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.     

Development of the rat septohippocampal projection: Tracing with DiI and electron microscopy of identified growth cones

The factors determining the development of specific fiber tracts in the central nervous system as well as the interactions of growth cones with the surrounding micromilieu are largely unknown. Here we investigated the ontogenetic development of the septohippocampal projection in the rat with the lipophilic carbocyanine dye DiI which is transported anterogradely and retrogradely in neurons and can be applied to fixed embryonic tissue. Photoconversion of anterogradely labeled fibers allowed us to study individual growth cones by electron microscopy. The first axons originating from the septal complex were found in the hippocampus as early as on embryonic day (ED) 19, reaching the fimbrial pole of the hippocampus on ED 18. However, on ED 17 we consistently found retrogradely labeled cells in the hippocampus, indicating that the development of the hippocamposeptal projection precedes that of the septohippocampal projection. On ED 19, the majority of the axons directed toward the hippocampal formation passed the hippocampus and grew further into the subicular complex and entorhinal cortex. These axons gave off collaterals that invaded the hippocampus proper. A fairly adult pattern of the septohippocampal projection was reached on postnatal day 10, although many growth cones were still found. A comparative analysis of individual growth cones found in the fimbria and the hippocampus proper revealed no striking differences in their morphology. Electron microscopic analysis showed that growth cones in the fimbria were mainly contacted by other axons, whereas growth cones in the hippocampus had contact with all available elements. This may indicate that growing septohippocampal fibers are guided by axons of the earlier formed hippocamposeptal projection. In the hippocampus proper, other cues, probably derived from the target itself, may guide the septhippocampal axons to their appropriate target cells. © 1993 Wiley-Liss, Inc.     

Multiple ephrins regulate hippocampal neurite outgrowth

Increasing evidence indicates that the eph family of ligands and receptors guides the formation of topographic maps in the brain through repulsive interactions. For example, we have recently found that in the hippocamposeptal system, the ligand ephrin-A2, which is expressed in an increasing gradient from dorsal to ventral septum, selectively induces pruning of topographically inappropriate medial hippocampal axons. The recent detection of ephrins A3 and A5, as well as A2, in the septum raised critical functional questions. Do the ligands act combinatorially, ensuring appropriate three-dimensional spatiotemporal projection, or do they exert entirely distinct actions in addition to guidance mechanisms? To approach these alternatives, we cloned mouse ephrin-A2 and compared the activities of the three ligands. Here, we show that these ligands reduce the number of hippocampal neurites in a similar fashion. The effect was regionally specific; medial hippocampal neurites were reduced 1.5- to 1.8-fold, whereas lateral hippocampal neurites were not significantly affected, conforming to topographic projection in vivo. Furthermore, we found that ephrins regulated neurite number in a stage-specific fashion, affecting E19 hippocampal neurites more than E16 neurites. Our observations suggest that all three septal ephrins, A2, A3, and A5, play spatiotemporally specific roles in guiding topographic projections from the hippocampus.     

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.     

Somatospiny neurons in the rat lateral septal area are synaptic targets of hippocamposeptal fibers: a combined EM/Golgi and degeneration study.

The mediolateral part of the lateral septal area (LSA) is a common target of hippocamposeptal afferents, neuropeptide containing, catecholaminergic, cholinergic, and GABAergic pericellular baskets of different origins. This specific innervation pattern as well as electrophysiological data concerning this area suggest a convergent input from different sources to particular LSA neuron populations. Light and electron microscopy combined with Golgi impregnation and acute anterograde degeneration techniques following transection of the fimbria-fornix were employed to determine whether LSA neurons with hippocampal input have any characteristic and distinctive morphological signs. About 20% of all Golgi impregnated LSA neurons were found to have somatic spines. All of these somatospiny neurons are synaptic targets of hippocamposeptal fibers. The degenerated hippocamposeptal boutons establish asymmetric synaptic contacts on their soma, somatic and dendritic spines, and on dendritic shafts. Somatospiny neurons located in the most medial and dorsal parts of the LSA seem to project toward the medial septum while all of the others appear to send descending fibers to ventral areas. Somatospiny neuron axons occasionally give out recurrent collaterals. Quantitative analysis on the spatial distribution of the somatospiny neurons revealed that practically all of them are encountered in the mediolateral division of the LSA. This area includes the lateral part of the intermediolateral septal nucleus and adjacent lateral portions of the dorsolateral and the ventrolateral septal nuclei.     

