Development of the hippocamposeptal projection in the rat

We analyzed the development of the hippocamposeptal projection and the morphology of the neurons giving rise to this projection. The fluorescent tracer Dil was injected into the septal region or the hippocampus in fixed brains of embryonic and early postnatal rats. Anterogradely labeled hippocampal axons first reached the septal region at E16. They ran along the midline of the brain, thereby approaching the medial septum. Axons to the lateral septum were first observed around E18/19. The lateral septum is partly innervated by collaterals of axons that travel to the medial septum. The projection to the lateral septal nuclei becomes more massive during early postnatal stages, whereas that to the medial septum becomes smaller. Cells in the medial septum retrogradely labeled by injection into the hippocampus were first observed at E18. Thus, the hippocamposeptal projection is established earlier than the septohippocampal projection. The first hippocampal projection neurons are nonpyramidal neurons that appear to pioneer the pathway to the septum. Pyramidal cell axons follow this first cohort of axons into the medial septum. Pyramidal cells could be retrogadely labeled from the medial septum during the perinatal period but then diminished in number. At P10, only nonpyramidal cells were labeled by medial septal injections. This indicates that the pyramidal component of this projection is transient and is removed shortly after birth. However, as is known from ther studies, hippocampal pyramidal cells give rise to a powerful projection to the lateral septum in adult animals. Our results show that there is a considerable remodeling of the projection from the hippocampus to the septum during ontogenetic development. © 1995 Willy-Liss, Inc.     

Coordinated functions of Netrin-1 and Class 3 secreted Semaphorins in the guidance of reciprocal septohippocampal connections

An essential characteristic of the CNS function is the formation of reciprocal connections between brain areas. Although the mechanisms controlling the establishment of neuronal connections are being determined, very little is known about the development of reciprocal connections, which often course along identical pathways. Here, we show that Netrin-1, expressed along the fimbria, chemoattracts both septohippocampal and hippocamposeptal fibers. Moreover, we show that both Semaphorins 3A and 3F expressed in regions nearby the septum prevent the growth of septal axons into these regions. Blocking experiments with recombinant ecto-Neuropilins indicate that both Semaphorins 3A and 3F act cooperatively in the repulsion of septal axons. Furthermore, netrin-1-deficient mice develop a reduced septohippocampal projection. We conclude that the coordinated actions of Netrin-1 and Semaphorins 3A and 3F cooperate in the development of septohippocampal and hippocamposeptal connections, indicating that the same molecular cues serve the construction of reciprocal connections in both directions of growth.     

The organization of the embronic and early postnatal murine hippocampus. II. Development of entorhinal,commissural, and septal connections studied with the lipophilic tracer DiI

We have analyzed the early development of the main hippocampal afferents in the mouse. Following injections of the lipophilic tracer 1–1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine perchorate (DiI) in the entorhinal cortex, entorhinal axons were observed for the first time inthe hippocampus at E15, in the white matter, At E17, entorhinal fibers arborized within the stratum lacunosum-moleculare. At subsequent stages entorhinal axons formed dense networks that were restricted to their appropriate termination zone in the lacunosum-moleculare. The first axons invading the fascia dentata were noticed at E19, their density increasing at later stages. These axons were mainly present in the outer molecular layer. This onset of entorhinohippocampal projections was corroborated by retrograde labeling data after injections in the hippocampus. Commissural fibers first entered the contralateral hippocampus at E18, their number increasing at the following stages. Commissural axons arborized within the stratum oriens and radiatum in the hippocampus proper. In the fascia dentata, the earliest commissural fibers were seen at P2, terminating in the inner zone of the molecular layer and in the hilus. We conclude that developing entorhinal and commissural axons show a high degree of laminar specificity from the earliest stages of formation, which is compatible with the notion that distinct subsets of early maturing neurons populating the hippocampal plexiform layers may attract particular fiber systems. Hippocamposeptal fibers develop at E15, before the first septal fibers can be detected in the hippocampus. These early hippocamposeptal fibers originated from nonpyramidal neurons and terminated in the medial septal area, which is the main source of septal afferents to the hippocampus. In contrast, septohippocampal fibers were not seen in the hippocampus until E17. At perinatal stages, the hippocamposeptal connection reshapes, sending axons to the dorsolateral septal area as the innervation of the medial septum becomes less conspicuous. This sequence suggests that hippocampal neurons pioneer the formation of septohippocampal connections. © 1994 Wiley-Liss, Inc.     

