protein synthesis within PCs is required for LTD induction. Transcriptional inhibitors, actinomycin D and 5,6-dichloro-l-beta-D-ribofuranosyl-benzimidazole, also blocked the LTD induction, but this effect was apparent when 5-min pulses of the transcriptional inhibitors preceded the conjunctive stimulation by 30 min or more. This time lag of 30 min is presumed to be required for depletion of the protein(s) required for LTD induction. The presently observed effects of translational and transcriptional inhibitors on the LTD induction are of temporal characteristics corresponding to their depressant effects on the type-1 metabotropic glutamate-receptor (mGluR1)-mediated slow EPSPs in PCs as we have reported recently. An antagonist of mGluR1s [(RS)-1-aminoindan-1,5-dicarboxylic acid], however, did not block LTD induction when it was applied during the 10-min period following conjunctive stimulation, where translational inhibitors effectively blocked LTD induction. This discrepancy in time course suggests that the rapidly turned over protein(s) required for LTD induction is involved in a process occurring downstream of the activation of mGluR1s.

Rapidly turned over protein maintains metabotropic synaptic transmission in Purkinje cells.

It has generally been thought that protein synthesis is required for relatively slow cellular processes such as synaptogenesis and synaptic plasticity. In this study on rat cerebellar slices, we found that metabotropic glutamate receptor-mediated synaptic transmission to cerebellar Purkinje cells was quickly and persistently depressed by brief (5 min) applications of translational inhibitors, which were confirmed to induce quick and persistent depression of protein synthesis in cerebellar tissues. Brief applications of transcriptional inhibitors also depressed metabotropic synaptic transmission, but progressively over 1 h, presumably due to depletion of mRNAs in the dendrites. Results of this study indicate the presence of a unique protein(s) that is dynamically involved in metabotropic synaptic transmission.

Stimulus parameters for induction of long-term depression in in vitro rat Purkinje cells.

Long-term depression (LTD) was induced in rat cerebellar slices by conjunctive stimulation of parallel fibers (PFs) and climbing fibers (CFs) under perfusion of 20 microM picrotoxin. LTD was estimated by the reduction in the initial rising slope of EPSPs PF-induced in Purkinje cell dendrites. LTD-inducing efficacy was represented by both the average amount of depression and the probability of inducing depression greater than 25%, both measured at 40 min after the onset of conjunctive stimulation. Using 300 regularly recurring pulses given to both CFs and PFs with 0 ms interval, LTD was optimally induced at 1 Hz, and to lesser degrees at other frequencies. When the number of conjunctive stimuli at 1 Hz with zero CF-PF interval was varied from 50 to 500, 300 stimuli induced LTD most robustly. When CF-PF interval was varied while 300 pulses were given at 0.25-4 Hz, LTD was induced even when PF stimuli were delayed after CF stimuli by as much as 2 s, but it was inhibited when PF stimuli preceded CF stimuli by 10-100 ms. LTD was also induced by applying repeated short pulse trains to both CFs and PFs, but repeated application of a PF stimulus train immediately followed by a CF stimulus train as in classical conditioning was effectless. The present results suggest complex processes leading to LTD as a result of conjunctive CF and PF stimulation.

The conjunctive stimuli that cause long-term desensitization also predominantly induce c-Fos and Jun-B in cerebellar Purkinje cells.

Expression of immediate early genes (IEGs) was examined following long-term desensitization of cerebellar Purkinje cells. This form of desensitization, which may underlie synaptic long-term depression (LTD), was evoked by co-administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and 8-bromo cGMP (8-Br-cGMP). Among the IEGS examined with in situ hybridization and immunohistochemistry, combined application of 8-Br-cGMP and AMPA synergistically enhanced the expression of c-Fos and Jun-B in Purkinje cells. This may suggest a role for active transcriptional complexes such as AP-1 (c-Fos/Jun-B), which could be formed following conjoint inputs to Purkinje cells and which may help to establish cerebellar long-term plasticity.

