Response of seizure-induced newborn neurons in the dentate gyrus of adult rats to second episode of seizures

In this study we examined the unknown issue of whether seizure-induced newborn hippocampal neurons in freely moving adult rats are able to respond to pathophysiological stimuli in the same way as their neighboring neurons do. Three days after pentylenetrazol (PTZ)-induced generalized seizures, rats received 5-bromodeoxyuridine (BrdU) injections to label dividing cells, followed 4 weeks later by the second PTZ injection to induce second episode of generalized seizures. We observed that the first episode of PTZ-induced seizures resulted in a significant increase in the number of newborn neurons in the adult hippocampal dentate gyrus. In comparison with vehicle-injected control rats that exhibited no Fos immunoreactivity and mild glutamic acid decarboxylase 67 (GAD67) expression in the dentate granule cells, rats killed 2-6 h following the second PTZ injection showed intensive Fos and GAD67 expression in virtually all granule cells with or without BrdU double-labeling. These findings provide important evidence indicating that seizure-induced newborn neurons in freely moving adult rats are able to respond to pathophysiological stimuli in the same way as neighboring neurons do.     

Immunocytochemical mapping of Fos protein following seizures in gerbils indicates the activation of hippocampal neurons

The expression of the proto-oncogene, c-fos, and its protein, Fos, has been shown to be a useful marker for elevated levels of neuronal activity generated in the brain following different stimuli, including seizures. Since previous studies indicated hippocampal involvement in seizure activity in gerbils, Fos immunocytochemistry was used to determine whether hippocampal neurons become activated following environmentally induced seizures in this animal. Gerbils with maximal seizures showed many Fos-immunolabeled neurons in the granule cell layer and hilus of the dentate gyrus, as well as in CA3 and CA1 of the hippocampus. These gerbils had significantly greater numbers of Fos-immunolabeled dentate granule cells than gerbils with less severe seizures or no seizures. The number of dentate granule cells and CA3 pyramidal cells with Fos immunolabeling increased in an exponential manner with increased seizure severity. Many Fos-immunolabeled neurons were found in several regions of the neo- and paleocortex and in other limbic structures including the piriform cortex, cortical amygdaloid nucleus, and arcuate nucleus of the hypothalamus. These results indicate that hippocampal neurons are activated following seizures in a genetic model, and provide further proof that the hippocampal formation is involved in the circuitry for seizures in gerbils.     

Localization and induction of c-fos protein-like immunoreactive material in the nuclei of adult mammalian neurons

The distribution of the proto-oncogene product, c-fos protein, has been studied in tissue from adult rat brain using immunocytochemical techniques. Sections of rat brain were incubated with a polyclonal antibody to a synthetic fragment of the c-fos protein (M-peptide) and visualized for immunoreactivity using the avidin-biotin technique. Low levels of c-fos protein-like immunoreactivity were concentrated in the nucleus. Dark nuclear staining of cells was observed in the cerebral cortex, in hippocampal pyramidal neurons, in granule cells of the dentate gyrus and other regions. Neurons in the cat visual cortex also showed low levels of c-fos protein-like immunoreactivity. Pre-absorbing the antibody with the M-peptide abolished the immunostaining. Generalized seizures evoked by injection of pentylenetetrazol produced a massive induction of fos protein(s) in the piriform and cingulate cortices as well as the dentate gyrus of rats. These results demonstrate that c-fos protein-like immunoreactive materials is found within the nuclei of fully differentiated adult mammalian neurons at low basal levels and that activation of nerve cells leads to an induction of c-fos proteins.     

Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia

Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.     

Presence and induction of Fos B-like immunoreactivity in neural, but not non-neural, cells in adult rat brain

The presence and induction of Fos B, a novel growth factor-activated gene, was investigated in adult rat brain using an antiserum to Fos B and immunocytochemical methods. In normal rat brain immunoreactivity was detected in the nuclei of nerve cells scattered in the cerebral cortex, striatum, amygdala, hippocampus and dentate gyrus. This immunostaining was not present in adjacent brain sections incubated with anti-Fos B serum preadsorbed with the Fos B peptide. Furthermore, Fos B-like immunoreactivity was induced in neurons by two treatments (focal brain injury and haloperidol injection) that are known to induce Fos, however, whereas Fos levels returned to baseline 24 h after these treatments, Fos B-like immunoreactivity remained elevated at this time point. Also, although focal brain injury and rolipram injections induced Fos in ependymal and glial-like cells in rat brain, Fos B-like immunoreactivity was not detected in these non-neural cells. The implications of these results for the presence of Fos B in adult neurons is discussed.     

