Migration and nucleogenesis of mouse precerebellar neurons visualized by in utero electroporation of a green fluorescent protein gene

Neural migration is a critical step for accurate CNS development, but the molecular mechanisms that regulate migration, settlement and nucleogenesis remain largely unknown. The precerebellar neurons (PCNs), generated in the lower rhombic lip (LRL), migrate towards their destinations: some neurons form the pontine gray nucleus (PGN) and reticulotegmental nucleus (RTN) in the ipsilateral pons, while others form the lateral reticular and external cuneate nuclei in the contralateral medulla after crossing the midline. Here, by introducing an EGFP gene into a unilateral LRL of mouse embryos by in utero electroporation, we specifically labeled and tracked the PCNs in vivo. We found that a substantial number of the labeled neurons crossed the midline and formed PGN/RTN on the contralateral side. In addition, we found that a subpopulation of the interpolar subnucleus of the spinal trigeminal nucleus, which projects the axons to the cerebellum, was one of the PCNs derived from the LRL. Furthermore, because the electroporated mice were born and grew up healthy, we could visualize the PCNs and their mossy fibers in the adult brain. Therefore, the EGFP labeling of PCNs can be applied to studying the physiology of the mossy fiber system as well as PCN development in embryos.     

Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system--II. Brainstem, cerebellum and spinal cord

Multiple nuclei and fiber tracts in the adult rat brainstem and spinal cord were found to contain nerve growth factor receptor-related protein, as recognized by the monoclonal antibody 192-IgG. Both cholinergic and non-cholinergic sensory and motor regions demonstrated immunoreactive cell bodies and fibers. Nerve growth factor receptor-immunoreactive cells were seen in the mesencephalic nucleus of trigeminal nerve, superior colliculus, parabrachial, prepositus hypoglossal, raphe, dorsal and ventral cochlear, interstitial nucleus of the vestibular nerve, ambiguus and reticular nuclei, cerebellum and ventral spinal cord. Immunoreactive cells resembling neuroglia were distributed subpially along the superior colliculus. Intracerebroventricular injection of colchicine resulted in significantly increased nerve growth factor receptor immunoreactivity in all previously positive neurons and especially in certain neurons of the cochlear and ambiguus nuclei. It also resulted in the visualization of receptor immunoreactivity in certain neurons which were normally non-immunoreactive including cerebellar Purkinje cells, neurons of the central gray, locus coeruleus, facial, dorsal motor vagal and hypoglossal nuclei. In normal animals, nerve growth factor receptor-immunoreactive fibers and varicosities occurred in the trigeminal nerve nuclei, pontine, vestibular, parabrachial, facial, hypoglossal, dorsal motor vagal, solitary, gracile and cuneate nuclei and spinal cord. Although most fiber-like immunoreactive structures were probably axons and nerve terminals, neuroglial or extracellular localizations could not be excluded in some areas. For example, the medial nucleus of the inferior olive and most cerebellar nuclei contained diffuse non-fibrillar receptor immunoreactivity. The presence of nerve growth factor receptor-like immunoreactivity in cell bodies and fibers of several sensory and motor areas of the adult rat brainstem, cerebellum and spinal cord suggests multifocal actions of nerve growth factor or a nerve growth factor-like substance. Although the degree of overlap between nerve growth factor receptor- and choline acetyltransferase-containing regions in the brainstem is not as great as in the forebrain, our findings suggest a potential influence of nerve growth factor or nerve growth factor-like substances on cholinergic systems outside the forebrain. Furthermore, the disparities which occur imply that non-cholinergic nerve growth factor receptor-containing neurons of the brainstem, cerebellum and spinal cord may be affected by such trophic substances.     

