Purkinje cell activity in rats following chronic treatment with harmaline

Harmaline and related alkaloids produce a fine, generalized motor tremor with a frequency of 8-14 Hz in many mammalian species. The tremor is though to be initiated by the synchronous activation of cells in the inferior olive. Repeated administration of the drug at tremorogenic doses results in the rapid development of tolerance in the rat. Since the generation of cerebellar cyclic 3',5'-guanosine monophosphate by harmaline or apomorphine is reduced in harmaline-tolerant rats, it is possible that the site of tolerance is the olivocerebellar system. The present study used extracellular single unit recording techniques to determine whether harmaline tolerance was associated with changes in the firing patterns of Purkinje cells in the cerebellar vermis of the rat. In non-tolerant animals, the majority (8/13) of Purkinje cells recorded in the vermis responded to harmaline with a rhythmic increase in complex spike rate and a prolonged suppression of simple spikes. In harmaline-tolerant animals, only one cell in 14 could be identified that showed this response. In these animals, a variety of responses not encountered in experimentally naive animals were observed. Since the complex spike activity of Purkinje cells is presumed to reflect the activity of climbing fibers originating in the cells of the inferior olive, the results of the studies reported here support the conclusion that a reduction in the synchronous activation of cells at the olivocerebellar level blocks the appearance of tremor in harmaline-tolerant animals.     

Effect of harmaline on cells of the inferior olive in the absence of tremor: differential response of genetically dystonic and harmaline-tolerant rats

The genetically dystonic rat is insensitive to the tremorogenic effects of harmaline. This behavioral deficit has been linked to a defect in the olivocerebellar pathway, since few Purkinje cells of dystonic rats show a normal increase in rhythmic complex spike activity following harmaline. In normal rats, the Purkinje cell response to harmaline and tremor are initiated by a rhythmic increase in neuronal firing in the caudal inferior olive. The present single unit recording study was conducted, therefore, to determine if the inferior olive of the dystonic rat is activated by harmaline. Olivary unit responses to harmaline were also examined in normal rats made tolerant to harmaline tremor. These rats are behaviorally insensitive to harmaline and also fail to display rhythmic complex spike activity but do not have the motor deficits of the mutant rats. The spontaneous firing rate of neurons in the caudal and rostral inferior olive of the dystonic rat was significantly slower than that of phenotypically normal littermates. Despite this, all cells recorded in the caudal portion of the medial accessory olive of both dystonic and normal rats showed increased rhythmic activity following harmaline injection. Thus, the failure of the mutants to show harmaline tremor is not due to a failure of the drug to activate cells in the olive. Rather, the data suggest a defect in the subsequent transmission of this information. Unlike the control and dystonic rats, harmaline-tolerant rats failed to show sustained rhythmic activity in the inferior olive. These findings suggest that chronic treatment with harmaline may interfere with harmaline tremor at the level of the inferior olive.     

Harmaline induced tremor III. A combined simple units,horseradish peroxidase,and 2-deoxyglucose study of the olivocerebellar system in the rat

Summary Purkinje cells were recorded extracellularly and mapped in the cerebellar cortex of the rat under tremogenic doses of harmaline. Four différent types of responses were encountered, of which two were considered as being responsible for the harmaline tremor. The latter had a regular firing pattern of complex spikes at 5 to 10 Hz and were mostly found in the vermis. Their number decreased in the more lateral region of the cerebellar cortex until they eventually disappeared. Horseradish peroxidase was injected into all the areas of the cerebellar cortex containing Purkinje cells with harmaline-induced activity. Labeled neurons were in all cases traced to the medial accessory olive. The metabolic activity of the inferior olive under harmaline was measured with 2-deoxyglucose. Increased labeling was only found in the medial accessory olive. Such an increase was demonstrated as being due to a direct effect of the drug on the inferior olivary neurons, indicating that the medial accessory olive is responsible for the harmaline tremor in the rat. Our results point out that, in the rat, there is an inverse relationship between serotoninergic innervation of a region in the inferior olivary nucleus and that with harmaline sensitivity, therefore a serotoninergic mechanism hypothesis for the harmaline tremor needs further investigation.Assistant from University Paris VII     

