Cerebellar nuclei and the nucleocortical projections in the rat: retrograde tracing coupled to GABA and glutamate immunohistochemistry.

The amino acids GABA and glutamate (Glu) are thought to be the principal substances in the central nervous system responsible for neuronal inhibition and excitation. Their distributions among the different neurons in a defined pathway may thus be indicative of the contributions of the cells to pathway function. Examples of such neurons are those of the cerebellar nuclei which, while regulating output from the Purkinje cells of the cerebellar cortex, are also found to project back to the cerebellar cortex. Immunohistochemical experiments were done to identify GABA and glutamate (Glu) containing cells in the adult rat cerebellar nuclei. Consecutive semithin and serial vibratome sections were incubated with antisera raised in rabbit against GABA and Glu. In semithin sections, only small neurons were intensely GABA immunoreactive (GABA-IR) (31.7%), and the majority (80.5%) were Glu immunoreactive (Glu-IR) of different sizes. Consistent with Glu being a metabolic precursor for GABA, 75.4% of the GABA-IR population colocalized Glu. In vibratome sections GABA-IR neurons showed some local differences in number, whereas the Glu-IR were uniformly distributed in the three nuclei studied. Measured mean diameters for these neurons showed a distinct size difference for the GABA- and Glu-IR with little overlap. Cerebellar nuclei neurons projecting to the cortex (nucleocortical neurons, NCN) were identified by locally preinjecting the retrograde transported WGA-apoHRP-colloidal gold complex in the cerebellar cortex. Vibratome sections of these cerebellar were silver intensified for the retrograde tracer and double labeled for GABA and Glu. Of the total number of identified NCN, 8.7% were GABA-IR (10 animals) and 47.7% Glu-IR (5 animals). Many retrograde labeled NCN in the core of the thick sections were immunonegative for both amino acids due to poor antibody penetration, thus underestimating the proportions of cells containing GABA and Glu. The size distributions for the GABA-IR and Glu-IR NCN were similar to those measured in non-retrograde labeled nuclei in thick sections. The conclusions reached are that GABA-IR neurons of the cerebellar nuclei, including the NCN, use GABA as the presumed inhibitory neurotransmitter and that Glu-IR neurons may use Glu or another excitatory neurotransmitter.

Nucleus medialis-nucleus interpositus interface: its olivary and cerebellocortical projections in the rat.

The nuclear target of the X zone of the cerebellar cortex was identified in rats as clusters of neurons scattered at the interface between the nuclei medialis (NM) and interpositus (NI). In a previous study, we had outlined these target neurons and termed them "interstitial cell groups" (icg). In order to determine whether the icg should be considered as part of either the NM or the medial NI, we analyzed two efferent pathways from the icg: their nucleocortical and nucleoolivary projections. These were compared to their homologues from the NM and the NI. This analysis is based on mapping retrograde cell labeling and anterograde terminal labeling following microinjections of tracers in either the cerebellar cortex, the cerebellar nuclei, or the inferior olive. Nucleocortical projections originating from the icg are of the three types described previously: a "reciprocal" projection to the ipsilateral X zone, a "nonreciprocal" projection to the ipsilateral A zone, and a "symmetrical" projection to the contralateral X zone. These features can be considered as the summed characteristics of the nucleocortical projections from the NM and from the medial NI. Nucleoolivary projections from the icg target the lateral-rostral portion of the dorsal accessory olive as well as the centrocaudal part of the medial accessory olive. These pathways converge with the nucleoolivary projections from the medial NI and from the NM, respectively. The icg receives olivary afferents from both the regions of the dorsal and medial accessory olives to which it projects. On the basis of similarities shown here between the two types of efferents originating from the icg and those from the NM as well as the medial NI, the icg may be regarded as a "mosaic" of neuron clusters alternately belonging to the NM and the medial NI. Therefore, the icg would be reciprocally connected with the inferior olive.

Brainstem efferents from the interface between the nucleus medialis and the nucleus interpositus in the rat.

In a previous report (Buisseret-Delmas et al. [1993] Neurosci. Res. 16:195-207), the authors identified the interface between the cerebellar nuclei medialis and interpositus as the origin of the nuclear output from cortical zone X. They named this nuclear interface the interstitial cell group (icg). In this study, the authors analyzed the icg efferents to the brainstem by using the anterograde and retrograde tracer biotinylated dextran amine. The main targets of these efferents are from rostral to caudal: 1) the accessory oculomotor nuclear region, essentially, the interstitial nucleus of Cajal; 2) the caudoventral region of the red nucleus; 3) a dorsal zone of the nucleus reticularis tegmenti pontis; 4) restricted regions of the four main vestibular nuclei; and 5) three restricted areas in the inferior olive, one that is caudal in the medial accessory subnucleus and two others that are rostral and caudal in the dorsal accessory subnucleus, respectively. These data support the notion that the icg contributes to the control of gaze-orientation mechanisms, particularly those that are related to the vestibuloocular reflex.