Morphology of embryonic hippocampus transplants and generation of long-distance extrinsic graft fiber connections to the lateral septum and the entorhinal cortex in the mature rat brain

Pyramidal neurons inside transplants of embryonic nervous tissue are capable of generating axonal extrinsic hippocampal fiber connections over considerable distances to appropriate target areas in the mature brain. The establishment of long-distance graft efferents to the lateral septum and to the entorhinal cortex was shown by retrograde transport of the tracers HRP and bisbenzimide which were injected into these areas after bilateral neurotoxic lesions of the hippocampus. Additional AChE-staining demonstrated the presence of an afferent cholinergic graft input mainly from the medial septum via the fornix. Morphological analysis of the transplants grafted as cell suspensions showed typical details of the original hippocampus cytoarchitecture with bands of pyramidal and granule cells.     

The topographical organization of the hippocampal projection to the septal area: a comparative neuroanatomical analysis in the gerbil, rat, rabbit, and cat

An experiment was performed to determine the origin of the projection from the hippocampus to the septal area in the subrimate mammalian nervous system. Lesions were made by aspiration or by radio frequency in 4 gerbils, 17 rats, 8 rabbits, and 7 cats. Survival times varied from 2–5 days. Tissues were stained principally with the Fink Heimer I method for identification of degenerating axons and their terminals. Following lesions destroying any one or more of the fields of the dorsal hippocampus of the gerbil, rat, rabbit, or cat, terminal degeneration was observed only in the medial septal area, olfactory tubercle, and adjacent portions of the diagonal band. In addition, lesions producing total destruction of all dorsal hippocampal fields also resulted in the presence of terminal degeneration restricted to the medial septal area. In contrast, superficial lesions of field CA1 of the ventral hippocampus produced terminal degeneration in the lateral septal area, nucleus accumbens, olfactory tubercle, and adjacent portions of the diagonal band. Similar findings were also observed following more widespread lesions of the ventral hippocampus which produced damage to other CA fields as well. Superficial lesions of the posterior crus of the hippocampus (i.e., a position midway between dorsal and ventral hippocampus) resulted in terminal degeneration localized to an intermediolateral region of the septum. Combined lesions of the dorsal hippocampus and fimbria produced widespread terminal degeneration in both the lateral and medial septum indicating that the axons contained within the fimbria arise only from the ventral hippocampus. Finally, lesions of the medial and lateral segments of the fornix of the cat produced terminal degeneration in the medial and lateral regions of the septum, respectively. These findings, collectively, indicate that the origin of the topographical projection to the medial and lateral septum are the dorsal and ventral hippocampus, respectively. This projection is unrelated to cytoarchitectonic fields within the hippocampus and is also invariant among the species considered in this study.     

Septal projections to nucleus incertus in the rat: Bidirectional pathways for modulation of hippocampal function

Projections from the nucleus incertus (NI) to the septum have been implicated in the modulation of hippocampal theta rhythm. In this study we describe a previously uncharacterized projection from the septum to the NI, which may provide feedback modulation of the ascending circuitry. Fluorogold injections into the NI resulted in retrograde labeling in the septum that was concentrated in the horizontal diagonal band and areas of the posterior septum including the septofimbrial and triangular septal nuclei. Double‐immunofluorescent staining indicated that the majority of NI‐projecting septal neurons were calretinin‐positive and some were parvalbumin‐, calbindin‐, or glutamic acid decarboxylase (GAD)?67‐positive. Choline acetyltransferase‐positive neurons were Fluorogold‐negative. Injection of anterograde tracers into medial septum, or triangular septal and septofimbrial nuclei, revealed fibers descending to the supramammillary nucleus, median raphe, and the NI. These anterogradely labeled varicosities displayed synaptophysin immunoreactivity, indicating septal inputs form synapses on NI neurons. Anterograde tracer also colocalized with GAD‐67‐positive puncta in labeled fibers, which in some cases made close synaptic contact with GAD‐67‐labeled NI neurons. These data provide evidence for the existence of an inhibitory descending projection from medial and posterior septum to the NI that provides a "feedback loop" to modulate the comparatively more dense ascending NI projections to medial septum and hippocampus. Neural processes and associated behaviors activated or modulated by changes in hippocampal theta rhythm may depend on reciprocal connections between ascending and descending pathways rather than on unidirectional regulation via the medial septum. J. Comp. Neurol. 523:565–588, 2015. © 2014 Wiley Periodicals, Inc.     