Harmaline-induced tremor

Summary Units were recorded extracellulary in the cat brainstem under the effect of tremogenic doses of harmaline. They were localized post mortem and the units discharging at the harmaline tremor frequency were mapped.Harmaline-sensitive neurons were found in the bulbo-pontine reticular formation, in particular, in the lateral reticular nucleus and the nucleus reticularis tegmenti pontis. The nucleus interpositus as well as the red nucleus also displayed numerous units discharging at the tremor frequency, indicating that the cerebello-interposito-rubro-spinal system controlling the flexor muscles participate in harmaline tremor.Participation of the oculomotor system in the harmaline-induced tremor was tested at the level of the vestibular neurons relaying the vestibulo-ocular reflex, the motoneurons, the eye muscles and the eye movements. No rhythmic discharge at the tremor frequency nor eye movements could be detected, indicating that harmaline tremor does not affect the oculomotor system.     

Abnormal spontaneous and harmaline-stimulated Purkinje cell activity in the awake genetically dystonic rat

The genetically dystonic rat is an autosomal recessive mutant with a movement disorder that closely resembles the generalized dystonias seen in humans. Abnormal activity of neurons within the cerebellar nuclei is critical to the dystonic rat motor syndrome. Increased glutamic acid decarboxylase activity, increased glucose utilization, and decreased muscimol binding within the cerebellar nuclei of the dystonic rat suggests that Purkinje cell firing rates are increased in these animals. However, under urethane anesthesia, Purkinje cell simple spike firing rates in dystonic rats were less than half the rates seen in normal littermates. In this study, both spontaneous and harmaline-stimulated single-unit Purkinje cell recordings were obtained from awake normal and dystonic rats. In striking contrast to previous results obtained under urethane anesthesia, there was no statistically significant difference in average Purkinje cell spontaneous simple spike frequency between dystonic and normal rats. Similar to previous studies obtained under urethane anesthesia, Purkinje cell spontaneous complex spike frequency was much lower in dystonic than in normal rats. Many Purkinje cells from dystonic rats, particularly those from the vermis or older animals, exhibited rhythmic bursting simple spike firing patterns. Cross-correlations showed that complex spikes produced less suppression of simple spikes in dystonic than in normal rats and harmaline-stimulated complex spike activity was, on average, faster and more rhythmic in normal than in dystonic rats. These findings indicate that olivocerebellar network abnormalities in the dystonic rat are not due to an inability of Purkinje cells to fire at normal rates and suggest that abnormal Purkinje cell bursting firing patterns in the dystonic rat are due to a defect in the pathway from the inferior olive to climbing fiber synapses on Purkinje cells.     

Ultrastructure of synapses in the cerebellar cortex after long-term activation of climbing fibres

Adult male rats were treated for 24 or 48 h with harmaline which selectively activates neurones in the inferior olivary nucleus which give rise to climbing fibres projecting to the cerebellar vermis. Electrophysiological studies have shown that harmaline-induced climbing fibre activity completely blocks the responses of the Purkinje cell to parallel fibre input. Morphometric analysis of the ultrastructure of climbing and parallel fibre synapses revealed no significant differences in morphology between vermis (experimental) and hemisphere (control). These findings indicate that the decreased responsiveness of Purkinje cells to parallel fibre inputs induced by increased climbing fibre activity over 24 or 48 h is not accompanied by any observable structural changes in the cerebellar cortex.     

Metabolic activity of intracerebellar nuclei in the rat: Effects of inferior olive inactivation

Summary Metabolic activity of the intracerebellar nuclei during cryoinactivation of the inferior olive was studied in the anaesthetized rat by using the ¹⁴C-2-deoxyglucose method. Single unit recording of Purkinje cells was simultaneously monitored in the cerebellar cortex.Local inactivation in the inferior olive resulted in regional suppression of complex spike discharges in the cerebellar cortex.An increased metabolic activity was observed in the cerebellar nuclei contralateral to the cryoinactivation site correlating the somatotopically arranged olivo-cerebello-nuclear circuit. This increase was shown to be due specifically to inactivation of the inferior olive, since it was not obtained in a rat in which the inferior olive was previously destroyed by neurotoxic doses of 3-acetylpyridine.The results are interpreted as being due to an increased presynaptic activity of the terminals of the Purkinje cells which fire simple spikes at high rates after climbing fibre deafferentation.     

