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Stress stimulates the adrenal medulla to rapidly secrete catecholamines (CAs), and the adrenal cortex to release progesterone (PROG), which may locally regulate stress-induced CA release. We used bovine chromaffin cells to investigate the effects of PROG on CA secretion. PROG dose-dependently inhibited CA secretion induced by nicotinic acetylcholine receptor (nAChR) agonist 1,1-dimethyl-4-phenlypiperazinium iodide (DMPP) up to 77%. Pre-incubation with PROG up to 1 h increased this inhibition. 3alpha,5alpha-Tetrahydroprogesterone (3alpha,5alpha-THP) and dexamethasone were less potent inhibitors. Patch-clamp techniques revealed that PROG co-applied with DMPP inhibited peak DMPP-induced current up to 68% and with 3 min pre-incubation inhibited both peak and integrated current up to approximately 95%. Monitoring of FURA-2 showed that PROG similarly inhibited parallel changes in intracellular-free Ca(++) concentration. PROG also inhibited CA secretion elicited by elevated K(+) (38%), and, in single cells, suppressed Ca(++) current evoked by step depolarization, inhibiting amplitude by 15%, and reducing the time constant of current decay during depolarization by 57%. In contrast to the immediate inhibition of nicotinic current, inhibition of Ca(++) current became statistically significant only after 1 min exposure to PROG. PROG did not inhibit secretion stimulated by high Ca(++) perfusion of permeabilized cells. These data suggest that PROG inhibits CA secretion from chromaffin cells predominantly by rapidly inhibiting nAChRs, and by gradually enhancing the inactivation of voltage-dependent Ca(++) channels (VDCCs), but not by affecting secretory processes downstream of Ca(++) influx. This study supports a role for adrenocortical PROG in the regulation of CA secretion during stress.

As part of an ongoing investigation devoted to understanding the pathogenesis of senile plaques, we employed histochemical and immunocytochemical techniques to examine the distribution and cytologic features of acetylcholinesterase (AChE), choline acetyltransferase (ChAT), somatostatin (SOM), neurotensin (NT) and substance P (SP) containing fibers and neurons within the amygdala of: (1) patients with Alzheimer's disease (AD); (2) age-matched non-demented controls (NC); and (3) a group of non-demented cases, who upon postmortem neuropathologic examination exhibited sufficient numbers of senile plaques to be classified as AD. This latter group was referred to as high plaque non-demented (HPND). For every case, the distribution of immunolabeled fibers and neurons were determined for each transmitter throughout the various subnuclei of the amygdala. In addition, in the AD and HPND cases the topographic distribution of senile plaques was determined throughout the amygdala using thioflavine-S and Bielschowsky silver methods. In the amygdala, the distribution and density of senile plaques were not bound by conventional cytoarchitectural groupings but rather were most dense in the ventromedial regions of the amygdala with decreasing density in dorsal and lateral directions. Importantly, the density and distribution of senile plaques failed to correlate with the normal topography and/or density of the various peptidergic or cholinergic fibers within the amygdala. The finding that plaques do not correlate with the topographic distribution of any specific transmitter system suggests that plaques likely do not arise from the degeneration of a single neurotransmitter system (i.e., the cholinergic system). However, the finding that in AD a transmitter is most markedly depleted in regions of greatest plaque density, suggests certain constituents of the plaque (e.g. beta-amyloid) may be contributing to the degeneration of local fibers. The extent to which a transmitter was depleted in AD patients varied considerably among those four investigated with the cholinergic and NT systems displaying the most dramatic reductions, followed by SP and SOM. Despite these differential reductions in fiber density, all four neurotransmitters were found localized within dystrophic neurites and in most instances these dystrophic neurites were associated with thioflavine-positive senile plaques. In contrast to the AD cases, the HPND cases were characterized by no significant reductions in immunolabeled fibers, although immunostained dystrophic neurites were very prevalent in the HPND cases. These data suggest that dystrophic neurites occur very early in the disease process and likely precede the actual loss of fibers when or if it occurs.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of axonal transport by vincristine applied to hypoglossal nerves 7 days after crush injury turns off the usual injured-induced expression of low affinity nerve growth factor receptor (p75NGFr). Vincristine applied proximal but not distal to the crush prevents p75NGFr induction. These results indicate that a continuing signal is axonally transported from the crush site that induces and maintains p75NGFr expression by injured motor neurons.