Role of class 3 semaphorins in the development and maturation of the septohippocampal pathway

In examining the role of Class 3 secreted semaphorins in the prenatal and postnatal development of the septohippocampal pathway, we found that embryonic (E14-E16) septal axons were repelled by the cingulate cortex and the striatum. We also found that the hippocampus exerts chemorepulsion on dorsolateral septal fibers, but not on fibers arising in the medial septum/diagonal band complex, which is the source of septohippocampal axons. These data indicate that endogenous chemorepellents prevent the growth of septal axons in nonappropriate brain areas and direct septohippocampal fibers to the target hippocampus. The embryonic septum expressed np-1 and np-2 mRNAs, and the striatum and cerebral cortex expressed sema 3A and sema 3F. Experiments with recombinant semaphorins showed that Sema 3A and 3F, but not Sema 3C or 3E, induce chemorepulsion of septal axons. Sema 3A and 3F also induce growth cone collapse of septal axons. This indicates that these factors are endogenous cues for the early guidance of septohippocampal fibers, including cholinergic and gamma-aminobutyric acid (GABA)ergic axons, during the embryonic stages. During postnatal stages, when target cell selection and synaptogenesis take place, np-1 and np-2 were expressed by septohippocampal neurons at all ages tested. In the target hippocampus, pyramidal and granule cells expressed sema 3E and sema 3A, whereas most interneurons expressed sema 3C, but few expressed sema 3E or 3A. Combined tracing and expression studies showed that GABAergic septohippocampal fibers terminated preferentially onto sema 3C-positive interneurons. In contrast, cholinergic septohippocampal fibers terminated onto sema 3E and sema 3A-expressing pyramidal and granule cells. The data suggest that Class 3 secreted semaphorins are involved in postnatal development. Moreover, because GABAergic and cholinergic axons terminate onto neurons expressing distinct, but overlapping, patterns of semaphorin expression, semaphorin functions may be regulated by different signaling mechanisms at postnatal stages.     

Nerve growth factor promotes collateral sprouting of cholinergic fibers in the septohippocampal cholinergic system of aged rats with fimbria transection.

Nerve growth factor (NGF) was injected intraventricularly into aged (24 months) rats with unilateral fimbria transection. Controls received intraventricular injections of cytochrome c. A quantitative analysis of acetylcholinesterase (AChE)-positive fibers was used to evaluate whether the NGF treatment can stimulate regeneration and reinnervation of the cholinergic axons in the septohippocampal system of aged rats with fimbria transection. A marked increase in the density of AChE-positive fibers was observed in the lateral septum, the dorsal fornix and the dorsal hippocampus of the NGF-treated animals, as compared to the controls. In the lateral septum, the increase was observed in the 2-month NGF-treated animals but not in the 15-day NGF-treated animals. In the dorsal fornix at the level of the dorsal hippocampus, the increase was observed on both the lesioned and unlesioned sides of both the 15-day and 2-month NGF-treated animals. In the denervated (lesioned side) hippocampus, the increase took place in the dorsal hippocampus but not in the ventral hippocampus of both the 15-day and 2-month NGF-treated animals. There was no recovery of AChE-positive fibers on the lesioned side of the fimbria distal to the lesion site even in the 2-month NGF-treated animals. These results demonstrate that intraventricular injections of NGF can stimulate collateral sprouting of intact cholinergic axons in the septohippocampal system and promote cholinergic reinnervation of the denervated hippocampus of aged rats with fimbria transection.     