Differential expression of VGLUT1 or VGLUT2 in the trigeminothalamic or trigeminocerebellar projection neurons in the rat

The vesicular glutamate transporters, VGLUT1 and VGLUT2, reportedly display complementary distribution in the rat brain. However, co-expression of them in single neurons has been reported in some brain areas. We previously found co-expression of VGLUT1 and VGLUT2 mRNAs in a number of single neurons in the principal sensory trigeminal nucleus (Vp) of the adult rat; the majority of these neurons sent their axons to the thalamic regions around the posteromedial ventral nucleus (VPM) and the posterior nuclei (Po). It is well known that trigeminothalamic (T-T) projection fibers arise not only from the Vp but also from the spinal trigeminal nucleus (Vsp), and that trigeminocerebellar (T-C) projection fibers take their origins from both of the Vp and Vsp. Thus, in the present study, we examined the expression of VGLUT1 and VGLUT2 in Vp and Vsp neurons that sent their axons to the VPM/Po regions or the cortical regions of the cerebellum. For this purpose, we combined fluorescence in situ hybridization (FISH) histochemistry with retrograde tract-tracing; immunofluorescence histochemistry was also combined with anterograde tract-tracing. The results indicate that glutamatergic Vsp neurons sending their axons to the cerebellar cortical regions mainly express VGLUT1, whereas glutamatergic Vsp neurons sending their axons to the thalamic regions express VGLUT2. The present data, in combination with those of our previous study, indicate that glutamatergic Vp neurons projecting to the cerebellar cortical regions express mainly VGLUT1, whereas the majority of glutamatergic Vp neurons projecting to the thalamus co-express VGLUT1 and VGLUT2.     

Further studies on the fiber connections of the central cervical nucleus in the cat

Summary The course and cerebellar termination of the axons of the cells in the central cervical nucleus (CCN) was studied in five cats after injections of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the C1-4 segments involving the CCN. In two cats a hemisection was performed ipsilateral to and above the injections in order to prevent transport of WGA-HRP from cerebellar-projecting neurons in lamina VI, just dorsal to the CCN. Labeled axons were found in the brain stem contralateral to the injections just lateral to the spinal trigeminal tract and the vestibular nuclei, and in the reticular formation. Some fibers terminated in the vestibular nuclei (especially group x), the reticular formation, and the inferior olive. The axons entered the cerebellum via the superior cerebellar peduncle. In the cerebellum mossy fiber terminals were found bilaterally in the deep vermal parts of lobules I–VIII. Only in cases without lesions were terminals found in the paramedian lobule ipsilateral to the injection, suggesting that neurons in lamina VI, but not in the CCN, project to the paramedian lobule. In the brain stem retrogradely labeled neurons (possible afferents to the CCN) were found in the vestibular (lateral, inferior and medial) nuclei, the reticular formation, and the trigeminal (spinal and mesencephalic) nuclei. The cerebellar distribution of mossy fiber terminals suggests that spinocerebellar fibers from lower parts of the spinal cord passing through the injection area do not transport WGA-HRP to their terminals.     

Organization of the pontine nuclei.

The pontine nuclei provide the cerebellar hemispheres with the majority of their mossy fiber afferents, and receive their main input from the cerebral cortex. Even though the vast majority of pontine neurons send their axons to the cerebellar cortex, and are contacted monosynaptically by (glutamatergic) corticopontine fibers, the information-processing taking place is not well understood. In addition to typical projection neurons, the pontine nuclei contain putative GABA-ergic interneurons and complex synaptic arrangements. The corticopontine projection is characterized by a precise but highly divergent terminal pattern. Large and functionally diverse parts of the cerebral cortex contribute; in the monkey the most notable exception is the almost total lack of projections from large parts of the prefrontal and temporal cortices. Within corticopontine projections from visual and somatosensory areas there is a de-emphasis of central vision and distal parts of the extremities as compared with other connections of these sensory areas. Subcorticopontine projections provide only a few percent of the total input to the pontine nuclei. Certain cell groups, such as the reticular formation, project in a diffuse manner whereas other nuclei, such as the mammillary nucleus, project to restricted pontine regions only, partially converging with functionally related corticopontine connections. The pontocerebellar projection is characterized by a highly convergent pattern, even though there is also marked divergence. Neurons projecting to a single cerebellar folium appear to be confined to a lamella-shaped volume in the pontine nuclei. The organization of the pontine nuclei suggests that they ensure that information from various, functionally diverse, parts of the cerebral cortex and subcortical nuclei are brought together and integrated in the cerebellar cortex.     

The efferent connections of the nuclei of the descending trigeminal tract in the mallard (Anas platyrhynchos L.)