Organisation of reciprocal connections between trigeminal and vestibular nuclei in the rat.

In order to study the connection patterns between the sensory trigeminal and the vestibular nuclei (VN), injections of anterogradely and/or retrogradely transported neuronal tracers were made in the rat. Trigeminal injections resulted in anterogradely labelled fibres, with an ipsilateral preponderance, within the VN: in the ventrolateral part of the inferior nucleus (IVN), in the lateral part of the medial nucleus (MVN), in the lateral nucleus (LVN) with a higher density in its ventral half, and in the superior nucleus (SVN), more in the periphery than in the central part. Moderate trigeminal projections were observed in the small vestibular groups f, x and y/l and in the nucleus prepositus hypoglossi. Additional retrogradely labelled neurones were seen in the IVN, MVN, and LVN, in the same regions as those receiving trigeminal afferents. Morphological analysis of vestibular neurones demonstrated that vestibulo-trigeminal neurones are relatively small and belong to a different population than those receiving projections from the trigeminal nuclei. The trigeminovestibular and vestibulo-trigeminal relationships were confirmed by tracer injections in the VN. The results show that, in the VN, there is sensory information from facial receptors in addition to those reported from the neck and body. These facial afferents complement those from the neck and lower spinal levels in supplying important somatosensory information from the face and eye muscles. The oculomotor connections of the respective zones of the VN receiving trigeminal afferents suggest that sensory inputs from the face, including extraocular proprioception, may, through this pathway, influence the vestibular control of eye and head movements.

Cerebellar afferences from the mesencephalic trigeminal nucleus in the rat.

The aim of the present study was to test for and characterize the organization of a direct projection from neurones of the trigeminal mesencephalic nucleus (Vme) to the cerebellum. WGA-HRP was used as a retrograde tracer following injections in the cerebellar cortex. The extent of each injection site within the sagittal zones was determined according to corticonuclear and olivocortical connections. Retrogradely labelled neurons were observed in the caudal part of the ipsilateral Vme only following vermal injection. The Vme projections reached exclusively the ipsilateral sagittal zone X in the anterior lobe, lobule VI and lobule IX. This identification was confirmed by anterograde labelling of mossy fibre terminals following a biocytin injection restricted to the Vme.

On the caudal extension of the X zone in the cerebellar cortex of the rat.

Following a selective injection of biotinylated dextran amine in the nuclear target (the interstitial cell groups, icg) of the X zone of the rat cerebellum, retrogradely labelled Purkinje cells (PCs) were found within a longitudinal strip of cortex, 250 microns in width, 1000 microns lateral to midline. This labelling delineates two compartments in the X zone, one rostral through lobules II-VI, and one caudal through lobules VIII-X. The whole rostrocaudal extent of the icg appears to be the target of PCs from both compartments without any apparent topographical organization.

Trigeminocerebellar and trigemino-olivary projections in rats.

Retrograde and anterograde neuronal tracers (HRP, biocytin, biotinylated dextran-amine) were used to study the organisation of trigeminocerebellar and trigemino-olivary connections, focusing on the connectivity between trigeminal nuclear regions and the sagittal zones of the rat cerebellar cortex. Trigeminocerebellar projections were bilateral, but mostly ipsilateral. Direct trigeminocerebellar fibres originated mostly in the principal trigeminal nucleus (VP) and pars oralis (Vo), pars interpolaris (Vi), and to a lesser extent in pars caudalis (Vc) of the spinal trigeminal nucleus. Consistent projections were found from the Vc to cerebellar lobules IX and X. The trigeminal fibres terminated in the cerebellum in an organised fashion. The ventral part of the VP, Vo and Vi projected to the medial A zone and the C3 and D2 subzones, whereas the dorsal part of the nuclei projected to the lateral A zone and the C2, D0 and D1 subzones. In lobules IX and X, the organisation was different. The medial half of the VP, Vo, Vi and Vc projected to the lateral aspects of these lobules whereas their lateral part projected to their medial aspects. Trigeminal projections to the deep cerebellar nuclei were also present. Projections to a given sagittal zone concomitantly reached its corresponding nuclear target. Trigemino-olivary projections were principally contralateral. The Vo, Vi and Vc projected to the rostromedial dorsal accessory olive, the adjacent dorsal leaf and the dorsomedial part of the ventral leaf of the principal olive, which are known to project subzones C3, D0 and D1 of the rat cerebellar cortex.

Synaptic connections of Purkinje cell axons with nucleocortical neurones in the cerebellar medial nucleus of the rat.

The cerebellar nucleocortical neurones may be part of a cortico-nucleocortical loop. It has not yet been demonstrated, however, whether they are directly afferented by Purkinje cell axons. This question has been addressed by using electron microscopic methods. WGA-HRP injections into the cerebellar vermis anterogradely labelled Purkinje cell terminals and retrogradely labelled nucleocortical neurones of the nucleus medialis. Postembedding GABA immunolabelling was used to double-labelled PC terminals and identified the GABA-immunoreactive nuclear neurones. Of the identified nucleocortical neurones, the majority were immunonegative, but a few were GABA-immunoreactive. Both types were in synaptic contact with identified Purkinje cell terminals.