Various types of non-pyramidal hippocampal neurons project to the septum and contralateral hippocampus

The morphology was studied of hippocampal neurons which had their somata in the hilus of the area dentata, and in stratum radiatum or stratum oriens of Ammon's horn, and which sent projections to the septum and contralateral hippocampus, respectively. The fluorescent marker Fast Blue was injected into the septum or contralateral hippocampus. Somata were then identified by their fluorescent label in slices of perfused brains. After intracellular injection of these somata with Lucifer Yellow, it was found that contralaterally projecting neurons were pyramidal cells, inverted fusiform and multipolar cells in CA3c, and stellate, fusiform and multipolar cells in the hilus. After septal injections, we identified two groups of aspiny stellate cells in the hilus; pyramidal basket cells, polygonal basket cells, horizontal basket cells in stratum oriens; and stellate cells in stratum radiatum of CA1 and CA3, as well as pyramid-like aspiny cells in stratum radiatum of CA1. These cells also had short locally arborizing axons, thus probably contributing to local circuits. Such cells may constitute a third class of hippocampal neurons combining the properties of principal cells and interneurons. These results support the opinion that the simple concept of separating hippocampal cells into projection neurons and local-circuit neurons needs reconsideration.     

Effects of elevated levels of nerve growth factor on the septohippocampal system in transgenic mice

Elevating target-derived levels of nerve growth factor (NGF) in peripheral organs of postnatal mammals is known to enhance the survival of postganglionic sympathetic neurons and to promote the terminal arborization of sympathetic axons within such NGF-rich target tissues. Although increasing levels of NGF in the central nervous system can ameliorate cholinergic function of damaged and aged neurons of the medial septum, it remains undetermined whether the postnatal development of this neuronal population and their projections that innervate the hippocampus are likewise affected by elevated levels of target-derived NGF. To address this question, the cholinergic septohippocampal pathway was examined in adult transgenic mice which display elevated levels of NGF protein production in the dorsal hippocampus during postnatal development. Adult transgenic mice possessed a cholinergic population of septal neurons ≈ 15% larger than that seen in age-matched control animals. Despite increased numbers of cholinergic septal neurons, as well as elevated levels of hippocampal NGF, the density of cholinergic septal axons in the outer molecular layer of the hippocampal dentate gyrus of adult transgenic animals was comparable with that found in wild-type controls. These results reveal that elevating levels of target-derived NGF during postnatal development can increase the population size of the cholinergic septal neurons but does not alter their pattern of afferent innervation in the hippocampus of adult mice.     

Evidence for target preferences by cholinergic axons originating from different subdivisions of the basal forebrain.

Possible target preferences of basal forebrain cholinergic neurons were studied in organotypic slice cultures. Cholinergic neurons in slices of medial septum or substantia innominata send axons into both hippocampus and neocortex when co-cultured together. However, septal cholinergic axons course through adjacent slices of neocortex to reach and branch densely in slices of hippocampus, but septal axons seldom grow beyond adjacent hippocampal tissue to reach neocortex. In contrast, cholinergic axons from substantia innominata commonly grow through hippocampus to reach neocortex, and also grow through neocortex to reach hippocampus, with similar branching densities in each target. The greater density of septal axonal branches in hippocampus than in neocortex suggests a preference of septal axons for the hippocampal target.     