Following fimbria/fornix transection, cells in the medial septum appear to undergo retrograde degeneration as shown by Nissl and acetylcholine esterase (AChE) staining. Recent studies immunocytochemical techniques have also demonstrated loss of choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr) labeling of neurons in this region. Whether the apparent loss of ChAT- and NGFr-positive neurons is the result of the actual death of these neurons, or is instead a loss of ChAT enzyme or NGFr expression below levels detectable by immunocytochemical methods, remains an unresolved issue. In order to address this question, rhodamine-labeled fluorescent latex microspheres were injected into the hippocampus where they were retrogradely transported to the cell bodies of the medial septum. Five days later these animals received either unilateral or bilateral fimbria/fornix lesions and were allowed to survive an additional 4 weeks. Compared to unlesioned control animals, unilaterally lesioned animals showed a 91% loss of fluorescently labeled cells and bilaterally lesioned animals showed a 93% loss. The inability to detect the fluorescent microspheres in the medial septum suggests that the majority of medial septal cells die after fimbria/fornix transection. ChAT and NGFr immunohistochemical staining were also performed. Cells stained for ChAT were reduced in number by 92% in animals with unilateral lesions and by 75% in animals with bilateral lesions, while NGFr-stained cells were reduced in number by 75% in animals with unilateral lesions and by 68% in animals with bilateral lesions. Of note, several cells were double-labeled for both fluorescent microspheres and either ChAT or NGFr after both unilateral or bilateral lesions, suggesting that not all hippocampally projecting cholinergic cells of the medial septum die after fimbria/fornix transection. Issues concerning cell dysfunction and death are discussed.

This work investigates whether nitric oxide production and lipid peroxidation contribute to the pathophysiology of ischemia and whether glycine and a novel Russian compound, Semax are neuroprotective via a mechanism involving the regulation nitric oxide (NO) and lipid peroxidation. In brief, nitric oxide and indices of lipid peroxidation were elevated following global ischemia. While glycine proved ineffective in reducing NO levels or ameliorating the neurological deficits following global ischemia, Semax proved to be highly effective in abating the rise in nitric oxide and restoring neurologic functioning.

Immunocytochemical techniques were employed to examine the changes in immunolabeling of the α-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the dentate gyrus 1, 3, 7, 14, 30, and 90 days after a unilateral perforant pathway lesion in the rat brain. Completeness of the lesion was confirmed following examination of Nissl-stained tissue sections at all times post-lesion and acetylcholinesterase (AChE)-stained sections 14, 30 and 90 days post-lesion, the latter providing evidence of compensatory sprouting of cholinergic fibers in the outer molecular layer of the dentate gyrus. Compared to the non-lesioned hippocampus there was no difference in the staining pattern of AMPA receptor subunits in the dentate gyrus of the deafferented hippocampus 1, 3, 7 and 14 days following lesioning of the perforant pathway. In contrast, 30 and 90 days post-lesion, GluR1 immunolabeling was increased in the outer molecular layer of the dentate gyrus (i.e., deafferented zone) ipsilateral to lesion. Likewise, GluR2/3 immunolabeling was increased within the same region although the intensity of the response was less than that which was observed for GluR1. These data suggest that the loss of the perforant pathway fibers results in a compensatory increase in GluR1 and to a lesser extent GluR2/3 immunolabeling of the outer molecular layer at 30 and 90 days post-lesion and further suggest that AMPA receptor subunits play a role in perforant pathway signal transduction.