Differential projections to the hippocampus by neurons of the medial septum and vertical limb of the diagonal band

Neurons of the medial septum and vertical limb of the diagonal band of Broca project topographically to the hippocampus through the fornix and fimbria, the supracallosal stria and via a ventral route through the amygdala. Injection of the fluorescent dyes Fluoro-Gold or Diamidino yellow into the fimbria-fornix retrogradely labeled neurons in the medial septum and in the ventromedial portion of the vertical limb of the diagonal band. Injection of True blue into the vicinity of the supracallosal stria labeled only neurons in the dorsalateral portion of the diagonal band. No double labeled neurons were observed. These results indicate that (1) neurons of the medial septum and the ventromedial diagonal band project to (or towards) the hippocampus via the fornix and fimbria, (2) neurons in the dorsolateral portion of diagonal project via the supracallosal stria, and (3) neurons of the medial septum and diagonal band do not send collaterals via both routes. The differential projection of the two groups of neurons may explain the differences in degenerative changes in the two nuclei after damage to their axons.     

Precocious invasion of the optic stalk by transient retinopetal axons

This study demonstrates that the fetal optic nerve contains a conspicuous population of transient retinopetal axons. Implants of the carbocyanine dye, DiI, were made into the retina or diencephalon of fetal ferrets to label the retinopetal axons retrogradely or anterogradely, respectively, and sections were immunostained for β-tubulin to label the early differentiating axons in the optic nerve. Dye implants into the optic nerve head, but not the retinal periphery, retrogradely labeled somata in the ventrolateral diencephalon, provided the implants were made before embryonic day (E) 30. When dye implants were made into the ventrolateral diencephalon, these same retinopetal axons were anterogradely labeled, coursing through the optic nerve but never invading the retina. The axons course as 2–5 fascicles from their cells of origin and turn laterally to enter the optic nerve where it joins the future hypothalamus. The retinopetal cells can be retrogradely labeled as early as E20, before optic axons have left the retina. The optic nerve and fiber layer are immunoreactive for β-tubulin on E24 and thereafter, whereas on E20 and E22, they are immunonegative. Yet at these early embryonic ages, immunopositive fascicles of axons course from the diencephalon into the optic stalk, confirming the precocious nature of the retinopetal projection. Implants of dye made into the future optic nerve head at these very early stages also retrogradely label retinopetal cells in the future chiasmatic region. These cells are distributed primarily on the side ipsilateral to the midline, but a few can be found contralateral to it. Both these, as well as the retinopetal axons arising from the ventrolateral diencephalon, may serve a transient guidance function for later developing optic axons. © 1995 Wiley-Liss, Inc.     

Hippocampal Cajal-Retzius cells project to the entorhinal cortex: retrograde tracing and intracellular labelling studies

Cajal-Retzius (CR) cells are characteristic horizontally orientated, early-generated transient neurons in the marginal zones of the neocortex and hippocampus that synthesize the extracellular matrix protein reelin. They have been implicated in the pathfinding of entorhino-hippocampal axons, but their role in this process remained unclear. Here we have studied the axonal projection of hippocampal CR cells. Following injection of the carbocyanine dye DiI into the entorhinal cortex of aldehyde-fixed rat embryos and young postnatal rats, neurons in the outer molecular layer of the dentate gyrus and stratum lacunosum-moleculare of the hippocampus proper with morphological characteristics of CR cells were retrogradely labelled. In a time course analysis, the first retrogradely labelled CR cells were observed on embryonic day 17. This projection of hippocampal CR cells to the entorhinal cortex was confirmed by retrograde tracing with Fast Blue in new-born rats and by intracellular biocytin filling of CR cells in acute slices from young postnatal rat hippocampus/entorhinal cortex and in entorhino-hippocampal slice cocultures using infrared videomicroscopy in combination with the patch-clamp technique. In double-labelling experiments CR cells were identified by their immunocytochemical staining for reelin or calretinin, and their interaction with entorhino-hippocampal axons labelled by anterograde tracers was analysed. Future studies need to investigate whether this early transient projection of hippocampal CR cells to the entorhinal cortex is used as a template by the entorhinal axons growing to their target layers in the hippocampus.     

