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The basilar pontine nucleus (PN) is the key relay point for the cerebrocerebellar link. However, the projection pattern of pontocerebellar mossy fiber axons, which is essential in determining the functional organization of the cerebellar cortex, has not been fully clarified. We reconstructed the entire trajectory of 25 single pontocerebellar mossy fiber axons labeled by localized injection of biotinylated dextran amine into various locations in the PN and mapped all their terminals in an unfolded scheme of the cerebellum in 10 mice. The majority of axons (20/25 axons) entered the cerebellum through the middle cerebellar peduncle contralateral to the origin, while others entered through the ipsilateral pathway. A small number of axons (1/25 axons) had collaterals terminating in the cerebellar nuclei. Axons projected mostly to a combination of lobules, often bilaterally, and terminated in multiple zebrin (aldolase C) stripes, more frequently in zebrin-positive stripes (83.9%) than in zebrin-negative stripes, with 66.5 mossy fiber terminals on the average. Axons originating from the rostral (plus medial and lateral), central and caudal PN mainly terminated in the paraflocculus, crus I and lobule VIb-c, in the simplex lobule, crus II and paramedian lobule, and in lobules II-VIa, VIII and copula pyramidis, respectively. The results suggest that the interlobular branching pattern of pontocerebellar axons determines the group of cerebellar lobules that are involved in a related functional localization of the cerebellum. In the hemisphere, crus I may be functionally distinct from neighboring lobules (simple lobule and crus II) in the mouse cerebellum based on the pontocerebellar axonal projection pattern., (© 2019 Wiley Periodicals, Inc.)

The cerebellum is involved in the control of movement, emotional responses, and reward processing. The tree shrew is the closest living relative of primates. However, little is known not only about the systematic nomenclature for the tree shrew cerebellum but also about the detailed neurochemical characterization and afferent projections. In this study, Nissl staining and acetylcholinesterase histochemistry were used to reveal anatomical features of the cerebellum of tree shrews (Tupaia belangeri chinensis). The cerebellar cortex presented a laminar structure. The morphological characteristics of the cerebellum were comprehensively described in the coronal, sagittal, and horizontal sections. Moreover, distributive maps of calbindin-immunoreactive (-ir) cells in the Purkinje cell layer of the cerebellum of tree shrews were depicted using coronal, sagittal, and horizontal schematics. In addition, 5th cerebellar lobule (5Cb)-projecting neurons were present in the pontine nuclei, reticular nucleus, spinal vestibular nucleus, ventral spinocerebellar tract, and inferior olive of the tree shrew brain. The anterior part of the paramedian lobule of the cerebellum (PMa) received mainly strong innervation from the lateral reticular nucleus, inferior olive, pontine reticular nucleus, spinal trigeminal nucleus, pontine nuclei, and reticulotegmental nucleus of the pons. The present results provide the first systematic nomenclature, detailed atlas of the whole cerebellum, and whole-brain mapping of afferent projections to the 5Cb and PMa in tree shrews. Our findings provide morphological support for tree shrews as an alternative model for studies of human cerebellar pathologies., (© 2018 Wiley Periodicals, Inc.)

The pontine nuclei (PN) are the largest of the precerebellar nuclei, neuronal assemblies in the hindbrain providing principal input to the cerebellum. The PN are predominantly innervated by the cerebral cortex and project as mossy fibers to the cerebellar hemispheres. Here, we comprehensively review the development of the PN from specification to migration, nucleogenesis and circuit formation. PN neurons originate at the posterior rhombic lip and migrate tangentially crossing several rhombomere derived territories to reach their final position in ventral part of the pons. The developing PN provide a classical example of tangential neuronal migration and a study system for understanding its molecular underpinnings. We anticipate that understanding the mechanisms of PN migration and assembly will also permit a deeper understanding of the molecular and cellular basis of cortico-cerebellar circuit formation and function.

The cerebellum receives extensive disynaptic input from the neocortex via the basal pontine nuclei, the neurons of which send mossy fiber (MF) axons to the granule cell layer of the contralateral cerebellar hemisphere. Although this cortico-cerebellar circuit has been implicated in tasks such as sensory discrimination and motor learning, little is known about the potential role of MF morphological plasticity in the function of the cerebellar granule cell layer. To address this issue, we labeled MFs with EGFP via viral infection of the basal pons in adult rats and performed in vivo two-photon imaging of MFs in Crus I/II of the cerebellar hemisphere over a period of several weeks. Following the acquisition of baseline images, animals were housed in control, enriched, or deprived sensory environments. Morphological dynamics were assessed by tracing MF axons and their terminals, and by tracking the stability of filopodia arising from MF terminal rosettes. MF axons and terminals were found to be remarkably stable. Parameters derived neither from measurements of axonal arbor geometry nor from the morphology of individual rosettes and their filopodial extensions significantly changed under control conditions over 4 weeks of imaging. Increasing whisker stimulation by manipulating the sensory environment or decreasing such stimulation by whisker trimming also failed to alter MF structure. Our studies indicate that pontine MF axons projecting to Crus I/II in adult rats do not undergo significant structural rearrangements over the course of weeks, and that this stability is not altered by the sustained manipulation of whisker sensorimotor experience.

Sensorimotor integration is crucial to perception and motor control. How and where this process takes place in the brain is still largely unknown. Here we analyze the cerebellar contribution to sensorimotor integration in the whisker system of mice. We identify an area in the cerebellum where cortical sensory and motor inputs converge at the cellular level. Optogenetic stimulation of this area affects thalamic and motor cortex activity, alters parameters of ongoing movements and thereby modifies qualitatively and quantitatively touch events against surrounding objects. These results shed light on the cerebellum as an active component of sensorimotor circuits and show the importance of sensorimotor cortico-cerebellar loops in the fine control of voluntary movements.

Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI:http://dx.doi.org/10.7554/eLife.00400.001.

We investigated the role of histone methyltransferase Ezh2 in tangential migration of mouse precerebellar pontine nuclei, the main relay between neocortex and cerebellum. By counteracting the sonic hedgehog pathway, Ezh2 represses Netrin1 in dorsal hindbrain, which allows normal pontine neuron migration. In Ezh2 mutants, ectopic Netrin1 derepression results in abnormal migration and supernumerary nuclei integrating in brain circuitry. Moreover, intrinsic topographic organization of pontine nuclei according to rostrocaudal progenitor origin is maintained throughout migration and correlates with patterned cortical input. Ezh2 maintains spatially restricted Hox expression, which, in turn, regulates differential expression of the repulsive receptor Unc5b in migrating neurons; together, they generate subsets with distinct responsiveness to environmental Netrin1. Thus, Ezh2-dependent epigenetic regulation of intrinsic and extrinsic transcriptional programs controls topographic neuronal guidance and connectivity in the cortico-ponto-cerebellar pathway.

