Chronic antidepressant treatment and central 5-HT synapses.

The present studies have shown that chronic antidepressant treatment with desipramine, imipramine, zimelidine and alaproclate led to adaptive changes in both pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptor mechanisms which appear to result in 5-HT sub- or supersensitivity development depending upon the 5-HT nerve terminal systems analyzed. The results underline the heterogeneity of the central 5-HT neurone systems in their responses to chronic antidepressant treatment. An involvement of 5-HT comodulators is postulated in the actions of antidepressants on central 5-HT synapses. Finally, the results point to the importance of 5-HT neurone systems as targets for the action for antidepressant drugs and they may mediate at least some of the therapeutic activity of antidepressant drugs.

Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive neurons in the rostral medulla oblongata of the adult and old male rat.

In the present paper the neuronal systems of the medulla oblongata containing phenylethanolamine-N-methyltransferase- and neuropeptide Y-like immunoreactivity have been characterized in adult (3-month-old) and old (24-month-old) male rats. The phenylethanolamine-N-methyltransferase and neuropeptide Y-immunoreactive neurons have been visualized by means of immunocytochemistry (peroxidase-antiperoxidase technique) and analysed in a quantitative fashion by means of morphometrical (phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell groups) and microdensitometrical (phenylethanolamine-N-methyltransferase-immunoreactive cell groups) approaches developed on the IBAS II image analyser (Zeiss-Kontron). During aging there is (a) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive neuropil for both the C1 and C2 adrenaline cell groups; (b) a reduction in the area covered by the phenylethanolamine-N-methyltransferase-immunoreactive cell bodies, which is highly significant only for the C2 cell group; (c) a decrease in the area covered by the phenylethanolamine-N-methyltransferase-positive cell cluster for both C1 and C2 cell groups; (d) a decrease in the degree of phenylethanolamine-N-methyltransferase immunoreactivity present in the C1 and C2 cell groups; (e) a decay of neuropeptide Y immunoreactivity in the C1 and C2 groups, while the C3 group is unaffected by aging as evaluated by number of phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive cell body profiles. These results indicate heterogeneities in the responses of the adrenaline-neuropeptide Y cell groups to the aging process. The possible functional consequences of aging-induced changes in the cardiovascular adrenergic neurons are discussed, especially in relation to development of hypertension.

Morphometrical and microdensitometrical studies on phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive nerve terminals and on glucocorticoid receptor-immunoreactive nerve cell nuclei in the paraventricular hypothalamic nucleus in adult and old male rats.

The phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive nerve terminal profiles and the glucocorticoid receptor-immunoreactive nuclear profiles have been characterized in the parvocellular part of the paraventricular hypothalamic nucleus of the adult (3 month) and the old (24 month) male rat. The phenylethanolamine-N-methyltransferase-, neuropeptide Y- and glucocorticoid receptor-immunoreactive structures have been demonstrated by means of the indirect immunoperoxidase procedure and analysed in a quantitative way by means of morphometrical and microdensitometrical approaches using both semiautomatic and automatic image analysis. During aging there is (a) a marked reduction in the number of neuropeptide Y-immunoreactive profiles, a moderate reduction of phenylethanolamine-N-methyltransferase-immunoreactive profiles and a small reduction in the number of glucocorticoid receptor-immunoreactive profiles without a significant change in the evenness of distribution of such profiles as evaluated by means of Gini's index; (b) a loss of the significant correlation in the distribution of the glucocorticoid receptor- and phenylethanolamine-N-methyltransferase-immunoreactive profiles at the two most caudal levels analysed (A5150 and A5270 micron) while a significant correlation developed between these two distributions at a more rostral level (A5400 micron); (c) a substantial decline in the overlap area of the glucocorticoid receptor- and phenylethanolamine-N-methyltransferase-immunoreactive profiles at four out of five rostrocaudal levels analysed; (d) a marked reduction in the density-intensity of the neuropeptide Y-immunoreactive profiles and a small significant reduction in the density-intensity of the phenylethanolamine-N-methyltransferase-immunoreactive profiles without any associated changes in the intensity of the glucocorticoid receptor-immunoreactive profiles. Furthermore, three-dimensional reconstructions of the overall distribution of the glucocorticoid receptor-, phenylethanolamine-N-methyltransferase- and neuropeptide Y-immunoreactive structures have been made in the paraventricular hypothalamic nucleus of the adult male rat. The present results indicate a reduction of neuropeptide Y- and phenylethanolamine-N-methyltransferase-immunoreactive nerve terminal profiles in the parvocellular part of the paraventricular hypothalamic nucleus during aging. These results may in part reflect a loss of neuropeptide Y-like peptides in phenylethanolamine-N-methyltransferase-immunoreactive nerve terminals of the paraventricular hypothalamic nucleus, favouring our view that during aging the modulatory peptides may be lost, leading to a loss of

