Synaptophysin immunoreactivity in Pick's disease: comparison with Alzheimer's disease and dementia with Lewy bodies

Frontotemporal lobe atrophy is a hallmark of Pick's disease (PiD), however, the underlying pathobiology of the neuronal losses is unknown. Synaptic losses have been described in Alzheimer's disease (AD) and correlate with the severity of dementia, however few studies of synaptic integrity have been done to determine whether synaptic loss also contributes to symptoms in non-AD dementias. To begin to assess synaptic integrity in other types of dementia, we examined the site of termination of the hippocampal perforant pathway, the major source of afferent tracts to the hippocampus. We determined immunoreactivity for the synaptic-terminal specific protein synaptophysin in the outer molecular layer of the hippocampal dentate gyrus (OMDG) in eight PiD, nine AD, nine dementia with Lewy bodies (DLB), and seven control cases. Quantitative data were obtained using an Image-Pro automated image analysis system. In AD and PiD, synaptophysin immunoreactivity was visibly reduced in the OMDG Densitometric analysis confirmed that there were statistically significant differences among groups in synaptophysin immunoreactivity when comparing the OMDG to the adjacent inner molecular layer of the hippocampal dentate gyrus (IMDG) (p = 0.002). These differences were present between PiD and both the control and DLB groups. The AD group also showed a reduction in synaptophysin immunoreactivity compared with DLB and control groups. In contrast, perforant pathway synaptic losses in DLB were minimal. Our data supports the hypothesis that focal synaptic losses occur in PiD and AD and may contribute to the cognitive deficits in both conditions.     

Memory deficits in amyotrophic lateral sclerosis patients with dementia and degeneration of the perforant pathway A clinicopathological study

Degeneration of the perforant pathway, not extensively surveyed so far in amyotrophic lateral sclerosis (ALS) with dementia, was found in eight out of twelve autopsied patients with clinically detectable dementia. Because the severity of degeneration of the entorhinal cortex and that of spongiosis of the outer half of the molecular layer of the dentate gyrus were correlated in these eight patients, it is suspected that the degeneration of the perforant pathway may explain these concomitant lesions. This was further corroborated by occasional involvement of the parahippocampal white matter and subiculum, other components of this pathway. Moreover, six of them manifested clinically detectable memory deficits and three of them exhibited amnesia or episodic memory impairments similar to Alzheimer's disease (AD). Abnormal intensity restricted the dentate gyrus on brain magnetic resonance imaging in a severe case looks like the degeneration of the molecular layer. This involvement of the perforant pathway in ALS patients and its correlation to memory deficits should be taken in account for evaluation of dementia.     

Hippocampal plasticity in Alzheimer's disease: changes in highly polysialylated NCAM immunoreactivity in the hippocampal formation

The highly polysialylated neural cell adhesion molecule (PSA-NCAM) is one of the most promising molecules that contributes to plasticity in the central nervous system. We evaluated PSA-NCAM immunoreactivity in the hippocampal formation of Alzheimer's disease (AD) patients. We found significant increases over control levels in the optical density ratios of PSA-NCAM immunoreactivity in the outer molecular layer/granule cell layer (ODoml/grl) and in the inner molecular layer/granule cell layer (ODiml/grl) in the dentate gyrus of AD patients. The optical density of the granule cell layer in the dentate gyrus did not differ significantly between AD patients and control subjects. However, the number of PSA-NCAM-immunopositive infragranule cells was higher in the AD group compared with control subjects. The major finding in the CA1, subiculum and entorhinal cortex of AD patients was the disorganization of PSA-NCAM-immunoreactive fibres. These results indicate that neuronal remodelling occurs, especially in the dentate gyrus of patients with AD.     

Reactive synaptogenesis assessed by synaptophysin immunoreactivity is associated with GAP-43 in the dentate gyrus of the adult rat.