The efferent and intranuclear connections of the nuclei of the descending trigeminal tract of the mallard have been studied with lesion methods, and by axonal transport techniques following injections of tritiated leucine, and of horseradish peroxidase.The large subnucleus oralis neurons, including those belonging to the nucleus of the ascending glossopharyngeal tract, have proven to be the sole origin of trigeminocerebellar connections. The cerebellar afferents are of the mossy fiber type, and terminate predominantly in lobules V, VI and VII, and possibly, lobule IV. Trigeminocerebellar projections are ipsilateral except for the vermal area.Subnucleus interpolaris is the main source of intratrigeminal fibers that terminate in subnucleus oralis and the ventral part of the main sensory nucleus. These intranuclear connections are bilateral, but the medium-celled caudal part of subnucleus interpolaris in particular contains the majority of bi- and/or contralaterally projecting neurons. Additionally, the small cells in the rostral part of subnucleus interpolaris project ipsilaterally upon the parabrachial region, and upon the lateral reticular formation.Projections upon the parabrachial region furthermore emanate bilaterally from layer I of the rostral subnucleus caudalis. A minor part of layer I neurons sends its axons contralaterally along with those of the dorsal column nuclei toward the thalamic nucleus dorsolateralis posterior. Associated with the medial lemniscus, contralateral termination is also present in the lateral part of the ventral lamella of oliva caudalis, in the marginal zone of nucleus mesencephalicus lateralis, pars dorsalis and immediately surrounding intercollicular grey and, finally, in the nucleus intercalatus thalami. Furthermore, a bilaterally descending projection from subnucleus caudalis upon layers I and II of the rostral cervical cord was observed. Close to their origin subnucleus caudalis neurons project upon the adjoining caudal part of the lateral reticular formation.     

Immunohistochemical localization of glutamate, glutaminase and aspartate aminotransferase in neurons of the pontine nuclei of the rat

The pontine nuclei form the key relay nuclei in the cerebropontocerebellar pathway. Although a great deal of information is available regarding the anatomy of this region, the identity of the neurotransmitter(s) contained in the neurons of the pontine gray are not known. The aim of the present investigation is to utilize immunohistochemical techniques to determine whether glutamate, a putative excitatory transmitter, and the enzymes responsible for its metabolism, are found in pontine neurons. Both glutaminase, an enzyme which converts glutamine to glutamate, and aspartate aminotransferase, an enzyme which is involved in the interconversion between glutamate and aspartate, have been proposed to be markers of neurons which use excitatory amino acids as neurotransmitters. The present study utilizes a monoclonal antibody against carbodiimide-fixed glutamate and polyclonal antisera against glutaminase and aspartate aminotransferase in conjunction with the indirect peroxidase technique or the peroxidase-labeled biotin-avidin procedure to localize glutamatergic neurons in the pontine nuclei of the rat. Numerous neurons in all subdivisions of the pontine nuclei were found to contain carbodiimide-fixed glutamate-like immunoreactivity, glutaminase-like immunoreactivity or aspartate aminotransferase-like immunoreactivity. Horseradish peroxidase was injected into the cerebellum of four rats for use with a combined retrograde transport-immunohistochemical procedure. Double-labeled neurons were observed in all subdivisions of the pontine nuclei, indicating that pontine neurons which contain glutamate-like immunoreactivity project to the cerebellum. Based on the hypothesis that increased levels of glutamate, glutaminase and aspartate aminotransferase reflect a transmitter role for glutamate, the present data raise the possibility that glutamate may be a major neurotransmitter of pontocerebellar fibers.     

Central projection of proprioceptive afferents arising from maxillo-facial regions in some animals studied by HRP-labeling technique