Harmaline-induced climbing fiber activation causes amino acid and peptide release in the rodent cerebellar cortex and a unique temporal pattern of Fos expression in the olivo-cerebellar pathway

Cerebellar climbing fibers have a unique relationship with the dendritic tree of cerebellar Purkinje cells and have been proposed as a key input in establishing long-term plastic changes in the cerebellar cortex. Although both glutamate and aspartate and a number of neuropeptides have been implicated as climbing fiber-released neurotransmitters/neuromodulators, the in vivo release of these substances during climbing fiber stimulation remains to be demonstrated. In the present study, climbing fibers were activated with harmaline and rats or mice were implanted with a microdialysis probe or a microperfusion probe, respectively, to measure amino acid or peptide release. Additional rats were euthanized at various timepoints post-harmaline injection and Fos immunocytochemistry was used to visualize the activation pattern of the inferior olive, cerebellar cortex and deep nuclei over time. Fos expression was first detected in the inferior olive at 15 min post-harmaline injection followed by expression in the deep cerebellar nuclei (30 min) and then in the cerebellar cortex (1 h). Between 2 and 6 h Purkinje cells expressing Fos were found in variable numbers in both the vermal and paravermal regions and there was a distinct parasagittal-banding pattern in the vermal region. Of several amino acids measured following harmaline administration only glutamate and aspartate levels increased significantly in the first dialysate sample compared to preharmaline levels and their release was blocked by prior lesion of the inferior olive. Citrulline also increased following climbing fiber stimulation, but this occurred in the second and third dialysate samples and may reflect nitric oxide production. Four peptides were examined in cerebellar microperfusates following climbing fiber stimulation. Only corticotropin releasing factor (CRF), calcitonin gene related peptide (CGRP) and bradykinin were significantly increased compared to pre-harmaline levels. These results suggest that glutamate, aspartate, CRF and CGRP are released from climbing fibers during activation of the olivocerebellar system.     

The climbing fibers of the cerebellar cortex,their origin and pathways in cat

Summary The sources and pathways of the climbing fibers to the cerebellar posterior vermis were studied with combined electrophysiological and anatomical methods in cats.Recording from identified cerebellar Purkinje cells, monosynaptic climbing fiber (CF) responses have been obtained both for stimulation of the inferior olive (IO) and various parts of the brain stem (BS). CF responses were found to be of three types, IO only, BS only or both IO and BS. However the responses to BS stimulation were very few in number in comparison with IO or IO and BS types of responses. The latencies of the responses were shorter for the BS cases consistent with their distance from the cerebellum.A comparison of latencies and the relative responsiveness of the different area of the brain stem which were studied, indicate that part of the CF ascend through the pontine region and enter the cerebellum by way of the medium and superior peduncles. This finding is confirmed by the results of anatomical studies in which degenerating fibers were found in the molecular layer (using the Nauta technique) after lesion of the brachium pontis but not after lesions of the medial portion of the pons. Similarly, injection of radioactive leucine into the pontine nuclei failed to show any labeled fibers in the molecular layer.Horseradish peroxidase (HRP) was injected into localized regions of the posterior vermis after total bilateral destruction of the inferior peduncles. Large numbers of positive, marked cells were still found in the inferior olive.It is concluded that nearly all, if not all, the climbing fibers originate in the inferior olive and that they ascend to the cerebellum by way of all the peduncles.     

Long term modification of cerebellar inhibition after inferior olive degeneration

Summary The long term effects of inferior olive destruction on the activities of the Purkinje cells and their target neurones in the cerebellar nuclei were studied in the rat. Careful observations were also made of motor behaviour throughout the study. Albino rats were injected with 3-acetylpyridine to produce a neurotoxic destruction of the inferior olive and then were used for acute recording experiments at 1–2 days, 5–7 days, 12–18 days, 35–38 days, 75–97 days and 230–252 days. After degeneration of the inferior olive, there was an initial period lasting for a few days, characterized by a high firing frequency of Purkinje cells associated with a very low level of activity of the neurones in the cerebellar nuclei. During this period, there was a deep depression of motor activity. A period of adaptation follows during the first month, characterized by a slow recovery of the initial firing frequency of the cerebellar units and a gradual recovery of spontaneous locomotion; nevertheless the firing pattern and motor behaviour remain abnormal. From one month on the unit activities disturbances and the motor deficiencies stabilize. The hypothesis is advanced that Purkinje cell inhibition on their target neurones, which increases during the initial period, gradually diminishes during the adaptation time, and then stabilizes to a subnormal state.     

