Nutritional parameters modify muricidal behavior of male Wistar rats: preventive effects of amino acids and 4' Cl diazepam.

In male Wistar rats fed a diet enriched in polyunsaturated fatty acids and starch (PUFA+S), the percentage of muricidal (Mu) rats increased to 82% within 60 days. Mu rats had higher serum triglyceride levels and lower cholesterol levels than non-Mu rats. Water intake decreased in all rats on the PUFA+S diet concurrently with the increase in the proportion of Mu rats; protracted water restriction in rats fed standard diet also increased the percentage of Mu rats. In the offspring of two Wistar females fed the PUFA+S diet, the proportion of young Mu rats was 67%. When the PUFA+S diet was replaced with standard diet, the induced Mu behavior was not reversed. PK11195 (6 mg/kg i.p.), clonazepam (0.2 mg/kg i.p.), and flumazenil (15 mg/kg i.p.) were ineffective in reversing the induced Mu behavior, whereas 4'-chlorodiazepam (5 mg/kg i.p.) or muscimol (0.5 mg/kg i.p.) caused reversals of 63% or 50%, respectively. A 5-hydroxytryptophan overload (60 mg/kg i.p.) also reversed Mu behavior by 71%. All reversal effects were temporary. Pretreatment with yeast for 7 days before the PUFA+S diet was given prevented induction for more than 90 days on the PUFA+S diet, while similar pretreatment 4'Cl-diazepam resulted in 71% prevention of induction. The results are analyzed in terms of the involvement of endozepin, vasopressin, and serotonin receptors, and of possible genetic parameters.

Regional distribution of benzodiazepine binding sites in the human newborn and infant hypothalamus. A quantitative autoradiographic study.

Using in vitro quantitative autoradiography and [3H]flunitrazepam we examined the rostrocaudal distribution of benzodiazepine binding sites in the human neonate/infant hypothalamus. The autoradiographic analysis shows the presence of a heterogeneous distribution throughout the rostrocaudal extent of this brain structure. High [3H]flunitrazepam binding corresponds primarily to the diagonal band of Broca and the preoptic region. The labelling in the preoptic region showed a rostrocaudal increase, contrasting in that with the other hypothalamic structures. Intermediate densities were present in the septohypothalamic, suprachiasmatic, periventricular and paraventricular nuclei as well as in the mammillary complex. Low binding was observed in the other hypothalamic structures. The benzodiazepine binding sites analyzed belong mostly to type II receptors. In an attempt to unravel possible differences related to age, we compared the autoradiographic distribution in three postnatal age ranges. The topographical distribution of these binding sites was almost identical in each period analyzed. We found, however, that benzodiazepine binding is generally low in the neonatal period and a tendency in increasing densities is observed during development. Taken together, these results provide evidence for a large distribution of benzodiazepine binding sites in neonate/infant hypothalamus, suggesting their implication in the development of this brain structure and the maintenance of its various functions.

Cholesterol and triglyceride levels in the serum of muricidal male Wistar rats: indices of mitochondrial benzodiazepine receptors?

Cholesterol and triglyceride levels were studied in the serum of aggressive muricidal and non-muricidal male Wistar rats. The muricidal behavior was either spontaneous or induced by a long-term isolation or by adrenalectomy. Cholesterol levels were slightly higher in the whole population of muricidal rats; this was mainly observed in spontaneously and in adrenalectomized muricidal rats, as compared to non-muricidal rats of the same series. As regards triglyceride levels, they were significantly higher in the whole population of muricidal rats, mainly in isolation- and adrenalectomy-induced muricidal rats; the ratio of triglycerides to body weight was higher in the serum of muricidal rats of all series. The possible significance of these results is discussed in light of the data of the literature and related to the functional role of either mitochondrial benzodiazepine receptors or serotonin.

Benzodiazepine binding sites in the human hypothalamus. Autoradiographic study.

Using in vitro labelling and autoradiographic techniques, we have analyzed the fine and the detailed distribution of benzodiazepine binding sites in the post-mortem human hypothalamus. Binding sites were labelled in mounted tissue sections from adult brains, using the selective high affinity ligand [3H]-Flunitrazepam. A heterogeneous distribution of benzodiazepine binding sites was found throughout the rostrocaudal extent of human hypothalamus. The autoradiographic labelling was shown in the three hypothalamic parts, i.e., anterior, mediobasal and posterior levels. At the anterior level, the highest densities were present in the diagonal band of Broca, the preoptic area (medial and lateral parts) and the septohypothalamic nucleus. At the mediobasal hypothalamic level, the highest densities were mainly localized in the ventromedial nucleus, whereas the other structures were moderately labelled with [3H]-Flunitrazepam. The mammillary complex as well as the posterior hypothalamic area represented the most heavily labelled structures in the posterior hypothalamus. The results obtained in this study, indicate the presence of a large and heterogeneous distribution of benzodiazepine binding sites in human adult hypothalamus. This could support their implication in the control of distinct neural functions (like neuroendocrine role).