Protein kinases and phosphatase inhibitors mediating long-term desensitization of glutamate receptors in cerebellar Purkinje cells.

Long-term desensitization of the AMPA-selective glutamate receptors in Purkinje cells was examined in rat cerebellar slices by means of the wedge recording method. It was not induced by application of AMPA alone, but occurred regularly when slices were conditioned by perfusion with 0.5 mM 8-bromo-cGMP (but not cAMP derivatives) or the protein phosphatase inhibitors, okadaic acid and calyculin A. Phorbol esters also showed a similar effect. The 8-bromo-cGMP desensitization was antagonized by KT5823, an inhibitor of protein kinase G, while the effect of calyculin A was inhibited by polymyxin B, H-7, or K252a. These results suggest that AMPA receptors are persistently desensitized due to concerted action of both an agonist and an enzymatic system involving protein kinases G and C and a protein phosphatase inhibitor.

Messengers mediating long-term desensitization in cerebellar Purkinje cells.

Signal transduction for the characteristic long-term desensitization of glutamate receptors in Purkinje cells was investigated with wedge recordings from rat cerebellar slices. Long-term desensitization was induced specifically in the AMPA-selective subtype of glutamate receptors following brief exposure to 100 microM quisqualate. It was abolished either by treatment of the rat with pertussis toxin or by perfusion of a slice with BAPTA-AM, L-NMMA, hemoglobin, or inhibitor of PKG. Brief application of AMPA alone did not cause desensitization, but in combination with t-ACPD, sodium nitroprusside, or 8-bromo-cGMP, AMPA produced desensitization similar to that induced by quisqualate. These results indicate that the desensitization arises from activation of AMPA receptors in association with activation of metabotropic glutamate receptors, the latter leading to Ca2+ elevation to nitric oxide (NO) production to cGMP synthesis, and eventually to activation of PKG.

Receptor subtypes involved in, and time course of, the long-term desensitization of glutamate receptors in cerebellar Purkinje cells.

Desensitization of quisqualate (QA)-specific glutamate receptors of Purkinje cells was examined by 'wedge' recording from cerebellar slices. The desensitization was induced by conditioning with bath application of 100 microM QA for 4 min, and was represented by reduced responsiveness to testing QA applications. Bath application of 10 microM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) for 1 min at a time had no effect on inducing desensitization, but AMPA responsiveness was reduced by the 4-min QA conditioning to the same extent as QA responsiveness. These results indicate that QA-induced desensitization in Purkinje cells takes place specifically in the ionotropic subtype of QA receptors which AMPA stimulates selectively, and further suggest that another metabotropic subtype of QA receptors which AMPA does not stimulate plays a role in inducing the desensitization. By using AMPA applications as neutral testings, the desensitization was followed for 2 h without sign of recovery. For intervals longer than 2 h, AMPA responsiveness was compared with responsiveness to a 1-min application of 3 mM aspartate (AS) as a control, since the latter was not affected by QA conditioning. The AMPA/AS responsiveness ratio remained reduced throughout 10 h after 4-min QA conditioning without sign of recovery.

Long-term desensitization of quisqualate-specific glutamate receptors in Purkinje cells investigated with wedge recording from rat cerebellar slices.

Responses of Purkinje cells to quisqualate and aspartate were examined in rat cerebellar slices with a 'wedge' recording technique. Conditioning with bath application of 100 microM quisqualate for 1 or 4 min frequently induced reduction of Purkinje cell responses to testing bath application of 100 microM quisqualate. Test responses obtained at 5-30 min intervals were 79% after 1 min conditioning and 64% after 4 min conditioning, relative to conditioning responses. Since there was no correlation between the degree of reduction and the conditioning-testing interval, the effect of conditioning appeared to be sustained over 30 min. No such desensitization was observed with 3 or 5 mM aspartate. The presently demonstrated desensitization of quisqualate-specific glutamate receptors could represent a central mechanism of the 'long-term depression' type of synaptic plasticity in Purkinje cells.