Organization of projection neurons of the hippocampus

The origin of the fornix system was investigated by the horseradish peroxidase (HRP) technique. Small injections of HRP into the septal region resulted in labeling of neurons in both the pyramidal and nonpyramidal layers. Labeled neurons in the stratum pyramidale were seen in CA3 (bilaterally), CA2, CA1, and the adjacent subicular complex. Different patterns of uptake were observed in those hippocampal subfields. Numerous neurons in stratum oriens also displayed HRP positivity. These neurons were both polymorphic and spindle shape. The neurons were mainly restricted to the ventral portions of the hippocampus but labeled neurons were also seen in more dorsal levels. Furthermore, scattered neurons in stratum radiatum also displayed HRP positivity. These data demonstrate that some neurons generally considered to be interneurons are indeed projection neurons of the hippocampus.     

Types and synaptic connections of hippocampal inhibitory neurons reciprocally connected with the medial septum

The morphological properties and connectivity of γ-aminobutyric acid (GABA)ergic hippocampal cells projecting to the medial septum (HS cells) were examined in the rat. Two types of HS cells are located in different layers of the hippocampus: sparsely-spiny cells are in CA1–3 str. oriens and CA3 str. radiatum, where recurrent axons of pyramidal cells arborize. Densely-spiny HS cells with spiny somata are located in the termination zone of granule cell axons. In the hilus, intermediate morphologies can also be found. HS cells receive GABAergic medial septal afferents in all layers where they occur, thus the connectivity of the septum and the hippocampus is reciprocal at cell level. HS cells receive extremely dense innervation, sparsely-spiny cells are innervated by ∼19 000 excitatory inputs, while densely-spiny cells get an even larger number (∼37 000). While 14% of the inputs are inhibitory for the sparsely-spiny cells, it is only 2.3% in the case of densely-spiny cells. Because a high proportion (up to 54.5% on somata and 27.5% on dendrites) of their GABAergic inputs derived from labelled septal terminals, their predominant inhibitory input probably arises from the medial septum. CA1 area HS cells possessed myelinated projecting axons, as well as local collaterals, which targeted mostly pyramidal cell dendrites and spines in str. oriens and radiatum. The synaptic organization suggests that by sampling the activity of large populations of principal cells HS cells can reliably broadcast hippocampal activity level to the medial septum.     

Septo-hippocampal networks in chronic epilepsy

The medial septum inhibits the appearance of interictal spikes and seizures through theta rhythm generation. We have determined that medial septal neurons increase their firing rates during chronic epilepsy and that the GABAergic neurons from both medial and lateral septal regions are highly and selectively vulnerable to the epilepsy process. Since the lateral septal region receives a strong projection from the hippocampus and its neurons are vulnerable to epilepsy, their functional properties are probably altered by this disorder. Using the pilocarpine model of temporal lobe epilepsy we examined the pilocarpine-induced functional alterations of lateral septal neurons and provided additional observations on the pilocarpine-induced functional alterations of medial septal neurons. Simultaneous extracellular recordings of septal neurons and hippocampal field potentials were obtained from chronic epileptic rats under urethane anesthesia. Our results show that: (1) the firing rates of lateral septal neurons were chronically decreased by epilepsy, (2) a subset of lateral septal neurons increased their firing rates before and during hippocampal interictal spikes, (3) the discharges of those lateral septal neurons were well correlated to the hippocampal interictal spikes, (4) in contrast, the discharges of medial septal neurons were not correlated with the hippocampal interictal spikes. We conclude that epilepsy creates dysfunctional and uncoupled septo-hippocampal networks. The elucidation of the roles of altered septo-hippocampal neuronal populations and networks during temporal lobe epilepsy will help design new and effective interventions dedicated to reduce or suppress epileptic activity.     