Evaluation of endozepine-like immunoreactivity in the frontal cortex of suicide victims.

Diazepam-binding inhibitor (DBI) and its processing products, including the octadecaneuropeptide (ODN), are polypeptides called endozepines which have multiple biological effects, including regulation of mitochondrial steroidogenesis and modulation of GABA-gated chloride channels. Concentrations of ODN-like immunoreactivity (ODN-Li) were measured by radioimmunoassay in the frontal cortex of nine drug-free suicides and nine drug-free sudden-death victims. The level of ODN-Li was higher in the right than in the left frontal cortex, in both suicide (p < 0.05) and control (p < 0.02) subjects. No significant differences were found between suicides and controls either in the right and left cortex, or when considering the gender and the post-mortem diagnosis of depression.

Prevention of ACTH- and adrenalectomy-induced muricidal behavior: by benzodiazepinic ligands.

We investigated the effect of treatment with central (neuronal and glial) benzodiazepine binding site-active molecules on ACTH- or adrenalectomy (ADX)-induced muricidal behavior in male Wistar rats. Pretreatment (IP) with either flumazenil or clonazepam prevented the subsequent induction of ADX-induced behavior, but only flumazenil protected against ACTH-induced behavior; posttreatment in both cases induced no significant modifications. Using 4'-chloro-diazepam or PK 11195, both pre- and posttreatment afforded protection, the effect lasting longer (> 1 week) than that induced by flumazenil or clonazepam (2 days). Pretreatment with the GABAA agonist, muscimol, also resulted in complete protection, whereas posttreatment had only a slight effect.

Induction of muricidal behavior by ACTH or adrenalectomy in young male Wistar rats.

ACTH administered IP induces a muricidal behavior in 52% of male Wistar rats that do not express it before; such behavior is also observed spontaneously in 68% of adrenalectomized animals. This change in behavior is characterized by an exceptionally long duration (several months in some animals), and by its prevention by previous or substitutive treatments with dexamethasone. Data obtained using hypophysectomized or adrenalectomized animals with or without ACTH treatments suggest that the muricidal-inducing effect of ACTH might have, at least partially, a central origin.

Long-term isolation of Wistar rats alters brain monoamine turnover, blood corticosterone, and ACTH.

In this work, we investigated neurochemical parameters in the brain of male Wistar rats after isolation times (13 weeks) longer than those previously reported with this strain: a large majority of animals became muricidal under these conditions. Changes in monoamines turnover in hippocampus, cortex, and cerebellum and, in the blood, ACTH, and corticosterone were investigated. Monoamine turnover was analysed using two different approaches: first, by measuring neurotransmitter and metabolite levels and second, by measuring rate of accumulation of the precursor after decarboxylase. Both methods revealed a significant increase in catecholamine turnover in the three regions studied after the 13-week isolation; in contrast, only a modest elevation of 5-hydroxytryptophan accumulation was obtained in cortex and cerebellum of isolated rats. We also observed a decrease in corticosterone levels in blood concomitant with an increase of ACTH.

Benzodiazepine binding sites and their modulators in hippocampus of violent suicide victims.

Benzodiazepine binding sites were studied by autoradiography in several hippocampic layers in brains of drug-free violent suicide victims (hanging) and matched controls. Kd was increased in suicides, and when brain sections from control subjects were incubated in the bath fluid that had previously served to incubate sections from suicides, Kd was increased in the same way. Results are discussed in terms of possible modulators of benzodiazepine binding sites, mainly tissue GABA and amino acid concentrations.

Quantitation by gas chromatography with selected-ion monitoring mass spectrometry of "natural" diazepam, N-desmethyldiazepam and oxazepam in normal human serum.

