GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus

The distribution of Ca2+-binding protein, parvalbumin (PV), containing neurons and their colocalization with glutamic acid decarboxylase (GAD) were studied in the rat hippocampus and dentate gyrus using immunohistochemistry. PV immunoreactive (PV-I) perikarya were concentrated in the granule cell layer and hilus in the dentate gyrus and in the stratum pyramidale and stratum oriens in the CA3 and CA1 regions of the hippocampus. They were rare in the molecular layer of the dentate gyrus, in the stratum radiatum and in the stratum lacunosum-moleculare of the hippocampus. PV-I axon terminals were restricted to the granule cell layer, the stratum pyramidale and the immediately adjoining zones of these layers. Almost all PV-I neurons were also GAD immunoreactive (GAD-I), whereas only about 20% of GAD-I neurons also contained PV. The percentages of GAD-I neurons which were also immunoreactive for PV were dependent on the layer in which they were found; i.e. 40-50% in the stratum pyramidale, 20-30% in the dentate granule cell layer and in the stratum oriens of the CA3 and CA1 regions, 15-20% in the hilus and in the stratum lucidum of CA3 region and only 1-4% in the dentate molecular layer and in the stratum radiatum and the stratum lacunosum-moleculare of the CA3 and CA1 regions. PV-I neurons are a particular subpopulation of GABAergic neurons in the hippocampal formation. Based on their morphology and laminar distribution, they probably include basket cells and axo-axonic cells.     

Gap junction protein in rat hippocampus: light microscope immunohistochemical localization

An affinity-purified antibody against a 27-kD rat liver gap-junctional protein (GJP) was used to determine the distribution of GJP immunoreactivity in sections of rat hippocampus. Four heterogeneously distributed GJP-immunostaining patterns were observed. The two most common were punctate immunoreactive elements ranging in size from 0.3 to 0.7 microns and networks of immunoreactive varicose fibers coursing in a variety of directions within the various hippocampal layers and ranging in length from a few microns up to 200 microns. The density of punctate immunostaining was highest within a portion of the stratum pyramidale, at the border between the stratum pyramidale and stratum oriens, and at the border between the molecular and granule cell layers of the dentate gyrus. Moderate to low densities were observed in other hippocampal areas. Immunoreactive fibers were most concentrated within the border portions of the stratum pyramidale and oriens, moderately distributed in the stratum radiatum and the remaining part of the stratum oriens, and sparse in the alveus. In the dentate gyrus, fiber networks were most evident at the border between the granule cell and molecular layers and very unevenly distributed in the molecular layer. The two other patterns observed included intense filamentous immunostaining within a small number of neuronal perikarya located mainly in the stratum pyramidale of areas CA2 and CA3, but rarely in area CA1 or the dentate gyrus, and diffuse immunostaining of small cell bodies dispersed throughout the hippocampus but most numerous in the vicinity of the stratum pyramidale and in the alveus. All of these immunostaining patterns were seen at all rostrocaudal hippocampal levels. These results suggest that if GJP-immunoreactive fibers and neurons observed in the hippocampus have the capacity to form gap junctions, then electrotonic transmission may constitute an important means of information processing within this structure.     

GFR alpha-1 is expressed in parvalbumin GABAergic neurons in the hippocampus

Glial cell line derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. GDNF binds with high affinity to GDNF-family receptor alpha-1 (GFR alpha-1). This receptor is expressed in different areas of the brain, including the hippocampus and dentate gyrus. By using in situ hybridization and immunohistochemistry, we found that 19% to 37% of glutamic acid decarboxylase (GAD) expressing neurons co-expressed GFR alpha-1 in the hippocampus. GFR alpha-1/GAD co-expression was found mainly in the stratum (s) pyramidale (29-37%) and s. oriens (20-25%). Further characterization of GFR alpha-1 expressing interneurons, based on their calcium-binding protein immunoreactivity, demonstrated that many parvalbumin (PV) immunoreactive neurons express GFR alpha-1 in the s. pyramidale of CA1 (72%), CA2 (70%) and CA3 (70%) subfields of the hippocampus. GFR alpha-1/PV double labeled neurons were also detected in the s. oriens of CA1 (52%), CA2 (27%) and CA3 (36%) subfields. The expression of GFR alpha-1 in principal neurons and in a specific sub-population of GABAergic neurons (PV-containing neurons) suggest that GDNF might modulate, in a selective manner, functions of the entire adult hippocampus.     