Characterization of medial septal glutamatergic neurons and their projection to the hippocampus

The two neuronal populations that have been typically investigated in the septum use acetylcholine and GABA as neurotransmitters. The existence of noncholinergic, non-GABAergic, most likely glutamatergic septal neurons has recently been reported. However, their morphological characteristics, numbers, distribution, and connectivity have not been determined. Furthermore, the projection of septal glutamatergic neurons to the hippocampus has not been characterized. To address these issues, subpopulations of cholinergic and GABAergic neurons were identified by immunohistochemistry. In addition, the retrograde tracer fluorogold was injected into the hippocampus to determine the characteristics of a glutamatergic septo-hippocampal projection. Our work revealed that although glutamatergic neurons are found throughout the septum, they concentrate in medial septal regions. Using stereological probes, approximately 16,000 glutamatergic neurons were estimated in the medial septal region. Triple immunostaining showed that most glutamatergic neurons do not immunoreact with cholinergic or GABAergic neuronal markers (anti-ChAT or anti-GAD67 antibodies, respectively). Fluorogold injections into CA1, CA3, and dentate gyrus of the hippocampus showed that septal glutamatergic neurons project to each of these hippocampal regions, forming approximately 23% of the septo-hippocampal projection. Most cell bodies of septo-hippocampal glutamatergic neurons were located in the medial septum. The remaining cell bodies were found in the diagonal band. This data shows that glutamatergic neurons constitute a significant neuronal population in the septum and that a subpopulation of these neurons projects to hippocampal regions. Thus, the septo-hippocampal projection needs to be reconsidered as a three neurotransmitter pathway.     

Efferent connections of the hippocampal formation in the rat.

In this investigation the projections of the hippocampal formation to the septal area and hypothalamus were studied in the rat with the combined use of 3H-amino acid radioautography and horseradish peroxidase histochemistry. The results indicate that all of the fibers which project to the hypothalamus and the majority of fibers which project to the septum arise from the subicular cortex and not from hippocampal pyramidal cells. The projection to both of these areas are topographically organized along the longitudinal axis of the hippocampal formation. Specifically, fibers from subicular cortical cells situated at the septal end of the hippocampal formation which project through the medial part of the dorsal fornix terminate in the dorsomedial quadrant of the lateral septal nucleus and in the dorsal portion of the pars posterior of the medial mammillary nucleus. Fibers from progressively more posteroventral levels of the hippocampal formation which project through more lateral portions of the dorsal fornix and fimbria terminate in progressively lateral and ventral quadrants of the lateral septal nucleus and in progressively more ventral portions of the pars posterior. Concerning the specific origin of the fornix system, fibers from only the prosubiculum and subiculum project through both the pre- and postcommissural fornix. Hippocampal pyramidal cells from all CA fields have a restricted projection through the precommissural fornix and terminate in the caudal half of the septum while the presubiculum projects solely through the postcommissural fornix. The medial corticohypothalamic tract (MCHT) was found to arise from cells located in anterior ventral levels of the subicular cortex. Fibers from this tract appeared to be distributed throughout the pericellular region of the entire ventromedial extent of the hypothalamus from the level of the suprachiasmatic nucleus through the level of the medial mammillary nucleus. In this way, the mammillary bodies receive input from the subicular cortex via two routes: the descending column of the fornix and the MCHT.     

Organization of the septal region in the rat brain: cholinergic-GABAergic interconnections and the termination of hippocampo-septal fibers

This study deals with two characteristic cell types in the rat septal complex i.e., cholinergic and GABAergic neurons, and their synaptic connections. Cholinergic elements were labeled with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Antiserum against glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was employed to identify GABAergic perikarya and terminals, by using either the peroxidase-antiperoxidase (PAP) technique or a biotinylated second antiserum and avidinated gold or ferritin. With these contrasting immunolabels we have studied the cholinergic-GABAergic interconnections in double-labeled sections of intact septal regions and the GABAergic innervation of medial septal area cholinergic neurons in sections taken from animals 1 week following lateral septal area lesion. In other electron microscopic experiments we have studied cholinergic and GABAergic neurons in the septal complex for synaptic contacts with hippocamposeptal fibers, which were identified by anterograde degeneration following fimbria-fornix transection. Our results are summarized as follows: (1) GAD-positive terminals form synaptic contacts on ChAT-immunoreactive dendrites in the medial septum/diagonal band complex (MSDB), (2) surgical lesion of the lateral septal area resulted in a dramatic decrease of the number of GABAergic boutons on MSDB cholinergic neurons, (3) cholinergic terminals establish synaptic contacts with GAD immunoreactive cell bodies and proximal dendrites in the MSDB as well as in the lateral septum (LS), (4) degenerated terminals of hippocampo-septal fibers were mainly observed in the LS, where they formed asymmetric synaptic contacts on dendrites of GABAergic neurons and on nonimmunoreactive spines. We did not observe degenerated boutons in contact with ChAT-positive dendrites or cell bodies in the MSDB. From these results and from data in the literature we conclude that excitatory hippocampo-septal fibers activate GABAergic cells, and as yet unidentified spiny neurons in the LS, which may control the discharge of medial septal cholinergic neurons known to project back to the hippocampal formation.     