Using biochemical techniques we determined concentrations of the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3 in the entorhinal cortex of patients with Alzheimer's disease pathology and age-matched controls. Tangle density was also determined in anatomically matched samples and correlated with GluR1 and GluR2/3 receptor concentration. In Alzheimer's disease brain, Western blot analysis revealed average reductions of 43% and 38% for GluR1 and GluR2/3, respectively. Based on previous immunohistochemical studies, we infer that the majority of protein reduction was due to decreases in GluR1 and GluR2/3 immunolabeled elements in the more superficial layers of the entorhinal cortex (layers II and III). These layers of the entorhinal cortex contained numerous neurofibrillary tangles in Alzheimer's disease, but neither GluR1 nor GluR2/3 protein concentration correlated significantly with tangle density. We hypothesize that the decrease in specific glutamate receptor subunits, particularly GluR2/3, may contribute to the vulnerability of neurons in the entorhinal cortex via mechanisms involving calcium conductance through AMPA-selective channels.

The present work employed immunocytochemical techniques and examined the distribution and cytological features of the AMPA receptor subunits, GluR2/3 and GluR1 within the entorhinal cortex of non-demented elderly (NC), patients with neuropathological and clinical verification of Alzheimer's disease (AD) and patients without a clinical history of dementia yet exhibiting sufficient quantities of senile plaques to meet neuropathological criteria of Alzheimer's disease (HPND). In NC cases, GluR2/3-immunolabeled neurons were abundantly distributed throughout layers II, III, V and VI of the entorhinal cortex. In contrast, GluR1-positive cells were comparatively sparse in number and largely restricted to layers V and VI. In AD, GluR2/3- and GluR1-labeled neurons were markedly reduced. Similarly, adjacent Nissl-stained tissue sections revealed substantial cell loss in the entorhinal cortex thus providing a reasonable explanation for the loss of these receptor subunits. Importantly, a dramatic loss of GluR2/3- and GluR1-immunolabeled neurons is also observed in the HPND cases, although examination of Nissl-stained tissue sections reveals little if any evidence of cell loss. The latter data suggest that a 'down-regulation' of these receptor subunits occurs prior to the actual loss of these cells. Furthermore, we hypothesize that the decrease of specific AMPA receptor subunits may influence neuronal vulnerability via a mechanism involving increased intracellular calcium and the destabilization of intracellular calcium homeostasis.

Within the amygdala of elderly subjects and patients with Alzheimer's disease (AD), we recently found evidence suggesting amyloid beta-protein (A beta P) deposition occurs before the appearance of dystrophic neurites. Moreover, these data suggested dystrophic neurites initially lack evidence of cytoskeletal pathology although with time and further maturation, the dystrophic neurites display an altered cytoskeleton as evidenced by their immunoreactivity to Alz-50 and paired-helical filaments (PHF). These findings are of particular relevance to our understanding of the sequence of pathologic events in AD and thus it has become important to determine whether these events are unique to the amygdala or are representative of a more general pattern which can be found throughout the brain. Using a battery of antibodies to markers that are characteristic of AD pathology (i.e., A beta P, PHF, and Alz-50), three peptidergic neurotransmitters (neurotensin, somatostatin, and substance P), and one neurotransmitter biosynthetic enzyme (choline acetyltransferase), we examined the entorhinal cortex (EC) of three groups of subjects (AD, normal elderly, and a group of nondemented elderly with numerous senile plaques). The EC was studied, in part, because it is well recognized as a brain region displaying severe and, most importantly, early pathologic changes. Like the amygdala, we found evidence that amyloid beta-protein immunoreactive (A beta P-IR) and thioflavine-S-positive senile plaques occur within the EC prior to the appearance of transmitter-, Alz-50-, or PHF-immunoreactive dystrophic neurites. We also observed transmitter-immunoreactive dystrophic neurites in the absence of Alz-50 or PHF-immunolabeled dystrophic neurites and transmitter- and Alz-50-IR dystrophic neurites in the absence of those containing PHF. Collectively, these findings were similar to those seen within the amygdala and thus reinforced the concept that A beta P deposition is the primary event in plaque pathology, and this deposition is subsequently followed by the appearance of dystrophic neurites which retain their transmitter phenotype yet lack an altered cytoskeleton. With time, these dystrophic neurites develop cytoskeletal alterations and become immunoreactive to Alz-50 and PHF.