Nerve growth factor-like immunoreactive profiles in the primate basal forebrain and hippocampal formation

The distribution of nerve growth factor (NGF), the prototypic neurotrophin, within the basal forebrain and hippocampal formation of young adult monkeys and aged humans was characterized with and affinity purified polyclonal β-NGF antibody raised against mouse β-NGF. In the basal forebrain of both primates, a granular NGF-like immunoreactive (ir) reaction product was observed within neurons of the medial septum, nucleus of the diagonal band, and nucleus basalis of Meynert. NGF-like immunoreactivity exclusively colocalized within p75 NGF receptor (NGFR) containing basal forebrain neurons. The intensity of NGF immunolabeling varied between cell bodies. Many NGF-ir perikarya were highly immunoreactive. In other basal forebrain neurons, NGF-like immunoreactivity was either undetectable or minimally expressed. In the hippocampus of both species, NGF-like immunoreactivity was mainly localized within the hilus of the dentate gyrus and within CA3 and CA2 hippocampal subfields. A marked diminution in NGF-like staining was seen in CA1. Within the hippocampal formation, NGF-like immunoreactivity was heaviest within the neuropil of stratum radiatum, intermediate in stratum oriens, and lightest in stratum pyramidal. NGF-like immunoreactivity was not found within the granule or pyramidal cells of the dentate gyrus and hippocampal formation, respectively. These findings demonstratre the presence of an NGF-like antigen in association with monkey and human magnocellular basal forebrain neurons and within their hippocampal target sites. This lends support to the hypothesis that NGF is internalized from sources located within target regions of the primate cholinergic basal forebrain neurons and is retrogradely transported to these cell bodies where the NGF trophic effect likely occurs.     

Basal expression and induction of glutamate decarboxylase GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus

The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two “fast-acting” amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects. © 1996 Wiley-Liss, Inc.     

c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress

The induction of c-fos mRNA was assessed using Northern blots and in situ hybridization in adult rats administered hypertonic saline (HS) and isotonic saline (IS). HS induced c-fos mRNA in magnocellular paraventricular nucleus (PVNm), parvocellular paraventricular nucleus (PVNp), supraoptic nucleus (SON), and lamina terminalis (LMT). This occurred within 5 min, peaked at 30-60 min, and disappeared by 180 min. Fos protein, detected using a specific monoclonal antibody, was maximal at 1-2 hr and disappeared 4-8 hr after HS administration. This confirms observations showing that the c-fos gene response is transient even in the presence of a continuing stimulus. In contrast, Fos-like immunoreactivity (FLI), detected using two polyclonal antisera, was observed in PVNm, PVNp, SON, and LMT for 1-24 hr during continuous osmotic stimulation. Moreover, FLI was observable in these structures for 7 d in rats administered HS and allowed to drink water ad libitum beginning 24 hr later. At times greater than 8 hr, FLI presumably represents Fos-related antigens (FRA), proteins immunologically and functionally related to Fos, whose expression is much more prolonged than authentic Fos following the osmotic stimulus. In addition to induction of c-fos expression in regions specifically involved in osmotic regulation, HS injections also induced c-fos in many other forebrain regions. In order to assess the induction of c-fos mRNA due to the "stress" of the injections, rats injected with isotonic saline were compared to uninjected controls. Isotonic saline injections induced c-fos mRNA in the PVNp, anterior hypothalamus, suprachiasmatic nucleus, cingulate gyrus, neocortex, ventral lateral septal nucleus, piriform cortex, hippocampal pyramidal and dentate granule neurons, paraventricular and intralaminar thalamic nuclei, bed nuclei of stria terminalis, cortical and medial amygdaloid nuclei, and other structures. In accord with other work, we interpret this pattern of c-fos expression to result from the stress of handling and injections. Since Fos and FRA probably bind to the promoters of target genes and regulate their expression, they likely mediate biochemical changes in the cells activated by the osmotic and stressful stimuli. Whereas the Fos signal is transient, FRA may act on target genes for the duration of the stimulus or longer.     