The organization of the reciprocal connections between the subiculum and the entorhinal cortex in the cat: I. A neuroanatomical tracing study

The connections between the subiculum (SUB) and the entorhinal cortex (EC) were studied in the cat with retrograde and anterograde tracing techniques. Injections of the retrogradely transported tracer WGA-HRP at different levels along the septotemporal axis of the subiculum result in labeled neurons predominantly in the medial entorhinal cortex (MEA) in the superficial layers II and III. In the deep layers labeled cells are found more widespread over the EC. The superficially located labeled EC neurons are topographically distributed in a lateromedial gradient, which corresponds to a septotemporal gradient along the longitudinal axis of the subiculum. This organization of the EC-SUB projection system could be substantiated by the use of injections anterogradely transported radioactively labeled amino acids in EC. The SUB to EC projections were investigated with the anterograde transport of WGA-HRP and with radioactively labeled amino acids that were injected at different levels along the septotemporal axis of the subiculum. This results in a patch of anterogradely labeled fibers and terminals in MEA, predominantly in layers II and III, with a wider band of label in the deep layers. Again, a topographical distribution along the lateromedial axis of the EC corresponding to the septotemporal axis of the SUB was observed. Contralateral reciprocal connections between EC and SUB are also present, and exhibit a similar topographical organization.     

Direct synaptic projections to esophageal motoneurons in the nucleus ambiguus from the nucleus of the solitary tract of the rat

Neurons of the nucleus of the solitary tract (NTS) serve as interneurons in swallowing. We investigated the synaptology of the terminals of these neurons and whether they project directly to the esophageal motoneurons in the compact formation of the nucleus ambiguus (AmC). Following wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) injection into the NTS, many anterogradely labeled axodendritic terminals were found in the neuropil of the AmC. The majority of labeled axodendritic terminals (89%) contained round vesicles and made asymmetric synaptic contacts (Gray's type I), but a few (11%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II). More than half of the labeled terminals contacted intermediate dendrites (1-2 μm diameter). There were no retrogradely labeled medium-sized motoneurons, but there were many retrogradely labeled small neurons having anterogradely labeled axosomatic terminals. A combined retrograde and anterograde transport technique was developed to verify the direct projection from the NTS to the esophageal motoneurons. After the esophageal motoneurons were retrogradely labeled by cholera toxin subunit B conjugated HRP, the injection of WGA-HRP into the NTS permitted ultrastructural recognition of anterogradely labeled axosomatic terminals contacting directly labeled esophageal motoneurons. Serial sections showed that less than 20% of the axosomatic terminals were labeled in the esophageal motoneurons. They were mostly Gray's type I, but a few were Gray's type II. In the small neurons, more than 30% of axosomatic terminals were labeled, which were exclusively Gray's type I. These results indicate that NTS neurons project directly not only to the esophageal motoneurons, but also to the small neurons which have bidirectional connections with the NTS. J. Comp. Neurol. 381:18-30, 1997. © 1997 Wiley-Liss, Inc.     

Direct synaptic connections of axons from superior colliculus with identified thalamo-amygdaloid projection neurons in the rat: Possible substrates of a subcortical visual pathway to the amygdala

The aim of the present study was to identify synaptic contacts from axons originating in the superior colliculus with thalamic neurons projecting to the lateral nucleus of the amygdala. Axons from the superior colliculus were traced with the anterograde tracers Phaseolus vulgaris leucoagglutinin or the biotinylated and fluorescent dextran amine “Miniruby.” Thalamo-amygdaloid projection neurons were identified with the retrograde tracer Fluoro-Gold. Injections of Fluoro-Gold into the lateral nucleus of the amygdala labeled neurons in nuclei of the posterior thalamus which surround the medial geniculate body, viz. the suprageniculate nucleus, the medial division of the medial geniculate body, the posterior intralaminar nucleus, and the peripeduncular nucleus. Anterogradely labeled axons from the superior colliculus terminated in the same regions of the thalamus. Tecto-thalamic axons originating from superficial collicular layers were found predominantly in the suprageniculate nucleus, whereas axons from deep collicular layers were detected in equal density in all thalamic nuclei surrounding the medial geniculate body. Double-labeling experiments revealed an overlap of projection areas in the above-mentioned thalamic nuclei. Electron microscopy of areas of overlap confirmed synaptic contacts of anterogradely labeled presynaptic profiles originating in the superficial layers of the superior colliculus with retrogradely labeled postsynaptic profiles of thalamo-amygdaloid projection neurons. These connections may represent a subcortical pathway for visual information transfer to the amygdala. J. Comp. Neurol. 403:158–170, 1999. © 1999 Wiley-Liss, Inc.     

