The role of calcium signaling in early axonal and dendritic morphogenesis of rat cerebral cortex neurons under non-stimulated growth conditions.

The effects of depolarizing stimuli on neurite outgrowth have been shown to depend on an influx of extracellular calcium. However, the role of calcium under non-stimulated growth conditions is less well established. Here we investigated the contribution of calcium signaling to early neuronal morphogenesis of rat cerebral cortex neurons at three levels by blocking L-type voltage sensitive calcium channels, by depleting intracellular calcium or by blocking myosin light chain kinase. Detailed quantitative morphological analysis of neurons treated for 1 day revealed that depletion of intracellular calcium strongly decreased the density of filopodia, arrested axonal outgrowth and strongly decreased dendritic branching. Preventing calcium influx through L-type voltage sensitive calcium channels and blocking of myosin light chain kinase activity selectively decreased dendritic branching. Our observations support an essential role for basal intracellular calcium levels in axonal elongation. Furthermore, under non-stimulated conditions calcium entry through L-type voltage sensitive calcium channels and myosin light chain kinase play an important role in dendritic branching.

Metabolic activity of the human ventromedial nucleus neurons in relation to sex and ageing.

The ventromedial nucleus (VMN) in animals is involved in a number of sexually dimorphic behaviors, including reproduction, and is a well-documented target for sex steroids. In rats and in lizards, it is also characterized by the presence of structural sexual dimorphisms. In the present study, we determined whether the metabolic activity of human ventromedial nucleus neurons was sex- or age-related. The size of the immunocytochemically defined Golgi apparatus (GA) and cell profiles were determined as measures for neuronal metabolic activity in 12 male and 16 female control brains sub-divided into four groups with the dividing line being the age of 50. It appeared that the size of the GA relative to cell size was 34% larger in young women (<50 years old) than in young men and was 25% larger in elderly men (> or = 50 years old) than in young men. In addition, the GA/cell size ratio correlated significantly with age in men and not in women. Our data suggest that androgens play an inhibitory role with respect to the metabolic activity of the human VMN neurons.

Decreased hippocampal metabolic activity in Alzheimer patients is not reflected in the immunoreactivity of cytochrome oxidase subunits.

In the present study we have compared histochemically determined cytochrome oxidase activity with the levels of immunocytochemically stained cytochrome oxidase subunits (CO II and CO IV) and ATP synthase in the human hippocampus in relation with Alzheimer's disease. Cytochrome oxidase activity was significantly reduced in all hippocampal areas of Alzheimer patients. The protein levels of subunits II and IV were not different between control subjects and Alzheimer patients. Additionally, it was observed that the active cytochrome oxidase is evenly distributed over both cell bodies and neuropil, while a relatively large pool of inactive enzyme or precursors is limited to the neuronal somata. Further, in Alzheimer patients the CO IV immunoreactivity decreased with age, whereas in control subjects it increased with age. Our results suggest that the assembly of cytochrome oxidase or the processing of its subunits may be impaired.

Male-to-female transsexuals have female neuron numbers in a limbic nucleus.

Transsexuals experience themselves as being of the opposite sex, despite having the biological characteristics of one sex. A crucial question resulting from a previous brain study in male-to-female transsexuals was whether the reported difference according to gender identity in the central part of the bed nucleus of the stria terminalis (BSTc) was based on a neuronal difference in the BSTc itself or just a reflection of a difference in vasoactive intestinal polypeptide innervation from the amygdala, which was used as a marker. Therefore, we determined in 42 subjects the number of somatostatin-expressing neurons in the BSTc in relation to sex, sexual orientation, gender identity, and past or present hormonal status. Regardless of sexual orientation, men had almost twice as many somatostatin neurons as women (P < 0.006). The number of neurons in the BSTc of male-to-female transsexuals was similar to that of the females (P = 0.83). In contrast, the neuron number of a female-to-male transsexual was found to be in the male range. Hormone treatment or sex hormone level variations in adulthood did not seem to have influenced BSTc neuron numbers. The present findings of somatostatin neuronal sex differences in the BSTc and its sex reversal in the transsexual brain clearly support the paradigm that in transsexuals sexual differentiation of the brain and genitals may go into opposite directions and point to a neurobiological basis of gender identity disorder.

Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy.

The main goal of this study was to develop a better light microscopic procedure for quantitative study of the cellular co-localization of neuropeptides in adult human brain tissue. To reach this goal, we opted for a method (proved to be optimal on rat brain) in which sections were double immunolabeled with two different fluorophore-conjugated secondary antibodies and analyzed with a confocal laser scanning fluorescence microscope. One of our main problems faced was a strong autofluorescence of the sections, mainly caused by lipofuscin granules normally present in adult human brain tissue, which made any analysis of specific fluorescence impossible. This problem could be solved by staining the sections after immunolabeling with the dye Sudan Black B, which completely blocked this autofluorescence. The complete optimized procedure that we eventually developed can be summarized as follows. After a relatively short fixation time (6-14 days) in 4% freshly depolymerized paraformaldehyde, the resected brain tissue can best be stored in a 30% sucrose solution supplemented with 0.05% NaN3 at 4C. Stored under these conditions, cryosections from the tissue still reveal good histology and allow successful immunocytochemical staining after a period of 6 months. Double immunolabeling is done by incubating cryo- or paraffin sections in a mixture of two primary antibodies directed against the targeted antigens, followed by incubation with two different fluorophore-conjugated secondary antibodies. Amplification with a biotinylated secondary antibody followed by fluorophore-conjugated streptavidin is possible. Finally, the sections are stained with Sudan Black B, mounted in plain 80% Tris-buffered glycerol, and studied by confocal laser scanning fluorescence microscopy. Sections processed in this way are well suited for qualitative and quantitative analyses of co-localized neuropeptides in human brain tissue.

Lack of association between depression and loss of neurons in the locus coeruleus in Alzheimer disease.

BACKGROUND: Depression, one of the most frequent psychiatric disturbances in Alzheimer disease (AD), is proposed to have its neurobiological basis in neuron loss in the noradrenergic locus coeruleus, although this is not the case in idiopathic depression. METHODS: We performed image analyzer-assisted morphometry of the locus coeruleus in 6 depressed, 6 transiently depressed, and 6 nondepressed patients with AD and in 8 control subjects, emphasizing longitudinal psychiatric evaluations and matching for the clinical and neuropathological severity of dementia. RESULTS: The mean (+/-SD) number of pigmented neurons in the locus coeruleus in controls (11 607+/-946) was higher than in patients with AD, regardless of being depressed (5165+/-928; P=.001), transiently depressed (5647+/-1163; P=.003), or nondepressed (3717+/-661; P=.001). No significant difference was found in the number of pigmented neurons between patients with AD who were depressed, transiently depressed, and nondepressed. Patients who had depression at the onset of AD had a higher pigmented neuron number than other patients with AD. CONCLUSIONS: We confirmed the loss of pigmented neurons in the locus coeruleus of patients with AD; however, no supplementary loss of pigmented neurons in the locus coeruleus was found in patients with depression and AD. This finding resembles the situation in idiopathic depression, but is in contrast with earlier studies on depression in AD.

Depolarization stimulates lamellipodia formation and axonal but not dendritic branching in cultured rat cerebral cortex neurons.

Electric activity is known to have profound effects on growth cone morphology and neurite outgrowth, but the nature of the response varies strongly between neurons derived from different species or brain areas. To establish the role of electric activity in neurite outgrowth and neuronal morphogenesis of rat cerebral cortex neurons, cultured neurons were depolarized for up to 72 h and quantitatively analyzed for changes in axonal and dendritic morphology. Depolarization with 25 mM potassium chloride induced a rapid increase in lamellipodia in almost all growth cones and along both axons and dendrites. Lamellipodia formation was dependent on an influx of extracellular calcium through L-type voltage-sensitive calcium channels. Prolonged depolarization for 24 h induced an increase in total axonal length, mainly due to an increase in branching. After three days of depolarization axonal outgrowth was largely the same as in control neurons, suggesting accommodation of the growth cones to chronic depolarization. Dendrites showed very little change during the first three days in culture, and dendritic length or branching were not affected by depolarization. Thus, in early cerebral cortex neurons depolarization specifically stimulates axonal outgrowth through increased branching. This increase in branching may be a consequence of the earlier increase in lamellipodia formation. In contrast, early dendrites seem to be unable to translate the increase in lamellipodia into changes in outgrowth or branching. This difference between axons and dendrites could be due to differences in the stabilization of the tubulin cytoskeleton.