IMMUNOCYTOCHEMICAL LOCALIZATION OF ENKEPHALIN-LIKE IMMUNOREACTIVITY IN NEURONS OF HUMAN HIPPOCAMPAL FORMATION: EFFECTS OF AGEING AND ALZHEIMER''S DISEASE

The localization of neurons containing enkephalin-like immunoreactivity was studied within the human hippocampal formation. It was present in numerous dentate granule cells and a few larger neurons within the dentate molecular layer. In hippocampus proper, numerous pyramidal cells also demonstrated it, especially in field H1 of Rose (1926) and subiculum. No difference attributable to ageing or to dementia of the Alzheimer type was seen in this distribution. Post-mortem delay was the major factor affecting the intensity of its immunocytochemical localization to hippocampal cells and nonspecific background staining, due to binding of the secondary antiserum, increased directly with this. Some dendrites of dentate granule cells with immunoreactivity were found entering senile (neuritic) plaques in tissue from cases of Alzheimer's disease. These dendrites appeared morphologically normal.     

Secretoneurin: A marker in rat hippocampal pathways

Secretoneurin is a 33-amino acid peptide, generated in brain by proteolytic processing of secretogranin II. The distribution of secretoneurin-like immunoreactivity and secretogranin II mRNA was investigated in the hippocampus of the rat. Secretogranin II mRNA was found in high concentrations throughtout the granule cell and pyramidal cell layers and in many local neurons, notably in the hilus of the dentate gyrus. The general distributional pattern of secretoneurin-like immunoreactivity was characterized by a prominent staining in the area of the terminal field of mossy fibers with an obvious staining in the infrapyramidal area of CA3 and a strongly immunopositive band in the inner third of the molecular layer of the dentate gyrus. Lesions of the granule cells by local injection of colchicine significantly reduced secretoneurin-like immunoreactivity in the terminal field of mossy fibers, but not in the inner molecular layer of the dentate gyrus. On the other hand, destruction of interneurons of the dentate gyrus (mossy cells and certain γ-aminobutyric acid-ergic interneurons) by kainic acid—induced seizures was associated with a reduction of secretoneurin-like immunoreactivity in the inner molecular layer of the dentate gyrus. However, 30 days after kainic acid—induced seizures, a strongly secretoneurin-immunoreactive band reappeared in this area, which at this late time point is due to sprouting of mossy fibers collaterals. Our experiments suggest a widespread distribution of secretoneurin-like immunoreactivity in neurons of the hippocampal formation with a preferential localization in excitatory pathways including associational/commissural fibers originating from secretoneurin-containing mossy cells. J. Comp. Neurol. 377:29-40, 1997. © 1997 Wiley-Liss, Inc.     

The indusium griseum and anterior hippocampal continuation in the rat

The morphology and connections of the indusium griseum (IG) and anterior hippocampal continuation (AHC) suggest that this cortex contains analogues to several portions of the hippocampal formation. Whereas the outer neuronal layer of this cortex is made up of cells which are similar in structure to the neurons of the granule cell layer of the dentate gyrus, the three successively deeper layers contain morphological analogues to the neurons of the dentate hilus, Ammon's horn, and the subiculum, respectively. The neurons within each of these four layers of the AHC and IG have afferent and efferent connections which are quite similar to the connections of their hippocampal counterparts. Thus, the granule cells of the IG and AHC receive laminar inputs from the entorhinal cortex, the IG-AHC itself, and the supramammillary region. Each of these three classes of inputs ends at successively more proximal positions on the dendritic tree of these granule cells. Other inputs to this region include those from the septal nuclei and the olfactory bulb. The deeper layers of the IG and AHC receive several inputs, including those from the thalamic and septal nuclei and the entorhinal cortex. The efferent cell bodies of the IG and AHC are segregated in such a way that the granule cells appear to give rise to only short connections, while the hilar cells project to the granule cells, the intermediate pyramidal neurons project to other portions of the IG and AHC and to the olfactory bulb, and the deep pyramidal neurons project to the diencephalon. These results demonstrate that the IG-AHC is a continuation of the hippocampal formation.     

Parvalbumin- and calbindin D28k-immunoreactive neurons in the hippocampal formation of the macaque monkey.

