Regional distribution of 5-HT transporters in the brain of wild type and `Purkinje cell degeneration' mutant mice: a quantitative autoradiographic study with [H]citalopram

The neurological mutant `Purkinje cell degeneration' (pcd) is characterized by a primary degeneration of Purkinje cells, as well as by retrograde and secondary partial degeneration of cerebellar granule cells and inferior olivary neurons, and can be considered as an animal model of human degenerative ataxias. The serotonin (5-HT) innervation was examined in wild type and pcd mice, by quantifying 5-HT uptake sites, or transporters, using [<sup>3</sup>H]citalopram binding autoradiography. In both wild type and pcd mutants, the highest densities of 5-HT transporters were in mesencephalic and rostral pontine regions, in limbic structures, in hypothalamus and in discrete thalamic divisions, while the lowest labelling was found in cerebellum and brainstem reticular formation. In pcd mice, although [<sup>3</sup>H]citalopram labelling was higher in cerebellar cortex and deep cerebellar nuclei, when binding densities were corrected for surface area, the up-regulation of 5-HT transporters was present only in deep cerebellar nuclei. Also, higher labelling was found in nuclei raphe dorsalis and medialis, in ventral divisions of rostral neostriatum, caudal neostriatum, rostral globus pallidus, posteromedial amygdaloid nucleus, septum, olfactory tubercles, vertical limb of Broca's diagonal band, periventricular, latero-ventral and medio-ventral thalamic nuclei, medial geniculate nucleus, anterior hypothalamus and entorhinal cortex. The results indicate a relative integrity of the 5-HT innervation, but with a reorganization of serotoninergic terminals in the cerebellum, in particular in the deep cerebellar nuclei. This suggests that in progressive cerebellar degeneration, as found in the pcd mutant, the modified 5-HT system may still participate in motor functions by exerting an overall modulation of excitatory amino acid neurotransmission, but the availability of 5-HT may be altered in defined brain targets, as is the case for other spontaneous cerebellar mutants, in particular for the `Lurcher' mutant mouse, a model of human olivopontocerebellar atrophy.     

Regional brain distribution of noradrenaline uptake sites, and of alpha1-alpha2- and beta-adrenergic receptors in PCD mutant mice: a quantitative autoradiographic study

The mouse "Purkinje cell degeneration" (pcd) is characterized by a primary loss of Purkinje cells, as well as by retrograde and secondary partial degeneration of cerebellar granule cells and inferior olivary neurons; this neurological mutant can be considered as an animal model of human degenerative ataxia. To determine the consequences of this cerebellar pathology on the noradrenergic system, noradrenaline transporters as well as alpha1-, alpha2- and beta-adrenergic receptors were evaluated by quantitative ligand binding autoradiography in adult control and pcd mice using, respectively, [3H]nisoxetine, [3H]prazosin, [3H]idazoxan and [3H]CGP12177. In cerebellar cortex and deep nuclei of pcd mutants, [3H]nisoxetine labelling of noradrenaline transporters was higher than in control mice. However, when binding densities were corrected by surface area, they remained unchanged in the cerebellar cortex but associated with 25% and 40% lower levels of labelling of alpha1 and beta receptors, as well as a very important increase (275%) of alpha2 receptors. In deep cerebellar nuclei, surface corrections did not reveal any changes either in transporter or in receptor densities. Higher densities of [3H]nisoxetine labelling were found in several regions related with the cerebellum, namely inferior olive, inferior colliculus, vestibular, reticular, pontine, raphe and red nuclei, as well as in primary motor and sensory cerebral cortex; they may reflect an increased noradrenergic innervation related to motor adjustments for the cerebellar dysfunction. Increased [3H]nisoxetine labelling was also measured in vegetative brainstem regions and in dorsal hypothalamus, implying altered autonomic functions and possible compensation in pcd mutants. Other changes found in extracerebellar regions affected by the mutation, such as thalamus and the olfactory system implicated both noradrenaline transporters and adrenergic receptors. In contrast to the important alterations of the noradrenergic system in cerebellar cortex, the lack of receptor changes in deep cerebellar nuclei suggests that local adaptations may be sufficient to minimize the consequence of the cerebellar atrophy on motor control. An intense labelling by [3H]idazoxan of the inner third of the molecular layer was a novel, albeit unexplained finding, and could represent a postsynaptic subset of alpha2-adrenergic receptors.     