Electron microscopic study of the rubrocerebellar projection in the cat

Rubral neurons sending axons to the cerebellar anterior interpositus nucleus (AIN) in the cat were identified light microscopically by labeling them with horseradish peroxidase (HRP). The synaptic organization of these rubral neurons and of their afferents from the cerebral motor cortex and the AIN was also analyzed electron microscopically by combined anterograde degeneration and retrograde HRP-labeling techniques. In the light microscopic study, either HRP or a mixture of HRP and kainic acid was injected into the AIN. Both of the injections resulted in retrograde labeling of rubrocerebellar projection neurons in the red nucleus on the contralateral side. The labeled neurons were distributed throughout the rostrocaudal extent of the red nucleus: some lay in clusters. Most labeled neurons were small to medium-sized, although some were large. The injection of HRP into the AIN also resulted in anterograde labeling of cerebellorubral projection fibers terminating in a wider area of the red nucleus on the contralateral side of the injection, whereas the injection of a mixture of HRP and kainic acid showed no anterograde labeling of fibers or terminals. In one set of electron microscopic observations, HRP injections into the AIN were combined with ablation of the motor cortex. Degenerating axon terminals were occasionally found to synapse with both dendrites and neuronal somata labeled with HRP retrogradely. In another set of electron microscopic observations, a mixture of HRP and kainic acid was injected into the AIN in order to label rubrocerebellar projection neurons retrogradely and to bring about degeneration in the cerebellorubral projection fibers anterogradely. Abundant degenerating axon terminals were observed to make axosomatic synaptic contacts with rubral neurons labeled with HRP retrogradely and also with unlabeled rubral neurons. These results indicate that cerebrorubrocerebellar and rubrocerebellorubral monosynaptic circuitries exist which constitute one of the cerebrocerebellar linkages, as well as those linkages via the inferior olivary complex and the pontine nuclei.     

Development of the transient ipsilateral retinotectal projection in the chick embryo: A numerical flourescence-microscopic analysis

The ipsilateral retinotectal projection in the developing chick was examined by using rhodamine-B-isothiocyanate (RITC)as an anterograde and retrograde vital marker for the retinal ganglion cells and their axons. Staining of the entire retina following intravitreal RITC injection between incubation days 3 and 16 revealed a small number of anterogradely labeled fibers in the optic tract and the anterior half of the optic tectum ipsilateral to the injection site. The total number of ipsilaterally projecting fibers was estimated to be about 2,000 on developmental day 9. The ipsilateral projection totally disappeared after day 15. The arrangement of fibers within the ipsilateral projection was examined by local anterograde RITC staining of localized retinal regions between days 9 and 10. The projection was retinotopically organized along the dorsoventral axis such that fibers of dorsal retinal origin projected on the ventral tectal half, whereas fibers of ventral retinal orgin projected on the dorsal tectal half. The localization of ipsilaterally projecting ganglion cell bodies was examined by retrograde RITC staining during days 9 and 15. Ganglion cells of all four quadrants of the central retina contributed to the production of the ipsilateral projection. The ipsilaterally growing retinotectal fibers did not represent collaterals of contralaterally projecting retinotectal axons. We assume that the tendency of early growing retinotectal axons to grow straight, as well as the ability of axonal growth cones to “sample” the environment, lead to a crossing of axons to the contralateral side. Ipsilateral projections would therefore represent “pathfinding errors.” Explanations for the elimination of the ipsilateral retinotectal projection are discussed.     

Nonserotonergic projection neurons in the midbrain raphe nuclei contain the vesicular glutamate transporter VGLUT3

The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network.     

The pallidostriatal projection in the rat: a recurrent inhibitory loop?