Reactive synaptogenesis and terminal proliferation are known to occur in the dentate gyrus of the rat hippocampus following removal of specific afferents. In the present study we have examined the relation of synaptophysin immunoreactivity to the immunohistochemical staining pattern of GAP-43, a putative marker of neuritic growth. Within the molecular layer of the normal dentate gyrus, synaptophysin immunolabeling shows a trilaminar pattern, with the inner and outer layers having the greatest density of staining. Within the first week following denervation, there was a significant decrease in the staining density in the outer two-thirds of the molecular layer, followed by a moderate recovery at 14 days and 80% recovery by 30 days. This pattern is consistent with the time course of denervation and reinnervation in this system as determined previously by electron microscopy. By comparison, the staining pattern for GAP-43 in the intact dentate gyrus showed the middle and outer thirds of the molecular layer to be less densely stained than the inner third. Within a week following deafferentation, the outer two-thirds of the molecular layer displayed decreased levels of GAP-43 immunoreactivity, followed by recovery to normal levels by 30 days. By 84 days postlesion, patterns of both synaptophysin and GAP-43 immunostaining reflected an increased width of the inner molecular layer. Laser confocal imaging of double-immunolabeled sections at 14 days postlesion showed a 370% increase in the number of GAP-43-positive terminals in the molecular layer as compared to unoperated controls. Many of these GAP 43-positive terminals were synaptophysin negative. We conclude that GAP-43 may play a role in the synaptic remodeling that occurs in the denervated rat hippocampus and that quantitative morphometry of synaptophysin immunolabeling accurately reflects the fate of presynaptic terminals in this model of degeneration and reinnervation.     

Astrocyte hypertrophy in the Alzheimer's disease hippocampal formation

In Alzheimer's disease (AD), neuritic plaques are often found in the hippocampal dentate gyrus along the boundary between inner and outer molecular layers. The dentate outer molecular layer in AD also exhibits axon sprouting in response to an early loss of entorhinal neurons. The relationship between the laminar arrangement of plaques and the sprouting remains unclear. In experimental entorhinal lesions in the rat, the denervated dentate outer molecular layer demonstrates hypertrophic astrocytes which may provide trophic support for the sprouting response. It is not known whether an equivalent astrocyte response occurs in AD or whether this response is related to the distribution of plaques. We used immunohistochemical staining for glial fibrillary acidic protein (GFAP) to demonstrate reactive astrocytes in the hippocampus in AD patients and age-matched controls. These results were compared to the astrocyte response to an experimental entorhinal lesion in the rat. Quantitative and qualitative analyses demonstrated a significant increase in GFAP-positive hypertrophic astrocytes in the dentate outer molecular layer in AD compared to controls. These astrocytes were randomly distributed within the outer layer and did not parallel the distribution of neuritic plaques. In the entorhinal-lesioned rat, reactive hypertrophied astrocytes also showed a selective distribution within the denervated outer molecular layer. Our results further support the similarity of the hippocampal response in AD and experimental entorhinal lesion but do not explain the laminar distribution of neuritic plaques along the denervated zone.     

A quantitative analysis of synaptogenesis in the molecular layer of the dentate gyrus in the rhesus monkey.

Quantitative electron microscopy was used to study synapse formation in the molecular layer of the dentate gyrus in rhesus monkeys ranging in age from embryonic day 62 to adult. Four to eight radial probes, consisting of a series of overlapping electronmicrographs and extending across the full thickness of the molecular layer were made in each specimen. Synaptic density (normalized to volume of neuropil) increased significantly during the last half of gestation, reaching adult levels at the time of birth. However, new synapses were added during infancy, resulting in an apparent peak in density at between 4 and 5 months of age. This increase was followed by a decline in the synaptic density over the next 5 months, to levels comparable to that of the newborn. In addition to synaptic density, synapse type (symmetric, asymmetric), location (on dendritic shafts or spines), and laminar distribution in the developing molecular layer was determined. The decrease in synaptic density is unlikely to be due to 'dilution' caused by an increase in molecular layer volume since no increase in the volume of the dentate gyrus could be detected during this period. Our calculations suggest that a selective overproduction of asymmetrical, axo-spinous synapses occurs during infancy. Finally, synaptic density was significantly greater in the middle third of the molecular layer suggesting that synaptic exuberance may be related to entorhinal input.     

Naturally occurring degrading events in axon terminals of the dentate gyrus and stratum lucidum in the spiny mouse (Acomys cahirinus) during maturation, adulthood and aging.