Calcium-binding proteins calbindin D28k (CaBP) and parvalbumin (PV) were localized in neurons of the monkey hippocampal formation. CaBP immunoreactivity is present in all granule cells and in a large proportion of CA1 and CA2 pyramidal neurons, as well as in a distinct population of local circuit neurons. In the dentate gyrus, CaBP-immunoreactive nongranule cells are present in the molecular layer and in the hilar region, but they do not include the pyramidal basket cells at the hilar border. In the Ammon's horn, CaBP-positive, nonpyramidal neurons are more frequent in the CA3 area than in any other parts of the hippocampal formation. They are concentrated in the strata oriens and pyramidale of areas CA1-3, whereas only a few small neurons were found in the strata lucidum and radiatum of CA3 and in the stratum moleculare of the CA1 area. PV is exclusively present in local circuit neurons both in the dentate gyrus and in Ammon's horn. In the dentate gyrus the presumed basket cells at the hilar border exhibit PV immunoreactivity. In the hilar region and molecular layer only a relatively small number of cells are immunoreactive for PV. Most of these PV-positive cell bodies are located in the inner half of the molecular layer, with occasional horizontal cells at the hippocampal fissure. In Ammon's horn, strata oriens and pyramidale of areas CA1-3 contain a large number of PV-positive cells. There are no PV-immunoreactive cells in the strata lucidum, radiatum, or lacunosum moleculare. The CaBP- and PV-containing neurons form different subpopulations of cells in the monkey hippocampal formation. With the exception of a basket cell type in the monkey dentate gyrus, the CaBP- and PV-positive cell types were found to be remarkably similar in rodents and primates.     

Extranuclear estrogen receptor beta immunoreactivity is on doublecortin-containing cells in the adult and neonatal rat dentate gyrus

In adult female rats, estrogen receptor (ER) activation, particularly of ERbeta, promotes hippocampal neurogenesis. We previously reported that extranuclear ERbeta immunoreactivity (ir) in adult rats is on cellular profiles in or near the granule cell layer, which is the location of newly generated cells. During development, cells in or near the granule cell layer transiently express high levels of estrogen binding and nuclear ERs. Thus, we sought to determine if extranuclear ERbeta is in newly generated cells in adult and neonatal rat dentate gyrus. Sections from the dentate gyrus of adult proestrus or postnatal day 7 and 14 female rats were dual-labeled for ERbeta and the new-cell marker doublecortin (DCX) and examined by electron microscopy. DCX-containing neurons were found in the subgranular hilus in adult rats and were more widespread throughout the granule cell layer and hilus of neonatal rats. In both adults and neonatal rats, ERbeta immunoreactivity was found in a subset of DCX-labeled neurons. Electron microscopic examination of the adult dentate gyrus revealed that most perikarya with DCX-ir had the morphological characteristics of granule cells, although a few resembled interneurons. Dendrites with DCX-ir also were observed. In both adults and neonates, DCX-labeled neuronal perikarya and dendrites contained ERbeta-ir; ERbeta-ir usually was aggregated near the plasma membrane, mitochondria or endoplasmic reticula. ERbeta-ir was in glial profiles that apposed DCX-labeled perikarya and dendrites. These findings are consistent with data showing that estrogens can exert non-genomic effects directly and indirectly on newly generated cells in neonatal and adult rat dentate gyrus.     

Effects of adult-generated granule cells on coordinated network activity in the dentate gyrus

Throughout the adult life of most mammals, new neurons are continuously generated in the dentate gyrus of the hippocampal formation. Recent work has documented specific cognitive deficits after elimination of adult hippocampal neurogenesis in rodents, suggesting that these neurons may contribute to information processing in hippocampal circuits. Young adult-born neurons exhibit enhanced excitability and have altered capacity for synaptic plasticity in hippocampal slice preparations in vitro. Still, little is known about the effect of adult-born granule cells on hippocampal activity in vivo. To assess the impact of these new neurons on neural circuits in the dentate, we recorded perforant-path evoked responses and spontaneous network activity from the dentate gyrus of urethane-anesthetized mice whose hippocampus had been focally X-irradiated to eliminate the population of young adult-born granule cells. After X-irradiation, perforant-path responses were reduced in magnitude. In contrast, there was a marked increase in the amplitude of spontaneous γ-frequency bursts in the dentate gyrus and hilus, as well as increased synchronization of dentate neuron firing to these bursts. A similar increase in gamma burst amplitude was also found in animals in which adult neurogenesis was eliminated using the GFAP:TK pharmacogenetic ablation technique. These data suggest that young neurons may inhibit or destabilize recurrent network activity in the dentate and hilus. This unexpected result yields a new perspective on how a modest number of young adult-generated granule cells may modulate activity in the larger population of mature granule cells, rather than acting solely as independent encoding units.     