The pallidostriatal projection in the rat was investigated employing the PHA-L tracing technique. Following iontophoretic injections into the lateral aspect of the globus pallidus external segment, the ipsilateral striatum showed patches of dense anterograde labeling separated by areas containing sparse anterograde labeling and isolated retrogradely labeled neurons. The densely labeled patches did not correspond to any known compartments of the striatum. The retrogradely labeled neurons consistenly showed similar distribution of morphological features reminiscent of striatal type II projection neurons. As all projection neurons of the striatum and all pallidal neurons are GABAergic, the complementary pattern of anterogradely and retrogradely labeled profiles from the globus pallidus suggest a possible mechanism whereby a horizontal inhibition may be exerted on groups of striatal neurons via the striato-pallido-striatal pathway.     

Extensive reinnervation of the hippocampus by embryonic basal forebrain cholinergic neurons grafted into the septum of neonatal rats with selective cholinergic lesions

Reconstruction of the septohippocampal pathways by axons extending from embryonic cholinergic neuroblasts grafted into the neuron-depleted septum has been explored in the neonatal rat by using a novel lesioning and grafting protocol. Neonatal ablation of the basal forebrain cholinergic projection neurons, accompanied by extensive bilateral cholinergic denervation of the hippocampus and neocortex, was produced at postnatal day (PD) 4 by 192 immunoglobulin (IgG)-saporin intraventricularly. Four days later, cholinergic neuroblasts (from embryonic day 14 rats) were implanted bilaterally into the neuron-depleted septum by using a microtransplantation approach. The results show that homotopically implanted septal neurons survive and integrate well into the developing septal area, extending axons caudally along the myelinated fimbria-fornix and supracallosal pathways that are able to reach the appropriate targets in the denervated hippocampus and cingulate cortex as early as 4 weeks postgrafting. Moreover, the laminar innervation patterns established by the graft-derived axons closely resembled the normal ones and remained essentially unchanged up to at least 6 months, which was the longest postoperative time studied. The reinnervating fibers restored tissue choline acetyltransferase activity (up to 50% of normal) in the dorsal hippocampus and the parietooccipital cortex. Retrograde labeling with Fluoro-Gold from the host hippocampus combined with immunocytochemistry confirmed that most of the projecting neurons, indeed, were cholinergic. The results suggest that the graft-host interactions that are necessary for target-directed axon growth are present in the septohippocampal system during early postnatal maturation. Thus, the present approach may contribute to overcome the functional limitations inherent in the use of ectopically placed intrahippocampal transplants. © 1996 Wiley-Liss, Inc.     

HRP study of cerebellar corticonuclear-nucleocortical topography of the dorsal culminate lobule--lobule V--in a prosimian primate (Galago): with comments on nucleocortical cell types

The distribution of retrogradely labeled cerebellar nucleocortical (NC) cells and anterogradely labeled corticonuclear (CN) fibers was investigated in a prosimian primate (Galago) by means of horseradish peroxidase as a tracer. Iontophoretic and pressure injections were made in the cortex of lobule V and the resultant patterns of label were determined in the cerebellar nuclei. Following iontophoretic injections in vermal (zone A), intermediate (zones C1, C3), and lateral (zone D) cortices, retrogradely labeled cells were present in medial (NM), anterior interposed (NIA), and lateral (NL) cerebellar nuclei, respectively. Larger injections that involved A-C2 zones resulted in NC label in NM, medial NIA, and throughout the posterior interposed (NIP) nucleus. Retrogradely labeled NC cells were usually found in areas of their respective nuclei that also contained anterogradely filled CN axons. In addition, retrogradely labeled cells were seen contralateral to some injection. Contralateral NC cells were found mainly in the NM and NIP and seemed to be labeled in response to injections that involved zones A, C2, and possibly x on the opposite side. No contralateral CN labeling was seen. It appears that the NC projections of lobule V follows a basic zonal (sagittal) orientation and that most are reciprocal to CN fibers arising from the same cortical area. There is evidence of zonal heterogeneity in the ipsilateral NC projection. Iontophoretic injections placed in adjacent zones resulted in markedly different numbers of retrogradely labeled NC cells in their respective nuclei. Also, after pressure injections that involved two or more adjoining zones, the number of labeled NC cells was large in one nucleus but minimal in an adjacent nucleus. These data suggest that different cerebellar cortical zones have quantitatively different NC input; this may relate to specific functional demands placed on each nucleus and its corresponding cortical zone. On the basis of their known connections, it is hypothesized that there are at least three and possibly four categories of NC cells. Ipsilateral reciprocal NC cells are found in, or on the periphery of, CN terminal fields formed by axons originating from the same cortical area to which the NC cells project. Ipsilateral nonreciprocal NC cells are located outside the CN terminal field and may even be found in an adjacent nucleus; these are fewer in number than the reciprocal population. Contralateral NC cells are found in the opposite cerebellar nuclei and appear to be topographically related to the ipsilateral contingent as well as to the injection site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that cholera toxin B subunit (CTb) can be avidly taken up and transported by fibers of passage