The naturally occurring dynamics of presynaptic axon terminals were investigated in the dentate gyrus and stratum lucidum of the spiny mouse (Acomys cahirinus) during maturation, adulthood and aging. A sensitive and selective silver-staining technique was applied to analyze neuronal lysosome accumulation (LA), indicating synaptic degradation during development. LA was quantified by counting silver grains in the inner third and outer two thirds of the molecular layer, granular layer, and the infragranular layer of the dentate gyrus, and in the strata oriens, pyramidale, lucidum and radiatum of the medial and distal regio inferior on postnatal days 21, 28, 95, 730, and 1,460. In young and adult animals, LA was most abundant within the inner molecular layer. When animals grew older, LA densities obviously decreased in the inner molecular layer but increased in the outer molecular layer. Within the stratum lucidum only the distal regio inferior showed an extremely high LA density on postnatal day 21, dramatically decreasing thereafter and reaching adult low values during the first postnatal month. By electron microscopy in the inner molecular layer we found LA in large synaptic boutons and small terminals both with distinct synaptic contact zones. Degrading presynaptic profiles may further accumulate dense bodies, zones with completely disorganized cytoplasm, and lamellarly organized whorled membrane debris. In the distal regio inferior comparable phenomena were observed in typical mossy fiber boutons. Despite these degrading events, no electron-dense degenerating terminals were found. These results on naturally occurring nondegenerative synaptic degradation are discussed with current concepts of synaptic turnover and remodelling in the developing, adult and aging brain.     

Senile dementia of the Alzheimer type: is there a correlation between entorhinal cortex and dentate gyrus lesions?

Summary Senile plaques (SP) are one of the neuropathological hallmarks of senile dementia of the Alzheimer type (SDAT). In 14 patients affected with SDAT (over 74 years of age), thioflavine S, Tau and acetylcholinesterase (AChE) stainings demonstrated an increased density of SP in the outer two thirds of the dentate gyrus molecular layer. However, a wide range of SP density was observed among the cases. The molecular layer of the dentate gyrus is one of the termination site of the perforant pathway that originates in layers II and III of the entorhinal cortex. We have found that the number of AChE-, thioflavine, S-and Tau-positive SP that accumulate in the dentate gyrus is positively correlated with the density of thioflavine S-stained neurofibrillary tangles in layers II and III of the entorhinal cortex. In contrast, a similar correlation is not found when using Tau immunolabeling of the entorhinal tangles. These observations show an association between the accumulation of AChE-positive SP in the dentate molecular layer and the lesions of the perforant pathway. Furthermore, they suggest that the density of SP in the dentate gyrus correlates with the late stages of neurofibrillary tangles formation (thioflavine S positive), but not with the early stages (Tau positive).Supported in part by a grant from Bayer-Pharma, France     

Increased synapsin I immunoreactivity during long-term potentiation in rat hippocampus

The level of synapsin I, a synaptic vesicle protein and marker for synaptic activation, was studied 8 h after long-term potentiation (LTP) induction in rat hippocampus in vivo. Quantitative immunohistochemical analysis revealed that synapsin I increased significantly in the molecular layer of the dentate gyrus and the stratum lucidum of CA3, suggesting activation of the granule cells by synaptic input to the dentate gyrus, and persistent enhancement of glutamate release from the axon terminals of the dentate granule cells.     

Synapse alterations in the hippocampal-entorhinal formation in Alzheimer's disease with and without Lewy body disease

We quantified by microdensitometry the immunoreactivity (IR) to monoclonal antibodies (SP6, SP12, SP15 and SP18) against various synaptic proteins in the molecular layers of the dentate gyrus, CA4, CA3, CA1, subiculum and entorhinal cortex in Alzheimer's disease (AD), Lewy body variant of AD (LBV) and diffuse Lewy body disease (DLBD). A significant decrease in SP6 IR was observed in almost all regions in AD (28.4–70.1%, mean 41.3%), LBV (19.0–42.5%, mean 26.8%) and DLBD (19.9–31.7%, mean 27.1%) compared to controls. In addition, SP6 IR in the outer molecular layer of the dentate gyrus was strongly correlated with tangle count in the entorhinal cortex (r = −0.70, P < 0.002), suggesting loss of perforant pathway projection. Although the decrease in SP12 and SP15 IR was less pronounced, the mean values were decreased in dementia. Furthermore, SP12 and SP15 labeled a large number of neuritic plaques, and SP15 occasionally stained cortical LBs. The present findings indicate (i) that in the hippocampal-entorhinal formation, the decrease of synapse protein IR in AD is more severe than that in LBV and DLBD, (ii) that synaptic markers detect a subset of dystrophic neurites in the plaques and (iii) that synapse proteins are involved in the formation of cortical LBs.     