20 ICR mice (adult females) were used for analyzing the axonal projection of the trigeminal mesencephalic tract neurons and 10 Japanese shrew-moles for analyzing that of the snout proprioceptive neurons. The HRP-labeled axons were found to be ipsilaterally terminated in the trigeminal motor nucleus, supratrigeminal nucleus and trigeminal main and spinal tract nuclei, lateral pontine-medullary reticular formation, vagal dorsal motor and hypoglossal nuclei and the lamina V of the C2 spinal cord segments. No HRP-labeled axons were found in the facial and solitary nuclei and the cerebellum. Also, the functional localization of the trigeminal mesencephalic tract neurons was analyzed with the retrograde tracers of fluorescent compounds injected into the jaw-closing muscles having spindles. The fluorescent-labeled neurons were found to be intermingled throughout the nucleus and clustered at the caudal level of the nucleus. Also, double or triple fluorescent-labeled neurons were not observed in the nucleus. The HRP-labeled axon bundle of the facial proprioceptive neurons are divided rostro-caudally into the shorter ascending and the longer descending roots, both running closely dorsal to the trigeminal spinal tract nucleus. The ascending root lies adjacently dorsal to the spinal tract nucleus, giving off the terminal fibers to it, to the wider area of the dorso-medial pontine nuclei and finally to the cerebellar nuclei. At the level of the facial inner genu, it turns medially to meet the facial nerve root, giving off the terminal fibers to the facial motor nucleus and to the raphe nuclei and to the opposite nuclei bilaterally. The HRP-labeled descending root takes the course caudally at least down to the C3 segment of the spinal cord, giving off the terminal fibers to the spinal tract nucleus, the nuclei of the IXth, Xth and XIIth cranial nerves and the pontine-medullary reticular formation. In the spinal cord, it descends bilaterally through the posterior fascicles, giving off the terminal fibers to the dorsal and ventral horns.     

Distribution of premotor neurons for orbicularis oculi motoneurons in the cat, with particular reference to possible pathways for blink reflex

After injecting horseradish peroxidase into the facial nucleus regions containing orbicularis oculi motoneurons, labeled neuronal cell bodies were found in the lateral medullary reticular formation, pretectal olivary nucleus, sensory trigeminal nuclei, lateral and medial parabrachial nuclei, ventromedial reticular formation medial to the facial nucleus, red nucleus and its surroundings, anterior horn of the upper cervical cord, medullary raphe nuclei, oculomotor nucleus and its surroundings, nuclei of Darkschewitsch, Cajal and Edinger-Westphal, ventral part of the midbrain central gray, pontine tegmentum, lateral vestibular nucleus and deep layers of the superior colliculus.     

Mosaic distribution of phosphate-activated glutaminase-like immunoreactivity in the rat striatum.

The dorsal and ventral striatum of mammals has been known to be organized in a mosaic manner, referred to as "patches" and "matrix" of the caudatoputamen. The present study was primarily attempted in order to reveal the relationship of glutamatergic neuronal components to the mosaic organization in the rat striatum by using a monoclonal antibody to phosphate-activated glutaminase, a major synthetic enzyme of transmitter glutamate. Antibodies against glutamate decarboxylase and choline acetyltransferase were also used as the markers for GABAergic and cholinergic neuronal components, respectively. Glutaminase immunoreactivity was seen in a number of large- and a few medium-sized neurons in the caudatoputamen, nucleus accumbens and olfactory tubercle. The large neurons with glutaminase immunoreactivity were observed in the neuropil of the caudatoputamen and nucleus accumbens; glutaminase immunoreactivity was particularly marked in the neuropil of island-like patchy areas although it was seen throughout the neuropil of the nuclei. In the caudatoputamen, island-like areas with marked glutaminase immunoreactivity exhibited less marked choline acetyltransferase immunoreactivity than the surrounding background region, and were thus considered to correspond to the patches. The mosaic distribution of glutamate decarboxylase immunoreactivity in the caudatoputamen seemed identical with that of glutaminase immunoreactivity. However, in the nucleus accumbens, the mosaic pattern of neuropil labeling for glutaminase was neither consistent with that for glutamate decarboxylase nor that for choline acetyltransferase, suggesting the presence of non-GABAergic glutaminase-containing nerve terminals in the nucleus. In an attempt to clarify the origin of neuropil labeling for glutaminase in the striatum, lesions were made in the regions sending projection fibers to the caudatoputamen and nucleus accumbens. After placing lesions in the cerebral cortex, glutaminase immunoreactivity was decreased in neuropil of the caudatoputamen, but the mosaic pattern remained. Lesions which were placed in the intralaminar thalamic nuclei, amygdaloid body, globus pallidus or substantia nigra produced no substantial change in glutaminase immunoreactivity in the caudatoputamen and nucleus accumbens. After injection of kainic acid into the caudatoputamen or nucleus accumbens, glutaminase immunoreactivity in the neuropil of the affected regions was decreased to lose the mosaic pattern, indicating that neuronal components with glutaminase immunoreactivity in the neuropil of the patches were mainly of intrinsic origin. In summary, possible axon terminals containing glutaminase were observed with mosaic patterns in the caudatoputamen and nucleus accumbens, in which large cholinergic and medium-sized non-cholinergic neurons were immunoreactive for glutaminase. In the caudatoputamen, glutaminase immunoreactivity in neuropil was more marked in the patches than in the matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutamate immunoreactivity in the rat basilar pons: light and electron microscopy reveals labeled boutons and cells of origin of afferent projections.