It has been reported that cholera toxin B subunit (CTb) is a sensitive neuronal tracer with unique features. However, the possible uptake of CTb by non-terminal fibers passing through the injection site has not been examined thoroughly. In the present study, small iontophoretic injections (current = + 2 μA) of CTb were made in the olivocerebellar pathway in the rat ventrolateral medulla. A large number of retrogradely labeled neurons were seen in the contralateral inferior olive. In addition, prominent anterogradely labeled climbing fibers/terminals were found in the cerebellum ipsilateral to the injection site. This study, in contrast to previous report(s), indicates that CTb can be taken up avidly by fibers of passage and transported both anterogradely and retrogradely.     

Retinotectal terminals in the superior colliculus of the rabbit: a light and electron microscopic analysis

The projection from the retina to the controlateral superior colliculus was studied light and electron microscopically by means of anterogradely transported horseradish peroxidase and tetramethylbenzidine histochemistry as well as light microscopically by experimental degeneration and [3H]-leucine autoradiography. Labeled boutons were found in stratum zonale (SZ) and in stratum griseum superficiale (SGS), but not in stratum opticum (S0). The number of boutons was maximal in a narrow zone in SZ about 25 to 100 micron below the surface. The labeled boutons contained numerous round vesicles and predominantly pale mitochondria. They usually formed asymmetrical synapses and contacted dendrites or boutons. Occasionally, labeled boutons were observed whose cytological features were different from those generally associated with retinotectal axons. In general, labeled boutons in SZ contained fewer mitochondria than those from SGS. Labeled myelinated axons were found throughout SGS, in the lowest part of SZ, and in SO. In upper and middle SGS they were small while in lower SGS and SO also large fibers were found.     

Axonal connections from posterior paralaminar thalamic neurons to basomedial amygdaloid projection neurons to the lateral entorhinal cortex in rats

Stimulation of amygdaloid nuclei and emotionally relevant stimuli are known to influence the induction and maintenance of long-term potentiation in the hippocampal formation and the formation of long-term declarative memories. Because the thalamic projection from the posterior paralaminar thalamic nuclei is an important sensory afferent projection to amygdaloid nuclei mediating the fast acquisition of fear-potentiated behavior, we were interested in verifying whether this projection establishes synaptic contacts on amygdala neurons that project to the hippocampal formation. Thalamic afferents were labeled with the anterograde tracer Phaseolus vulgaris leucoagglutinin and amygdalo-hippocampal neurons were identified by injection of the retrograde tracer Fluorogold into the lateral entorhinal cortex. A massive overlap of both projection systems was observed especially in the anterior basomedial nucleus of the amygdala. Light microscopic examination revealed that single anterogradely labeled boutons were in close apposition to retrogradely labeled neurons suggesting synaptic contacts. The occurrence of such synaptic contacts was confirmed with electron microscopy. However, despite the massive overlap of anterogradely labeled axons and retrogradely labeled neurons observed at the light microscopic level, electron microscopy revealed that only 10% of all labeled profiles make direct contacts on each other; anterogradely labeled boutons predominantly contacted unlabeled profiles but synapses with direct contact between labeled profiles were rare. Altogether the findings demonstrate that the thalamic connection with the basomedial nucleus of the amygdala may represent an anatomical substrate for modulating amygdala output to the hippocampal formation.