Decreased expression of calretinin in the cerebral cortex and hippocampus of SOD1G93A transgenic mice

In the present study, we investigated the changes of calretinin (CR) expression in the central nervous system of SOD1G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). In wild-type SOD1 (wtSOD1) transgenic mice, many CR-immunoreactive neurons were found in all cortical regions. In the cerebral cortex of SOD1G93A transgenic mice, the number and staining intensity of CR-positive neurons were decreased. In the hippocampal formation, layer-specific alterations in the staining intensity of CR-immunoreactive neurons were observed in the CA1-3 areas and dentate gyrus. In wtSOD1 transgenic mice, CR-immunoreactive neurons with long processes were found in the stratum oriens and stratum radiatum of CA1-3 areas, and heavily stained band-like molecular layer was prominent in the dentate gyrus. CR immunoreactivity was decreased in each layer of CA1-3 areas and dentate gyrus of SOD1G93A transgenic mice. The first demonstration of decreased immunoreactivity for CR in the cerebral cortex and hippocampus of SOD1G93A transgenic mice may provide insights into the pathogenesis of motor neuron degeneration in human ALS although further quantitative studies are needed.     

GFRα-1 is expressed in parvalbumin GABAergic neurons in the hippocampus

Glial cell line derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. GDNF binds with high affinity to GDNF-family receptor α-1 (GFRα-1). This receptor is expressed in different areas of the brain, including the hippocampus and dentate gyrus. By using in situ hybridization and immunohistochemistry, we found that 19% to 37% of glutamic acid decarboxylase (GAD) expressing neurons co-expressed GFRα-1 in the hippocampus. GFRα-1/GAD co-expression was found mainly in the stratum (s) pyramidale (29–37%) and s. oriens (20–25%). Further characterization of GFRα-1 expressing interneurons, based on their calcium-binding protein immunoreactivity, demonstrated that many parvalbumin (PV) immunoreactive neurons express GFRα-1 in the s. pyramidale of CA1 (72%), CA2 (70%) and CA3 (70%) subfields of the hippocampus. GFRα-1/PV double labeled neurons were also detected in the s. oriens of CA1 (52%), CA2 (27%) and CA3 (36%) subfields. The expression of GFRα-1 in principal neurons and in a specific sub-population of GABAergic neurons (PV-containing neurons) suggest that GDNF might modulate, in a selective manner, functions of the entire adult hippocampus.     

Somatostatin-containing neurons in rat organotypic hippocampal slice cultures: Light and electron microscopic immunocytochemistry

Light and electron microscopic immunocytochemical techniques were used to study the interneuron population staining for somatostain (SRIF) in cultured slices of rat hippocampus. The SRIF immunoreactive somata were most dense in stratum oriens of areas CA1 and CA3, and in the dentate hilus. Somatostain immunoreactive cells in areas CA1 and CA3 were characteristically fusiform in shape, with dendrites that extended both parallel to and into the alveus. The axonal plexus in areas CA1 and CA3 was most dense in stratum lacunosum-moleculare and in stratum pyramidale. Electron microscopic analysis of this area revealed that the largest number of symmetric synaptic contacts from SRIF immunoreactive axons were onto pyramidal cell somata and onto dendrites in stratum lacunosum-moleculare. In the dentate gyrus, SRIF somata and dendrites were localized in the hilus. Hilar SRIF immunoreactive neurons were fusiform in shape and similar in size to those seen in CA1 and CA3. Axon collaterals coursed throughout the hilus, projected between the granule cells and into the outer molecular layer. The highest number of SRIF synaptic contacts in the dentate gyrus were seen on granule cell dendrites in the outer molecular layer. Synaptic contacts were also observed on hilar neurons and granule cell somata. SRIF synaptic profiles were seen on somata and dendrites of interneurons in all regions. The morphology and synaptic connectivity of SRIF neurons in hippocampal slice cultures appeared generally similar to intact hippocampus. © 1994 Wiley-Liss, Inc.     

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.     