Increases in sulphated glycoprotein-2 mRNA levels in the rat brain after transient forebrain ischemia or partial mesodiencephalic hemitransection.

Sulphated glycoprotein-2, thought to be involved in programmed cell death in peripheral organs, has been detected at increased levels in brain degenerative states. In this paper we have investigated the regional and cellular expression of this protein during development of brain lesion. With this aim sulphated glycoprotein-2 mRNA levels were studied in models of ischemic (transient forebrain ischemia) or mechanical (partial mesodiencephalic hemitransection) brain injuries using in situ hybridization histochemistry. Marked increases in sulphated glycoprotein-2 mRNA were observed in lesioned brains in both models. In addition, we report a shift in the regional distribution of positive cells in both lesion models 1-7 days post-lesion. After transient forebrain ischemia, sulphated glycoprotein-2 mRNA increases were always localized in selectively vulnerable regions (caudate-putamen, hippocampal formation), showing a temporal change in the pattern of intraregional distribution from less to more lesioned parts. In the case of mechanical lesion at 1 day, increased labelling had a widespread distribution on the lesioned side and was also observed on the intact side near the midline. In contrast, at 7 days increased labelling was restricted to regions directly lesioned (either areas whose input and/or output connections were severed by the transection or areas which were directly affected by the mechanical lesion). Analysis at the cellular level revealed that at both time intervals and in both lesion models most cell bodies overlain by dense clusters of specific grains were non-neuronal cells. The distribution patterns and their change over time suggest that at least some of these cells are inflammatory and phagocytic cells. The majority of degenerating neuronal cells after ischemia did not show increased levels of sulphated glycoprotein-2 mRNA. However, seven days after hemitransection and at all time intervals after transient ischemia, some cells clearly identifiable as neurons exhibited increased sulphated glycoprotein-2 mRNA levels.

Spermidine/spermine N1-acetyltransferase mRNA levels show marked and region-specific changes in the early phase after transient forebrain ischemia.

Considerable evidence points to an involvement of natural polyamines (putrescine, spermidine and spermine) in trophic regulation of brain tissue. Spermidine/spermine N1-acetyltransferase is the key enzyme in the interconversion pathway which leads to the formation of spermidine and putrescine from spermine and spermidine, respectively. In the present paper we have studied using in situ hybridization histochemistry the levels of spermidine/spermine N1-acetyltransferase mRNA in the rat central nervous system after transient forebrain ischemia. In the first hours after the insult, a modest increase in spermidine/spermine N1-acetyltransferase mRNA levels was observed in ependymal cells and other non-neuronal cells of all telencephalic and diencephalic regions. In addition, major increases in spermidine/spermine N1-acetyltransferase mRNA levels were observed in regions selectively vulnerable to the ischemic insult, such as striatum, hippocampus and cerebral cortex, during the first day post-reperfusion. The time course and extent of labelling increase were subregion- and cell-specific. At the cellular level, the labelling appeared markedly increased in neurons (8-10 fold in ventromedial striatum and CA1 region) and, to a lesser extent, in non-neuronal cells. The increase in SSAT mRNA levels was not directly related to cell degeneration, as it was detected in both some vulnerable and some resistant cell populations. However, the peak increase of SSAT labelling was precocious in resistant neurons (such as those of ventromedial striatum and dentate gyrus granular layer) and delayed or very limited in vulnerable neurons (such as those of CA1 pyramidal layer and dorsolateral striatum). The increase in spermidine/spermine N1-acetyltransferase may contribute to the increase in putrescine and decrease in spermidine levels observed after ischemia and gives further support to the notion that polyamine metabolism in the early phase after lesion is oriented towards putrescine production. This phenomenon could be relevant in determining the prevalence of neurotrophic vs. neurotoxic effects of polyamines.

