Morphological and functional studies on the serotoninergic innervation of the inferior olive

The inferior olive of the cat has, with fluorescence histochemistry, been shown to receive a rich serotoninergic innervation. The distribution of this innervation agrees with the topography of spinal afferent termination as well as the olivo-cerebellar climbing fiber projection. This indicates that different olivary compartments are under different serotoninergic influence. The serotoninergic innervation of the dorsal accessory nucleus (DAO) of the inferior olive of the rat has been identified with electron microscopic radioautography after labelling with 3H-serotonin. The serotoninergic varicosities contain microcanaliculi, tubular-vesicular organelles and large granular vesicles. Few of the serotoninergic varicosities engage in typical synaptic junctions. However, non-junctional varicosities often display other ultrastructural indications of polarity and directed transmitted release. Electrophysiological results indicate that the harmaline-induced tremor, as well as the tremor component of the "serotonin-syndrome", depends on the serotoninergic innervation of the inferior olive. Thus, the sensitivity of different olivary compartments to the induction of rhythmic, synchronous activity by harmaline parallels the distribution of serotoninergic innervation. Neurotoxic destruction of the serotoninergic innervation leads to decreased sensitivity to harmaline. Further, the serotonin receptor agonist 5-methoxy-N,N-dimethyltryptamine, as well as monoamine oxidase inhibition + L-tryptophan loading, leads to rhythmic mass climbing fiber activity in the cerebellum and whole body tremor. A neuromodulatory effect of serotonin on the olivary action potential mechanisms is proposed.     

Origin, distribution and organization of the serotoninergic innervation in the inferior olivary complex of the rat

The serotoninergic innervation of the inferior olivary complex of the rat was studied using a specific immunohistochemical technique. Fibers and varicosities positive for serotonin were present throughout the nucleus. The densest varicosities were found in the lateral portion of the dorsal accessory olive and the caudal portion of the medial accessory olive. The rostral medial accessory olive and the principal olive were sparsely populated with labeled elements. Ultrastructurally, labeled profiles were found in close opposition to small dendrites and to olivary cell bodies, but they did not display any synaptic specialization. Labeled perikaria were found in the periolivary regions, some of them located laterally to the olivary complex are responsible for the serotoninergic innervation of the dorsal accessory olive; some others located dorsally and medially in the nucleus raphe obscurus and raphe pallidus were responsible for the innervation of the medial accessory olive.     

Anatomical distribution and physiological effects of enkephalin in rat inferior olive

The opioid peptide enkephalin was evaluated as a possible synaptic transmitter in the inferior olivary complex using anatomical and physiological techniques. With standard immunohistochemical procedures, fibres and terminals containing enkephalin-like immunoreactivity were found to be heterogenously distributed in the inferior olive of the rat. Enkephalin-like immunoreactivity was found in the medial and dorsal accessory nuclei, but was sparse in the principal nucleus. Iontophoresis or pressure microapplication of Leu⁵-enkephalin, or a peptidase-resistant enkephalin analogue, depressed both spontaneous and amino acid-evoked firing of inferior olivary complex cells. The effect appeared to be postsynaptic, since the excitatory effects of iontophoretically-applied dl-homocysteic acid were suppressed. No selective effect on a calcium-mediated component of the action potential was seen. We conclude that enkephalin may act as a neurotransmitter or neuromodulator at afferent synapses in the inferior olive.     

大鼠下橄榄复合体至小脑旁绒球和绒球的投射：HRP逆行追踪的研究

应用辣根过氧化物酶（ＨＲＰ）做逆行标记,以四甲基联苯胺（ＴＭＢ）,为呈色剂,对成年大鼠下橄榄复合体到小脑分级球和线球的纤维投射进行了详细的研究。结果显示旁线球和线球接受对侧下橄榄复合体的纤维投射。其中,绒球只接受背帽（ＤＣ）和内侧副橄榄核（ＭＡＯ）嘴段的投射;旁绒球接受ＭＡＯ、橄榄主核（ＰＯ）及少量腹外延伸（ＶＬＯ）的投射。但是,未观察到由下橄榄复合体的背侧副橄榄核（ＤＡＯ）到旁绒球或绒球的投射。此外,对大鼠橄榄旁绒球和橄榄绒球投射的带型以及下橄榄与绒球和旁线球的功能做了相关的讨论。     