Precise temporal and spatial sequences of synaptogenesis were demonstrated in 172 human foetuses and neonates post-mortem in transverse paraffin sections of pons and cerebellar vermis and hemispheres, using synaptophysin immunoreactivity of this protein of synaptic vesicular walls. The pontine nuclei exhibit a transitory patchy pattern not predicted from the uniform histology and reminiscent of the corpus striatum; synaptic vesicle reactivity appears at 20 weeks and is uniform by 34 weeks. In the cerebellar cortex, the vermis matures sooner than the cerebellar hemispheres and the paravermal portions earlier than the lateral folia. The earliest synapses occur around the somata of Purkinje neurons and later in the internal granular layer, but synaptic glomeruli are not well formed until after 26 weeks. The normal patterns here shown, together with earlier data of the Guillain-Mollaret triangle, provide controls for the interpretation of synaptic delay or precociousness and other pathological patterns in malformations, genetic/metabolic conditions and prenatal acquired insults affecting the human foetus., (Copyright © 2013 S. Karger AG, Basel.)

The cerebellum dedicates a majority of the brain's neurons to processing a wide range of sensory, motor, and cognitive signals. Stereotyped circuitry within the cerebellar cortex suggests that similar computations are performed throughout the cerebellum, but little is known about whether diverse precerebellar neurons are specialized for the nature of the information they convey. In vivo recordings indicate that firing responses to sensory or motor stimuli vary dramatically across different precerebellar nuclei, but whether this reflects diverse synaptic inputs or differentially tuned intrinsic excitability has not been determined. We targeted whole-cell patch-clamp recordings to neurons in eight precerebellar nuclei which were retrogradely labeled from different regions of the cerebellum in mice. Intrinsic physiology was compared across neurons in the medial vestibular, external cuneate, lateral reticular, prepositus hypoglossi, supragenual, Roller/intercalatus, reticularis tegmenti pontis, and pontine nuclei. Within the firing domain, precerebellar neurons were remarkably similar. Firing faithfully followed temporally modulated inputs, could be sustained at high rates, and was a linear function of input current over a wide range of inputs and firing rates. Pharmacological analyses revealed common expression of Kv3 currents, which were essential for a wide linear firing range, and of SK (small-conductance calcium-activated potassium) currents, which were essential for a wide linear input range. In contrast, membrane properties below spike threshold varied considerably within and across precerebellar nuclei, as evidenced by variability in postinhibitory rebound firing. Our findings indicate that diverse precerebellar neurons perform similar scaling computations on their inputs but may be differentially tuned to synaptic inhibition.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by polyglutamine-expanded ataxin-3. SCA3 neurodegeneration is found in the pontine nuclei and cerebellum. Polyglutamine-expanded ataxin-3-Q79 caused apoptotic death of cerebellar and pontine nuclei neurons by upregulating mRNA expression of pro-apoptotic Bax and activating mitochondria-mediated apoptotic cascade. Following various cellular stresses, transcription factor p53 promotes apoptotic neuronal death by enhancing the transcription of pro-apoptotic genes including Bax and PUMA. In the present study, cellular and animal models of SCA3 were used to test the hypothesis that mutant polyglutamine ataxin-3 upregulates Bax expression of cerebellar and pontine nuclei neurons by augmenting transcriptional activity of p53. Electrophoretic mobility shift assay (EMSA) indicated that p53 binding activity to Bax promoter sequence was significantly enhanced in cultured cerebellar neurons expressing mutant ataxin-3-Q79 and pontine nuclei and cerebellum of SCA3 transgenic mice expressing ataxin-3-Q79. The mRNA level of PUMA, a p53-inducible pro-apoptotic gene, was increased in the cerebellum and pontine nuclei of SCA3 transgenic mice and cultured cerebellar neurons expressing ataxin-3-Q79. Mutant polyglutamine ataxin-3 increased the protein level of active phospho-p53(Ser15) in cerebellar and pontine nuclei neurons without affecting mRNA or protein level of p53. Intraperitoneal administration of p53 inhibitor pifithrin-α significantly ameliorated neuronal death in the pontine nuclei of SCA3 transgenic mice. Our results suggest that polyglutamine-expanded ataxin-3 upregulates mRNA expression of Bax and PUMA and causes apoptotic death of affected neurons by enhancing phosphorylation and transcriptional activity of p53., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The primate lobulus petrosus (LP) of the cerebellar paraflocculus receives inputs from visual system-related pontine nuclei, and projects to eye movement-related cerebellar nuclei. To reveal a potential involvement of LP in oculomotor control, we lesioned LP unilaterally by local injections of ibotenic acid in three Macaca fuscata. We examined the effects of lesion on eye movements evoked by step (3 degrees )-ramp (5-15 degrees/s) moving target. To step-ramp moving target, the monkeys showed an initial slow eye movement and later a small catch-up saccade, which was followed by the post-saccadic pursuit nearly matching to the velocity of the ramp target motion. After LP lesioning, the velocity of post-saccadic pursuits in the ipsiversive and down-ward directions decreased by 20-40% in all three monkeys. These deficits lasted for at least 1 month, and some recovery was observed. In the amplitudes of catch-up saccades, no consistent changes were seen among the three monkeys after LP lesioning. These results suggest an involvement of LP in the primate smooth pursuit eye movement control.

The cerebral cortex conveys major input to the granule cell layer of the cerebellar hemispheres by way of the pontine nuclei. Cerebrocortical projections terminate in multiple, widely distributed clusters in the pontine nuclei. This clustered organization is thought to provide the transition between the different organizational principles of the cerebrum and cerebellum, and indicates that parallel processing occurs at multiple sites in the pontine nuclei. At a cellular level, however, it is unknown whether individual cerebropontine neurons target pontocerebellar cells located in different clusters or not. We have employed anterograde axonal tracing and 3D computerized reconstruction techniques to characterize the branching pattern and morphology of individual cerebropontine axons from the primary somatosensory cortex (SI). Our findings show that 43% of the cerebrobulbar fibers arising from SI whisker representations provide two or three fibers entering the pontine nuclei, whereas 39% have only one fiber, and the remaining 18% do not project to the pontine nuclei. Thus, it appears that a majority of cerebropontine axons originating in SI whisker representations diverge to contact multiple, separated pontocerebellar cells. Further, 84% of the somatosensory cerebropontine fibers are collateral branches from cerebrobulbar and/or cerebrospinal parent fibers, while 16% are direct cerebropontine projections without a further descending projection. A range of thicknesses of the fibers entering the pontine nuclei were observed, with collaterals of corticobulbar fibers having the smallest diameter. Taken together, these findings may be related to previously described separate cerebropontine transmission lines with different properties.