GABAergic neurons containing CCK-8-like and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus

The coexistence of cholecystokinin-octapeptide-like (CCK-L) and/or vasoactive-intestinal-polypeptide-like immunoreactive (VIP-LI) materials and glutamate decarboxylase (GAD) was studied in the rat hippocampus and dentate gyrus by means of immunohistochemistry. Consecutive 40-micron-thick sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for these peptides and GAD in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell, incubated in two different antisera. Almost all of the CCK-LI neurons were also GAD immunoreactive, whereas only about 10% of the GAD-immunoreactive neurons were CCK-LI. The percentages of GAD-immunoreactive neurons which were also immunoreactive for CCK were dependent on the laminar area in which they were found: i.e., 15-20% in the stratum radiatum of the hippocampus, about 10% in the stratum pyramidale, and about 6% in the stratum oriens. In contrast to the CCK-LI neurons, only about 40% of the VIP-LI neurons were identified to be also GAD immunoreactive, which might correspond to only part of the GAD-immunoreactive neurons. Furthermore the coexistence of VIP-LI and CCK-LI materials was recognized in about 10% of the CCK-LI neurons or about 35% of the VIP-LI neurons, indicating that some GABAergic neurons (presumably about 1%) in the rat hippocampus and dentate gyrus may contain both CCK-LI and VIP-LI materials.     

Age-related changes in the distribution of nitrotyrosine in the cerebral cortex and hippocampus of rats

A wealth of indirect evidence implicates oxidative damage of cellular constituents in aging, as well as in the pathogenesis of the neurodegenerative diseases of later years. In the present study, we have determined age-related changes in the distribution of 3-nitrotyrosine (3-NT) in the cerebral cortex and hippocampus of rats. In adult rats, no 3-NT-immunoreactive cells were found in the cerebral cortex and hippocampus, whereas 3-NT immunoreactivity was significantly increased in aged rats. Some pyramidal cells of CA3 area and granule cells of the dentate gyrus highly expressed 3-NT in aged rats. Many interneurons located within stratum pyramidale and stratum oriens of CA1 were strongly immunoreactive for 3-NT. Our first demonstrations of increased 3-NT in the cerebral cortex and hippocampus during aging implicate these areas as sites for functionally significant oxidative damage. The mechanisms underlying the increased immunoreactivity for 3-NT, and the functional implications of this increase, require elucidation.     

Age-related changes of gamma-aminobutyric acid transaminase immunoreactivity in the hippocampus and dentate gyrus of the Mongolian gerbil

We investigated the age-related changes of gamma-aminobutyric acid transaminase (GABA-T, a GABA degradation enzyme) in the hippocampus and dentate gyrus of the gerbil at postnatal month 1 (PM 1), PM 3, PM 6, PM 12, and PM 24. Age-related changes of GABA-T immunoreactivity were distinct in the hippocampal CA1 region and in the dentate gyrus. GABA-T immunoreactivity was weak at PM 1, but at PM 3, it had increased significantly, and then increased further. Between PM 6 and PM 12, strong GABA-T immunoreactivity was found in nonpyramidal cells (GABAergic) in the stratum pyramidale of the CA1 region, and at PM 6, strong GABA-T immunoreactivity was found in neurons of the dentate gyrus subgranular zone. At PM 24, CA1 pyramidal cells showed strong GABA-T immunoreactivity. Western blot analysis showed a pattern of GABA-T expression similar to that shown by immunohistochemistry at various ages. In conclusion, our results suggest that the age-related changes of GABA-T provide important information about the aged brain with GABA dysfunction.     

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.     

Calretinin/PSA-NCAM immunoreactive granule cells after hippocampal damage produced by kainic acid and DEDTC treatment in mouse

There is a dramatic increase in the number of lightly immunoreactive calretinin cells in the granular layer of the dentate gyrus of the mouse hippocampus 1 day after excitotoxic injury using kainic acid combined with the zinc chelator diethyldithiocarbamate. At 7 days after treatment, these cells are strongly immunoreactive for calretinin and for the polysialated form of the glycoprotein neural cell adhesion molecule (PSA-NCAM). The reexpression of calretinin and PSA-NCAM after treatment corresponds well with the loss of input from the damaged hilar mossy cells. These cells could be considered immature granule cells since they are immunoreactive to markers for immature cells such as PSA-NCAM, and are not immunoreactive to calbindin D28k and neuronal nuclear specific protein NeuN (present in mature granule cells), or GABA (present in interneurons). Ultrastructural analysis of these cells indicates that they are immature. Labelling of cell proliferation with 5-bromo-2'-deoxyuridine (BrdU) shows that by day 1 no calretinin immunoreactive cell of the dentate gyrus corresponds to newly generated cells. By day 7 only 6% of the calretinin immunoreactive cells in the dentate gyrus are marked for BrdU. Our data indicate that the CR/PSA-NCAM immunoreactive cells of the dentate gyrus, in spite of their immature characteristics, are not the products of reactive neurogenesis. These cells could represent a reservoir of pre-existing not completely differentiated granule cells that react to damage.     