Nerve cell clusters in dorsal striatum and nucleus accumbens of the male rat demonstrated by glucocorticoid receptor immunoreactivity.

Glucocorticoid receptor-immunoreactive nerve cells have been analysed in the dorsal striatum and nucleus accumbens of the rat by means of a monoclonal antibody against rat liver glucocorticoid receptor. Glucocorticoid receptor immunoreactivity was present in the nuclei of the vast majority of the striatal nerve cells. The analysis of sections stained with glucocorticoid receptor antibody and cresyl violet showed that around 90% of the entire striatal neuronal population contained glucocorticoid receptor immunoreactivity. By means of the double immunoperoxidase technique evidence was provided that somatostatin- and choline acetyltransferase-immunoreactive nerve cells in the striatum do not contain glucocorticoid receptor immunoreactivity. The density of glucocorticoid receptor-immunoreactive nerve cells in the grey matter and the presence of clusters of glucocorticoid receptor-immunoreactive nerve cells have been investigated in three fields located in the medial and central dorsal striatum and nucleus accumbens at the coronal level A 8620 microns according to the König and Klippel atlas using computer-assisted image analysis. Every aggregate containing three or more glucocorticoid receptor-immunoreactive nerve cells, which had an intercenter distance less than the mean diameter (10-11 microns) of the striatal cells, was considered an island. A higher density of both glucocorticoid receptor-immunoreactive nerve cell nuclei and islands was found in the nucleus accumbens with respect to dorsal striatal areas. The most frequent island formed consisted of three to ten nerve cells both in dorsal striatum and nucleus accumbens. Furthermore, some nucleus accumbens islands contained up to 100 nerve cells, whereas in the dorsal striatum the maximum number of glucocorticoid receptor-immunoreactive nerve cells per island ranged from 50 to 60. The present procedure proved to be a sensitive method to reveal clusters of chemically identified structures and provided evidence for a basic cytoarchitectonic organization of the dorsal striatum and nucleus accumbens of the rat. This paper also demonstrated that the vast majority, but not all, striatal nerve cells contained glucocorticoid receptor immunoreactivity, and thus may be under the control of circulating glucocorticoids. In fact, only small transmitter-identified neuronal populations, such as somatostatin- and choline acetyltransferase-immunoreactive nerve cells, were devoid of glucocorticoid receptor immunoreactivity.

Aspects of neural plasticity in the central nervous system-I. Computer-assisted image analysis methods.

Microdensitometric and morphometric techniques have been developed to quantitatively characterize cell groups and terminal populations of transmitter-identified neuronal systems. Various microdensitometric methods implemented on the image analyzer or on the scanning microdensitometer were introduced and compared. On this basis a technique to assess the half-life of dopamine stores determined by quantitative immunocytochemistry has been developed. The problem of relative and absolute quantification of microdensitometric analysis of immunocytochemical preparations is discussed here. A method has been developed for the study, both in 2- and 3-dimensions, of the overall features of the profile distribution in a defined neuroanatomical area. An approach to determine the degree of uniformity of a certain profile distribution is also proposed. Furthermore, methods for the evaluation of the codistribution of two or more different types of profiles and to characterize the morphometric features of patches of profiles in a certain region are presented. All these quantitative morphological approaches were tested in relevant preparations of the central nervous system.