The granule cell layer of the cerebellar hemispheres contains a patchy and noncontinuous map of the body surface, consisting of a complex mosaic of multiple perioral tactile representations. Previous physiological studies have shown that cerebrocerebellar mossy fibre projections, conveyed through the pontine nuclei, are mapped in registration with peripheral tactile projections to the cerebellum. In contrast to the fractured cerebellar map, the primary somatosensory cortex (SI) is somatotopically organized. To understand better the map transformation occurring in cerebrocerebellar pathways, we injected axonal tracers in electrophysiologically defined locations in Sprague-Dawley rat folium crus IIa, and mapped the distribution of retrogradely labelled neurons within the pontine nuclei using three-dimensional (3-D) reconstructions. Tracer injections within the large central upper lip patch in crus IIa-labelled neurons located centrally in the pontine nuclei, primarily contralateral to the injected side. Larger injections (covering multiple crus IIa perioral representations) resulted in labelling extending only slightly beyond this region, with a higher density and more ipsilaterally labelled neurons. Combined axonal tracer injections in upper lip representations in SI and crus IIa, revealed a close spatial correspondence between the cerebropontine terminal fields and the crus IIa projecting neurons. Finally, comparisons with previously published three-dimensional distributions of pontine neurons labelled following tracer injections in face receiving regions in the paramedian lobule (downloaded from http://www.rbwb.org) revealed similar correspondence. The present data support the coherent topographical organization of cerebro-ponto-cerebellar networks previously suggested from physiological studies. We discuss the present findings in the context of transformations from cerebral somatotopic to cerebellar fractured tactile representations.

The organization of pontocerebellar projections to the paravermis and hemisphere of the posterior cerebellum of the rat was studied in relation to the organization of climbing fibers. Small injections of cholera toxin subunit B were placed in the cerebellar cortex at locations predetermined by evoked climbing fiber potentials from selected body parts or based on coordinates. The injection site was characterized with respect to the zebrin pattern and by the distribution of retrogradely labeled neurons in the inferior olive. The following zones were studied: hindlimb-related zones C1 and C2 of lobule VIII; forelimb-related zones C1, C2, and D0/D1 of the paramedian lobule; and face-related zones A2 of the paramedian lobule and C2 and D0 of crus 2B. The results show that the distribution of pontine neurons is closely related to the climbing fiber somatotopy. Injections centered on face-related zones result in distribution of pontine neurons within the pontine core region. Forelimb regions surround this core, whereas hindlimb regions are mostly supplied by caudal pontine regions and by a single patch of more rostrally located neurons. This distribution fits well with published data on the somatotopy of the corticopontine projection from the rat primary somatosensory cortex. However, apart from differences in the participation of ipsilaterally projecting cells, the distribution of pontine neurons does not change significantly when the injection covers different zones of the same lobule such as C1 and C2 of lobule VIII; C1, C2, and D0/D1 of the paramedian lobule; A2 of the paramedian lobule; and C2 and D0 of crus 2B.

Previous studies often considered the basilar pontine nuclei (BPN) and the nucleus reticularis tegmenti pontis (NRTP) as relays of a single cerebro-(ponto)-cerebellar pathway. Conversely, the different cortical afferences to the BPN and the NRTP, as well as the anatomical and functional features of the cerebellopetal projections from these pontine nuclei, support the different, and for some aspect, complementary arrangement of the cerebrocerebellar pathways relayed by the BPN or NRTP. Both the BPN and the NRTP are innervated from the cerebral cortex, but with regional prevalence. The NRTP is principally innervated from motor or sensori-motor areas while the BPN are principally innervated from sensory, mainly teloceptive, and associative area. Projections from sensory-motor areas were also traced to the BPN. The BPN and NRTP project to all parts of the cerebellar cortex with a similar pattern. In fact, from single areas of them projections were traced to set of sagittal stripes of the cerebellar cortex. In variance to such analogies, the projections to the cerebellar nuclei differed between those traced from the NRTP and from BPN. In fact, BPN and NRTP have private terminal areas in the cerebellar nuclei with relatively little overlaps. The BPN innervated the lateroventral part of the nucleus lateralis and the caudoventral aspect of the nucleus interpositalis posterioris. The NRTP principally innervated the mediodorsal part of the nucleus lateralis, the nucleus interpositalis anterioris, the nucleus medialis. Since the single cerebellar nuclei have their specific targets in the extracerebellar brain areas, it follows that the BPN and the NRTP, passing through their cerebellar nuclei relays, are devoted to control different brain areas and thus likely to play different functional roles. From single pontine regions (of both BPN and NRTP) projections were traced to the cerebellar cortex and to the cerebellar nuclei. In some cases these projections reached areas which are likely anatomically connected (by Purkinje axons). This pattern of the pontine projections was termed as coupled projection. In some other cases, the projections reached areas of the cerebellar cortex but not the nuclear regions innervated by them. We termed this as uncoupled projection. The existence of both coupled and uncoupled projections, open new vistas on the functional architecture of the pontocerebellar pathway. More in detail, this study showed the different quantitative and topographic distribution of the coupled and uncoupled projections visualized in the cerebellar projections from BPN and NRTP. All these evidences strongly support the anatomical and the functional differences that characterise the cerebrocerebellar pathways relayed by the BPN and the NRTP.

During development, precerebellar neurons migrate dorsoventrally from the rhombic lip to the floor plate. Some of these neurons cross the midline while others stop. We have identified a role for the slit receptor Rig-1/Robo3 in directing this process. During their tangential migration, neurons of all major hindbrain precerebellar nuclei express high levels of Rig-1 mRNA. Rig-1 expression is rapidly downregulated as their leading process crosses the floor plate. Interestingly, most precerebellar nuclei do not develop normally in Rig-1-deficient mice, as they fail to cross the midline. In addition, inferior olivary neurons, which normally send axons into the contralateral cerebellum, project ipsilaterally in Rig-1 mutant mice. Similarly, neurons of the lateral reticular nucleus and basilar pons are unable to migrate across the floor plate and instead remain ipsilateral. These results demonstrate that Rig-1 controls the ability of both precerebellar neuron cell bodies and their axons to cross the midline.

We aimed to clarify the overall distribution of glycinergic neurons in the midbrain, pons, and cerebellum in rats, using in situ hybridization for mRNA encoding glycine transporter 2 (GLYT2), which reliably detects glycinergic cell bodies. We combined this method with in situ hybridization for mRNA encoding glutamic acid decarboxylase isoform 67 (GAD67), and have presented for the first time global and detailed views of the distribution of glycinergic neurons in relation to GABAergic neurons. In addition to this single-detection study, we performed double-detection of GLYT2 mRNA and GAD67 mRNA to determine the distribution of neurons co-expressing these mRNAs. We have shown that many areas of the brainstem and cerebellum, not only areas where previous immunohistochemical studies have specified, involve double-labeled neurons with GLYT2 and GAD67 mRNAs. In particular, when lightly labeled GLYT2 mRNA-positive neurons were distributed within the area of GAD67 mRNA-positive neurons, almost all such GLYT2 mRNA-positive neurons were GAD67 mRNA-positive. Areas or neuron groups expressing exclusively GLYT2 mRNA or GAD67 mRNA were rather limited, such as the superior colliculus, nucleus of the trapezoid body, and Purkinje cells. The present study suggests that the corelease of glycine and GABA from single neurons is more widespread than has been reported.