Immunohistochemical localization of N-methyl-D-aspartate receptor subunits in the adult murine hippocampal formation: evidence for a unique role of the NR2D subunit

NMDA receptors were immunopurified from adult mouse forebrain and screened by immunoblotting. NR1 was co-associated with NR2A, NR2B and NR2D but not NR2C, nor was NR2C detected in adult mouse hippocampal membranes. The anatomical distribution of NR1, 2A, 2B and 2D was mapped in the adult murine hippocampal formation. NR1-like immunoreactivity was localised to cell bodies of pyramidal neurons, granule cells and hilar cells of the dentate gyrus. Apical dendrites of the CA subfields and hilar cells were also immunopositive. NR2A- and NR2B-like immunoreactivity essentially co-localised with that of NR1 implying co-assembly of all three subunits in this brain structure. NR2D-like immunoreactivity was distinct, being totally excluded from pyramidal, granule and hilar cell bodies. Strong, punctate staining was restricted to the oriens layer of CA1 and the stratum lucidum of CA3 consistent with labelling of presynaptic receptors. Less intense staining was also observed in the internal third of the molecular layer of the dentate gyrus.     

Serotonergic reinnervation reverses lesion-induced decreases in PSA-NCAM labeling and proliferation of hippocampal cells in adult rats

Serotonin (5-HT) is believed to play a role in structural plasticity in the adult brain, and cell adhesion molecules may be involved in such adaptive processes. The present study sought to determine the effects of 5-HT denervation and reinnervation of the hippocampal formation on the expression of glial and neuronal markers and neurogenesis in adult rats. Injections of 5,7-dihydroxytryptamine (5,7-DHT) in the dorsal and medial raphe nuclei, producing a partial loss of 5-HT neurons, induced rapid and transient increases in glial fibrillary acidic protein immunoreactivity indicative of a reactive gliosis, but no changes in the S100beta or tenascin-C normally secreted by astroglial cells. In contrast, as long as the hippocampal formation was deprived of 5-HT innervation, significant decreases were observed in the number of granule cells expressing the highly polysialylated form of the neural cell adhesion molecule (PSA-NCAM) as well as the PSA-NCAM staining of the hilus in the dentate gyrus. Similar decreases in the number of newly generated granule cells labeled with bromodeoxyuridine were also detected during this time. All these effects were reversed later, when the hippocampal formation was reinnervated by 5-HT fibers. These results indicate that 5-HT is one factor which may regulate the number of granule cells proliferating in the adult dentate gyrus and thereafter expressing PSA-NCAM immunoreactive at the level of cell bodies, dendrites, and axonal paths (mossy fibers). They emphasize the critical role played by 5-HT in the neuronal organization of the hippocampus.     

Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus

The distribution of enkephalin-like immunoreactivity in the hippocampal formation of the rat was analyzed. Two specific projection systems are described. The first emerges from the hilus of the dentate gyrus and appears to terminate with notably large boutons on the proximal apical and, to a lesser extent, basal dendrites of hippocampal regio inferior pyramidal cells. This projection corresponds in source, position, and character to the hippocampal mossy fiber system. The second axonal population enters the temporal hippocampal formation from the medial wall of the subicular complex and follows the hippocampal fissure to occupy stratum lacunosum-moleculare of the hippocampus proper and the distal third of the dentate gyrus molecular layer; this pattern corresponds to the distribution of afferent input from the lateral entorhinal cortex and/or perirhinal area. Lesions of the hilus or retrohippocampal area caused a selective depletion of immunoreactivity in the mossy fiber fields and molecular layers of the dentate gyrus, respectively. Enkephalin-like immunoreactivity was found within the somata of three types of hippocampal neurons: (1) granule cells of the dentate gyrus, (2) occasional pyramidal shaped cells of field CA1 stratum pyramidale, and (3) varied scattered interneurons. Of this last group, two types of interneurons were consistently seen. The first occupy the border between stratum radiatum and stratum lacunosum-moleculare and extend processes at right angles to the long axis of the pyramidal cell dendrites, whereas the second lie within stratum radiatum of field CA1 and extend processes in alignment with the long axis of the pyramidal cell dendrites. Cells containing enkephalin-like immunoreacactivity were also observed in the subiculum and retrohippocampal region, most notably including layers II and III of the lateral entorhinal cortex-perirhinal area—the probable source of extrinsic immunoreactive input to the hippocampal formation. Intraventricular colchicine treatment intensified the immunoreactive staining of some hippocampal neurons but did not reveal any cell types not seen to be labeled in untreated rats.     