Aspects of neural plasticity in the central nervous system-V. Studies on a model of transient forebrain ischemia in male Sprague-Dawley rats.

A morphological and functional characterization of the four-vessel occlusion model of transient (30 min) forebrain ischemia has been carried out. The rats were classified as fully ischemic when an isoelectric pattern of electroencephalographic activity was present within 5 min of the occlusion of carotid arteries. Otherwise they were considered as partially ischemic rats. The modifications of cerebral blood content during and after the ischemic insult were assessed by a histochemical method which visualizes red blood cells in cerebral vessels. The periods of increase and decrease of red blood cell content were found to correspond to previous reports of post-ischemic hyper- and hypoperfusion. Neuronal damage was assessed by a quantitative analysis of Nissl stained preparations of cingulate cortex, dorsal hippocampus and striatum. The signs of morphological damage were quantified by means of computer-assisted image analysis of Nissl preparations. The highest vulnerability to the ischemic insult was demonstrated in the pyramidal layer of the hippocampal CA1 field and in the lateral striatum. Arterial blood pressure measurements were performed during the ischemic and post-ischemic periods, demonstrating a peak increase of arterial blood pressure within 2 min after carotid artery occlusion, followed by a slow decrease towards basal levels during the ischemic period and a full recovery within 15 min of reperfusion. Ischemic rats were tested in a neurological test battery and in a passive avoidance task. While a full recovery of the relatively simple tasks of the neurological test battery was attained within 14 days of reperfusion, a highly significant impairment of passive avoidance behavior was still present 15 days after the ischemic insult. Finally, a discriminant analysis was applied to separate, on the basis of non-invasive techniques (neurological tests and hot plate), the group of completely ischemic rats from that of partially ischemic rats.

Regional increases in ornithine decarboxylase mRNA levels in the rat brain after partial mesodiencephalic hemitransection as revealed by in situ hybridization histochemistry.

Changes in the level of ornithine decarboxylase mRNA after partial mesodiencephalic hemitransection were evaluated in various regions of the rat brain by means of in situ hybridization histochemistry coupled with computer-assisted image analysis. On days 1 and 2 after the lesion, increased accumulation of ornithine decarboxylase mRNA was observed on the lesioned side in various telencephalic regions (e.g. neostriatum and frontoparietal cortex), and in the pars compacta of the substantia nigra. Both in the frontoparietal cortex and substantia nigra a decreasing gradient of ornithine decarboxylase mRNA activation was observed going far from the site of the lesion. Seven days after the operation, ornithine decarboxylase mRNA levels returned to control values on the lesioned side but increased in some regions, such as the frontoparietal cortex, on the intact side. The present results demonstrate that the parent cell body biosynthetic machinery is activated by the mechanical lesion of the axons at the level of ornithine decarboxylase gene expression. The increase of ornithine decarboxylase mRNA is not as large as the enhancement in ornithine decarboxylase activity previously shown, suggesting that the response to the lesion may also involve changes in the rate of translation of ornithine decarboxylase mRNA and/or in the rate of degradation of ornithine decarboxylase protein.

Studies on neuropeptide Y-catecholamine interactions in the hypothalamus and in the forebrain of the male rat. Relationship to neuroendocrine function.