The GABAergic neurones of the cerebellar nuclei in the rat: projections to the cerebellar cortex.

The presence of gamma-aminobutyric acid (GABA) in the neurones of the cerebellar nucleocortical pathway is here reported. The pathway was identified by retrograde tracer and the GABA content was revealed immunohistochemically. It was found that most of the neurones giving rise to the reciprocal, non-reciprocal and symmetrical projections are indeed GABA-immunoreactive. They were observed in all the subdivisions of the nucleus medialis, of the nucleus interpositus and of the nucleus lateralis sending axons respectively to the sagittal zones A, C1-3 and D of the cerebellar cortex. The nucleus vestibularis lateralis and the related sagittal zone B were devoid of such projections.

Projection from trigeminal nuclei to neurons of the mesencephalic trigeminal nucleus in rat.

The mesencephalic trigeminal nucleus contains cell bodies of primary somatic sensory neurons that innervate the head region. The neurons resemble dorsal root ganglion cells but a striking difference is the presence of synaptic boutons in the nucleus. The present report demonstrates with anterograde tracers, the existence of a direct trigeminal projection from secondary sensory neurons of the principal and spinal nuclei to the mesencephalic nucleus. Our observations strongly suggest that synaptic contact may be established on the cell bodies as well as on the neurites of the mesencephalic neurons. These pathways could play a modulatory role in the processing of sensory afferent information and in the control of orofacial and/or oculomotor functions.

Connections between the cerebellar nucleus interpositus and the vestibular nuclei: an anatomical study in the rat.

Interposito-vestibular connections were analysed, using the anterograde and retrograde tracer biotinylated dextran amine. The interposito-vestibular projections mainly arise from medial portions of the cerebellar nuclei interpositi anterior (NIA) and posterior (NIP), and reach each of the main vestibular nuclei, ipsilaterally. The highest density of projections is found throughout nucleus vestibularis lateralis. Fibres also reach the peripheral part of nucleus superior, the caudal part of nucleus inferior, and the lateral part of nucleus medialis. Some fibres also reach groups I, x and f. Contralaterally, few fibres reach zones of the vestibular nuclei symmetric to the ipsilateral projection. A small, reciprocal, vestibulo-interposed projection is sent from the vestibular nuclei onto NIA-NIP. Possible influences of the interposito-vestibular projections upon the major targets of the vestibular nuclei, spinal motoneurones and oculomotor neurones, are discussed.

Colocalization of calbindin and GABA in medial nucleus of the trapezoid body of the rat.

Using immunocytochemical methods, both calbindin and GABA were found to be colocalized in the somas of all the cells of the medial nucleus of the trapezoid body (NMTB) of the rat auditory system. In the lateral superior olive (LSO), calbindin was also found in the terminals but not in the cells. Some terminal labelling was found in the medial superior olive (MSO). GABA was also found in the somas of some cells in both LSO and MSO, but most of the labelling was in terminals. In the rat, calbindin appears to be more involved in a pathway that detects interaural intensity differences.

Serotonergic projections from the nodose ganglia to the nucleus tractus solitarius: an immunohistochemical and double labeling study in the rat.

Possible projections of serotonin (5-HT)-immunoreactive neurons in the nodose ganglia (NG) to the nucleus tractus solitarius (NTS) were investigated in the rat using a double labeling method combining retrograde transport and 5-HT immunohistochemistry. After injection of a complex of colloidal gold-apo-horseradish peroxidase into the medio-caudal and commissural parts of the NTS, most of the 5-HT-immunoreactive neurons were found to be labelled by the gold complex. The present study provides direct evidence for the existence, in the rat, of a serotonergic NG-NTS system. This system may be involved in the regulation of blood pressure and vigilance states.

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.

[Anatomy and development of the visual system of Pleurodeles poiretti]. / Anatomie et developpement du système visuel de Pleurodèles poiretti.

After a deposit of HRP crystals in one eye of larval and adult Pleurodeles and the PAP immunocytochemical procedure, the organization of the visual system was described at stages 36, 45; 52 and postmetamorphic (according to Gallien and Durocher, 1975) and in the adult. At hatching stage, the retinopreoptic and the accessory pathways differentiate, soon after the retino-thalamico-pretectal pathway appears. The tectum differentiates later and a true retinotectal bundle can be seen only after the end of metamorphosis. In the whole, the pattern of the normal system is very simple in Pleurodeles especially as regard the ipsilateral fibres which never reach the tectum. This rudimentary organization lead us to conclude that the Pleurodeles and the Protopterus visual patterns are very similar.

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

Purkinje cells were recorded extracellularly and mapped in the cerebellar cortex of the rat under tremogenic doses of harmaline. Four different 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.