Afferents of the caudal fastigial nucleus in a New World monkey (Cebus apella)

Summary The afferents of the fastigial nucleus (FN) were studied in two capuchin monkeys (Cebus apella) one of which had received a unilateral injection of horseradish peroxidase in the caudal FN, and a second monkey which received a control injection that involved the lateral caudal FN but extended into the cerebellar white matter between the FN and posterior interposed nucleus (PIN). All of the sources of FN afferents were found to be labeled bilaterally. In addition to the restricted distribution of labeled Purkinje cells in lobules VI and VII of the posterior lobe vermis (oculomotor vermis), retrogradely labeled cells were present in the dorsolateral pontine nucleus (DLPN), dorsomedial pontine nucleus (DMPN), nucleus reticularis tegmenti pontis (NRTP), pontine raphe (PR), paramedian nucleus reticularis pontis caudalis (NRPC), nucleus prepositus hypoglossi (NPH), subnucleus b of the medial accessory olivary nucleus (sbMAO), and vestibular complex (VC). The second (control) injection appeared to confirm a proposed (Langer et al. 1985b) projection from the flocculus to the basal interstitial nucleus. The results are discussed in terms of the functional relationships of the FN to the frontal eye field and oculomotor-related brainstem structures involved in the production of saccadic and smooth pursuit eye movements.Abbreviations AIN anterior interposed nucleus, cerebellum - BC brachium conjunctivum (sup. cerebellar peduncle) - BIN basal interstitial nucleus, cerebellum - BPN basilar pontine nuclei - CS corticospinal (pyramidal) tract - DBC decussation of brachium conjunctivum - DLPN dorsolateral pontine nucleus (Nyby and Jansen 1951) - DMPN dorsolateral pontine nucleus - DN dentate nucleus, cerebellum - DNV descending nucleus of the trigeminal complex (C.N. V) - FN fastigial nucleus, cerebellum - IC inferior colliculus - ICP inferior cerebellar peduncle - IVN inferior vestibular nucleus - LCN lateral (external) cuneate nucleus - LPN lateral pontine nucleus - MAO medial accessory nucleus of the inferior olivary complex - ML medial lemniscus - MLF medial longitudinal fasciculus - MRF medullary reticular formation (nuc. retic. gigantocellularis) - MVN medial vestibular nucleus - NI nucleus intercalatus of Staderini of the perihypoglossal complex - NPH nucleus prepositus hypoglossi - NRPC nucleus reticularis pontis caudalis - NRPO nucleus reticularis pontis oralis - NRTP nucleus reticularis tegmenti pontis - PIN posterior interposed nucleus - PR pontine raphe - sbMAO subnucleus b, medial accessory olivary nucleus - SC superior colliculus - sbPVG supragenial peri- (IVth) ventricular gray - SVN superior vestibular nucleus - VIn abducens nerve (C.N. VI) - XIIn hypoglossal nerve (C.N. XII)     

A temporal analysis of the origin and distribution of serotoninergic afferents in the cerebellum of pouch young opossums

Summary In the present study, a temporal analysis of the pattern of distribution of serotoninergic fibers and varicosities within the cerebellum of pouch young opossums was carried out. Particular attention was focused on animals ranging in age from postnatal day (PD) 21-PD 72, because there is a transient expression of serotonin immunoreactivity in the cerebellar cortex during that interval. Between PD 1–33, there is a progressive increase in serotoninergic immunoreactivity throughout the cerebellar cortex. After PD 33, there is a decrease in the relative number of immunostained fibers followed by a reorganization into the adult pattern of distribution.A double labeling paradigm, in which horseradish peroxidase, used as a retrograde marker, combined with serotonin immunohistochemistry was employed to localize serotoninergic neurons that project to the developing cerebellum. Initially (PD 9), serotoninergic cells in the medullary reticular formation and dorsolateral pontine tegmentum are double labeled. After PD 77, only neurons in the medullary reticular formation were double labeled.The course taken by serotoninergic axons from the brainstem to the cerebellum also was analyzed. Between PD 1 and PD 42, serotoninergic axons enter the cerebellum via four different routes: 1) the inferior cerebellar peduncle; 2) a pathway located lateral and rostral to the inferior cerebellar peduncle; this bundle of serotonin axons contains immunoreactive fibers that also enter the tectum (this tract is referred to as the tecto-cerebellar bundle in this report); 3) the medial aspect of the superior cerebellar peduncle; and 4) the tela choroidea. After PD 40, the latter two pathways are the primary routes by which serotoninergic fibers enter the cerebellum. The loss of serotoninergic fibers in the first two pathways coincides with the decrease in serotoninergic immunoreactivity seen in the cerebellar cortex described above.In summary, the results suggest that the serotoninergic projection to the opossum's cerebellum is remodelled during development. It is proposed that the serotonin fibers present at early stages of development may play a role in regulating specific events in cerebellar maturation. In contrast, the serotoninergic axons which have a more restricted pattern of distribution later in development, and in the adult, likely modulate neuronal activity within the cerebellum.Abbreviations BP basilar pons - CB cerebellum - CF cephalic flexure - CN cerebellar nuclei - CRI crus I - CRII crus II - DAO dorsal accessory olive - DN dentate nucleus - EGL external granule cell layer - F flocculus - FP primary fissue - ICP interior cerebellar reduncle - IO inferior olive - IOC inferior olivary complex - IV fourth ventricle - LS lobus simplex - MCP middle cerebellar peduncle - MED medulla - MID midbrain - PF pontine flexure - PFL paraflocculus - PML paramedian lobule - PN pontine nuclei - PT pontine tegmentum - RA raphe - RGc _v nucleus reticularis gigantocellularis pars ventralis - SO superior olive - SV superior medullary velum - TC tela choroidea - TE tectum - VII facial nucleus - Roman numerals I–X cerebellar lobules     