Fine structural analysis of calcitonin gene-related peptide in the mouse inferior olivary complex

Climbing fiber afferents to the cerebellum, from the inferior olivary complex, have a powerful excitatory effect on Purkinje cells. Changes in the responsiveness of olivary neurons to their afferent inputs, leading to changes in the firing rate or pattern of activation in climbing fibers, have a significant effect on the activation of cerebellar neurons and ultimately on cerebellar function. Several neuropeptides have been localized in both varicosities and cell bodies of the mouse inferior olivary complex, one of which, calcitonin gene related peptide (CGRP), has been shown to modulate the activity of olivary neurons. The purpose of the present study was to investigate the synaptic relationships of CGRP-containing components of the caudal medial accessory olive and the principal olive of adult mice, using immunohistochemistry and electron microscopy. The vast majority of immunoreactive profiles were dendrites and dendritic spines within and outside the glial boundaries of synaptic glomeruli (clusters). Both received synaptic inputs from non-CGRP labeled axon terminals. CGRP was also present within the somata of olivary neurons as well as in profiles that had cytological characteristics of axons, some of which were filled with synaptic vesicles. These swellings infrequently formed synaptic contacts. At the LM level, few, if any, CGRP-immunoreactive climbing fibers, were seen, suggesting that CGRP is compartmentalized within the somata and dendrites of olivary neurons and is not transported to their axon terminals. Thus, in addition to previously identified extrinsic sources of CGRP, the widespread distribution of CGRP within olivary somata and dendrites identifies an intrinsic source of the peptide suggesting the possibility of dendritic release and a subsequent autocrine or paracrine function for this peptide within olivary circuits.     

Detailed organization of cerebello-olivary projections in the cat. An autoradiographic study

Several small injections of tritiated leucine were placed in regions of the cerebellar nuclei and sites of distribution in the olive were revealed by autoradiography. Different survival periods from a few hours to several days were utilized and high or low specific activity tracers were evaluated. A general pattern of distribution whereby the dentate, anterior and posterior interposed respectively project to principal, dorsal accessory and medial accessory olives was confirmed. Several new details of distributions from regions of the interposed and dentate nuclei to parts of the olivary subdivisions were demonstrated. The pattern of representations is complex and distributed in three dimensions. It is very precise and approximates a point to point representation. However, these projections which appear relatively dense do not represent the only link between the cerebellar nuclei and olive. With survival periods of a few hours, additional lighter projections distributed according to a different pattern can be revealed. A small ipsilateral projection was observed and it is suggested that it is made of collaterals of the crossed fibers.     

Role of olivary electrical coupling in cerebellar motor learning

The level of electrotonic coupling in the inferior olive is extremely high, but its functional role in cerebellar motor control remains elusive. Here, we subjected mice that lack olivary coupling to paradigms that require learning-dependent timing. Cx36-deficient mice showed impaired timing of both locomotion and eye-blink responses that were conditioned to a tone. The latencies of their olivary spike activities in response to the unconditioned stimulus were significantly more variable than those in wild-types. Whole-cell recordings of olivary neurons in vivo showed that these differences in spike timing result at least in part from altered interactions with their subthreshold oscillations. These results, combined with analyses of olivary activities in computer simulations at both the cellular and systems level, suggest that electrotonic coupling among olivary neurons by gap junctions is essential for proper timing of their action potentials and thereby for learning-dependent timing in cerebellar motor control.     