The molecular cues that regulate neurite morphology within the target environment are key to the formation of complex neural circuitry. During development of the ponto-cerebellar projection, pontine fibers sprout and form elaborate arbors within the inner cerebellar layer prior to arrival of their target cells, the cerebellar granule neurons. Here, we describe the biochemical fractionation of two granule neuron-derived factors that stimulate elaboration of pontine neurites. These factors were identified using a dissociated pontine bioassay and biochemically fractionated from granule cell (GC) conditioned medium (GCCM). One of the factors, STIM1, is a protein with a molecular weight greater than 30 kDa that is distinct from known neurotrophins. The other, STIM2, is a small, protease-resistant molecule with an estimated molecular weight below 1 kDa. We show that these factors stimulate pontine neurite elongation both independently and cooperatively and thus may contribute to the formation of elaborate pontine arbors within the cerebellar cortex.

Neuronal responses that were observed during smooth-pursuit eye movements were recorded from cells in rostral portions of the nucleus reticularis tegmenti pontis (rNRTP). The responses were categorized as smooth-pursuit eye velocity (78%) or eye acceleration (22%). A separate population of rNRTP cells encoded static eye position. The sensitivity to pursuit eye velocity averaged 0.81 spikes/s per degrees /s, whereas the average sensitivity to pursuit eye acceleration was 0.20 spikes/s per degrees /s(2). Of the eye-velocity cells with horizontal preferences for pursuit responses, 56% were optimally responsive to contraversive smooth-pursuit eye movements and 44% preferred ipsiversive pursuit. For cells with vertical pursuit preferences, 61% preferred upward pursuit and 39% preferred downward pursuit. The direction selectivity was broad with 50% of the maximal response amplitude observed for directions of smooth pursuit up to +/-85 degrees away from the optimal direction. The activities of some rNRTP cells were linearly related to eye position with an average sensitivity of 2.1 spikes/s per deg. In some cells, the magnitude of the response during smooth-pursuit eye movements was affected by the position of the eyes even though these cells did not encode eye position. On average, pursuit centered to one side of screen center elicited a response that was 73% of the response amplitude obtained with tracking centered at screen center. For pursuit centered on the opposite side, the average response was 127% of the response obtained at screen center. The results provide a neuronal rationale for the slow, pursuit-like eye movements evoked with rNRTP microstimulation and for the deficits in smooth-pursuit eye movements observed with ibotenic acid injection into rNRTP. More globally, the results support the notion of a frontal and supplementary eye field-rNRTP-cerebellum pathway involved with controlling smooth-pursuit eye movements.

Novel neuroanatomic approaches for investigating topographic maps at a systems level include combined use of sensitive neural tracing techniques and computerized methods for three-dimensional reconstruction from serial sections. Application of these methods have allowed discovery of new principles of topographic organization in the rat pontine nuclei. The pontine nuclei are intercalated in the large pathways connecting the cerebral cortex to the cerebellum. In rat, cerebropontine projections are characterized by multiple delineated terminal fields. It is generally accepted that these projections are topographically organized. The presence of widespread axonal clusters in the pontine nuclei is typically interpreted to represent a complex scheme of organization. In recent anatomic investigations of somatosensory corticopontine projections in young and adult rats, a somatotopic distribution of axonal clusters, concentrically organized in an inside-out fashion, has been reported. This review summarizes the topographic principles of organization proposed for somatosensory corticopontine projections, and discusses the possibility that widely segregated corticopontine terminal fields are located inside lamellar volumes. This organizational pattern may be explained by mechanisms operative during development, and resembles the patterns of organization previously described in cat and monkey. Possible implications of this architecture are discussed in relation to map transformations from the cerebral cortex to the cerebellum.

In this study, we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections from the pontine nuclei (PN) to the rostral (rPML) and caudal (cPML) parts of cerebellar paramedian lobule in the rabbit, known to be the forelimb and hindlimb regions, respectively. Following injections of fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) within rPML and cPML, respectively, substantial numbers of FB and DY single labeled neurons were found in the dorsolateral, paramedian, lateral and peduncular pontine nuclei bilaterally with a very clearcut contralateral preponderance. No labeling was observed in the ventral pontine nucleus. Extensive areas of overlap of FB or DY labeled neurons indicated that no somatotopical relationship existed in projection from PN to the two functionally different PML target regions. In addition, a small number of double FB + DY labeled neurons was detected in the common areas of FB and DY single labeling in PN. These neurons give rise to pontocerebellar projections to rPML and cPML simultaneously by way of axonal collaterals and thus they may play a role in the coordination of unilateral forelimb and hindlimb movements.

As an approach toward understanding the molecular mechanisms of neuronal differentiation, we utilized DNA microarrays to elucidate global patterns of gene expression during pontocerebellar development. Through this analysis, we identified groups of genes specific to neuronal precursor cells, associated with axon outgrowth, and regulated in response to contact with synaptic target cells. In the cerebellum, we identified a phase of granule cell differentiation that is independent of interactions with other cerebellar cell types. Analysis of pontine gene expression revealed that distinct programs of gene expression, correlated with axon outgrowth and synapse formation, can be decoupled and are likely influenced by different cells in the cerebellar target environment. Our approach provides insight into the genetic programs underlying the differentiation of specific cell types in the pontocerebellar projection system.

This study aimed to investigate the trajectory of fibres from the pontine nuclei that reach the two sides of the cerebellum. Injections of biotinylated dextran amine (BDA) were made within the basilar pontine nuclei (BPN) and the nucleus reticularis tegmenti pontis (NRTP) in one side of rats with electrolytic injury of the middle cerebellar peduncle (MCP), ipsilateral or contralateral to the side of injection. Fibres were traced from the pontine nuclei (BPN and NRTP) to both sides of the cerebellum passing through the respective MCPs. The study carried out in rats with injury to one peduncle showed projections segregated to the half-side of the cerebellum innervated by the intact peduncle. The laterality observed was confirmed by a retrograde tracer study. In fact, injections of different fluorescent tracers in rats with injury of single MCP showed that in the pontine nuclei only cell bodies stained by the tracer injected in the half-cerebellum ipsilateral to the intact peduncle. Finally, similar injections (i.e. different fluorescent tracers in symmetric areas of the cerebellar cortex) in the cerebellum of intact brain rats showed that BPN and NRTP differ for the laterality of their projections. In fact, 82% of BPN cells project contralaterally and 18% ipsilaterally, whereas 60% of NRTP cells project contralaterally and 40% ipsilaterally. In conclusion, this study showed that the MCPs receive fibres from the pontine nuclei of both sides and project to the ipsilateral half of the cerebellum and that different contingents of projections to the two sides of the cerebellum arise from BPN and NRTP.