The site of origin of calcitonin gene-related peptide-like immunoreactive afferents to the inferior olivary complex of the mouse.

The intent of the present study is to define the brainstem nuclei which give rise to CGRP-immunolabeled afferents to the inferior olivary complex of the mouse. A technique which combines retrograde transport of fluorescent microspheres with immunohistochemistry was used to address this question. In the present study, intensely labeled CGRP neurons were localized within several cranial nerve nuclei including the hypoglossal, facial, oculomotor, motor nucleus of the trigeminal nerve and nucleus ambiguus, as well as in the parabrachial nucleus, locus coeruleus and medullary and pontine reticular formation. In addition, lightly labeled CGRP neurons were identified within the deep cerebellar nuclei, the inferior olivary complex, lateral reticular nucleus, medial and lateral vestibular nuclei, nucleus Darkschewitsch, interstitial nucleus of Cajal, the central gray area adjacent to the third ventricle, and the zona incerta. The origin of the projection to the inferior olivary complex primarily arises from the deep cerebellar nuclei, the locus coeruleus, and the central gray matter of the mesodiencephalic area. In addition, a small CGRP input is derived from the superior and lateral vestibular nuclei as well as the zona incerta. In conclusion, we have identified several extrinsic sources of CGRP to the inferior olivary complex and have localized it within afferents that have been shown to have either excitatory (mesodiencephalic nuclei) or inhibitory (cerebellar nuclei) effects on olivary circuits. The presence of CGRP in these functionally diverse brainstem and cerebellar afferents suggests that the peptide may act as a co-transmitter to modulate the activity of olivary neurons.     

The afferent connections of the inferior olivary complex in rats. An anterograde study using autoradiographic and axonal degeneration techniques

Autoradiographic and axonal degeneration techniques were employed to determine the distribution patterns of inferior olivary afferents whose origins were determined using the horseradish peroxidase method.⁷⁰ The Fink-Heimer stain for degenerating axons was used following lesions of the cerebral cortex and spinal cord, while brainstem and cerebellar afferents were mapped by tritiated leucine autoradiography.After unilateral lesions of the mid-thoracic spinal cord, degenerating axons were observed within the subnuclei a and b of the caudolateral medial accessory olive and in the caudolateral dorsal accessory olive. Degeneration after upper cervical cord lesions extended more rostrally and medially within the same olivary subdivisions.Several nuclei within the caudal brainstem projected to the inferior olivary complex. The dorsal column nuclei distributed fibers primarily contralaterally to the lateral part of the dorsal accessory olive and to the caudolateral part of the medial accessory olive; the spinal trigeminal nucleus projected contralaterally to the rostromedial dorsal accessory olive; the medial and inferior vestibular nuclei projected to the ipsilateral subnuclei b, c, and β of the medial accessory olive and to the contralateral dorsomedial cell column; the nucleus prepositus hypoglossi sent fibers to the subnuclei c and β, the dorsal cap and the ventrolateral outgrowth; the lateral reticular nucleus projected to the subnucleus a of the caudolateral medial accessory olive bilaterally; and the reticular formation distributed fibers to the dorsal accessory olive contralaterally and to the β subnucleus ipsilaterally.Study of inferior olivary complex afferents from the deep cerebellar nuclei showed a projection from the fastigial nucleus to the β subnucleus and the ventrolateral outgrowth. The dentate and interpositus nuclei demonstrated topographic connections from these nuclei to the principal olive and accessory olives, respectively. All cerebellar connections were predominantly contralateral.Analysis of mesencephalic and diencephalic areas also demonstrated several inferior olivary complex afferent systems: the caudal pretectum and the superior colliculus projected to the subnucleus c contralaterally and the dorsal lamella of the principal olive ipsilaterally; the nucleus of the optic tract sent fibers to the dorsal cap; the lateral deep mesencephalic nucleus distributed fibers to the ipsilateral dorsal accessory olive and β subnucleus; the medial terminal nucleus of the accessory optic tract projected ipsilaterally to the ventrolateral outgrowth; and several areas including the medial deep mesencephalic nucleus, periaqueductal gray, the nucleus of Darkschewitsch, the subparafascicular nucleus, the rostral red nucleus and the prerubral field all projected ipsilaterally to the principal olive, rostral medial accessory olive, ventrolateral outgrowth and, to a lesser extent, the caudal medial accessory olive, dorsal cap and β subnucleus.Lesions of the frontal cortex produced axonal degeneration primarily ipsilaterally within many olivary subdivisions, especially the medial dorsal accessory olive and the caudomedial medial accessory olive.Although some notable differences in the distribution and laterality of fibers are described, our findings generally corroborate several earlier reports which used different techniques on a variety of species. Inferior olivary afferents from functionally related areas typically demonstrated similar distribution patterns within the subdivisions of the inferior olivary complex. These patterns suggest a functional localization within the inferior olivary complex which may facilitate an understanding of afferents from areas whose functions are not clearly known.     