The process of reinnervation in the dentate gyrus of the adult rat: A quantitative electron microscopic analysis of terminal proliferation and reactive synaptogenesis

The present study defined the time course of terminal proliferation (the growth of presynaptic processes) and reactive Synaptogenesis in the dentate gyrus of the adult rat. Quantitative electron microscopic analyses were carried out in the dentate gyrus 2, 4, 6, 8, 10, 12, 14 days and 7 months after destruction of the ipsilateral entorhinal cortex and in the contralateral (control) dentate gyrus. At each survival interval, counts were made from photographic montages of (1) terminals (presynaptic processes with or without contacts with postsynaptic elements), (2) intact synapses, (3) degenerating synapses, (4) degeneration (degenerating presynaptic processes), and (5) multiple synapses (terminals making more than one synaptic contact). Terminal density was initially reduced to about 13% of control in the middle molecular layer at 2 and 4 days postlesion, and to about 26% of control in the outer. The density of terminals began to increase between 4 and 6 days postlesion, reaching a plateau by day 12. Synapse density was reduced to about 8% and 12% of control in the middle and outer molecular layer respectively. Synapse density increased about 5-fold between 8 and 12 days postlesion, but continued to increase in the period between 14 days and 7 months postlesion. At 2 days postlesion, the number of intact terminals that are lost corresponds to the number of degenerating presynaptic processes. This correspondence is not present at 4 days postlesion, however, suggesting a rapid removal of degenerating terminals. In contrast, even at 2 days post-lesion, the number of intact synapses that are lost does not correspond to the number of degenerating synapses. Between 2 and 10 days postlesion, the number of postsynaptic specializations is about 60% of control, but recovers slightly by 12-14 days postlesion. Qualitative and quantitative evidence suggested a collapse of spines into configurations that resembled shaft synapses. There appeared to be a deformation of degenerating presynaptic processes resulting in the appearance of multiple synapse configurations prior to reinnervation. The combined results suggest that terminal proliferation precedes reactive Synaptogenesis in the dentate gyrus by 2-4 days, that terminal proliferation is essentially complete by 12 days while reactive Synaptogenesis continues, and that multiple synapses arise at least in part as a result of a deformation of degenerating presynaptic processes rather than as a consequence of the induction of additional contacts on existing presynaptic terminals.     

Synaptic loss reflected by secretoneurin‐like immunoreactivity in the human hippocampus in Alzheimer's disease

Secretoneurin is a recently described peptide derived by endoproteolytic processing from secretogranin II, previously named chromogranin C. In this study, we have investigated the distribution of secretoneurin-like immunoreactivity in the human hippocampus in controls and in Alzheimer's disease patients, and compared the staining pattern to that of calretinin. Secretoneurin-like immunoreactivity is present throughout the hippocampal formation. At the border of the dentate molecular layer and the granule cell layer, a band of dense secretoneurin immunostaining appeared. In this part, as in the area of the CA2 sector, the high density of secretoneurin-immunoreactivity coincided with calretinin-like immunoreactivity. The mossy fibre system displayed a moderate density of secretoneurin-immunoreactivity. In the entorhinal cortex, a particularly high density of secretoneurin-immunoreactivity was observed. The density of secretoneurin-like immunoreactivity was significantly reduced in the innermost part of the molecular layer and in the outer molecular layer of the dentate gyrus in Alzheimer's disease. For calretinin-like immunoreactivity, a less pronounced decrease was found in the innermost part of the molecular layer. About 40–60% of neuritic plaques were secretoneurin-immunopositive. This study shows that secretoneurin is distinctly distributed in the human hippocampus and that significant changes of secretoneurin-like immunoreactivity occur in Alzheimer's disease, reflecting synaptic loss.     