Activity of Hippocampal CA1 Neurons in Alzheimer's Disease is not Affected by the Presence of Adjacent Neuritic Plaques.

Alzheimer's disease (AD) is neuropathologically characterized by neuritic plaques (NPs) and neurofibrillary tangles and functionally by a decreased metabolic rate of neurons. Our previous studies showed that in brain areas which are extensively affected by plaques and tangles, i.e. the CA1 area of the hippocampus and the hypothalamic tuberomamillary nucleus, neuronal protein synthetic ability is significantly lower in AD patients than in controls. However, the presence of tangles as shown by Bodian staining appeared not to be directly related to decreased protein synthetic ability of neurons. In order to study to what extent the metabolic function of neurons might be affected by the other neuropathological hallmark of AD, i.e. NPs, which are presumed to contain neurotoxic compounds, we studied eight severely demented AD patients matched for the ApoE genotype (ApoE 3/3). Using an image analysis system, the size of the neuronal Golgi apparatus (GA) and of the cell profile area was measured as a parameter for protein synthetic activity in the CA1 area of these patients. NPs were stained by Bodian, and subsequently the distance of each neuron with an immunostained GA to the nearest NP was measured. Our results showed that neither NP density nor the distance between NPs and neurons correlated with the protein synthetic ability of neurons as judged by the size of the GA. Based on these results we suggest that in AD the presence of NPs and decreased neuronal protein synthetic ability are basically two independent phenomena

Quantitative estimation of estrogen and androgen receptor-immunoreactive cells in the forebrain of neonatally estrogen-deprived male rats.

Using quantitative immunocytochemical procedures, the total number of estrogen and androgen receptors was estimated in a large number of hypothalamic and limbic nuclei of male rats, in which brain estrogen formation was inhibited neonatally by treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione. The highest densities of estrogen receptor immunoreactivity were observed in the periventricular preoptic area and the medial preoptic area. Neonatally estrogen-deprived males showed a higher estrogen receptor immunoreactivity than control males in the periventricular preoptic area and the ventrolateral portion of the ventromedial nucleus of the hypothalamus, i.e. those brain areas in which sex differences have been reported, with female rats showing a greater estrogen binding capacity than male rats. The highest densities of androgen receptor immunoreactivity were found in the septohypothalamic nucleus, the medial preoptic area, the posterior division of the bed nucleus of the stria terminalis and the posterodorsal division of the medial amygdaloid nucleus. No significant differences in distribution or total numbers of androgen receptors were found between neonatally estrogen-deprived males and control males. These findings suggest that neonatal estrogens, derived from the neural aromatization of testosterone, are involved in the sexual differentiation of the estrogen receptor system in the periventricular preoptic area and the ventromedial hypothalamus. The role of neonatal estrogens in the development of the forebrain androgen receptor system is less clear.

Unchanged amounts of vasopressin mRNA in the supraoptic and paraventricular nucleus during aging and in Alzheimer's disease.