Immunohistochemical methods that employed a polyclonal antiserum directed against a glutamate-hemocyanin conjugate were utilized to examine the rat basilar pontine nuclei at both light and electron microscopic levels in order to identify putative glutamatergic neural elements. A large number of cells ranging in size from 11 to 32 microns in diameter and present in all subdivisions and at all rostrocaudal levels of the basilar pons exhibited intense glutamate immunoreactivity. Immunoreactive punctate structures, confirmed by electron microscopy to be axon terminals, were homogeneously distributed throughout the pontine neuropil, although a somewhat greater accumulation was apparent medially at mid-levels of the basilar pons and laterally at more caudal levels. Immunolabeled axons were also present throughout the pontine nuclei. In order to demonstrate possible extrinsic sources of glutamate-immunoreactive axon terminals within the pontine gray, injections of wheat germ agglutinin-horseradish peroxidase were made directly into the basilar pons. Tissue was then evaluated for the presence of retrogradely transported wheat germ agglutinin-horseradish peroxidase and the same tissue sections processed for glutamate immunocytochemistry. Following this combined protocol, neuronal somata exhibiting both wheat germ agglutinin-horseradish peroxidase and glutamate immunoperoxidase reaction products were observed within layer Vb of the cerebral cortex, zona incerta, the dentate nucleus of the cerebellum, nucleus paragigantocellularis of the medullary reticular formation, and the dorsal column nuclei. Such double-labeled cells were considered to represent glutamatergic neurons that provide axonal projections to the basilar pons. Ultrastructural studies of the pontine nuclei confirmed the presence of glutamate immunogold labeling in dendrites, neuronal somata, axons, and axon terminals. Immunoreactive boutons contained round vesicles and primarily formed asymmetric synapses at various postsynaptic loci which included glutamate-immunolabeled dendritic profiles and somata. These results suggest that glutamatergic basilar pontine neurons form one segment of a multisynaptic pathway involving glutamatergic afferents to the basilar pons, glutamatergic pontocerebellar projection neurons, and the glutamatergic granule cells of the cerebellar cortex.     

大鼠三叉神经本体觉中枢通路中VGluT1样阳性胞体和终末的生后发育、分布以及与PAG样阳性神经元的联系

本研究应用免疫组织化学和免疫荧光组织化学技术,在光镜和激光共聚焦显微镜下观察了大鼠脑干内三叉神经本体觉中枢通路中I型囊泡膜谷氨酸转运体(VGluT1)样阳性神经元胞体和纤维终末的生后发育、表达和分布以及与磷酸激活的谷氨酰胺酶(PAG)样阳性神经元之间的联系。结果显示:(1)新生(P0)大鼠所有的三叉神经中脑核(Vme)神经元的胞体内均可观察到VGluT1的表达,3d(P3)时,VGluT1的表达最强,之后至P11,VGluT1的表达随着发育逐渐下降。P11后,在Vme神经元的胞体内未检测到VGluT1样免疫阳性产物,但在阴性胞体周围可观察到VGluT1样免疫阳性终末分布;(2)生后0d,三叉神经脊束核吻侧亚核背内侧部及其邻接的外侧网状结构(Vodm-LRF)、三叉神经感觉主核背内侧部(Vpdm)和三叉上核尾外侧部(Vsup-CL)内已可观察到VGluT1样阳性纤维终末分布。生后7d,VGluT1样阳性纤维终末开始在三叉神经运动核腹侧区(AVM)、上橄榄核背侧区(ADO)出现;(3)激光共聚焦显微镜下,于生后12d在Vme、Vodm和Vpdm内和生后21d在Vsup-CL内分别观察到一些VGluT1样阳性终末与PAG样阳性神经元的胞体或树突形成密切接触。以上结果提示:VGluT1可能与生后发育过程中大鼠脑干内三叉神经本体感觉中枢通路的兴奋性联系的建立密切相关。     