Neuropeptide Y-catecholamine interactions have been analyzed within the hypothalamus and in the forebrain of male rats by means of immunocytochemistry in combination with morphometry, quantitative histofluorimetry on catecholamine fluorescence in discrete catecholamine nerve terminal systems, biochemical analysis of catecholamines as well as by studies on serum levels of adenohypophyseal hormones vasopressin, adrenocortical hormones and angiotensin II using radioimmunoassay determinations. (1) Morphologic and morphometrical evidence indicates the existence of separate populations of neuropeptide Y and tyrosine hydroxylase immunoreactive nerve cell bodies in the parvo- and magnocellular components of the arcuate nucleus respectively. In addition, a significant codistribution of NPY immunoreactive nerve terminals and tyrosine hydroxylase immunoreactive nerve cell bodies were demonstrated in the ventrolateral part of the magnocellular component of the arcuate nucleus. (2) Immunocytochemical studies on the distribution of tyrosine hydroxylase, phenyl ethanolamine-N-methyltransferase and neuropeptide Y immunoreactive nerve terminal networks in the peri- and paraventricular hypothalamic nucleus indicated that these types of immunoreactive nerve terminals densely innervate the medial and anterior parvocellular part of the paraventricular hypothalamic nucleus and anterior periventricular hypothalamic nucleus. From studies on the pattern of terminal distribution results have been obtained compatible with the view that neuropeptide Y or a neuropeptide Y related peptide can be a comodulator in noradrenaline and adrenaline nerve terminal networks of these regions. (3) Acute intraventricular injections of neuropeptide Y (1.25 nmol) do not change dopamine and noradrenaline levels in any hypothalamic and telencephalic dopamine and noradrenaline nerve terminal system analyzed with the exception of the anteromedial frontal cortex, in which area a significant increase in the dopamine levels was observed as revealed biochemically. (4) By means of the tyrosine hydroxylase inhibition method it was possible to show that acute intraventricular injection of NPY (1.25 nmol) increased dopamine utilization in the medial and lateral palisade zone of the median eminence and in the anteromedial frontal cortex and reduced noradrenaline utilization in the parvocellular part of the paraventricular hypothalamic nucleus, while dopamine utilization was not influenced in the nucleus caudatus putamen, nucleus accumbens or in the tuberculum olfactorium. (5) In the intraventricular experiments reported above neuropeptide Y (1.25 nmol, 1 h) reduced the serum levels of thyreotropin stimulating hormone, prolactin and luteinizing hormone and increased serum corticosterone, adrenocorticotrophin, vasopressin, angiotensin II and aldosterone levels. The presence of the tyrosine hydroxylase inhibitor by itself, increased corticosterone, adrenocorticotrophin and aldosterone serum levels and reduced serum luteinizing hormone levels. Neuropeptide Y together with the tyrosine hydroxylase inhibitor further enhanced the adrenocorticotrophin, angiotensin II and aldosterone serum levels seen with the inhibitor, but could no longer produce its excitatory and inhibitory effects on serum corticosterone and luteinizing hormone levels, respectively. Vasopressin serum levels were increased to the same extent in the absence or presence of tyrosine hydroxylase inhibition. The present morphological, neurochemical and functional studies indicate that neuropeptide Y given intraventricularly inhibit the secretion of prolactin, luteinizing and thyreotropin stimulating hormones probably by activation mainly of neuropeptide Y receptors located in the somadendritic region of the arcuate DA cell bodies, leading to increased activity in inhibitory tubero-infundibular dopamine neurons. In addition, it is suggested that the ability of neuropeptide Y to increase adrenocorticotrophin and corticosterone secretion is at least in part related to its ability to reduce noradrenaline turnover in the parvocellular part of the paraventricular hypothalamic nucleus, rich in corticotrophin releasing factor immunoreactive nerve cell bodies. It is speculated that neuropeptide Y as a comodulator in the noradrenaline nerve terminals in this area may enhance the excitatory actions of noradrenaline on the corticotrophin releasing factor immunoreactive nerve cells. Such an action will lead to increases of corticotrophin releasing factor neuronal activity and of adrenocorticotrophin hormone secretion producing a feedback response, which may reduce noradrenaline turnover exclusively in this nucleus as was observed in the present experiments. The increase in aldosterone may be induced by the increased adrenocorticotrophin serum levels but the increase in vasopressin secretion and in angiotensin II serum levels may be secondary to the hypotensive activity of neuropeptide Y. Finally, it is suggested that neuropeptide Y mechanisms can increase dopamine synthesis and release in the anteromedial frontal cortex. Thus, neuropeptide Y mechanisms may participate in the control of cortical functions at least partly by regulating the cortical dopamine neurotransmission.