立体学在分析人延髓脑桥毛细血管网密度的初步应用

采用立体学方法对5例人延髓脑桥毛细血管密度作了观测。结果表明:核团的血管比纤维束的丰富;血管密度高的核团包括了脑桥核、下橄榄核簇等与小脑相连系的核团,及楔束核、上橄榄核和展神经核;纤维束中以被盖中央束的血管密度最高。     

Transsynaptic degeneration in the inferior olivary nucleus associated with pontine glioblastoma

A case of glioblastoma arising in the pons of a 14-year-old boy in whom transsynaptic degeneration was found in the inferior olivary nucleus is reported. The tumor occupied most of the pons including the tegmental tract and invaded into the midbrain, medulla oblongata, cerebellar peduncles, thalamus, basal ganglia, and meninges. The right inferior olivary nucleus was devoid of the tumorous lesion, but many neurons were severely vacuolated. An immunohistochemical study using glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), and S-100 protein was performed. GFAP and S-100 protein were positive in the reactive glia of the nucleus and NSE gave a faint reaction in some degenerated neurons. These degenerative changes found in neurons of the inferior olivary nucleus were considered to be transsynaptic degeneration due to the destruction of the tegmental tract at the pons and of cerebellar peduncles by invasive pontine glioblastoma.     

The GABAergic cerebello-olivary projection in the rat

Summary Immunocytochemical detection of glutamate decarboxylase (GAD), the predominant biosynthetic enzyme of gamma-aminobutyric acid (GABA), reveals the presence of a dense GABAergic innervation in all parts of the inferior olive. One brain center that provides a substantial projection to the inferior olive is the cerebellar nuclei, which contain many small GABAergic neurons. These neurons were tested as a source of GABAergic olivary afferents by combining retrograde tract tracing with GAD immunocytochemistry. As expected from previous studies, injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the inferior olive retrogradely label many small neurons in the interposed and lateral cerebellar nuclei and the dorsal part of the lateral vestibular nucleus, and fewer neurons in the ventro-lateral region of the medial cerebellar nucleus. These projections are predominantly crossed and are topographically arranged. The vast majority, if not all, of these projection neurons are also GAD-positive. The relative contribution of this projection to the GABAergic innervation of the inferior olive was tested by lesion of the cerebellar nuclei, or the superior cerebellar peduncle. Within 10 days the lesion eliminates most GAD-immunoreactive boutons in the principal olive, the rostral lamella of the medial accessory olive, the ventrolateral outgrowth, and the lateral part of the dorsal accessory olive ventral fold. Thus, the effectiveness of this depletion demonstrates that the cerebellar nuclei provide most of the GABAergic innervation to regions of the inferior olive known to receive a cerebellar projection. Moreover, when the lateral vestibular nucleus is damaged, the dorsal fold of the dorsal accessory olive is depleted of GABAergic boutons. The synaptic relations that boutons of the GABAergic cerebello-olivary projection share with olivary neurons were investigated at the electron microscopic level by GAD-immunocytochemistry, anterograde degeneration of the cerebellar axons or anterograde transport of WGA-HRP. All of these methods confirm that GABAergic, cerebello-olivary axon terminals contain pleomorphic vesicles, and synapse on various portions of olivary neurons, and especially on dendritic spines within glomeruli, often in very close proximity to the gap junctions that characteristically couple the dendritic profiles. These results demonstrate four major points: that virtually all of the GABAergic, and presumably inhibitory, neurons of the cerebellar and dorsal lateral vestibular nuclei are projection neurons; that a large portion of the inferior olive receives GABAergic afferents from the cerebellar nuclei; that a portion of the dorsal accessory olive receives GABAergic afferents from the dorsal lateral vestibular nucleus; and that cerebello-olivary fibers often synapse near gap junctions, and therefore could influence electrical coupling of olivary neurons.Abbreviations aMAO subnucleus a of MAO - beta beta nucleus - bMAO subnucleus b of MAO - cMAO subnucleus c of MAO - dc dorsal cap - DC dorsal cochlear nucleus - dfDAO dorsal fold of DAO - dlh dorsal lateral hump of cerebellar nuclei - dIPO dorsal lamella of PO - Gia gigantocellular reticular nucleus - dmcc dorsomedial cell column - GABA gamma-aminobutyric acid - GAD glutamate decarboxylase - HRP horseradish peroxidase - icp inferior cerebellar peduncle - IC inferior colliculus - Inf infracerebellar nucleus - IntA anterior interposed cerebellar nucleus - IntP posterior interposed cerebellar nucleus - Lat lateral cerebellar nucleus - LRt lateral reticular nucleus - LSO lateral superior olive - LVe lateral vestibular nucleus - MAO medial accessory olive - Med medial cerebellar nucleus - Me5 mesencephalic trigeminal nucleus - MVe medial vestibular nucleus - PFl paraflocculus of the cerebellar cortex - PO principle olive - RMg raphe magnus - rMAO rostral lamella of MAO - rs rubrospinal tract - scp superior cerebellar peduncle - SuVe spinal vestibular nucleus - SuVe superior vestibular nucleus - vfDAO ventral fold of DAO - vlo ventrolateral outgrowth - vlPO ventral lamella of PO - Y Y, y vestibular nucleus - WGA wheatgerm agglutinin This paper is dedicated to Professor Fred Walberg on the occasion of his 70th hirthdav     