The relationship between axons derived from the prearcuate region and pontine neurons projecting to contralateral cerebellar hemispheric lobule VII, lobulus petrosus (LP) of the paraflocculus or the dorsal paraflocculus (DPFl) was investigated in the monkey. The frontopontine axons were labeled with biotinylated dextran and pontocerebellar neurons with cholera toxin subunit B or fast blue. Labeled frontopontine axons and terminals were seen in the dorsal, medial, paramedian and dorsolateral parts of the pontine nuclei. The distribution of the labeled frontopontine axons overlapped that of labeled neurons projecting to hemispheric lobule VII but did not overlap that of labeled neurons projecting to LP or DPFl. The pathway from the prearcuate region to hemispheric lobule VII may provide an anatomical substrate for the involvement of hemispheric lobule VII in voluntary eye movements.

Patients with pathological laughter and crying (PLC) are subject to relatively uncontrollable episodes of laughter, crying or both. The episodes occur either without an apparent triggering stimulus or following a stimulus that would not have led the subject to laugh or cry prior to the onset of the condition. PLC is a disorder of emotional expression rather than a primary disturbance of feelings, and is thus distinct from mood disorders in which laughter and crying are associated with feelings of happiness or sadness. The traditional and currently accepted view is that PLC is due to the damage of pathways that arise in the motor areas of the cerebral cortex and descend to the brainstem to inhibit a putative centre for laughter and crying. In that view, the lesions 'disinhibit' or 'release' the laughter and crying centre. The neuroanatomical findings in a recently studied patient with PLC, along with new knowledge on the neurobiology of emotion and feeling, gave us an opportunity to revisit the traditional view and propose an alternative. Here we suggest that the critical PLC lesions occur in the cerebro-ponto-cerebellar pathways and that, as a consequence, the cerebellar structures that automatically adjust the execution of laughter or crying to the cognitive and situational context of a potential stimulus, operate on the basis of incomplete information about that context, resulting in inadequate and even chaotic behaviour.

In the primary somatosensory cortex (SI), the body surface is mapped in a relatively continuous fashion, with adjacent body regions represented in adjacent cortical domains. In contrast, somatosensory maps found in regions of the cerebellar hemispheres, which are influenced by the SI through a monosynaptic link in the pontine nuclei, are discontinuous ("fractured") in organization. To elucidate this map transformation, the authors studied the organization of the first link in the SI-cerebellar pathway, the SI-pontine projection. After injecting anterograde axonal tracers into electrophysiologically defined parts of the SI, three-dimensional reconstruction and computer-graphic visualization techniques were used to analyze the spatial distribution of labeled fibers. Several target regions in the pontine nuclei were identified for each major body representation. The labeled axons formed sharply delineated clusters that were distributed in an inside-out, shell-like fashion. Upper lip and other perioral representations were located in a central core, whereas extremity and trunk representations were found more externally. The multiple clusters suggest that the pontine nuclei contain several representations of the SI map. Within each representation, the spatial relationships of the SI map are largely preserved. This corticopontine projection pattern is compatible with recently proposed principles for the establishment of subcortical topographic patterns during development. The largely preserved spatial relationships in the pontine somatotopic map also suggest that the transformation from an organized topography in SI to a fractured map in the cerebellum takes place primarily in the mossy fiber pontocerebellar projection., (Copyright 2000 Wiley-Liss, Inc.)

The cerebellar flocculus has been implicated in vestibulo-oculomotor control. One major central input to this structure originates from brainstem cells in the paramedian tract (PMT), whose function is unknown. Here it is reported that PMT cells in the pons carry vestibular and eye movement signals and their pharmacological inactivation produces a leaky integrator combined with vestibular imbalance. The results suggest that PMT cells provide the cerebellum with sensory and motor signals that are essential for velocity-to-position integration, a common premotor process that is required in all motor systems.

The linear Purkinje cell density in the cerebellar vermis was investigated in a small cohort of adult onset schizophrenic men with well-documented hippocampal abnormalities. There were no differences in comparison with age-matched controls. This negative finding indicates that vermian abnormalities undoubtedly seen in some schizophrenic patients may constitute a subsyndrome, possibly related to autistic disorders in which cerebellar abnormalities are well corroborated.

If numbers matter, the projection that connects the cerebral cortex to the cerebellum is probably one of the most-important pathways through the CNS. Its extensive development as one ascends the phylogenetic scale parallels that of the cerebral hemispheres and the cerebellum, and it accompanies improvements in motor skills, suggesting that this system might have a decisive role in the generation of skilled movement. This article focuses on the pontine nuclei (PN), which are intercalated in the cerebro-cerebellar pathway, a large nuclear complex in the ventral brainstem of mammals, whose raison d'être has as yet not been examined. By considering recent morphological and electrophysiological findings, this article argues that the PN are an interface that is needed to accommodate the grossly different computational principles governing the cerebral cortex and the cerebellum.

We have studied the anatomic organization of corticopontine neurons in the monkey cytoarchitectonic areas 3a, 3b, 1, and 2. The purpose was to provide information about the composition of somatosensory cortical influence on cerebellar operations. Large tracer injections were made in the pontine nuclei. Retrogradely labeled neurons were confined to cortical layer 5, with the largest cell bodies located in area 3a and the smallest in area 3b. The distribution of labeled cells was quantitatively recorded and displayed in three-dimensional reconstructions and in flat maps. We have: (1) compared the average densities of labeled cells among the cytoarchitectonic areas, and (2) outlined the distribution of labeled cells within the cortical map of the body surface representation. The average density of labeled cells was considerably higher in areas 3a, 1, and 2, compared to area 3b. This finding suggests that areas 3a, 1, and 2 are more engaged in cerebellar operations than area 3b. We found marked density gradients of labeled cells within areas 3b, 1, and 2, but not within area 3a. When the density maps from areas 3b, 1, and 2 were superimposed on previously published somatotopic maps, we found higher average densities of corticopontine neurons in regions representing the trunk and proximal limbs, than in regions representing the distal forelimb. Thus, the distal forelimb representation, which is known to be strongly emphasized in terms of cortical volume, appears not to be correspondingly emphasized in the corticopontine projection.