Immunocytochemical characterization of hippocamposeptal projecting GABAergic nonprincipal neurons in the mouse brain: a retrograde labeling study

The neurochemical contents of hippocamposeptal projecting nonprincipal neurons were examined in the mouse brain by using retrograde labeling techniques. We used the immunofluorescent multiple labeling method with a confocal laser-scanning microscope. First of all, the hippocamposeptal projecting nonprincipal neurons were glutamic acid decarboxylase 67-immunoreactive (IR), i.e., these hippocamposeptal projecting nonprincipal neurons were immunocytochemically GABAergic in the mouse brain. Next, most (93.0%) of the hippocamposeptal projecting GABAergic neurons were somatostatin-like immunoreactive (SS-LIR). The SS-LIR hippocamposeptal projecting neurons were frequently found in the stratum oriens of the CA1 and CA3 regions, and were also occasionally found in the stratum radiatum, stratum lucidum, and stratum pyramidale of the CA3 region. They were also frequently found in the dentate hilus. On the other hand, at least 40.6% of SS-LIR neurons in the hippocampus projected to the medial septum. Next, 38.0% of hippocamposeptal projecting GABAergic neurons were calbindin D28K (CB)-IR. Although the distribution of the CB-IR hippocamposeptal projecting neurons was generally similar to that of the SS-LIR projecting neurons in Ammon's horn, they were never seen in the dentate hilus. At least 22.1% of CB-IR GABAergic neurons in the hippocampus projected to the medial septum. Furthermore, 5.8% of hippocamposeptal projecting GABAergic neurons were parvalbumin-IR, which were most always found in Ammon's horn. Finally, no hippocamposeptal projecting GABAergic neurons were neuronal nitric oxide synthase-IR nor calretinin-IR. These results indicate that the SS-LIR neurons play a crucial role in the hippocamposeptal projection of the mouse brain, and they are also assumed to be involved in the theta oscillation of the mouse hippocampus.     

Labeling of pyramidal and nonpyramidal neurons with lectin Vicia villosa during postnatal development of the guinea pig

Labeling of cortical neurons with a lectin, Vicia villosa (VVA), was investigated in guinea pigs aged 1 day old to adult. Lectin histochemistry revealed a perineuronal sheath, which outlined the cell bodies, apical dendrites, and axon initial segments, in distinct populations of pyramidal and nonpyramidal neurons. Their laminar positions were segregated, with the pyramidal neurons confined to layer 5 and the nonpyramidal neurons distributed mainly in layers 3–5. The VVA-labeled substance(s) was detected at the interfaces between neurons and cellular elements present in the perisynaptic region, including glial processes and fine axons. However, it was excluded from synaptic clefts of presynaptic terminals. Double immunohistochemistry revealed that most of the VVA-labeled neurons were also labeled perineuronally with a monoclonal antibody, Cat 301, and vice versa. Dendritic patterns of the VVA-labeled pyramidal neurons were studied further by intracellular injection of Lucifer yellow into fixed slices. Apical dendrites had a considerable thickness before arborizing into a few daughter branches in layer 3 or 4, suggesting a morphological resemblance to intrinsic, bursting pyramidal neurons defined physiologically in vitro. During postnatal development, there was a global spatiotemporal pattern in the onset of VVA labeling of the cortical neurons. The labeling progressed from medial and posterior cortical areas, which are closely related to the hippocampal formation, to more lateral and anterior areas, which are less closely related. The labeling patter thus tends to follow the order of the phylogenetical development of the isocortex. J. Comp. Neurol. 389:453–468, 1997. © 1997 Wiley-Liss, Inc.