Sprouting of central noradrenergic fibers in the dentate gyrus following combined lesions of its entorhinal and septal afferents

Virtually all of the afferents to the hippocampal formation undergo collateral sprouting after removal of adjacent afferent systems. However, the central noradrenergic (NA) afferents, which demonstrate a remarkable propensity for regeneration and sprouting in other regions of the brain, have not been found to sprout in the denervated hippocampal formation. The present study was designed to determine if the pattern of innervation by NA fibers in the dentate gyrus of adult rats can be altered by interruption of the other major afferents. The innervation pattern of NA fibers was examined in the dentate gyrus 4 weeks after removal of the ipsilateral and/or contralateral entorhinal afferents and/or transection of the fimbria-fornix and supracallosal stria. The noradrenergic identity of the fibers was indicated by immunoreactivity for dopamine beta hydroxylase (DBH) and peripheral sympathetic fibers were demonstrated by immunoreactivity for nerve growth factor receptor (NGFr), which did not stain cholinergic fibers in this application. In control brains, the noradrenergic innervation of the dentate molecular layer was light and uniform across the width of the layer. Transection of the perforant path (ipsilateral entorhinal afferents) or ventral hippocampal commissure (contralateral entorhinal afferents) resultd in a significant increase in innervation density in the outer half of the molecular layer, and the combination of these two lesions produced the greatest increase. In those brains with transection of the ipsilateral and contralateral entorhinal afferents, the denervated dentate gyrus had a nearly twofold increase in density of DBH-immunoreactive fibers within the outer half of the molecular layer. These fibers tended to course parallel to the pial surface rather that perpendicular as in control sections. Transection of the fimbria-fornix alone had no affect on the innervation pattern of DBH-ir fibers in the molecular layer. When the fimbria-fornix was transected in combination with both of the other lesions, an overall increase in innervation density occurred, but there was no further increase in the difference between the inner and outer halves of the molecular layer. No NGFr-immunoreactive fibers were observed in the molecular layer in any of the brains, indicating that the DBH-immunoreactive fibers in this region were not of peripheral origin. It is concluded that removal of the ipsi- and contralateral entorhinal afferents to the dentate gyrus results in the sprouting of central NA fibers in the outer half of the molecular layer. © 1994 Wiley-Liss, Inc.     

Corticotropin-releasing factor immunoreactive neurons persist throughout the brain in Alzheimer's disease

Corticotropin-releasing factor (CRF) immunoreactivity was examined in the hippocampal formation, cerebellum and hypothalamus of normal aged and Alzheimer's disease (AD) brains. Immunoreactive non-pyramidal neurons were located in the polymorphic layer of the dentate gyrus and CA fields. A plexus of CRF terminals was seen in the supragranular layer of the dentate gyrus. In AD, occasional senile plaques contained CRF-immunoreactive fiber terminals but the pattern of staining was otherwise unchanged aside from a suggestion of increased staining intensity. Similarly, the pattern of immunoreactive cerebellar climbing fibers and paraventricular hypothalamic neurons was preserved in AD brains aside from increased perikaryal staining intensity in the hypothalamus.     

Neuronal inputs to hippocampal formation in Alzheimer's disease and in parkinsonism-dementia complex on Guam

Summary This study concerns the expression of synaptophysin in the hippocampal formation of normal controls, of patients with Alzheimer's disease (AD) and of patients with parkinsonism-dementia complex on Guam (P-D complex). A monoclonal antibody was used to visualize synaptophysin, an integral component of presynaptic vesicle membranes. In the normal controls, a strong synaptophysin immunoreactivity was seen in the stratum pyramidale, stratum radiatum and stratum lacunosum-moleculare of the hippocampus proper, in the subiculum and in the molecular layer of the dentate gyrus. In the dentate gyrus molecular layer, the reaction product was distributed in a laminar fashion. By contrast, in AD and in P-D complex a significant decrease in immunoreactivity was observed in all hippocampal strata, and especially of the hippocampal subfield CA1 and the subiculum. In both diseases, synaptophysin expression was also diminished in the molecular layer of the dentate gyrus of all patients examined, with the inner portion exhibiting almost normal and the outer portion a strikingly reduced synaptophysin immunoreactivity.     