Aspects of neural plasticity in the central nervous system-VI. Studies of the effects of gangliosides on brain metabolic lesions.

The effects of gangliosides have been studied in two models of metabolic insult (insulin-induced hypoglycemia and transient forebrain ischemia) of the central nervous system. In the severe hypoglycemia experiments lactate extracellular fluid levels were evaluated by means of a microdialysis probe implanted in the frontoparietal cortex. Ganglioside GM1, given either peripherally (10 mg/kg, i.p.) or intracerebrally (2 x 10(?4) M, via the microdialysis probe) 2 h before insulin injection, was able to reduce the decay of the perfusate levels of lactate induced by the insulin injection. In the same animal model peripheral, but not central, administration of GM1 reduced the hypoglycemia-induced increase of cerebral blood flow and increased the survival time observed after the insulin injection. In the experiments on transient forebrain ischemia, a GM1 derivative, AGF2 (5 mg/kg/day, i.p.), was administered chronically, starting 5 days before or the day after the ischemic insult. With both treatment schedules a similar protective effect was observed in a neurological test battery and in the step-through latency in a passive avoidance test.

Effects of chronic treatment with uridine on striatal dopamine release and dopamine related behaviours in the absence or the presence of chronic treatment with haloperidol.

Uridine (15mg/kg/day, i.p.), haloperidol (1mg/kg/day, i.p.), uridine (15mg/kg/day, i.p.) plus haloperidol (1mg/kg/day, i.p.) or saline have been chronically administered to Sprague-Dawley male rats. Following 1 week of wash-out, the effects of these treatments on basal striatal dopamine (DA) release as well as on the DA release induced by an acute haloperidol challenge (2mg/kg, i.p.) were studied by means of intracerebral microdialysis. Behavioural tests such as haloperidol-induced catalepsy or apomorphine-induced stereotypics were also performed 4-7 days after drug withdrawal. The chronic treatment with uridine alone or associated with haloperidol markedly reduced DA release induced by an acute haloperidol challenge. The behavioural studies also indicated a change in DA-related behaviours in these conditions. The animals chronically treated with uridine showed significant increases in the stereotypy scores and in the catalepsy induced by an acute haloperidol challenge with respect to saline treated rats. The present results indicate that a chronic uridine treatment is able to potentiate the reduction of the striatal DA transmission induced by acute and chronic haloperidol treatment. This finding suggests the possibility to reduce the neuroleptic dose in the treatment of schizophrenia by combining neuroleptic and uridine treatments, thus making it possible to relieve some of the side effects of neuroleptic therapy.

Neuropeptide Y produces anxiolytic effects in spontaneously hypertensive rats.

The sedative and anxiolytic effects of intracerebroventricular administration of neuropeptide Y (NPY) were studied in spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto (WKy) rats using the two-compartment exploratory test, and in the open-field test after habituation. In the two-compartment tests, NPY produced anxiolytic effects by increasing the exploratory activity in SHR at a dose (0.25 nmol) lower than the minimal effective dose in WKy rats (1.25 nmol). In SHR, anxiolytic effects were observed for the whole NPY dose range (0.25-5.0 nmol), whereas in normotensive WKy rats the highest dose (5.0 nmol) failed to increase exploratory activity. The open-field test showed reduced locomotor activity and rearings in WKy rats when injected with 5.0 nmol NPY. These effects were not observed in SHR. The absence of sedative effects and the higher sensitivity to the anxiolytic effects of NPY in SHR are suggestive of a genetically determined difference in central NPY systems involved in behavioral adaptation that may be relevant for the development of hypertension.