TRANSSYNAPTIC DEGENERATION IN THE INFERIOR OLIVARY NUCLEUS ASSOCIATED WITH PONTINE GLIOBLASTOMA

A case of glioblastoma arising in the pons of a 14-year-old boy in whom transsynaptic degeneration was found in the inferior olivary nucleus is reported. The tumor occupied most of the pons including the tegmental tract and invaded into the midbrain, medulla oblongata, cerebellar peduncles, thalamus, basal ganglia, and meninges. The right inferior olivary nucleus was devoid of the tumorous lesion, but many neurons were severely vacuolated. An im-munohistochemical study using glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), and S-100 protein was performed. GFAP and S-100 protein were positive in the reactive glia of the nucleus and NSE gave a faint reaction in some degenerated neurons. These degenerative changes found in neurons of the inferior olivary nucleus were considered to be transsynaptic degeneration due to the destruction of the tegmental tract at the pons and of cerebellar peduncles by invasive pontine glioblastoma. ACTA PATHOL. JPN. 35: 1495–1500, 1985.     

Comparative effects of lesions to the ponto-cerebellar and olivo-cerebellar pathways on motor and spatial learning in the rat

Emerging evidence supports the role of the cerebellum in motor learning and previous studies have also shown that olivary projections to the cerebellum are involved in motor learning. Since the pontine nuclei make up the other main relay centre in the cerebro-cerebellar pathway, the purpose of the present study was to verify the involvement of the ponto-cerebellar pathway in motor and spatial learning, by comparing these functions in intact animals and in rats with selective injury of the olivary or pontine neurons. Two groups of rats were used: the first was treated with 3-acetylpyridine to destroy the inferior olivary complex, the second received electrolytic lesions of the middle cerebellar peduncle to interrupt the ponto-cerebellar pathway. Control and lesioned rats were then submitted to three tasks: unrotated rod, rota-rod at 20 r.p.m., and Morris water maze. In the first task both 3-acetylpyridine-treated rats and rats with lesions of the middle cerebellar peduncle showed static equilibrium deficiencies. Through training, however, they reached the maximal score attained by the controls. The rats submitted to the rota-rod at 20 r.p.m. obtained scores significantly inferior to the controls. The Morris water maze results indicated that the lesion of inferior olivary complex and middle cerebellar peduncle both alter learning of the spatial task. These findings show that both the ponto- and olivo-cerebellar pathways are involved in learning complex motor sequences and spatial tasks. Since both projections converge onto Purkinje cells, our results suggest an integration of these two pathways in the cerebellar control of learning mechanism.     