Sources of mossy and climbing fiber inputs to the flocculus (FL), ventral paraflocculus (VP) and/or dorsal paraflocculus (DP) were identified in the vestibular ganglion, medulla oblongata and pons of 19 Wistar rats after 26 local injections of horseradish peroxidase, wheat-germ agglutinin-conjugated horseradish peroxidase, fast blue or diamidino yellow into the FL, VP and/or DP. There were large differences in the sources of mossy fibers to the FL and VP/DP. Labeled neurons after injections into the FL were observed mainly in the ipsilateral vestibular ganglion, bilaterally in the vestibular and prepositus hypoglossal nuclei, and in the caudal part of the nucleus reticularis tegmenti pontis. Labeled neurons were rarely observed in the pontine nuclei after localized injections into the FL. By contrast, after injections into the VP and/or DP, numerous labeled neurons were observed in the pontine nuclei with a contralateral predominance and in the rostral part of the nucleus reticularis tegmenti pontis bilaterally, but not in the vestibular nuclei in either side. Sources of climbing fibers to the FL and paraflocculus were completely contralateral to the injection side. After injection into the FL, labeled neurons were observed in the caudal dorsal cap and ventrolateral outgrowth of the inferior olivary nucleus. After injections into the VP, labeled neurons were observed mainly in the rostral dorsal cap, ventral medial accessory olivary nucleus (MAO) and caudal half of the ventral leaf of the principal olivary nucleus. After injections into the DP, labeled neurons were observed in the ventral MAO and caudal half of the ventral leaf of the principal olivary nucleus. These differences in the sources of mossy and climbing fiber inputs may suggest functional differences between the FL and VP/DP. The present results are consistent with our previous observations in monkey that the FL and VP/DP exhibit quite different mossy fiber input organizations.

In an in vitro model system based on purified target cerebellar granule neurons and explants of afferents, pontine mossy fiber afferents stop growing through contact-mediated mechanisms when they encounter granule neurons. Here we studied the developmental regulation of the stop signal posed by granule cells and the response of mossy fibers to the stop signal in two culture systems. Granule neurons presented in slices or as dissociated cells from postnatal day (P) 4 and P7 cerebellum were more potent in the arrest of P0 pontine neurites than younger (P0-P2) or older (up to P14) granule neurons. In contrast, pontine neurites at embryonic day (E) 18, during their period of normal growth toward the cerebellum, grew extensively on both cerebellar slices of all ages from P0 to P10 and dissociated P4 granule neurons. When E18 explants were maintained for 2 days before plating in medium conditioned by neonatal cerebellar cells, E18 pontine explants were rendered more responsive to the stop signal from P4 granule cells. These results indicate that the stop signal, and the response of afferents to it, are developmentally regulated. Moreover, factors within the target region may initiate these interactions.

Dendritic features of identified projection neurons in two precerebellar nuclei, the pontine nuclei (PN) and the nucleus reticularis tegmenti pontis (NRTP) were established by using a combination of retrograde tracing (injection of fluorogold or rhodamine labelled latex micro-spheres into the cerebellum) with subsequent intracellular filling (lucifer yellow) in fixed slices of pontine brainstem. A multivariate analysis revealed that parameters selected to characterize the dendritic tree such as size of dendritic field, number of branching points, and length of terminal dendrites did not deviate significantly between different regions of the PN and the NRTP. On the other hand, projection neurons in ventral regions of the PN were characterized by an irregular coverage of their distal dendrites by appendages while those in the dorsal PN and the NRTP were virtually devoid of them. The NRTP, dorsal, and medial PN tended to display larger somata and more primary dendrites than ventral regions of the PN. These differences, however, do not allow the differentiation of projection neurons within the PN from those in the NRTP. They rather reflect a dorso-ventral gradient ignoring the border between the nuclei. Accordingly, a cluster analysis did not differentiate distinct types of projection neurons within the total sample. In both nuclei, multiple linear regression analysis revealed that the size of dendritic fields was strongly correlated with the length of terminal dendrites while it did not depend on other parameters of the dendritic field. Thus, larger dendritic fields seem not to be accompanied by a higher complexity but rather may be used to extend the reach of a projection neuron within the arrangement of afferent terminals. We suggest that these similarities within dendritic properties in PN and NRTP projection neurons reflect similar processing of afferent information in both precerebellar nuclei.

The feedforward limb of the cerebrocerebellar system is directed largely through the corticopontine pathway. We determined the extent to which higher order corticopontine projections are derived from prefrontal associative cortices by injecting anterograde tracers into multiple prefrontal regions in rhesus monkeys. Most prefrontopontine projections were derived from the dorsolateral and dorsomedial convexities, including areas 8A, 46 dorsal, 9, and 10; and lighter projections arose from medial and ventrolateral cortices. These findings strengthen the observation that the cerebrocerebellar system incorporates associative cerebral regions, and they enhance the notion that the cerebellum participates in the organization of cognitive function.

Volkensin and ricin, either free or conjugated with colloidal gold, were injected into the cerebellar cortex of rats. The inferior olive and pontine nuclei were examined to verify the retrograde axonal transport of these two toxins, and the consequent neuronal damage. No evidence was obtained of a retrograde axonal transport of ricin in these pathways. Injection of gold-conjugated volkensin in the cerebellar cortex resulted in retrogradely labelled neurones in the inferior olive after 3 h, and in the pontine nuclei after 6 h. Degenerative changes were very severe in the retrogradely labelled neurones 48 h after the gold-conjugated volkensin injection. In the Nissl-stained material, neuronal degeneration started to be evident in the inferior olive 12 h, and in pontine nuclei 6 h, after volkensin injection. The neuronal degeneration in both the inferior olive and pons increased up to 4 days after the injection. These findings provide direct evidence of the retrograde axonal transport of volkensin in the central nervous system, and the time course of the consequent degenerative changes in the afferents to the cerebellar cortex.

Corticotropin-releasing factor (CRF) and CRF binding sites have been described in the cerebellum of several species, including the North American opossum (Didelphis marsupialis virginiana), the species used in the present study. Inotophoretic application of this peptide in the adult cerebellum enhances the spontaneous and amino acid-induced firing rate of Purkinje cells and overcomes the GABA-induced suppression of Purkinje cell activity. The present account provides immunohistochemical evidence for the localization of CRF in the North American opossum within developing axons and their growth cones prior to the formation of the Purkinje cell and granule cell layers. CRF mRNA is present on postnatal day (PD) 1 within the internal migratory stream of the ventral lateral medulla, which contains migrating olivary neurons, and within the ventral medulla in the region, where inferior olivary neurons first aggregate to form the inferior olivary complex. The olivary complex can first be identified on PD2 and is well defined by PD3. CRF-immunoreactive axons are evident within the cerebellar primordium on PD4 and penetrate the nascent Purkinje cell layer between PD14 and PD26. By PD26, CRF-immunoreactive puncta are organized within the Purkinje cell layer as parasagittal bands. Thus, olivary neurons express CRF mRNA prior to the time that the first CRF-labeled axons are present in the cerebellar anlage (PD4), suggesting that olivary axons are among the first to reach the developing cerebellum. Coincident (PD1-3) with the early transcription of CRF mRNA in the inferior olive, cells in the medullary reticular formation (PD1) and locus coeruleus (PD2) also transcribe CRF mRNA. These brainstem sites also could provide CRF-immunoreactive axons to the developing cerebellum; however, based on the results of this study and correlative data reported in the literature, we propose that the primary source of early-arriving CRF fibers is the inferior olivary complex. The early arrival of CRF-containing axons in the cerebellum prior to synaptogenesis and migration of both granule cells and Purkinje cells suggests a role for this peptide in target recognition and synaptic organization.