Localized expression of BMP and GDF mRNA in the rodent brain.

Expression of BMP- and GDF-related factors within the transforming growth factor-beta (TGF-beta) superfamily was examined in the rat and mouse brain by in situ hybridization. Strong signals were obtained in neurons for GDF-1 and GDF-10. GDF-1 is expressed at postnatal day 6 in the cerebral cortex, hippocampal CA1 through CA3 neurons, while only weakly expressed by cells in the dentate gyrus. Granule cells and neurons in the polymorph layer of the dentate gyrus are GDF-1-positive, as are the majority of neurons in the cortex. GDF-10 shows a distinct pattern of expression: At P6, strong labelling was seen in the superficial layers of cortex, notably in the posterior cingulate cortex, and in CA3 and dentate gyrus. From postnatal day 21, GDF-1 expression is strong in the hippocampus, cortex, and thalamic nuclei, while GDF-10 expression becomes restricted to the granule cell layer in the dentate gyrus. In contrast, OP-1 expression is restricted throughout development to cells of the medial habenular nucleus, choroid plexus, and leptomeninges. The markedly different expression patterns of these BMPs suggest they serve separate functions in the brain.     

Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus.

The basal expression of the protein products of the inducible immediate early genes (IEGs), Fos, Jun, and Krox 24, was investigated in rat hippocampus using immunocytochemical visualization methods with antisera specific for Fos only, Fos and the Fos-related antigens (FRAs), the Jun family, and Krox 24 (previously described as TIS 8, egr-1, NGF-IA or zif 268). In the normal adult rat brain basal levels of Jun, Krox 24 and Fos-related antigens but not Fos were seen within the hippocampus. More specifically very high basal levels of Jun were seen in the dentate granule cells with high basal Krox 24 levels seen in the CA1-subiculum region of the rat hippocampus. Basal FRAs but not Fos-positive cells were seen at low levels in the dentate granule cells. The implications of these results to the functioning of IEG proteins in hippocampal neurons is discussed.     

Proportion of parvalbumin-positive basket cells in the GABAergic innervation of pyramidal and granule cells of the rat hippocampal formation

Recent studies have indicated that hippocampal GABAergic neurons in both the dentate gyrus and Ammon's horn contain immunoreactivity for the calcium-binding protein parvalbumin (PARV). Although the distribution of PARV-positive neurons has been previously described, detailed quantitative electron microscopic studies of the PARV-positive axon terminals in the hippocampal formation are lacking. In the present study, immunocytochemical methods were used to localize PARV-positive neurons and axon terminals to determine their similarity to GABAergic neurons. The PARV-positive cells and axon terminals are associated closely with the pyramidal and granule cell layers. In agreement with previous studies, the morphology of PARV-positive neurons is similar to that of GABAergic cells, including the basket cells of both the dentate gyrus and Ammon's horn. The PARV-positive axon terminals form exclusively symmetric synapses with somata, dendrites, dendritic spines, and axon initial segments. However, these terminals represent only a portion of the total number of terminals that form symmetric synapses. Quantitative results indicate that only 32-38% of the total number of terminals forming symmetric axosomatic synapses with principal cells of the dentate gyrus and Ammon's horn are PARV positive. Together with previous findings from light microscopic double-labeling studies, these data indicate that the PARV-positive terminals arise from a subpopulation of GABAergic hippocampal neurons. Finally, it is important to note that the terminal plexus of PARV-positive hippocampal axons overlaps at all postsynaptic sites with a plexus of PARV-negative axons.