Do pontocerebellar mossy fibres give off collaterals to the cerebellar nuclei? An experimental study in the cat with implantation of crystalline HRP-WGA

In 15 cats with implantations of crystalline HRP-WGA in the cerebellar nuclei and tetramethylbenzidine histochemistry, the pontine nuclei were carefully examined for presence of retrogradely labelled cells. Findings in the nucleus reticularis tegmenti pontis and the inferior olive, both known to project to the cerebellar nuclei, served as controls for effectiveness of uptake and transport. After implantations restricted to the lateral cerebellar nucleus in 5 cats altogether two labelled cells were found in the contralateral pontine nuclei in regions receiving afferents from the lateral nucleus. In contrast, many labelled cells occurred in the nucleus reticularis tegmenti pontis and the inferior olive. After implantations in 5 cats restricted to the posterior or anterior interposed nuclei, altogether only one labelled cell was found in the pontine nuclei, while many labelled cells occurred in the inferior olive. The nucleus reticularis tegmenti pontis contained a small number of retrogradely labelled cells after implantations in the anterior interposed nucleus, but none after implantations restricted to the posterior interposed nucleus. After implantations restricted to the medial (fastigial) nucleus, no retrogradely labelled cells were found in the pontine nuclei and nucleus reticularis tegmenti pontis (although many were present in the inferior olive). The present findings support earlier conclusions based on anterograde tracing methods that the cerebellar nuclei receive very few, if any, afferents from the pontine nuclei.     

Projections from the medial septum and diagonal band of Broca to the dorsal and central superior raphe nuclei: a non-cholinergic pathway

Summary The anatomical organization of projections from the medial septal nucleus (MS), and the vertical (VDB) and horizontal limb (HDB) of the diagonal band of Broca to the dorsal raphe nucleus (NRD) and the central superior raphe nucleus (RCS) of the rat were studied by anterograde [³H]-leucine, and True Blue and Fluoro Gold fluorescent retrograde tracing. Projections from the MS were found to enter the basal mesencephalon at the rostro-medial aspect of the pontine nuclei, curve dorsally and terminate throughout the RCS and in the caudal portion of the NRD. Fibers from the VDB were found to enter these raphe nuclei by two separate routes; some fibers reached the basal mesencephalon, curved dorsally and terminated in the RCS and NRD. Other fibers entered the pedunculopontine nucleus, curved medially and reached the NRD. Presumed terminal labelling was found overlaying the RCS and NRD throughout their rostro-caudal extensions. The brain stem projections from HDB entered the mesencephalon by the same routes as those from VDB, but the labelling over RCS was sparse, and the NRD labelling was preferentially distributed to the rostral portion of the nucleus. The present data indicate a crude topographic organization of the projections from the septal region to the NRD and RCS. In general, the distribution of presumed terminal labelling appeared to be more closely associated with the distribution of NRD and RCS 5-HT immunoreactive cell bodies, than with the cytoarchitectonically defined extensions of these raphe nuclei. By sequential evaluation of the distribution of retrogradely labelled and acetylcholine esterase-stained cells on the same section, and by selective tracing with radiolabelled choline, it appears that the vast majority, if not all, of the neurons in MS and diagonal band which project to the rostral raphe are non-cholinergic.Abbreviations CLi caudal linear raphe nucleus - DTg dorsal tegmental nucleus - flm medial longitudinal fasciculus - HDB horizontal limb of the diagonal band of Broca - IP interpeduncular nucleus - MS medial septal nucleus - NRD dorsal raphe nucleus - PAG periaqueductal gray - Pn pontine nucleus - R red nucleus - RCS central superior raphe nucleus - RMg raphe magnus nucleus - RPn pontine raphe nucleus - SN substantia nigra - VDB vertical limb of the diagonal band of Broca - VTg ventral tegmental nucleus - 5 trigeminal motor nucleus     

Organization of afferent connections to the lateral and interpositus cerebellar nuclei from the brainstem relay nuclei: a horseradish peroxidase study in the cat

Afferent projections to the lateral (dentate) and interpositus cerebellar nuclei from the brainstem relay nuclei were studied in cats using the horseradish peroxidase (HRP) method. In the first series of experiments, HRP was injected into the brachium pontis. Mossy fiber terminals were anterogradely labeled, predominantly in the lateral (hemispherical) part, moderately in the intermediate part, and slightly in the vermal part of the cerebellum. Besides these terminals in the cerebellar cortex, axon terminals labeled anterogradely were also found in the cerebellar nuclei. The labeled terminals appeared almost exclusively in the lateral nucleus and rarely in the interpositus nucleus. Cells labeled retrogradely were found both in the pontine nuclei and the tegmental reticular nucleus, but not in other brainstem nuclei. In the second series of experiments, HRP was injected into the lateral and interpositus nuclei, and retrograde labeling was examined in the brainstem relay nuclei. After HRP injection into the lateral nucleus, the number of labeled cells was significantly large in the pontine nuclei, but fairly small in the reticular or vestibular nuclei. The number of labeled cells was generally large in the inferior olive, mainly in the principal olive. After HRP injection into the interpositus nucleus, the number of labeled cells was moderate in the reticular or vestibular nuclei, but small in the pontine nuclei. The number of labeled cells in the inferior olive was also large, being distributed mainly in the accessory olives. These results indicate that the pontine nuclei and the principal olive provide major afferent inputs to the lateral nucleus, whereas the reticular nuclei, the vestibular nuclei and the accessory olives are the major afferent sources to the interpositus nucleus.     