Based on enzymatic activity, the localization and the identification of D-amino-acid oxidase-containing cells in rat whole brain was systematically studied in serial fixed sections. The oxidase activity was absent or scarce in the forebrain, was confined to the brain stem (midbrain, pons and medulla oblongata) and cerebellum, and its localization was extended to the spinal cord. In the brain stem the oxidase was mainly localized in the tegmentum, particularly in the reticular formation. The intense oxidase reactions were present in the red nucleus, oculomotor nucleus, trochlear nucleus, ventral nucleus of the lateral lemniscus, dorsal and ventral cochlear nuclei, vestibular nuclei, nuclei of posterior funiculus, nucleus of the spinal tract of the trigeminal nerve, lateral reticular nucleus, inferior olivary nucleus, and hypoglossal nucleus. In the cerebellum the activity in the cortex was much more intense than that in the medulla. In all the fields described above, the oxidase-containing cells were exclusively astrocytes including Bergmann glial cells, and neither neuronal components, endothelial cells, oligodendrocytes nor ependymal cells showed oxidase activity. These results indicated that the astrocytes regionally differentiated into two distinct types, one of which expressed oxidase in the midbrain, rhombencephalon and spinal cord, and the other which did not in the forebrain. The localization of the oxidase was inversely correlated with the distribution of free D-serine in mammalian brains (Nagata, Y., Horiike, K. and Maeda, T., Brain Res., 634 (1994) 291-295). Based on the characteristic localization of the oxidase-containing astrocytes, we discussed the physiological role of the oxidase.

The distribution and development of ferritin-containing cells were studied in the pons and cerebellum of human fetuses up to adults, using an immunohistochemical method. The predominant cell type labeled with antiserum to ferritin in the pons and cerebellum was morphologically oligodendrocytes. In the pons, positive cells appeared at 21 weeks of gestation in the reticular formation, and longitudinal and transverse fibers, and at 25 weeks of gestation in pontine nuclei. They increased with age from 33 weeks to infancy. In the cerebellum, positive cells appeared at 25 weeks of gestation in the cerebellar white matter and granular layer. They increased from 29 to 40 weeks of gestation in the white matter, and to childhood in the granular layer. The time of appearance of positive cells in the pons and cerebellum is earlier than in the cerebrum, like myelination. This development of ferritin-positive glia may be related to maturation of oligodendrocytes as well as being the basis of myelination.

This study deals with the three-dimensional arrangement of populations of pontocerebellar cell bodies projecting to the parafloccular complex. The fluorescent tracers rhodamine B isothiocyanate, fluoro-gold and fast blue were injected in either adjacent or separated cerebellar folia. A set of coordinates (x, y, z) was assigned to each retrogradely labelled cell and the total distribution reconstructed and displayed on a graphics workstation. At a large scale, we found that the majority of the cells of each labelled population (all projecting to the same folium) were confined to a lamella-shaped tissue volume. Each lamella extended from medial to lateral, and accordingly followed the curving of the pontine grey around the corticospinal and corticobulbar fibre tracts. At a smaller scale, i.e. within each lamellar subspace, the neurons belonging to one labelled population were distributed in aggregates of various shapes. To enable further analysis of the shapes of the intralaminar aggregates, we developed a computer program for unfolding of the lamellae, based on cubic B-spline approximation. The flattened reconstructions were three-dimensional polygonal windows, circumscribing the large majority of the labelled cell swarm (usually 70-80% of the total number of labelled cells in one population). The present findings, taken together with previous data on a gradual, rather than disjunctive, shift of pontocerebellar neuronal position in relation to a gradual shift of target region (Bjaalie et al., Anat. Rec.,231, 510-523, 1991), suggest that the cerebropontocerebellar system may be organized according to a set of fairly simple topographic rules.

In an attempt to estimate the relative importance of the various afferent systems impinging on the oculomotor regions of the posterior cerebellar vermis of rhesus monkeys in quantitative terms, we made small injections of the retrograde tracers fast blue, fluorogold, and cholera toxin into different parts of a region of the posterior vermis, spanning lobuli VI through VIII. We found that the vast majority of cells retrogradely labeled by injections of lobulus VII and its vicinity lay in the pontine nuclei (PN), the nucleus reticularis tegmenti pontis (NRTP), and subnuclei a and b of the medial accessory olive. The remaining retrogradely labeled cells were distributed among a number of other brainstem nuclei or regions including the paramedian pontine reticular formation (PPRF). A quantitative analysis showed that the projection from the NRTP to the posterior vermis was focused on lobulus VII. While the projection from the PN as a whole demonstrated a preference for the more caudal parts of the posterior vermis, a closer look at the different regions of the PN revealed that cells located in the dorsal parts of the PN showed the same preference for lobulus VII as cells in the NRTP. The dorsal PN are a major gateway for cortical input to the cerebellum, related to visual processing and visually guided eye movements. Conversely, the NRTP, likewise involved in visually guided eye movements, is much more dependent on subcortical afferents. The observed convergence of input derived from the dorsal PN and the NRTP in oculomotor lobulus VII therefore suggests that a major function of this part of the vermis might be the integration of cortical and subcortical signals important for visually guided eye movements.

The quail-chick chimaera system was used to investigate the origin of the various neuronal cell types of the cerebellum from the mesencephalic and metencephalic brain vesicles at the 11- to 14-somite stage in the avian embryo. We have already demonstrated that the cerebellum is derived from both the mesencephalic and metencephalic brain vesicles. The mesencephalic contribution to the cerebellum is restricted to a mediodorsal territory inserted as a V-shaped area into the primitive metencephalic vesicle through complex morphogenetic movements taking place from day 2 to day 4 of embryonic development. Here we report that the cerebellar presumptive territory extends along the anteroposterior axis, over the caudal half of the mesencephalon and the rostral half of the metencephalon. Along the dorsoventral axis, the cerebellar anlage is located in the alar plates at the exclusion of the roof and basal plates, i.e. lies in the lateral walls of the neuroepithelium in the area included between approximately 25 and 120 degrees with respect to the sagittal plane. We also report that the neuroepithelium corresponding to the cerebellar presumptive territory also yields other brain structures (e.g. part of the optic tectum in the mesencephalon). The external granular layer (EGL) arises only from the rostral metencephalon, undergoing extensive tangential movements which we have analysed in detail: the more ventral the position of cells in the metencephalic alar plates the more rostral and lateral is their position in the EGL. Finally, we discuss the fact that the cerebellar cortex, an integrative structure of the brain, arises from the alar plates of the neural tube. This is consistent with the general spatial organization of the neural anlage of the vertebrate embryo, in which this part of the neuroepithelium is devoted to the production of interneurons, whereas the basal plate and the neural folds yield motor structures and primary sensory neurons respectively.