Mapping of alpha-melanocyte-stimulating hormone-like immunoreactivity in the cat brainstem

The distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures was studied in the brainstem of the cat using an indirect immunoperoxidase technique. Immunoreactivity was observed in several brainstem nuclei of the cat in which no immunoreactivity had been previously reported. Immunoreactive fibres were observed in the following; the inferior central nucleus; the pontine gray nuclei; the K?lliker-Fuse nucleus; the motor trigeminal nucleus, the anteroventral cochlear nucleus; the abducens nucleus; the retrofacial nucleus; the superior, lateral, inferior, and medial vestibular nuclei; the lateral nucleus of the superior olive; the external cuneate nucleus; the nucleus of the trapezoid body; the postpyramidal nucleus of the raphe; the medial accessory inferior olive; the dorsal accessory nucleus of the inferior olive; the nucleus ambiguus; the principal nucleus of the inferior olive; the preolivary nucleus; the nucleus ruber; the substantia nigra; and in the area postrema. Our results point to a more widespread distribution of alpha-melanocyte-stimulating hormone-like immunoreactive structures in the cat brainstem than that reported in previous studies carried out in the same region of the cat, rat and humans.     

Signals from the cerebellum guide the pathfinding of inferior olivary axons

During development, inferior olivary axons cross the floor plate and project from the caudal to the rostral hindbrain, whence they grow into the cerebellar plate. We have investigated the axon guidance signals involved in the formation of this projection in vitro. When the cerebellar plate was grafted ectopically along the margin of the hindbrain in organotypic cultures, inferior olivary axons could pathfind to the ectopic cerebellum, establishing a topographically normal projection. Following rostrocaudal reversal of a region of tissue in the axon pathway between the inferior olive and the cerebellum, olivary axons still navigated towards the cerebellum. Moreover, olivary axons could cross a bridging tissue explant (spinal cord) to reach a cerebellar explant. In collagen gel cultures of inferior olive explants, olivary axon outgrowth increased significantly in the presence of cerebellar explants and axons deflected towards the cerebellar tissue. These results show that the cerebellum is a source of diffusible axon guidance signals for olivary axons. We also found that, in organotypic cultures, olivary axons which had crossed the floor plate showed an increased tendency to respond to cerebellar cues. Taken together, these results indicate that the cerebellum is the source of cues that are chemoattractant and growth-promoting for inferior olivary axons; prior exposure to the floor plate increases responsiveness to these cues.     

To beat or not to beat: a decision taken at the network level.

The cells of the inferior olivary nucleus, the sole source of the cerebellar climbing fibers, form a network of electrically coupled neurons. Experimental observations show that these neurons produce a large repertoire of electrical signals, among which sub-threshold oscillations of the membrane potential. Simultaneous recordings from pairs of neurons and optical imaging of voltage sensitive dyes show that sub-threshold activity occurs in synchrony throughout the network. The mechanism underlying the generation of the sub-threshold oscillations is not fully understood. Experimental observations suggest that the electrical coupling is essential but insufficient for their generation. Several theoretical mechanisms have been suggested to explain these observations. Up-to-date, the most realistic model is the heterogeneity model, that assumes a certain degree of heterogeneity among olivary neurons. The heterogeneity model proposes that sub-threshold oscillations are produced by electrical coupling of neurons with the same types of ionic conductances, but with different densities. The variability in channel densities yield neurons of different functional types. The main prediction of the model is that different functional types of neurons should be found in the inferior olive. Dynamic clamp experiments support this prediction.     

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.     

Hypertrophy of inferior olivary neurons: a degenerative, regenerative or plasticity phenomenon

After contralateral hemi-cerebellectomy, neurons in the cat inferior olive may either degenerate, appear unchanged (affected) or become hypertrophic. Morphological and physiological aspects of the latter two cell types are studied by means of intracellular recording and injection techniques and compared to normal olivary neurons. It is demonstrated that affected and hypertrophic olivary neurons can be activated by mesodiencephalic stimulation. Affected olivary neurons are morphologically very similar to normal cells. However, they may respond with long latency action potentials only to mesodiencephalic stimulation. Hypertrophic olivary neurons have an enlarged dendritic tree and soma. The soma and proximal dendrites are studded with spine-like processes. Their reaction to mesodiencephalic stimulation is very diverse and may consist of short and/or long latency action potentials that may or may not trigger dendritic spikes. It is argued that olivary hypertrophy does not present either a degenerative or regenerative state, but that both hypertrophic as well as affected olivary neurons can survive axotomy due to a strong and continuous electrotonic coupling, made possible by destruction of the GABAergic cerebellar afferents.     