Reorganization of the septohippocampal cholinergic fiber system in experimental epilepsy

The septohippocampal cholinergic neurotransmission has long been implicated in seizures, but little is known about the structural features of this projection system in epileptic brain. We evaluated the effects of experimental epilepsy on the areal density of cholinergic terminals (fiber varicosities) in the dentate gyrus. For this purpose, we used two distinct post‐status epilepticus rat models, in which epilepsy was induced with injections of either kainic acid or pilocarpine. To visualize the cholinergic fibers, we used brain sections immunostained for the vesicular acetylcholine transporter. It was found that the density of cholinergic fiber varicosities was higher in epileptic rats versus control rats in the inner and outer zones of the dentate molecular layer, but it was reduced in the dentate hilus. We further evaluated the effects of kainate treatment on the total number, density, and soma volume of septal cholinergic cells, which were visualized in brain sections stained for either vesicular acetylcholine transporter or choline acetyltransferase (ChAT). Both the number of septal cells with cholinergic phenotype and their density were increased in epileptic rats when compared to control rats. The septal cells stained for vesicular acetylcholine transporter, but not for ChAT, have enlarged perikarya in epileptic rats. These results revealed previously unknown details of structural reorganization of the septohippocampal cholinergic system in experimental epilepsy, involving fiber sprouting into the dentate molecular layer and a parallel fiber retraction from the dentate hilus. We hypothesize that epilepsy‐related neuroplasticity of septohippocampal cholinergic neurons is capable of increasing neuronal excitability of the dentate gyrus.     

Chromogranins as markers of altered hippocampal circuitry in temporal lobe epilepsy

Chromogranins are polypeptides which are widely expressed in the central nervous system. They are stored in dense core vesicles of nerve terminals, from where they are released upon stimulation. Using immunocytochemistry, we investigated the distribution of chromogranin A, chromogranin B, secretoneurin, and, for comparison, dynorphin in hippocampal specimens removed at routine surgery from patients with drug-resistant mesial temporal lobe epilepsy and in autopsy tissues from nonneurologically deceased subjects. In post mortem controls (n = 21), immunoreactivity for all 4 peptides (most prominently for chromogranin B and dynorphin) was observed in the terminal field of mossy fibers. For chromogranins, staining was observed also in sectors CA1 to CA3 and in the subiculum. Chromogranin B immunoreactivity was found in the inner molecular layer of the dentate gyrus, the area of terminating associational-commissural fibers. Secretoneurin and dynorphin immunoreactivity labeled the outer molecular layer and the stratum lacunosum moleculare of sectors CA1 to CA3, where projections from the entorhinal cortex terminate. In specimens with Ammon's horn sclerosis (n = 25), staining for all 3 chromogranins and for dynorphin was reduced in the hilus of the dentate gyrus. Instead, intense staining was observed in the inner molecular layer, presumably delineating terminals of sprouted mossy fibers. Specimens obtained from temporal lobe epilepsy patients without Ammon's horn sclerosis (n = 4) lacked this pronounced rearrangement of mossy fibers. In the stratum lacunosum moleculare of sector CA1, secretoneurin and dynorphin immunoreactivity was reduced in sclerotic, but not in nonsclerotic, specimens, paralleling the partial loss of fibers arising from the entorhinal cortex. Instead, presumably sprouted secretoneurin-immunoreactive fibers were found in the outer dentate molecular layer in sclerotic specimens. These changes in staining patterns for chromogranins and dynorphin mark profound plastic and functional rearrangement of hippocampal circuitry in temporal lobe epilepsy.     

Mossy fiber synaptic reorganization in the epileptic human temporal lobe

The distribution of the mossy fiber synaptic terminals was examined using the Timm histochemical method in surgically excised hippocampus and dentate gyrus from patients who underwent lobectomy of the anterior part of the temporal lobe for refractory partial complex epilepsy. The dentate gyrus of epileptic patients demonstrated intense Timm granules and abundant mossy fiber synaptic terminals in the supragranular region and the inner molecular layer. In contrast, the dentate gyrus of presenescent nonepileptic primates demonstrated no Timm granules in the supragranular region. In nonepileptic senescent primates, occasional very sparse supragranular Timm granules were results are morphological evidence of mossy fiber synaptic reorganization in the temporal lobe of epileptic humans, and suggest the intriguing possibility that mossy fiber sprouting and synaptic reorganization induced by repeated partial complex seizures may play a role in human epilepsy.     