Mu- and delta-opioid receptor binding peaks and kappa-homogeneity in the molecular layers of rat hippocampal formation

The sublaminar binding profiles of (D-Ala-NMe-Phe-Gly-ol)-enkephalin (DAGO), (2-D-penicillamine), 5-D-penicillamine)-enkephalin (DPDPE), and dynorphin A(1-8) (DYN) were studied in the CA1 subfield and dentate gyrus of rat hippocampal formation. Binding was assayed on cryomicrotome sections using coverslip autoradiographic and single grain counting techniques. DAGO, an agonist for mu-sites, had peak binding in the stratum pyramidale with a secondary peak in the distal part of the stratum radiatum. Binding of DAGO in the dentate gyrus was homogeneous. DPDPE, a delta-site agonist, also had peak binding in the stratum pyramidale, but there was no secondary peak in the molecular layer of the hippocampus. In the dentate gyrus, DPDPE binding was highest in the inner one-third of the molecular layer immediately adjacent to the granular cell layer. The endogenous opioid DYN had a laminar binding profile that mimicked that of DAGO. However, when tritiated DYN was coincubated with unlabeled DAGO and DPDPE, as much as 90% of DYN binding was blocked and remaining binding was homogenous though a small peak remained in the stratum pyramidale. The secondary peak of DAGO binding in the stratum radiatum corresponds to an area previously determined to contain processes immunoreactive for enkephalin and gamma aminobutyric acid. This correspondence suggests that opioid compounds may mediate disinhibition of the distal dendrites of hippocampal pyramidal neurons. In addition, DYN binding patterns indicate that its action in rat hippocampus is likely by both mu- and delta-receptors.     

Interneurons are the local targets of hippocampal inhibitory cells which project to the medial septum

A subset of GABAergic neurons projecting to the medial septum has long been described in the hippocampus. However, the lack of information about their local connectivity pattern or their correspondence with any of the well-established hippocampal interneuron types has hampered the understanding of their functional role. Retrograde tracing combined with immunostaining for neurochemical markers in the adult rat hippocampus showed that nearly all hippocampo-septal (HS) neurons express somatostatin (>95%) and, in the hilus and CA3 stratum lucidum, many contain calretinin (>45%). In contrast, in stratum oriens of the CA1 and CA3 subfields, the majority of HS neurons contain somatostatin (>86%) and calbindin (>73%), but not calretinin. Because somatostatin-positive hippocampal interneurons have been most extensively characterized in the stratum oriens of CA1, we focused our further analysis on HS cells found in this region. In 18-20-day-old rats, intracellularly filled CA1-HS cells had extensive local axon collaterals crossing subfield boundaries and innervating the CA3 region and the dentate gyrus. Electron microscopic analysis provided evidence that the axon terminals of CA1-HS cells form symmetrical synapses selectively on GABAergic interneurons, both locally and in the CA3 region. In addition, double retrograde labelling experiments revealed that many CA1-HS neurons of the dorsal hippocampus also have collateral projections to the ventral hippocampus. Thus, CA1-HS cells innervate inhibitory interneurons locally and in remote hippocampal regions, in addition to targeting mostly GABAergic neurons in the medial septum. This dual projection with striking target selectivity for GABAergic neurons may be ideally suited to synchronize neuronal activity along the septo-hippocampal axis.     

Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.     