A critical period for retinoic acid teratogenesis and loss of neurophilic migration of pontine nuclei neurons

Abnormalities in the pontine nuclei (PN) and inferior olive are hallmarks of human retinoic acid (RA) teratogenesis. This study shows that RA exposure of the mouse at a specific embryonic stage alters morphological structures that derive from the wall of the IVth ventricle to form components of the precerebellar system (the inferior olivary nucleus and the PN). The study employs both normal and a RAREhspLacZ transgenic RA reporter mouse. It is shown that abnormalities in the PN and inferior olive result from exposure at a critical period of embryonic day 9.5 and 10.5. The abnormalities in the PN are due to a failure in their usual neurophilic migration. The compact stream of cells that leads from the anterior rhombic lip to the ventral pons is instead scattered widely over the anterior medulla. Given that the RA exposure occurs after the resolution of rhombomere identity this suggests that teratogenic RA interferes with a regulatory event that overlays this original pattern.     

Multiunit activity of the inferior olive during a conditioned motor sequence]

Multinuit activity from the inferior olive was recorded in chronic cats during a learned motor task. The animals were trained to perform a succession of rapid flexion-extension arm movements alternating with two maintained postures. No significant differences were observed in the olivary activity during maintained postures. However an increase of activity occurred before the beginning of the flexion detected on the biceps EMG recordings. The first modifications of olivary activity occurred in synchrony with postural reorganization preceding the flexion. This latter involved primarily the triceps. The increase of activity took place during the execution of movement and ended after the reaching of the target.     

Ascending and descending projections to the inferior colliculus in the rat

The ascending and descending projections to the central nucleus of the inferior colliculus (IC) were studied with the aid of retrograde transport of horseradish peroxidase (HRP). HRP-labelled cells were found in contralateral cochlear nuclei, where the majority of different cell types was stained. Few labelled cells were observed in the ipsilateral cochlear nuclei. HRP-positive neurones were found in all nuclei of the superior olivary complex on the ipsilateral side with the exception of the medial nucleus of the trapezoid body, which was never labelled either ipsilaterally or contralaterally. The largest concentration of HRP-labelled cells was usually observed in the ipsilateral superior olivary nucleus. Smaller numbers of labelled cells were present in contralateral nuclei of the superior olivary complex. Massive projections to the inferior colliculus were found from the contralateral and ipsilateral dorsal nucleus of the lateral lemniscus and ipsilateral ventral nucleus of the lateral lemniscus. Many neurones of the central and external nuclei of the contralateral inferior colliculus were labelled with HRP. Topographic organisation of the pathways ascending to the colliculus was expressed in the cochlear nuclei, lateral superior olivary nucleus and in the dorsal nucleus of the lateral lemniscus. HRP--positive cells were found in layer V of the ipsilateral auditory cortex, however, the evidence for topographic organisation was lacking.     

Localization of alcohol and aldehyde dehydrogenases in the human spinal cord and brain

Location of aldehyde dehydrogenase (AldDG) and alcohol dehydrogenase (ADG) has been studied in 38 nuclei of the human brain. Neurons with a high AldDG activity predominate in the nucleus of the descending root of the trigeminal nerve, motor nuclei of the craniocerebral nerves (trigeminal, facial, abducent, blocking, sublingual, supraspinal), motor nuclei of the anterior horns of the spinal cord, lateral vestibular nucleus, posterior nucleus of the vagus nerve, pedunculopontine nucleus, superior salivary nucleus, and in the nucleus of Westphal-Edinger-Jacobovich. Neurons with a moderate AldDG activity predominate in the superior olivary complex, nucleus of the lateral loop, parabrachial (pigmented) mesencephalic nucleus and reticular lateral nucleus. A low enzymatic activity is specific for neurons of the pons proper, inferior vestibular nucleus, trapezoid body of the inferior olivary complex, dentate nucleus of the cerebellum, reticular nucleus of the tegmen of Bekhterev's pons and posterior nucleus of Gudden's suture. A high ADG activity is revealed in piriform neurons of the cerebellar cortex. Functional importance of ADG and AldDG activity in the brain is discussed.     