The paraventricular (PVN) and supraoptic nucleus (SON) demonstrate a striking stability with respect to cell numbers during aging and Alzheimer's disease (AD). Vasopressin (AVP) neurons even become activated during aging as judged from several parameters for neuronal activity, such as increased AVP plasma levels, enlarged nucleolar as well as cell size and an increased size of the Golgi apparatus in AVP-neurons. The activation possibly occurs as compensation for an age-related loss of AVP-receptors in the kidney. As a specific marker for AVP synthesis, we used quantitative in situ hybridization and estimated total amounts of AVP-mRNA in the entire SON and PVN of 14 control subjects and 14 AD patients that were matched for age, fixation time, postmortem delay and storage time of the tissue in paraffin. Following quantification, no differences were observed in total amounts of AVP-mRNA in the SON or PVN between young and old controls or between young and old AD patients, nor between the entire group of controls and AD patients. A significant negative correlation was found between the volume of the AVP-mRNA signal in the AD SON and age while the total amount of mRNA remained the same. This suggests a redistribution of cells or cell compartments in aging. A significant positive relation in both SON and PVN of AD patients was found between storage time of the paraffin-embedded tissue and the total amount of AVP-mRNA. A significant positive relation was present in the PVN, but not SON between pH of the cerebrospinal fluid, which is a marker for agonal state and the total amount of AVP mRNA. The present unchanged AVP-mRNA levels in SON and PVN confirm earlier observations on the stability of cell numbers in these nuclei in aging and AD. Although on the basis of other parameters, AVP-mRNA upregulation was expected, gradual, chronic stimulation over prolonged periods of time may, possibly, induce alternative mechanisms of regulation such as changes in translatability or in mRNA stability.

Modulatory role of catecholamines in the transsynaptic expression of c-fos in the rat medial prefrontal cortex induced by disinhibition of the mediodorsal thalamus: a study employing microdialysis and immunohistochemistry.

We studied the interaction of catecholaminergic and thalamic afferents of the medial prefrontal cortex (PFC) by analyzing the effects of catecholamine depletion on thalamus-induced c-fos expression in the PFC of freely moving rats. Thalamic projections to the PFC were pharmacologically activated by perfusing the GABA-A receptor antagonist bicuculline (0.03 mM or 0.1 mM) through a dialysis probe implanted into the mediodorsal thalamic nucleus. Bicuculline perfusion induced Fos-like immunoreactivity in the thalamic projection areas, including the PFC, and in the thalamic nuclei surrounding the dialysis probe. 6-Hydroxydopamine lesions of the ventral tegmental area causing a 70-80% depletion of catecholamines in the PFC did not influence the increase in the number of Fos-like immunoreactive nuclei in the prefrontal cortex in response to thalamic stimulation. However, densitometric image analysis revealed that the intensity of Fos-like immunoreactivity in the PFC of lesioned rats perfused with 0.1 mM bicuculline was higher than in correspondingly treated controls. The behavioral activity to bicuculline perfusion, an increase of non-ambulatory activity (0.03 mM) followed by locomotion and rearing (0.1 mM), was not changed in 6-hydroxydopamine-lesioned rats. It is suggested that the thalamically induced c-fos response is directly mediated by excitatory, presumably glutamatergic, transmission and not indirectly by an activation of catecholaminergic afferents of the PFC. The increase in the intensity of Fos-like immunostaining in strongly stimulated, catecholamine-depleted rats suggests that catecholamines modulate the degree to which thalamic activity can activate the PFC of awake animals.

Evidence from confocal fluorescence microscopy for a dense, reciprocal innervation between AVP-, somatostatin-, VIP/PHI-, GRP-, and VIP/PHI/GRP-immunoreactive neurons in the rat suprachiasmatic nucleus.

The rat suprachiasmatic nucleus (SCN) consists of several classes of neurons which can be identified by their transmitter content. Knowledge of putative interaction between these different cell types is essential in order to understand the possibilities of information processing within the SCN. The aim of the present study was therefore to obtain more information about the mutual innervation between the main cell classes in the rat SCN, viz. those containing the neuropeptides arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP) and somatostatin respectively. For this purpose, vibratome sections were double-immunolabelled for seven different peptide combinations and subsequently analysed by high-resolution confocal laser scanning fluorescence microscopy. Attention was focused on axosomatic appositions, the occurrence and frequency of which were quantitatively estimated. Our analysis of double-immunolabelled sections demonstrated that some of the VIP- and some of the GRP-immunoreactive nerve cells and endings showed colocalization. Assuming, on the basis of literature data, that VIP and PHI are always colocalized at the cellular level, the five main cell classes in the SCN appeared to be interconnected, at least axosomatically, in the following reciprocal way: AVP <--> VIP/PHI, AVP <--> GRP, AVP <--> somatostatin, somatostatin <--> VIP/PHI, somatostatin <--> GRP, VIP/PHI <--> GRP, VIP/PHI/GRP <--> GRP, VIP/PHI/GRP <--> VIP/ PHI. In addition to this heterologous axosomatic innervation, these cell groups also showed substantial homologous innervation. Supported by electron microscope data from the literature showing the existence of axodendritic synapses for some of these peptide combinations, our findings strongly suggest that the rat SCN comprises a complex synaptic network with strong interactive capabilities, which is probably a requisite for its biological clock function.