Swollen, bulbous-shaped (dystrophic) neurites are a common pathologic feature of Alzheimer's disease (AD) and represent one of the most abundant neuritic abnormalities within the brains of patients with this disease. In the present study, we sought to determine whether the dystrophic neurites which are observed in association with senile plaques are unique to AD or whether they are characteristic of a more generalized process of neuritic and/or neuronal degeneration which can be observed in other neurodegenerative diseases. To accomplish this, we examined post-mortem brain material from patients with AD, Parkinson's disease (PD), Parkinson's disease with associated AD, Parkinson's disease with dementia yet without AD pathology, Huntington's disease (HD), Pick's disease and normal age-matched controls (NC). Using a battery of antibodies to amyloid beta-protein (A beta P), paired-helical filaments (PHF), tyrosine hydroxylase, substance P, neurotensin, and somatostatin we found that immunolabeled dystrophic neurites of the type characteristically observed in AD, were seen only in cases and in brain regions where A beta P deposition was present. More specifically, brain areas known to display severe afferent and/or local degenerative changes such as the caudate and putamen in all three PD groups, the caudate in the HD cases, and the temporal cortex in the HD and Pick's cases were conspicuously free of these swollen neurites unless A beta P deposition was also present.(ABSTRACT TRUNCATED AT 250 WORDS)

Enkephalin and substance P-containing inputs to cholinergic perikarya were examined in the rat neostriatum using an ultrastructural immunocytochemical double-labeling protocol. Sections of rat neostriatum were double-labeled for either choline acetyltransferase (ChAT) and substance P or ChAT and enkephalin using silver intensified colloidal gold and peroxidase as labels. Regions containing both ChAT-positive neurons and peroxidase reaction product were identified in the light microscope prior to sectioning for electron microscopy. Substance P-containing terminals which contained round synaptic vesicles and made symmetrical synaptic contacts were commonly observed in the neostriatum. Substance P synapses onto ChAT-positive perikarya and dendrites were frequently observed: up to 5 synaptic contacts were observed onto a ChAT-positive dendrite. Enkephalin labeling was also seen in a population of axon terminals containing round synaptic vesicles and exhibiting symmetrical synaptic specializations. In contrast to substance P-containing terminals, relatively few synaptic contacts were observed onto ChAT-positive labeled perikarya and dendrites although enkephalin-labeled terminals were seen in frequent contact with perikarya and dendrites of unlabeled spiny neurons. Since enkephalin and substance P are contained within different populations of striatal spiny neurons, the results of the present study suggest that these two types of neurons differ in their intrinsic striatal connections.

Nerve transection causes decreased choline acetyltransferase (ChAT) expression and apperance of nerve growth factor receptor (NGFr) in hypoglossal motor neurons. Topical application of vincristine to the hypoglossal nerve blocks axonal transport of WGA for more than one week and causes loss of ChAT but no appearance of NGFr. These results indicate that loss of ChAT is related to interruption of axonal transport, but another signal induces de novo expression of NGFr.

The density of neurotensin immunoreactivity (NT-IR) was dramatically decreased in 6 of 12 amygdaloid nuclear subregions in patients with Alzheimer's disease (AD) compared to age-matched normals. Diminution of NT-IR was most pronounced in amygdaloid regions containing the greatest number of senile plaques. This contrasts to our previous findings of little, if any, loss of substance P or somatostatin immunoreactivity within these same regions. The present findings corroborate biochemical reports of a decrease in NT-IR in the AD amygdala and suggest that this peptide may be selectively affected relative to other neuropeptides.