Using three different retrograde tracing techniques, we analysed the broad distribution of ipsi-, contra- and bilateral NRTP neurons projecting to the cerebellum in the adult rat. The placement of the tracers into the cerebellar peduncles allowed us to determine the overall pattern of the NRTP-cerebellar projection. At the same time, the HRP "in vitro" technique permitted the study of the axonal profiles of the NRTP neurons within the pons until reaching the brachium pontis. Our observations confirm: i) A symmetrical organization of the NRTP-cerebellar projection, the number and location of ipsi- and contralateral NRTP neurons being very similar in all cases; ii) The only exception is represented by the rostralmost portions of the NRTP, in which the neurons show a cluster-complementary pattern; iii) The axons of the NRTP neurons cross the pontine midline at the dorsalmost aspect of the BPN, occupying a medial location within the brachium pontis.

Small injections of the retrograde tracer horseradish peroxidase were made into lobules V through X in the posterior cerebellar vermis of the rat. Labeled cells were counted in the auditory subdivisions of the pontine nuclei, the lateral and dorsolateral pontine nuclei. The results suggest that, within the posterior vermis, lobules VI, VII, and VIII together receive over 90% of projections from the lateral and dorsolateral pontine nuclei.

To reveal the organization and relative magnitude of connections from various parts of the cerebral cortex to the dorsal paraflocculus via the pontine nuclei, WGA-HRP was injected in the dorsal paraflocculus in conjunction with injection of the same tracer in various parts of the cerebral cortex in 17 cats. Termination areas of cortical fibres (anterogradely labelled) and pontine neurons projecting to the dorsal paraflocculus (retrogradely labelled) were carefully plotted in serial transverse sections. As an average of countings in ten cats, 90% of the labelled cells were found in the pontine nuclei contralateral to the injection, and the majority (70%) were located in the rostral half of the nuclei. The highest degree of overlap between anterograde and retrograde labelling was found after injections of the parietal association cortex (areas 5 and 7). In an experiment with double anterograde tracing, it was shown that both area 5 and 7 contribute substantially to the cerebral inputs to the dorsal paraflocculus. High degree of overlap also occurred after injections of several visual cortical areas (areas 17, 18, 19, 20 and the posteromedial lateral suprasylvian visual area, PMLS). Cases with injections restricted to individual visual areas indicate that they all contribute to the parafloccular input. Considerably less overlap occurred after injections of the primary sensorimotor region (SI, MI) and second somatosensory area (SII), while the supplementary motor area, the auditory cortex and gyrus cinguli probably have no or very restricted access to the dorsal paraflocculus. It is concluded that the dorsal paraflocculus has its major cortical input from the parietal association cortex and the visual cortical areas. Since all the various cortical regions studied project to largely different parts of the pontine nuclei, and overlap with neurons projecting to the dorsal paraflocculus takes place at numerous places, it follows that the pontine neurons projecting to the dorsal paraflocculus must consist of many subgroups differing with regard to their cortical input.

The pontine projection of the flocculus and adjacent ventral paraflocculus was investigated with antegrade and retrograde axonal tracer techniques. Injections of horseradish peroxidase into the floccular complex revealed subsets of labeled neurons in the nucleus reticularis tegmenti pontis, the nucleus raphe pontis and the medial lemniscus. Following injections of tritiated leucine in these subsets, the topographical distribution of labeled mossy fibers in the floccular complex was studied. Cells clustered in the central part of the nucleus reticularis tegmenti pontis project to the rostral flocculus and the rostral part of the caudal flocculus. The terminal field of cells in the nucleus raphe pontis and of cells associated with the lateral aspect of the medial lemniscus covered the same area. The number of mossy fiber terminals arising from these cells is small and concentrated in a medial position. The medial extension of the ventral paraflocculus and its most caudal sublobule do receive a very dense mossy fiber projection from cells associated with the medial edge of the medial lemniscus next to the rostral nucleus reticularis tegmenti pontis and beyond. Concomitantly, a collateral projection terminates in a restricted part of the uvula. Labeled mossy fiber terminals were never observed in the nodulus. The nucleus reticularis tegmenti pontis does not project to any part of the lower brain stem. The connections described in this paper are discussed in relation to the possible role of the nucleus reticularis tegmenti pontis as a relay nucleus in brain stem pathways transmitting visual information. It is concluded that in the cat this nucleus is an exclusively pre-cerebellar relay, not involved as a final link in the non-cerebellar pathway transmitting visual information to the vestibular nuclei.

In rainbow trout the glial cap cells ensheating the Mauthner cell axon cap form a more or less double-layered arrangement consisting of fibrous astrocytes, sometimes intermingled with an oligodendrocyte. All cells of the glial wall are linked together by many gap junctions. Especially the somata of the astrocytes are connected to each other by large symmetrical gap junctions. For this reason the glial cap cells might act as an (electro-) physiological unity. Morphologically, the high axon cap resistance above all results from the densely packed glial elements. Regarding their function the astro-astrocytic gap junctions might contribute to the formation of this barrier.

Following large injections of horseradish peroxidase - wheat germ agglutinin in the pontine nuclei, corticopontine neurons in areas 18 and 19 were quantitatively mapped and flat maps showing the distribution of retrogradely labeled cells were constructed. The areal borders were defined either cyto- and myeloarchitectonically or from standard retinotopic maps presented in frontal sections (Tusa et al. 1981). Maps of the retinotopic organization in areas 18 and 19 (Tusa et al. 1979) were transferred to the present flat maps. Thus, the number and distribution of pontine projecting cells could be correlated with the retinotopic organization. The cell density (number of labeled cells per mm2 cortex) is in both areas highest in the cortex representing the lower and upper visual periphery and decreases towards the representation of the retinal central area. However, since in both areas 18 and 19 the visual field representation is twisted and portions of the visual field are magnified, the actual number of cells is higher in the cortex representing the central area and the lower medial visual field than in other parts. The cortex representing the lower hemifield contains approximately 2/3 (mean, N = 4) of the corticopontine cells in both areas. The average density of corticopontine cells increases from area 17 through 18 to 19, but the total number of cells within each of the areas is about the same (area 17: 18000 cells, area 18: 13400 cells, area 19: 17200 cells; mean, N = 4; data on area 17 from Bjaalie and Brodal, 1983). In conclusion, areas 17, 18 and 19 contribute about equally in quantitative terms to the pontine nuclei. Furthermore, assuming that the corticopontine neurons transmit spatially relevant information, there is a moderate overrepresentation of central vision and the lower medial visual field in the pontine projection from areas 18 and 19. This visual field representation is remarkably similar to that found in the corticopontine projection from area 17 (Bjaalie and Brodal 1983).