A novel population of calretinin-positive neurons comprises reelin-positive Cajal-Retzius cells in the hippocampal formation of the adult domestic pig

Calretinin-containing neurons in the hippocampal formation, including the subiculum, presubiculum, parasubiculum, and entorhinal cortex, were visualized with immunocytochemistry. Calretinin immunoreactivity was present exclusively in non-principal cells. The largest immunoreactive cell population was found in the outer half of the molecular layer of the dentate gyrus and in the stratum lacunosum-moleculare of Ammon's horn. A proportion of these cells were also immunoreactive for reelin, a Cajal-Retzius cell marker. Similar calretinin-positive cells were found in the molecular layer of the subicular complex and entorhinal cortex. In the parasubiculum, a few immunoreactive bipolar and multipolar cells could be observed in the superficial and deep pyramidal cell layers. In the entorhinal cortex, bipolar and multipolar calretinin-positive cells were frequent in layer II, and large numbers of multipolar cells in layer V were immunoreactive. Electron microscopic analysis showed that somata of calretinin-positive cells contained either round nuclei with smooth nuclear envelopes or nuclei with multiple deep infoldings. Immunoreactive dendrites were smooth varicose, and the apposing axon terminals formed both symmetric and asymmetric synapses. Zonula adherentia were observed between calretinin-positive dendrites. Calretinin-positive axon terminals formed two types of synapses. Axon terminals with asymmetric synapses were found close to the hippocampal fissure, whereas axon terminals forming symmetric synapses innervated spiny dendrites in both the molecular layer of the dentate gyrus and in stratum lacunosum-moleculare of Ammon's horn. Calretinin-positive axon terminals formed both symmetric and asymmetric synapses with calretinin-positive dendrites. In conclusion, calretinin-positive neurons form two major subpopulations in the adult domestic pig hippocampus: (1) a gamma-aminobutyric acid (GABA)ergic subpopulation of local circuit neurons that innervates distal dendrites of principal cells in both the dentate gyrus and in Ammon's horn; and (2) Cajal-Retzius type cells close to the hippocampal fissure, as well as in the molecular layer of the subicular complex and entorhinal cortex.     

Nerve growth factor receptor immunoreactivity in the rat septohippocampal pathway: a light and electron microscope investigation.

Nerve growth factor receptor immunoreactivity in the septohippocampal pathway of adult Fischer 344 rats was assessed at the light and electron microscope level. The medial septum possesses immunoreactive somata, dendrites, axons, and terminals. Immunostained somata are either bipolar or multipolar in appearance. Dendritic processes of immunoreactive septal neurons are categorized into two groups: proximal dendrites with smooth plasma membranes and distal dendrites with numerous swellings. Immunoreactive axons within the septum are long and slender and do not possess varicosities. At the electron microscope level, immunoreactivity is confined predominantly to the plasma membrane of cell bodies and dendrites of septal neurons, as well as to the plasma membrane of axons and terminals. Both immunoreactive and nonimmunoreactive terminals that contain clear, spherical vesicles are observed contacting immunoreactive dendrites and somata. Although accumulations of vesicles are evident within these terminals at sites of contact, distinct synaptic specializations are difficult to distinguish due to the localization of reaction product on the apposing plasma membranes. Axons possessing immunoreactivity are also observed in the fimbria-fornix pathway, a major source of afferent inputs to the hippocampus. Immunoreactive axons and terminals are topographically organized in the hippocampal dentate gyrus. The density of immunostained axons and terminals is highest immediately adjacent to the granular layer. In comparison, a moderate density of immunoreactive axons is found in the outer molecular layer and a weak density in the inner molecular, granular, and polymorphic layers. Immunoreactivity is found on the plasma membrane of small unmyelinated axons and terminals aggregated into clusters throughout the dentate gyrus. Definitive examples of axosomatic and axodendritic synapses possessing immunoreactivity presynaptically are not observed. Immunoreactive profiles within the medial septum and hippocampus also circumfuse a small number of intracerebral vessels. Ultrastructural examination reveals that immunoreactivity is present within a narrowed extension of the subarachnoid space and appears to be closely associated with the plasma membrane of leptomeningeal cell processes. The present study provides direct evidence for the cellular distribution of nerve growth factor receptor immunoreactivity in the medial septum and dentate gyrus in the adult rat and offers new insight into the ultrastructural localization of nerve growth factor receptor among septal cholinergic neurons and their efferent projections to the hippocampus.