Spatial and temporal changes in natural and target deprivation-induced cell death in the mouse inferior olive

The survival of inferior olive neurons is dependent on contact with cerebellar Purkinje cells. There is evidence that this dependence changes with time. Because inferior olivary axons, called climbing fibers, already show significant topographical ordering in cerebellar target zones during late embryogenesis in mice, the question arises as to whether olive neurons are dependent on target Purkinje cells for their survival at this early age. To better characterize this issue, inferior olive development was studied in two transgenic mouse mutants, wnt-1 and L7ADT, with embryonic and early postnatal loss of cerebellar target cells, respectively, and compared to that in the well-studied mutant, Lurcher. Morphological criteria as well as quantitative measures of apoptosis were considered in this developmental analysis. Survival of inferior olive neurons is observed to be independent of Purkinje cells throughout embryogenesis, but dependence begins immediately at birth in both wild types and mutants. Thereafter, wild types and mutants show a rapid increase in olive cell apoptosis, with a peak at postnatal day 4, followed by a period of low-level, but significant, apoptosis that continues to at least postnatal day 11; the main difference is that apoptosis is quantitatively enhanced in the mutants compared to wild types. The multiphasic course of these effects roughly parallels the known phases of climbing fiber synaptogenesis. In addition, despite significant temporal differences among the mutants with respect to absolute numbers of dying cells, there are common spatial features suggestive of distinct intrinsic programs linking different olivary subnuclei to their targets.     

Morphological study on the inferior olivary nuclear complex of the donkey (Equus asinus)

This study provides basic data on the normal structure of the inferior olivary complex (IOC) of the donkey, Equus asinus, at the light microscopic level. In common with that of other mammals, the donkey IOC consisted of three major nuclei and four minor groups of cells. The former was comprised of the medial and dorsal accessory olives (MAO and DAO, respectively) and the principal olive (PO), and the latter was comprised of the dorsal cap, nucleus beta, ventrolateral outgrowth and dorsomedial cell column. The MAO had the longest rostral to caudal representation and formed the caudal pole of IOC. The DAO was located dorsally to the MAO in the caudal half of the IOC. In the rostral half, the DAO bended ventrally and merged with the dorsal lamella of PO. More rostrally, the DAO lost its connection with the dorsal lamella and then conversely connected with the ventral lamella of PO. The DAO formed the rostral pole of the IOC. The PO extended through the rostral half of the IOC. The dorsal cap was a small group of cells. Overall, the donkey IOC is similar to that of other mammals.     

Structure of Neuronal Impulse Trains of the Rat Inferior Olive under Conditions of Long-Lasting Vibrational Influence

In acute experiments on albino rats anesthetized with Nembutal (40 mg/kg, i.p.), we recorded the background impulse activity (BIA) generated by neurons of the inferior olive in the norm and after 5-, 10-, and 15-daylong vibrational influence (60 Hz, 2 h, daily). We characterized the distributions of neurons according to the regularity of impulse successions, their dynamics, and pattern of histograms of interspike intervals (ISIs); we also calculated the mean frequency of impulsation and the coefficient of variation of ISIs. It was demonstrated that the most significant shifts of the characteristics of BIA generated by neurons of the inferior olive were formed within the first 10 days of the vibrational influence. These shifts were observed mainly in the mean discharge frequency (increased within the initial period) and, to a lesser extent, in the intrinsic structure of impulse trains. Such shifts in the background activity of the inferior olive caused by long-lasting vibrational influence result, perhaps, from intensification of the influences of excitatory cerebellar/mesodiencephalic inputs to olivary neurons within the early periods of action of the above factor and prevalence of GABAergic influences within the later periods. It seems possible that, under such conditions, the characteristics of electrical synapses of the olivary neurons are also subjected to modification. Neirofiziologiya/Neurophysiology, Vol. 40, No. 4, pp. 340–347, July–August, 2008.