Colocalization of parvalbumin and somatostatin-like immunoreactivity in the mouse hippocampus: quantitative analysis with optical dissector

The colocalization of parvalbumin (PV) and somatostatin (SS)-like immunoreactivity was studied quantitatively in the mouse hippocampus, with particular reference to their areal and dorsoventral differences. The optical disector method was applied by using a confocal laser scanning microscope with immunofluorescent double-labeling. In the present study, we found a particular subpopulation of hippocampal nonprincipal neurons that contained both PV and SS-like immunoreactivity, i.e., PV-immunoreactive (IR)/SS-like immunoreactive (LIR) neurons. In the CA1 region, PV-IR/SS-LIR neurons were restricted to the stratum oriens (SO). In the CA3 region, they were scattered in the SO, stratum pyramidale (SP), and stratum radiatum (SR). However, they were rarely seen in the dentate gyrus (DG). The proportion of PV-IR/SS-LIR neurons in the PV-IR neurons or SS-LIR neurons was about 10% in the CA1 region, 15-30% in the CA3 region, 0-5% in the DG, and 10-20% in total. Laminar analysis revealed that the proportions of PV-IR/SS-LIR neurons in the PV-IR neurons were high in the SO (about 25%) of the CA1 region, and in the SO (about 50%) and SR (30-45%) of the CA3 region. The proportion of PV-IR/SS-LIR neurons in the SS-LIR neurons was low in the SO of the CA1 region (about 10%), but high in the SO (35-65%) and SR (35-45%) of the CA3 region. Morphologically, medium-sized horizontal fusiform and multipolar PV-IR/SS-LIR neurons were frequently observed, and they showed weak immunoreactivity for PV. Large-sized vertical bitufted and triangular PV-IR neurons lacked SS-like immunoreactivity, and most of them showed moderate to intense immunoreactivity for PV. In addition, we provide direct evidence that some PV-IR/SS-LIR neurons projected to the medial septum by using retrograde labeling with Fluoro-Gold injection. These observations indicate that PV-IR/SS-LIR neurons constitute a particular subpopulation of hippocampal nonprincipal neurons.     

Poor correlation between delayed neuronal death induced by transient forebrain ischemia,and immunoreactivity for parvalbumin and calbindin D-28k in developing gerbil hippocampus

In the normal developing hippocampus of the gerbil, parvalbumin-immunoreactive neurons first appear in the stratum pyramidale of CA3 at postnatal day 15 (P15), and in CA2 and hilus of the dentate gyrus from P21 onwards. Immunoreactive terminals also follow the same sequence from CA3 to CA1 to reach adult patterns by the end of the 1st month. Calbindin D-28k immunoreactivity is seen in the external part of the upper blade of the dentate gyrus at P5, and progresses to the granule cell and molecular layers of the whole gyrus by P15, except for a thin band of immature cells located at the base of the granule cell layer which are calbindin negative. Calbindin immunoreactivity in mossy fibers progresses from the external to the hilar region of CA3 during the same period. A few immunoreactive cells are also found in the stratum radiatum/lacunare of the CA3, but no calbindin-immunoreactive cells are observed in the CA1 and CA2 subfields. The adult pattern of calbindin immunoreactivity is reached at P21. Vulnerability following transient forebrain ischemia for 20 min was examined in the hippocampal formation of gerbils during postnatal development. No cellular damage was seen in animals aged 7 days. Dying cells were observed at the base of the granule cell layer of the dentate gyrus in animals aged 15, 21 and 30 days. Pyramidal cells in the CA3 subfield were also sensitive to ischemia in gerbils aged 15 days, and less frequently in animals aged 21 days. The adult pattern of cellular damage, characterized by selective vulnerability of the CA1 subfield, was seen from day 30 onwards. These findings show that the pattern of selective vulnerability following transient forebrain ischemia is different in young and adult gerbils, and suggest that little, if any, correlation exists between resistance to delayed cellular damage and parvalbumin and calbindin D-28k content in the hippocampus of young gerbils.Supported in part by grant FIS 93-131 and a grant from the Fundacio Pi i Synyer (to A.T.)     

Early maternal deprivation induces gender‐dependent changes on the expression of hippocampal CB1 and CB2 cannabinoid receptors of neonatal rats