An autoradiographic analysis of ascending projections from the medullary reticular formation in the rat

Ascending projections from the several nuclei of the medullary reticular formation were examined using the autoradiographic method. The majority of fibers labeled after injections of [3H]leucine into nucleus gigantocellularis ascended within Forel's tractus fasciculorum tegmenti which is located ventrolateral to the medial longitudinal fasciculus. Nucleus gigantocellularis injections produced heavy labeling in the pontomesencephalic reticular formation, the intermediate layers of the superior colliculus, the pontine and midbrain central gray, the anterior pretectal nucleus, the ventral midbrain tegmentum including the retrorubral area, the centromedian-parafascicular complex, the fields of Forel/zona incerta, the rostral intralaminar nuclei and the lateral hypothalamic area. Nucleus gigantocellularis projections to the rostral forebrain were sparse. Labeled fibers from nucleus reticularis ventralis, like those from nucleus gigantocellularis, ascended largely in the tracts of Forel and distributed to the pontomedullary reticular core, the facial and trigeminal motor nuclei, the pontine nuclei and the dorsolateral pontine tegmentum including the locus coeruleus and the parabrachial complex. Although projections from nucleus reticularis ventralis diminished significantly rostral to the pons, labeling was still demonstrable in several mesodiencephalic nuclei including the cuneiform-pedunculopontine area, the mesencephalic gray, the superior colliculus, the anterior pretectal nucleus, the zona incerta and the paraventricular and intralaminar thalamic nuclei. The main bundle of fibers labeled by nucleus gigantocellularis-pars alpha injections ascended ventromedially through the brainstem, just dorsal to the pyramidal tracts, and joined Forel's tegmental tract in the midbrain. With the brainstem, labeled fibers distributed to the pontomedullary reticular formation, the locus coeruleus, the raphe pontis, the pontine nuclei, and the dorsolateral tegmental nucleus and adjacent regions of the pontine gray. At mesodiencephalic levels, labeling was present in the rostral raphe nuclei (dorsal, median and linearis), the mesencephalic gray, the deep and intermediate layers of the superior colliculus, the medial and anterior pretectal nuclei, the ventral tegmental area, zona incerta as well as the mediodorsal and reticular nuclei of the thalamus. Injections of the parvocellular reticular nucleus labeled axons which coursed through the lateral medullary tegmentum to heavily innervate lateral regions of the medullary and caudal pontine reticular formation, cranial motor nuclei (hypoglossal, facial and trigeminal) and the parabrachial complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: II. Lateral reticular nucleus stimulation

Stimulation of mossy fibers arising from the pontine nuclei can be used as a conditioned stimulus (CS) during classical conditioning of the eyelid/nictitating membrane response (NM). In the present experiment we stimulated another source of mossy fibers, the lateral reticular nucleus (LRN), as a CS for NM conditioning. LRN stimulation was an effective CS, resulting in learning, and the conditioned response to LRN stimulation showed normal extinction. Unpaired presentation of CS and UCS did not result in pseudo-conditioning. Lesions of the cerebellar dentate-interpositus region abolished the conditioned response but left the unconditioned reflex response intact. We suggest that mossy fibers may normally carry CS information to the cerebellum.     

Substance P-containing neurons in the pontomesencephalic tegmentum of the human brain.