Regional brain variations of cytochrome oxidase activity and motor coordination in Lurcher mutant mice

Lurcher mutant mice are characterized by massive degeneration of cerebellar Purkinje cells and granule cells and by deficits in motor coordination. Regional brain variations of cytochrome oxidase (CO) activity were analyzed to identify those brain regions with abnormal metabolic activity as a secondary consequence of the cerebellar atrophy and to establish the relationship between CO activity and motor deficits. Lurcher mutants had higher CO activity in all three cerebellar deep nuclei than normal littermate controls of the same background strain. Higher CO activity was also found in Lurcher mutants in brain regions directly connected to the cerebellum, such as the lateral vestibular nucleus, the cochlear nucleus, the red nucleus, the ventrolateral thalamus, the dorsal raphe, the interpeduncular nucleus, and the inferior colliculus. By contrast, there was a sharp decrease in CO activity in the inferior olive. As for brain regions not directly connected to the cerebellum, higher CO activity was observed in the trigeminal motor nucleus and the CA1 molecular layer of the hippocampus, which highlights probable transsynaptic alterations as a secondary consequence of cerebellar atrophy. A positive correlation between CO activity in the red nucleus and latencies before falling in two motor-coordination tests indicates that a compensatory increase of metabolic activity in a cerebellar efferent region is associated with improved behavior. Received: 15 September 1997 / Accepted: 3 March 1998     

Ascending and intrinsic projections of the superior olivary complex in the cat

Summary The ascending and intrinsic projections of the superior olivary complex (SO) in the cat were investigated by injection of ³H-leucine and horseradish peroxidase (HRP) in SO and the inferior colliculus (IC), respectively. A topically arranged projection was demonstrated from the nucleus of the trapezoid body (NTB) to the ipsilateral lateral superior olivary nucleus (LSO) with a lesser connection in the opposite direction. The medial superior olivary nucleus (MSO) has a strictly ipsilateral projection, whilst LSO projects symmetrically through the lateral lemniscus (LL) of both sides, to end with topically arranged terminals in the ventrolateral part of the central nucleus of the inferior colliculus (CNIC). Terminal labelling found in the ventral and dorsal nuclei of LL (VNLL and DNLL) probably represents collaterals from bypassing fibres originating in MSO and LSO, respectively. These results were demonstrated by both techniques, whilst in addition the HRP method revealed an ipsilateral and a contralateral projection to IC from VNLL and DNLL, respectively.     

Vesicular acetylcholine transporter-immunoreactive axon terminals enriched in the pontine nuclei of the mouse

Information to the cerebellum enters via many afferent sources collectively known as precerebellar nuclei. We investigated the distribution of cholinergic terminal-like structures in the mouse precerebellar nuclei by immunohistochemistry for vesicular acetylcholine transporter (VAChT). VAChT is involved in acetylcholine transport into synaptic vesicles and is regarded as a reliable marker for cholinergic terminals and preterminal axons. In adult male mice, brains were perfusion-fixed. Polyclonal antibodies for VAChT, immunoglobulin G-peroxidase and diaminobenzidine were used for immunostaining. In the mouse brain, immunoreactivity was seen in almost all major cholinergic cell groups including brainstem motoneurons. In precerebellar nuclei, the signal could be detected as diffusely beaded terminal-like structures. It was seen heaviest in the pontine nuclei and moderate in the pontine reticulotegmental nucleus; however, it was seen less in the medial solitary nucleus, red nucleus, lateral reticular nucleus, inferior olivary nucleus, external cuneate nucleus and vestibular nuclear complex. In particular, VAChT-immunoreactive varicose fibers were so dense in the pontine nuclei that detailed distribution was studied using three-dimensional reconstruction of the pontine nuclei. VAChT-like immunoreactivity clustered predominantly in the medial and ventral regions suggesting a unique regional difference of the cholinergic input. Electron microscopic observation in the pontine nuclei disclosed ultrastructural features of VAChT-immunoreactive varicosities. The labeled bouton makes a symmetrical synapse with unlabeled dendrites and contains pleomorphic synaptic vesicles. To clarify the neurons of origin of VAChT-immunoreactive terminals, VAChT immunostaining combined with wheat germ agglutinin-conjugated horseradish peroxidase retrograde labeling was conducted by injecting a retrograde tracer into the right pontine nuclei. Double-labeled neurons were seen bilaterally in the laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus. It is assumed that mesopontine cholinergic neurons negatively regulate neocortico-ponto-cerebellar projections at the level of pontine nuclei.     