A method for rostrocaudal integration of morphometric information from transmitter-identified cell groups. A morphometrical identification and description of 5-HT cell groups in the medulla oblongata of the rat.

A method has been developed to integrate rostrocaudal information from morphometrically characterized 5-HT nerve cell groups visualized by means of the indirect immunofluorescence technique in single coronal sections of the neuroaxis. The present method has been applied to the 5-HT positive cells of the medulla oblongata of the rat. 5-HT cell body and cell group parameters were measured by the use of a semiautomatic image analyzer (Kontron, MOP AMO 2) plugged into an Apple II computer. The density distribution of 5-HT positive cells was also studied by dividing the area analyzed into unitary squares (about 120 X 120 micron) and then by considering the number of 5-HT cells falling in each of these squares. The existence of a 5-HT cell group was determined by testing the randomness of the 5-HT profiles per unitary square. The entire population of 5-HT positive cells as well as the 5-HT cell groups were described in terms of gravity center coordinates, mean maximal diameter and homogeneity index. A rostrocaudal representation of the gravity centers and of the dispersion of the 5-HT nerve cell groups around them provided an exact three-dimensional description of the respective locations of the 5-HT cell groups within the medulla oblongata. These methods can be applied to all types of transmitter-identified cell groups and at any level of the neuroaxis.

Principles for the morphological characterization of transmitter-identified nerve cell groups.

Principles for a morphometric description of transmitter identified nerve cell groups have been introduced, exemplified on the 5-HT nerve cell group of nucleus raphe dorsalis (B7 cell group), using immunocytochemical procedures to visualize 5-HT. Both cell body parameters (mean diameter, mean perimeter, mean area, shape factor) and cell group parameters (number of cells, mean free distance among cells, volume fraction, gravity centre, major axis slope with respect to the midline) have been considered. These parameters have been obtained by the use of a semiautomatic image analyzer (Kontron MOP AMO2) plugged into an Apple II computer. By the use of this system and of suitable computer programs, it is possible to give a Cartesian representation of 5-HT nerve cell bodies in a coronal section under study. The present work has mainly one aim. To detect whether subgroups exist within a transmitter-identified cell group. Two approaches have been introduced to obtain, on objective grounds, evidence whether or not a group consists of subgroups. The first of these approaches is based on differences in cell body density within the cell group, while the second approach is based on frequency distribution analysis. This second approach is mainly sensitive to shape changes of the cell group.

Neuroanatomical methods for the quantitative evaluation of coexistence of transmitters in nerve cells. Analysis of the ACTH- and beta-endorphin immunoreactive nerve cell bodies of the mediobasal hypothalamus of the rat.

A new statistical approach has been introduced to study in a quantitative way the coexistence of two neuromodulators in nerve cell bodies. The method has been exemplified on the ACTH-like and beta-endorphin-like immunoreactive nerve cell bodies of the mediobasal hypothalamus demonstrated by means of indirect immunofluorescence methodology. The method is based on the analysis of 3 adjacent sections which, in a random way, are stained with antiserum against neuromodulator 1, against neuromodulator 2 and with antisera against both neuromodulator 1 and neuromodulator 2. It could be shown that some neurons of the mediobasal hypothalamus do not contain at a detectable level both ACTH- and beta-endorphin-like immunoreactivity. The present method also involves an analysis of the two nerve cell groups by means of a morphometric procedure to collect additional information on the extent of coexistence. Thus, by this approach the gravity centers of the two cell groups can be calculated. The distances between the two gravity centers of the ACTH and beta-endorphin positive cell groups were significantly different at certain levels. The present method offers unique possibilities in increasing our understanding of the functional significance of coexistence of neuromodulators in one and the same nerve cell body since it makes possible a quantitative evaluation of coexistence. The usefulness of the present method is illustrated by the findings of a possible differential synthesis of ACTH- and beta-endorphin-like material in certain cell bodies of the mediobasal hypothalamus.