Raphe — Cerebellum interactions

Summary The effects of midbrain raphe stimulation and/or harmaline administration on cerebellar cell activities were examined in chloralosed rats.Cerebellar cortical cells were grouped into two categories. From a total sample of 68 cells, 48 were classified as Purkinje cells and the 20 others were unidentified.Midbrain raphe stimulation was found to inhibit for many sec the discharge of 40% of the Purkinje cells and 80% of the unidentified units. Other cells were unaffected, except 4 of them which were excited.Harmaline administration increased the CS firing rate of all Purkinje cells by inducing a rhythmic CS discharge at 7–12 Hz. Moreover, harmaline increased the discharge rate of unidentified units without inducing rhythmic activity. In both types of cerebellar cells a modulation of their firing pattern by periodical pauses at 0.1–0.4/s was noticed.These data are discussed in relation to the known influences of harmaline and cerebellar stimulation on raphe neurons. Taken in this context, results presented here confirm the existence of a modulatory influence of the raphe nuclei on the olivo-cerebellar circuitry. A general model of interactions is proposed.Supported by CNRS Research Grant ERA/272     

A specific harmaline-evoked increase in cerebellar 5′-nucleotidase activity

This study examines harmaline-induced changes in 5′-nucleotidase (5′-ND) activity in cerebellar fractions from rats with an intact inferior olive (IO) or prior destruction of the IO by 3-acetylpyridine (3-AP) intoxication. Harmaline markedly increased 5′-ND activity in the crude homogenate (P<0.05) and P2 fraction (P<0.001) of cerebella from rats with an intact IO. This increase was absent in the P1, P3 and S3 fractions and it was abolished by 3-AP olivectomy. It was also absent in basal ganglia P2 fractions. Since harmaline produces rhythmic complex spike discharges of Purkinje cells by activating IO neurons [4, 18], these data suggest that climbing fiber activation per se increases 5′-ND activity in the P2 fraction. This raises the possibility that a climbing fiber-induced local increase in 5′-ND activity at parallel fiber-Purkinje cell synapses results in a local increase in adenosine concentration. This may account for climbing fiber-evoked suppression of simple spike activity [12, 13, 28].     

Slow excitatory amino acid receptor-mediated synaptic transmission in turtle cerebellar Purkinje cells

1. The excitatory synaptic responses of turtle Purkinje cells to climbing and parallel fiber (CF and PF) stimulation have been studied by the use of intrasomatic and intradendritic recordings in intact cerebellum and brain stem-cerebellum preparations in vitro. 2. Activation of CF inputs from the cerebellar peduncle or the region of the inferior olive evoked complex spikes followed by slow excitatory postsynaptic potentials (EPSPs), both of which were evoked in an all-or-none fashion. 3. Single stimuli applied to the cerebellar molecular layer activated fast PF-mediated EPSPs; brief trains of PF stimuli (2-5 stimuli, 50-100 Hz) evoked volleys of fast EPSPs followed by a slow, long-lasting EPSP. The amplitude of the fast and slow PF-mediated EPSPs were both graded with stimulus intensity. 4. Slow EPSPs evoked both by CF and PF stimulation were associated with an increase in membrane conductance and were increased in amplitude by hyperpolarization. 5. The CF-evoked slow EPSP was profoundly attenuated by repetitive activation at interstimulus intervals of less than 15-20 s, whereas the PF-evoked slow EPSP was not reduced by repetitive activation. 6. The PF-evoked slow EPSP readily triggered dendritic pacemaker discharges when activated at or near resting membrane potential. The activation of this potential by phasic PF volleys may, therefore, provide an appropriate synaptic drive to cerebellar Purkinje cells to entrain the intrinsic pacemaker properties of these cells to cycles of motor activity. 7. Both slow synaptic potentials were blocked by the excitatory amino acid antagonists kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by DL-2-amino-5-phosphonovalerate (DL-AP5) or L-serine-O-phosphate (L-SOP). The PF-evoked slow EPSP was selectively antagonized by L-2-amino-4-phosphonobutyrate (L-AP4; 20-100 microM). 8. It is suggested that the CF- and PF-evoked slow EPSPs observed in this study represent a novel class of excitatory amino acid receptor-mediated slow synaptic potentials activated by Purkinje cell afferents, which may play a role in synaptic integration and motor pattern generation in the cerebellum.     