Descending projections from the subparafascicular thalamic nucleus to the lower brain stem in the rat

Summary In the course of our study on the neuronal connections of the subparafascicular nucleus (SPF) in the rat, descending projections from the SPF to the lower brain stem were examined by using the anterograde tracer PHA-L (Phaseolus vulgaris leukoagglutinin) and retrograde tracer WGA-HRP (horseradish peroxidase conjugated to wheat germ agglutinin). When PHA-L was injected into the magnocellular and/or parvicellular division of the SPF (SPFm and/or SPFp), presumed terminal labeling was seen, bilaterally with an ipsilateral dominance, in the mesencephalic and pontine central gray matter, peripheral shell regions of the inferior colliculus, cuneiform nucleus, and superior olivary complex (mainly in the superior paraolivary nucleus, and additionally in the nuclei of the trapezoid body). A few labeled axon terminals were also seen in the cochlear nuclei bilaterally with a contralateral dominance. In the second set of experiments, WGA-HRP was injected into the inferior colliculus, superior olivary complex, or cochlear nuclei. When WGA-HRP was injected into the peripheral shell regions of the inferior colliculus or the superior olivary complex, many labeled neuronal cell bodies were seen in the SPFm bilaterally with an ipsilateral dominance, and a moderate number of labeled neuronal cell bodies were observed in the SPFp (lateral SPF) bilaterally with an ipsilateral dominance. When WGA-HRP was injected into the cochlear nuclei, a moderate number of labeled neuronal cell bodies were observed in the SPFm and SPFp bilaterally with a contralateral dominance. The results indicate that the SPFm and SPFp (lateral SPF) of the rat send a considerable number of projection fibers to the lower brain stem. The target regions of these projection fibers include the auditory relay nuclei, such as the inferior colliculus, superior olivary complex, and cochlear nuclei.     

The nucleus reticularis tegmenti pontis and the adjacent rostral paramedian reticular formation: differential projections to the cerebellum and the caudal brain stem

Summary The projection of the nucleus reticularis tegmenti pontis and the adjacent tegmental area, to the caudal brain stem and the cerebellum were investigated by means of anterograde transport of tritiated leucine. The nucleus reticularis tegmenti pontis was found to be exclusively connected with the cerebellum. Mossy fiber terminals were absent only from lobule X and most abundant in lobule VII and the hemispheres with a slight contralateral predominance. The paramedian pontine reticular formation projects with bilateral symmetry to the cerebellar lobules VI, VII and the crura I and II, and heavily to the medial aspect of predominantly the ipsilateral reticular formation in the lower brain stem including specific targets as the nucleus reticularis paramedianus, the nucleus prepositus hypoglossi, the nucleus intercalatus, the nucleus of Roller, the nucleus supragenualis and the dorsal cap of the inferior olive. The nucleus vestibularis medialis receives a very weak projection. The connections are discussed in the light of their possible involvement in pathways for the execution of voluntary and reflex eye movements.Abbreviations bp brachium pontis - CBL cerebellum - cr I, II crus I, II - ct corpus trapezoides - dc dorsal cap of Kooy - dl dorsal lamina of the principal olive - FL flocculus - flm fasciculus longitudinalis medialis - gVII genu of the facial nerve - H VI hemisphere of lobule VI - IO inferior olivary nucleus - ll lemniscus lateralis - ml lemniscus medialis - NCS nucleus centralis superior - NIC nucleus intercalatus - NP nuclei pontis - NPH nucleus prepositus hypoglossi - NRaP nucleus raphe pontis - NRGc nucleus reticularis gigantocellularis - NRL nucleus reticularis lateralis - NRo nucleus of Roller - NRP nucleus reticularis paramedianus - NRPoC nucleus reticularis pontis caudalis - NRPoO nucleus reticularis pontis oralis - NRTP nucleus reticularis tegmenti pontis - NSG nucleus supragenualis - NVM nucleus vestibularis medialis - N VI nucleus abducens - n XII nervus hypoglossus - ped pedunculus cerebri - PFLD dorsal paraflocculus - PFLV ventral paraflocculus - PMD paramedian lobule - PPRF pontine paramedian reticular formation - vl ventral lamina of the principal olive - vlo ventrolateral outgrowth - X nucleus dorsalis vagi - XII nucleus hypoglossus - I-X lobules I to X     

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.