Microdialysis and luminescent probe: analytical and clinical aspects.

Immobilized enzymes are widely used in the clinical laboratory to assay several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable, disposable and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow sensors based on the use of nylon tube coil or epoxy methacrylate column as solid support. For in-vivo determination a suitable microdialysis probe inserted directly into brain or blood allows continuous measurement of extracellular lactate levels by means of a bioluminescent flow detector system. This procedure performs more measurements in the same time interval than other systems (HPLC), e.g. to give a detailed description of the effects of ischemia, or other pathological events, on lactate concentration in the brain.

Effects of polyamine synthesis blockade on neuronal loss and astroglial reaction after transient forebrain ischemia.

Polyamines and ornithine decarboxylase, the polyamine biosynthetic enzyme, have been demonstrated to increase in the early phase of several types of brain lesion. However, their role in the pathogenesis of tissue damage is still debated. In the present paper the effects of treatments with alpha-difluoromethylornithine, a suicide inhibitor of ornithine decarboxylase, have been investigated in a model of transient forebrain ischemia. Three treatment schedules were used: alpha-difluoromethylornithine treatment was either started 3 hr before and repeated 1 hr after the insult, or started at the time of the insult and continued for 3 or 7 days after post-ischemic reperfusion. The rats were sacrificed 4 hr, 7 or 40 days after reperfusion, respectively. The acute experiment demonstrated that alpha-difluoromethylornithine can reduce the increase of glial fibrillary acid protein immunoreactivity, an early marker of astroglial reaction, in ischemic striatum. Subchronic and chronic alpha-difluoromethylornithine treatments induced a worsening of the morphological outcome of the ischemic lesion. In caudate-putamen a trend for an increase of the area of neuronal loss was present after both treatments. In the hippocampal formation, a significant increase in the severity of neuronal lesion was observed in the mildly lesioned CA3 field. In addition, other alterations of lesioned tissue were observed in alpha-difluoromethylornithine-treated animals, including increases of non-neuronal cells at 7 and especially 40 days post-lesion in striatum and CA3 hippocampal field. In conclusion, present data indicate that ornithine decarboxylase activation after ischemic lesion is a crucial factor for survival of mildly lesioned neurons and proper tissue reaction to the ischemic lesion. The experiment on acute alpha-difluoromethylornithine treatment suggests that these effects may be, at least in part, related to putrescine-induced activation of astroglial cells in the early post-lesion period.

l-Glutamate reduces the affinity of [3H]N-propylnorapomorphine binding sites in striatal membranes.

l-Glutamate but not methyl-D-aspartate (NMDA) or quisqualate ( Quis ) (10(-6 M) in vitro with or without preincubation increased significantly the KD value of the [3H]N-propylnorapomorphine ( [3H]NPA) binding sites by 21 and 36% respectively in striatal membranes of rat without influencing the striatal [3H]spiperone binding sites. The number of striatal [3H]NPA binding sites was not changed by l-glutamate (10(-6) and 10(-5) M) in vitro. There may thus exist interactions between striatal glutamate receptors -- not related to excitatory amino-acid receptors of the NMDA or the QUIS type -- and high affinity striatal DA receptors.

Effects of chronic sino-aortic denervation in male rats on regional catecholamine levels and turnover and on neuroendocrine function.

A four week sino-aortic denervation produces an increase of adrenaline (A) turnover in the anterior but not the posterior hypothalamus and a selective reduction of dopamine stores in the anterior hypothalamus. Furthermore, the mean arterial blood pressure and heart rate are unchanged in conscious animals, and a selective increase in LH secretion is observed. The activation of a compensatory vasodepressor adrenergic mechanism in the anterior hypothalamus may in part be responsible for the maintenance of a normal mean arterial blood pressure after chronic sino-aortic denervation. The neurochemical changes in the hypothalamic area may also be related to the increase in LH secretion found after chronic sino-aortic denervation.