Stimulus-dependent activation of c-Fos in neurons and glia in the rat cerebellum

The intent of the present study was to use chemical or electrical stimulation of cerebellar afferents to determine how different stimulation paradigms affect the pattern of activation of different populations of neurons in the cerebellar cortex. Specifically, we analyzed immediate changes in neuronal activity, identified neurons affected by different stimulation paradigms, and determined the time course over which neuronal activity is altered. In the present study, we used either systemic (harmaline) or electrical stimulation of the inferior cerebellar peduncle (10 and 40 Hz) to alter the firing rate of climbing and mossy fiber afferents to the rat cerebellum and an antibody made against the proto-oncogene, c-fos, as a marker to identify activated neurons and glia. In control animals, only a few scattered granule cells express nuclear Fos-like immunoreactivity. Although no other cells show Fos-like immunoreactivity in their nuclei, Purkinje cells express Fos-like immunoreactivity within their somatic and dendritic cytoplasm in control animals. Within 15 min of chemical or electrical stimulation, numerous granule and glial cells express Fos-like immunoreactivity in their nuclei. Cells in the molecular layer express Fos-like immunoreactivity following harmaline stimulation in a time and lobule specific manner; they do not appear to be activated in the electrical stimulation paradigm. Following harmaline injections, there is an initial loss of Fos-like immunoreactivity in the cytoplasm of Purkinje cells; 90 min later, nuclear staining is observed in a few scattered Purkinje cells. Following electrical stimulation, the cytoplasmic staining in Purkinje cells is enhanced; it is never present in the nucleus. Data derived from this study reveal cell-specific temporal and spatial patterns of c-Fos activation that is unique to each paradigm. Further, it reveals the presence of an activity dependent protein in the cytoplasm of Purkinje cell somata and dendrites.     

Olivo-vestibular and cerebello-vestibular connections in albino rabbits

This study analyzes the organization of olivo-vestibular and cerebello-vestibular projections in rabbits. Iontophoretic injections of horseradish peroxidase, placed under physiological guidance into the superior, medial and lateral vestibular nuclei, produced retrogradely labeled neurons in the dorsal cap, ventrolateral outgrowth and lateral flexure of the principal olivary nucleus, the caudal half of the medial accessory olive and the caudal three-fourths of the dorsal accessory olive. This inferior olivary labeling was strictly contralateral. The same injections labeled groups of Purkinje cells in the ipsilateral cerebellar cortex, oriented perpendicular to the long axes of the folia of lobules I-V, VId-e and VIII-X of the vermis and the flocculus. The patterns of olivo-vestibular and cerebello-vestibular connections were consistent with the general hypothesis that inferior olivary axon collaterals project to both Purkinje cells and subcortical neurons inhibited by those Purkinje cells. In addition, the analysis of flocculo-nodular and dorsal cap-ventrolateral outgrowth projections to the medial and superior vestibular nuclei suggests that these connections are discrete at the level of a pool of olivary neurons which projects to functional pools of neurons in both cerebellar cortex, and in the vestibular nuclei. Thus, it is likely that inferior olivary projections define functional networks spanning cerebellar cortex and the vestibular nuclei.     

Inhibitory control of intracerebellar nuclei by the Purkinje cell axons

Summary In anaesthetized cats, synaptic events in cerebellar nuclei neurones were investigated with intracellular microelectrode techniques. These cells were identified by their antidromic activation along their axons and/or by their location in histological sections. In the cells of lateral nucleus IPSPs were induced monosynaptically during stimulation of the overlying hemispheral cortex of the cerebellum. In the cells of nuclei interpositus and fastigii, similar IPSPs were produced from the paravermal and vermal cortices, respectively. The postulate that the Purkinje cells exert an inhibitory action upon their target neurones thus applies not only to Deiters neurones, as previously proposed, but also to cells in the cerebellar nuclei. Stimulation of the cerebellar afferents at the inferior olive, the pontine nucleus and the lateral reticular nucleus produced EPSPs in cerebellar nuclei cells with relatively brief latencies, probably through axon collaterals of these afferents. The EPSPs were followed by IPSPs and slow depolarizations of disinhibitory nature, which, as studied previously in Deiters neurones, might be caused respectively by activation and subsequent depression of Purkinje cells through the cerebellar intracortical mechanisms.     