Differences in colocalization between Fos and PHI, GRP, VIP and VP in neurons of the rat suprachiasmatic nucleus after a light stimulus during the phase delay versus the phase advance period of the night.

Two groups of four rats each received a 15-minute light stimulus during the first part of the night (ZT14) and the second part (ZT19), respectively. After 45-60 minutes, the animals were killed by perfusion fixation. Adjacent Vibratome sections through the suprachiasmatic nucleus (SCN) were double-immunostained for the presence of peptide histidine isoleucine (PHI), gastrin releasing peptide (GRP) or vasoactive intestinal peptide (VIP) with Fos by using fluorophore-conjugated secondary antibodies. A few sections were triple-immunostained for PHI, GRP or VIP with vasopressin (VP) and Fos. Sections were analyzed with a confocal laser scanning microscope. It turned out that the ZT19 light stimulus induced 4.2 times more nuclear profiles in the SCN immunoreactive for Fos than the light stimulus given at ZT14. The SCN of control animals did not show any Fos immunoreactivity. After the ZT14 light stimulus, approximately 33% of the Fos profiles showed colocalization with a perikaryal profile immunoreactive for PHI, GRP or VIP, whereas at ZT19, this percentage had doubled to approximately 65%. After the light stimulus at ZT14, the relatively low Fos induction was numerically and proportionally most prominent in the PHI-immunoreactive perikarya. As compared with ZT14, the increase of Fos after the ZT19 light stimulus was most pronounced in the GRP-immunoreactive perikarya (21x) followed by VIP (15x) and PHI (5x). This outcome suggests that at least three different cell groups characterized by, respectively, PHI alone, GRP, and VIP fully or partly colocalized with PHI, play a prominent role during light-induced phase shifts: the PHI neurons during light-induced phase delays, the GRP and VIP/(PHI) neurons during light-induced phase advances.

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Image analyser-assisted morphometry of the locus coeruleus in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.

Several observations suggest that neuronal shrinkage rather than cell death is the major phenomenon in neurodegenerative diseases. In order to make this distinction, smaller cells should also be included in cell counts. Also, morphometric determination of total cell numbers of brain structures is required. Morphometry was performed on the locus coeruleus using a newly developed method to delineate this nucleus from five patients who had died with Alzheimer's disease, five with Parkinson's disease, five with amyotrophic lateral sclerosis and from five control subjects who had died from causes that would not have affected the locus coeruleus. The length and volume of the locus coeruleus and its total number of large pigmented neurons, small unpigmented neurons and glial cells were determined. Since reliable delineation of the boundaries of the locus coeruleus is a requirement for the determination of total cell numbers, an image analyser-assisted procedure was developed. In Alzheimer's disease we found an 82% decrease in the number of large pigmented neurons and a 39% decrease of small unpigmented neurons. In Parkinson's disease, we found a 39% decrease of large pigmented neurons but also a 44% (though not significant) increase of small unpigmented neurons, which is indicative of a shift from large pigmented neurons to small unpigmented neurons in Parkinson's disease. The large pigmented/small unpigmented neuron number ratio was greatly and significantly reduced in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. These findings support the hypothesis that the decrease of large pigmented neurons of the locus coeruleus in some neurodegenerative diseases is not entirely due to cell death, but rather to cell shrinkage and a loss of phenotype. This hypothesis may have consequences for the development of therapeutic strategies since atrophied cells can be activated. On the other hand our data confirm that, at least in Alzheimer's disease, large pigmented neurons do also undergo cell death.

Activation of vasopressin neurons in aging and Alzheimer's disease.