Long-term effects of 3-acetylpyridine-induced destruction of cerebellar climbing fibers on Purkinje cell inhibition of vestibulospinal tract cells of the rat.

The inhibitory action of Purkinje cells on vestibulospinal tract (VST) cells was examined in rats deprived of climbing fibers with 3-acetylpyridine (3-AP) intoxication. In order to resolve discrepancies raised in previous studies with various means, special efforts were devoted to directly estimate Purkinje cell inhibition at synaptic levels by using intracellular recording, to avoid sampling bias by using a systematic survey of VST cells in each rat, and to evaluate the time-dependence of the effects of climbing fiber deafferentation by regular testing at 10 day intervals until 160 days after 3-AP intoxication. As compared with 661 VST cells impaled in 15 control rats, 1771 VST neurons impaled in 29 3-AP-treated rats revealed four basic changes in the monosynaptic inhibitory postsynaptic potentials (IPSPs) induced by stimulation of Purkinje cell axons in the white matter of the cerebellar anterior lobe. First, the rate of IPSP occurrence among VST cells was 0.64 in control rats; at more than 10 days after 3-AP intoxication it decreased gradually, down to 0.37-0.38 at the 70th-81st days, and thereafter increased up to 0.53 by the 160th day. The rate of IPSP occurrence varied considerably between the rostral and caudal regions, and also between the dorsal and ventral divisions of the VST cell population, but its reduction after 3-AP intoxication occurred approximately in parallel in all divisions. Second, IPSPs evoked with standard 500 microA pulse stimuli were smaller in size on and after day 10. The reduction of IPSP size was by as much as 53% of control values at the 70th-101st days in the dorsal division, but no significant change occurred in the ventral division of the VST cell population. Third, the latency of the IPSPs was prolonged by about 0.25 ms on and after day 10. Analysis of the relationship between the IPSP latency and the dorsoventral location of VST cells in the medulla suggests that the major cause for the prolongation of IPSP latency is an increased synaptic delay at Purkinje cell axon terminals. Fourth, the cerebellar stimulation threshold for evoking IPSPs was almost always below 100 microA in control rats, but values of 100-250 microA were common after the 40th day. Thus, climbing fiber deafferentation exerts long-term influences on excitability of Purkinje cell axons, and on the connectivity and synaptic transmission from Purkinje cell axons to VST cells.

Eye field in the cerebellar flocculus of pigmented rabbits determined with local electrical stimulation.

The cerebellar flocculus was mapped with local stimulation techniques in alert pigmented rabbits. Triple-barrelled microelectrodes filled with solutions each containing one of three different dyes (Fast Green FCF, Pontamine Sky Blue and Nigrosine) were used for recording and stimulation. The H-zone from which local stimulation through the microelectrode induced abduction in the ipsilateral eye was visualized on the reconstructed model of the flocculus to span across major folia of the flocculus, forming a narrow strip 0.5-1.0 mm wide. The H-zone was flanked with two V-zones, rostral and caudal, from which downward eye movements were induced in the ipsilateral eye. The R-zone, from which clockwise rotation was induced in the contralateral eye, was extended rostrocaudally across the H- and V-zones. In addition to these eye movement-related zones, a restricted area specifically related to eye blinking was found in the rostroventral area of the flocculus.

Sites in the rabbit flocculus specifically related to eye blinking and neck muscle contraction.

In alert rabbits, the cerebellar flocculus was mapped for effects of local stimuli delivered through glass microelectrodes. Triple-barreled glass microelectrodes were used, each barrel of which was filled with solution containing one of three different dyes (Fast Green FCF, Pontamine Sky Blue and Nigrosine) for differentially labeling the sites exhibiting different stimulus effects. In addition to eye movements reported earlier, eye blinking and contraction of dorsal neck muscles were elicited from limited areas of the flocculus. Eye blinking sites were concentrated rostroventrally and neck muscle contraction sites caudoventrally within the flocculus. The present results suggest that the rabbit flocculus contains specific sites devoted to the control of eye blinking and neck posture.