We have employed immunohistochemical and computerized morphometric procedures to study substance P-containing neurons in the tegmentum of adult humans. An estimated 192,500 +/- 40,500 substance P-containing neurons were found in three main cytoarchitectural regions: the mesencephalic reticular formation, the central gray, and the pontine reticular formation. The morphology of the immunoreactive neurons varied according to the region in which they were found. On the basis of size alone two types of substance P-containing neurons, large and small, were readily distinguishable by eye and measurement. Within each of the three main regions it was possible to distinguish distinct subgroups using cell size, morphology and position. Large neurons were concentrated in the caudal midbrain (pedunculopontine tegmental nuclei), in the oral pontine reticular nucleus and in the lateral dorsal tegmental nucleus. In contrast, small neurons were concentrated in the rostral mesencephalic reticular formation (cuniform nuclei). Both small and large neurons were found in the midbrain and pontine raphe nuclei. In addition, small neurons were concentrated in discrete midline regions (the periaqueductal gray, the tegmental nuclei of the pontine central gray, and the interpeduncular nucleus). The findings suggest that the majority of neurons in the brainstem tegmental nuclei previously identified as cholinergic also contain substance P in humans.     

Analysis of the circuitry responsible for primary afferent depolarization in the trigeminal spinal nucleus caudalis of cats

Summary Depth analysis was performed on the field potential evoked by stimulation of the infraorbital nerve in the trigeminal spinal nucleus caudalis and the subjacent lateral reticular formation of cats. It was shown by dye marking of the recording positions that each subnucleus of the nucleus caudalis (subnucleus marginalis, gelatinosus and magnocellularis) and the reticular formation could be differentiated from one another by the characteristics of the peripherally evoked field potentials.Responses of neurons were extracellularly recorded in the subnuclei gelatinosus and magnocellularis of the nucleus caudalis and in the reticular formation to stimulation of the trigeminal sensory branches (the frontal, infraorbital and lingual nerves), the nucleus ventralis posteromedialis of the thalamus and the cerebral cortex. The properties of the neurons were studied in relation to their thresholds, latencies, receptive fields (sensory branches effective for spike generation) and frequency-following capacities. These responses were then compared with properties of the PAD induced in the fibers terminating in the nucleus caudalis by similar peripheral and central stimulation.It was found that the neurons in the subnucleus magnocellularis were the most likely candidates for the interneurons mediating the peripherally evoked disynaptic PAD in the trigeminal nerve fibers terminating in the nucleus caudalis.     

大鼠三叉神经脊束核尾侧亚核浅层内谷氨酸能、γ-氨基丁酸能和甘氨酸能终末的中枢起源

三叉神经脊束核尾侧亚核浅层（I、II层）内有较密集的谷氨酸样、γ-氨基丁酸样和甘氨酸样阳性终未。这些终未除分别来自外周初级传入或局部神经元外，还可能来自脑干的核团。为了探讨这些阳性终未的脑干起源部位，本研究用荧光金逆行追踪与免疫荧光组织化学染色相结合的双标技术对三叉尾侧亚核浅层内谷氨酸能、γ-氨基丁酸能和甘氨酸能终未在脑干的起源部位进行了追踪。结果表明：大鼠脑干向三叉尾侧亚核发出谷氨酸能、γ-氨基丁酸能和甘氨酸能投射的神经元主要位于构成下行抑制系统的中脑中央灰质、中缝大核及其周围的网状结构、中缝苍白核和中缝隐核。本研究的结果提示，除已知的5-羟色胺和P物质外，谷氨酸、γ-氨基丁酸和甘氨酸也可能参与下行抑制系统，对面口部伤害性刺激信息传递发挥调控作用。     

The cerebellar projection from the reticular formation of the brain stem in the rabbit

Summary By retrograde transport of horseradish peroxidase the reticulocerebellar projections were examined in twenty-six rabbits.After injections in the cerebellum retrogradely labeled neurons were more numerous in the caudal reticular formation (ventral and gigantocellular reticular nuclei) than in its rostral part (caudal and oral pontine reticular nuclei). The labeled cells were of all sizes, large, medium-sized and small. Giant cells were labeled only after injections in the posterior lobe vermis.After injections in the anterior lobe, the posterior vermis, the fastigial nucleus and the flocculus, retrogradely labeled neurons were found bilaterally in the ventral reticular nucleus, the gigantocellular reticular nucleus and the caudal pontine reticular nucleus. Some cases with posterior vermal and fastigial injections in addition showed labeled neurons bilaterally in the oral pontine reticular nucleus. There were no major side differences. The cases with injections in the anterior part of the paramedian lobule gave rise to only a few labeled cells in the gigantocellular reticular nucleus.Negative findings were consistently made in the mesencephalic reticular formation.