The supraoptic (SON) and paraventricular nuclei (PVN) of the human hypothalamus are production sites of vasopressin (AVP) and oxytocin (OXT). Although the hypothalamus is affected in Alzheimer's disease (AD), previous work has not only shown that in these two nuclei no neurons are lost, neither during aging nor in AD, but that the number of AVP-expressing neurons and their nucleolar size had even increased with age. These observations indicated that the peptide synthesis of the AVP neurons was activated in the oldest age-groups. Recently published, qualitative observations, using the area of the Golgi Apparatus (GA) as a sensitive parameter for neurosecretory activity, confirmed the activation of SON and PVN neurons with age in human; however, in this report the neurons were not identified according to their neuropeptide content. In the present quantitative study we determined whether the AVP neurons were indeed activated as a result of the aging process in controls and AD patients. We applied a polyclonal antiserum directed against the medial cisternae of the GA on formalin-fixed, paraffin-embedded tissue sections taken from the dorsolateral SON (dl-SON) of 10 controls and 10 AD patients, and performed our measurements in this area that is known to be predominantly occupied (90-95%) by AVP neurons. In addition, the sparse OXT cells present in the area of study, were excluded from the measurements on the basis of alternative sections stained for OXT. In the dl-SON, the area occupied by the GA and the cellular profile area per patient were quaNtified by means of image analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The monoclonal antibody Alz-50, used to reveal cytoskeletal changes in Alzheimer's disease, also reacts with a large subpopulation of somatostatin neurons in the normal human hypothalamus and adjoining areas.

The monoclonal antibody Alz-50 is directed against Alzheimer's disease-related modified tau proteins and reveals cytoskeletal changes, i.e. neurofibrillary tangles and dystrophic neurites. The present study shows that, in the hypothalamus of non-demented control subjects, this same antibody gives a distinctive staining pattern of a subpopulation of somatostatin neurons and beaded fibres. Furthermore, Alz-50 occasionally recognizes somatostatin-containing cell bodies and dystrophic neurite-like fibers in the (neuritic) senile plaques of AD patients. These observations have direct consequences for the interpretation of Alz-50 staining in diagnostic usage and for the assessment of Alzheimer's disease-like changes induced by beta-amyloid in experimental animal brains. On dot spotting, Alz-50 was found to bind to a number of fragments from the somatostatin precursor, of which somatostatin 15-28 stained best. Preadsorption of Alz-50 by somatostatin 15-28, as well as other specificity tests, failed, however, to provide any clue to the nature of the unknown compound(s) stained in the control hypothalamus.

Kindling induces time-dependent and regional specific changes in the [3H]muscimol binding in the rat hippocampus: a quantitative autoradiographic study.

To investigate possible changes in the GABAA receptor agonist site in the CA1 area and fascia dentata of rats kindled by stimulation of Schaffer collaterals, a quantitative autoradiographic study of the [3H]muscimol binding was carried out. Two kindled groups were studied, at 24 h (fully kindled stage) and at 28 days (long-term stage) after the last class V seizure. Several concentrations of [3H]muscimol were tested in the range of the high/intermediate (5-40 nM) and low-affinity (60-100 nM) binding sites. In the fully kindled group, the binding over the complete range of tested [3H]muscimol concentrations was significantly increased by 30-50% in the fascia dentata, while the binding was significantly decreased by 10-25% in the CA1 area. The high/intermediate-affinity binding was still significantly increased by 20-30% in the fascia dentata 28 days after the last seizure. In this long-term group there was still a significant decrease of 10-18% of the low-affinity binding in the CA1 area. These results show that kindling epileptogenesis induces long-lasting changes in the GABAA receptor agonist binding sites that are region specific. We hypothesize that the changes encountered at the fully kindled stage, i.e. increased binding in the fascia dentata and decreased binding in the CA1 area, may underly the electrophysiologically observed increased paired-pulse depression of field potentials in the former and the decreased paired-pulse depression in the latter area [Kamphuis et al. (1992) Neurosci. Lett. 141, 101-105; Kamphuis et al. (1988) Brain Res. 440, 205-215; Zhao and Leung (1991) Brain Res. 564, 220-229; Zhao and Leung (1992) Brain Res. 582, 163-167]. We conclude that the observed changes may not only contribute to the induction of kindling epileptogenesis but may also play a role in the maintenance of the kindled state.