Iontophoresis of the N-methyl-D-aspartic acid (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (2-APV) was found to suppress spontaneous bursting activity of layer V cortical neurones during stage 3/4 sleep in unrestrained, normally behaving rats. Iontophoresis of NMDA, on the other hand, increased cortical burst durations and increased the number of spikes per burst. 2-APV was found not to alter cells' responses to tactile stimulation or the generation of neuronal spindling activity during stage 2 sleep. These results provide the first evidence that NMDA receptors subserve a specific function in the neocortex of the behaving animal, by gene-rating burst activity in cortical neurones during stage 3/4 of the natural sleep state. The activation of NMDA/2-APV-sensitive cortical receptors by afferents from the anterior intralaminar nuclei in the generation of bursts by cortical cells during stage 3/4 sleep is discussed.

The ultrastructure of enkephalin-containing neurons and their capacity to take-up [3H]serotonin were examined in the area postrema. Untreated adult rats and rats with intraventricular infusions of 10(-4) M tritiated serotonin, 5-hydroxytryptamine [( 3H]5-HT) were perfused with 4% paraformaldehyde and 0.2-0.5% glutaraldehyde. Coronal Vibratome sections through the area postrema from both groups of animals were immunocytochemically labeled with an antiserum to leucine Leu5-enkephalin. The sections from the animals infused with the isotope subsequently were processed for autoradiography. Enkephalin-like immunoreactivity (ELI) was detected in perikarya, dendrites, axons and axon terminals most frequently located along the ventricular and ventrolateral portions of the area postrema. The labeled perikarya were few in number and were characterized by a thin rim of cytoplasm containing peroxidase immunoreactivity. Dendrites and terminals containing ELI formed synapses primarily with unlabeled axon terminals and dendrites, respectively. However, terminals containing ELI also formed synaptic junctions with other unlabeled axon terminals. Appositions between enkephalin-containing processes and modified glia were occasionally seen near the ventricular surface. In sections processed for both immunocytochemistry and autoradiography, approximately 5% of the terminals containing ELI showed uptake of [3H]5-HT. We conclude that neurons containing ELI are primarily, but not exclusively, associated with other intrinsic neurons or afferents in the rat area postrema and that some of the enkephalin-labeled terminals have the capacity to take-up serotonin. Specificity of uptake of [3H]5-HT in neurons containing endogenous serotonin and factors which may contribute to the low probability of detecting both peroxidase and autoradiographic markers in single sections are discussed.

Electron microscopic immunocytochemistry was employed to examine the ultrastructure of processes containing substance P-like immunoreactivity (SP-LI) in the area postrema of rat. SP-LI was present within axons and axon terminals which formed synapses with dendrites of intrinsic neurons in the rat area postrema. Specialized contacts between labeled processes and perivascular or ependymal glia cells were not observed. These data suggested that substance P may act as a neurotransmitter involved primarily with neuronal (axodendritic) interactions within the area postrema.

Alzheimer's disease or senile dementia of the Alzheimer type (SDAT) is a progressive neurodegenerative disease that is characterized pathologically by two types of microscopic lesions in the neocortex: the neurofibrillary tangle and neuritic plaque. The concentration of neuritic plaques is correlated with significant reductions in the level of specific neurotransmitter and neuropeptide systems in autopsied brains of patients with SDAT, including decreased amounts of the tetradecapeptide, somatostatin. The clinical effects of reduced cortical somatostatin activity in patients with SDAT is unclear, nor is it known whether somatostatinergic neurons participate in either lesion. In the present study we employed light microscopic immunocytochemistry to determine whether somatostatin-containing neurons participate in the formation of neuritic plaques. Examination of selected cortical regions from autopsied brains revealed 20-50% of all neuritic plaques contained somatostatin-positive profiles indicating that processes of somatostatinergic neurons are associated with neuritic plaque formation.