Early maternal deprivation (MD) in rats (24 h, postnatal day 9–10) is a model for neurodevelopmental stress. There are some data proving that MD affects the endocannabinoid system (ECS) in a gender‐dependent manner, and that these changes may account for the proposed schizophrenia‐like phenotype of MD rats. The impact of MD on cannabinoid receptor distribution in the hippocampus is unknown. The aim of this study is to evaluate the expression of CB₁ and CB₂ receptors in diverse relevant subregions (DG, CA1, and CA3) of the hippocampus in 13‐day‐old rats by immunohistochemistry and densitometry. MD induced a significant decrease in CB₁ immunoreactivity (more marked in males than in females), which was mainly associated with fibers in the strata pyramidale and radiatum of CA1 and in the strata oriens, pyramidale, and radiatum of CA3. In contrast, MD males and females showed a significant increase in CB₂ immunoreactivity in the three hippocampal areas analyzed that was detected in neuropil and puncta in the stratum oriens of CA1 and CA3, and in the polymorphic cell layer of the dentate gyrus. A marked sex dimorphism was observed in CA3, with females exhibiting higher CB₁ immunoreactivity than males, and in dentate gyrus, with females exhibiting lower CB₂ immunoreactivity than males. These results point to a clear association between developmental stress and dysregulation of the ECS. The present MD procedure may provide an interesting experimental model to further address the role of the ECS in neurodevelopmental mental illnesses such as schizophrenia. © 2008 Wiley‐Liss, Inc.     

Mossy Cells of the Rat Dentate Gyrus are lmmunoreactive for Calcitonin Gene-related Peptide (CGRP)

The neuropeptide calcitonin gene-related peptide (CGRP) was localized in the hippocampus and dentate gyrus of the rat by immunocytochemistry at the light and electron microscopic levels. Without colchicine treatment only faint neuropil labelling was found in the inner molecular layer of the dentate gyrus. Following colchicine treatment, a large number of neurons with numerous complex spines along the proximal dendrites were visualized in the hilus of the dentate gyrus, particularly in the ventral areas, and, in addition, staining of the inner molecular layer became stronger. Several CA3c pyramidal cells located adjacent to the hilar region in the ventral hippocampus also appeared to be faintly positive, although in most cases only their axon initial segments were labelled. Outside this region, the subicular end of the CA1 subfield contained occasional CGRP-positive non-pyramidal cells. The hilar CGRP-positive neurons were negative for parvalbumin, calretinin, cholecystokinin and somatostatin, whereas most of them were immunoreactive for GluR2/3 (the AMPA-type glutamate receptor known to be expressed largely by principal cells). Correlated electron microscopy showed that the spines along the proximal dendritic shafts indeed correspond to thorny excrescences engulfed by large complex mossy terminals forming asymmetrical synapses. Pre-embedding immunogold staining demonstrated that CGRP immunoreactivity in the inner molecular layer was confined to axon terminals that form asymmetrical synapses, and the labelling was associated with large dense-core vesicles. The present data provide direct evidence that CGRP is present in mossy cells of the dentate gyrus and to a lesser degree in CA3c pyramidal cells of the ventral hippocampus. These CGRP-containing principal cells terminate largely in the inner molecular layer of the dentate gyrus, and may release the neuropeptide in conjunction with their 'classical' neurotransmitter, glutamate.     

Deafferentation removes calretinin immunopositive terminals,but does not induce degeneration of calbindin D-28k and parvalbumin expressing neurons in the hippocampus of adult rats

Unilateral combined transections of the fimbriafornix and angular bundle in adult Fischer 344 rats were used to study the effects of deafferentation on hippocampal expression of calretinin, calbindin D-28k, and parvalbumin. Reflecting the widespread degeneration of synaptic contacts, immunostaining for glial fibrillary acidic protein 6 days after the lesions was increased in lacunosum-molecular and oriens layers of CA1, 2, and 3 in ipsi- and contralateral hippocampus and in the ipsilateral dentate gyrus outer molecular layer. At 21 days the immunoreactivity had decreased to control levels except for a still slightly increased signal in the oriens layer of CA1-3. At 6 and 21 days after the combined lesions the numbers of hippocampal neurons containing calretinin, parvalbumin, and calbindin D-28k was unaltered. The combined lesions abolished calretinin containing terminals in the dentate gyrus inner molecular layer on the deafferentated side. This could be reproduced by single unilateral fimbria-fornix transections, suggesting that the axons of these calretinin positive terminals project to the hippocampus through the fimbria-fornix. The most likely origin of the calretinin positive terminals are neurons in the supramammillary hypothalamic nucleus. Our findings demonstrate that the extensive lesion-induced synaptic rearrangements in the adult hippocampus do not induce degeneration of hippocampal neurons expressing calretinin, calbindin D-28k, and parvalbumin, but do remove calretinin containing terminals which reach their targets in the hippocampus through the fimbria-fornix. © 1994 Wiley-Liss, Inc.