Influence of inferior olive on flexor reflex activity

Summary In Wistar rats we have studied the effect of inferior olive lesion or activation on the threshold of a flexor reflex elicited by a nociceptive stimulus applied to the hindpaw. When the inferior olive is lesioned by means of 3-acetylpyridine, the threshold value is significantly decreased. A recovery occurs in 3–4 weeks. When the inferior olive is activated by means of harmaline, the threshold value is significantly increased. These experiments suggest the inferior olive activity exerts an inhibitory effect on flexor reflex activity. The recovery of the threshold value depends, probably, on the plastic reorganization of the cerebellar circuits, which occurs after inferior olive lesion.     

Effect of audiogenic seizures on activity of H and M forms of lactate dehydrogenase in neurons and neuroglia in various parts of the central nervous system

Cytospectrophotometric analysis showed that the ratio between activities of aerobic H forms of lactate dehydrogenase (LD) and the activity of its anaerobic M forms in cerebral cortical neurons and cerebellar Purkinje cells and also in their glial satellite cells of rats of the Krushinskii-Molodkina and Wistar strains is higher than in motoneurons in the anterior horns of the spinal cord and their perineuronal glia. In Krushinskii-Molodkina rats (with genetically determined high sensitivity to audiogenic seizures) epileptiform audiogenic seizures were accompanied by marked activation of both H and M forms of LD in the cortical neurons but by absence of changes in the perineuronal glia. In the spinal motoneurons, on the other hand, the activity of all forms of LD was unchanged, whereas in the neuroglial cells surrounding these neurons activity of the H forms of LD was definitely increased. During the seizures an increase in activity of M forms of LD was found in the cerebellar Purkinje cells but a decrease in their activity in the glial satellite cells.     

Inhibition of inferior olivary transmission by mesencephalic stimulation in the cat

Cerebellar climbing fiber responses (CFRs) evoked in anesthetized cats by stimulation of peripheral nerves, contralateral inferior olive and cerebellar white matter were investigated by recording unit activity and surface field responses in anterior lobe of cerebellar cortex. When nerve and olive stimulation was preceded at long intervals (greater than 35 ms) by weak electrical stimulation of an ipsilateral mesencephalic area close to the locus coeruleus and brachium conjunctivum, CFRs could be virtually abolished in the pars intermedia but not in the vermis. White-matter evoked CFRs were not affected; thus the site of the inhibition was the inferior olive.     

Termination and functional organization of the dorsal spino-olivocerebellar path

1. The spino-olivocerebellar path ascending through the dorsal funiculus (DF-SOCP) was investigated in decerebrate cats with the cord transected in the third cervical segment except for the dorsal funiculi. The climbing fibre responses evoked in Purkinje cells were studied by recording the mass activity at the cerebellar surface and by recording from single cells.2. The DF-SOCP forms a disynaptic path from the spinal cord to the cerebellar cortex as shown by latency measurements. Anatomical studies have recently demonstrated that the relays are in the rostral part of the dorsal funiculus nuclei and in the dorsal accessory olive.3. The DF-SOCP projects to sagittal zones in the pars intermedia and vermis of the anterior lobe. The somatotopical organization is predominantly transverse in the pars intermedia and predominantly longitudinal in the vermis.4. The olivary neurones in the DF-SOCP are activated by the flexor reflex afferents from wide receptive fields. The fields are restricted to one ipsilateral limb and the majority of the olivary neurones could be activated from all the nerves tested in this limb.5. Natural stimulation of receptors evoked excitation in about half of the olivary neurones investigated. This excitation was elicited by pressure against deep structures. Inhibitory effects were rarely observed.6. The dorsal and ventral spino-olivocerebellar paths are compared.