During the past five years, the literature has tended to prove the occurrence of "natural benzodiazepines" in tissues and biological fluids of non-medicated humans. Several have been identified but very few papers deal with their quantitation in biological material. We present here a method for the specific and sensitive measurement of serum levels of diazepam, N-desmethyldiazepam and oxazepam by gas chromatography with selected-ion monitoring mass spectrometry in twenty human volunteers without medication. Diazepam was found over the whole population, in the range 7.3-32.0 pg/ml, identical in males and females. The other two were present in only some individuals (1.0-7.6 pg/ml for N-desmethyldiazepam and 2.0-13.0 pg/ml for oxazepam). The origin (endogenous, dietary or microbial) of these substances is still to be elucidated.

Quantitative evaluation of octadecaneuropeptide-like immunoreactivity in hippocampus, cortex and cerebellum of long-term isolated male Wistar rats.

Quantitative evaluation of octadecaneuropeptide-like immunoreactivity (ODN-Li) was carried out in hippocampus, cortex and cerebellum of group-reared or three month isolated male Wistar rats, whether muricidal or not. ODN-Li was lower in cortex and cerebellum of isolated animals, (mainly in muricidal) than of group-reared ones. A similar trend was apparent in the hippocampus. No significant differences were observed between muricidal and non-muricidal animals. The results are discussed in terms of the involvement of the endogenous ligands of the GABA-benzodiazepine receptor complex in the integrations related to mood and behaviour.

Isolation-induced changes in radioligand binding to benzodiazepine binding sites.

Binding of [3H]flunitrazepam was studied in brain tissues of isolated Wistar rats and compared to group-reared animals. Modifications were observed in hippocampus and cortex (Kd increased) and in cerebellum (Bmax decreased) and when brain sections of control rats were incubated in the bath fluid that had served to incubate sections from isolated rats, a flattening of the saturation curve was observed. Results are discussed in terms of possible modulators of benzodiazepine binding sites, mainly tissue GABA concentrations.

Benzodiazepine receptor and neurotransmitter studies in the brain of suicides.

The characteristics of benzodiazepine binding sites (affinity, number heterogeneity) were studied on frozen sections of hippocampus of 7 suicides and 5 controls subjects, using biochemical and autoradiographic techniques. 3H flunitrazepam was used as ligand, clonazepam and CL 218,872 as displacing agents. Some neurotransmitters or their derivatives (GABA, catecholamines, hydroxy-indols) were evaluated quantitatively in parallel in the hippocampal tissue by liquid chromatography. We observed mainly an increase in the Ki of CL 218,872 subtype I binding sites in suicides, (7.48 +/- 1.7 to 17.24 +/- 1.7 nM, P less than 0.01), (m +/- SEM) and an increase in % of type I binding sites (30 +/- 4.2 to 42 +/- 2.5, P = 0.01). Among neurotransmitters, only norepinephrine differed significantly between controls and suicides (11.34 +/- 1.9 to 24.34 ng/g tissue, P = 0.02).

[Benzodiazepine receptors in the hippocampus of suicides]. / Etude des récepteurs des benzodiazépines dans l'hippocampe de suicidés.

The characteristics of benzodiazepine binding sites (distribution, number, affinity) were defined on frozen sections of suicide's hippocampus (death by hanging) labeled with 3H flunitrazepam and compared to data previously obtained on control brains. The study was carried out qualitatively by autoradiography (distribution) and quantitatively by a biochemical technique (number and affinity). As a whole, the characteristics of BZD binding sites were not modified in relation to controls, except for a very slight decrease in affinity of subtype I.

Autoradiographic and quantitative study of benzodiazepine-binding sites in human hippocampus.

Benzodiazepine-binding sites were studied on frozen sections of 5 human hippocampi, using autoradiographic and biochemical techniques. The affinity, density, distribution and heterogeneity (two types) of sites were investigated using [3H]flunitrazepam as a ligand, clonazepam or C1 218872 as displacing agents. The autoradiographic images evidence a differential distribution of the binding sites in the histologic layers of the hippocampus. Subtypes I and II coexist in the same proportion in the three layers exhibiting the highest densities of binding sites (stratum granulosum and pyramidale, deep layer of stratum radiatum). The Kd, Bmax and Ki values found here are analogous to those described in animal studies, but the anatomical distribution of the sites in human hippocampus seems to differ slightly from that previously described in that of the rat.

Anatomical distribution and characteristics of [(3)H]muscimol high affinity binding sites in the dorsolateral pontine tegmentum of the rat brain.

The pharmacological characterization and fine anatomical distribution of [(3)H]muscimol high affinity binding sites in the dorsolateral pontine tegmentum (region of locus coeruleus) of the rat was established using biochemical and autoradiographic techniques. On crude synaptosomal membranes obtained from microsamples of locus coeruleus, as well as on frozen sections of the dorsolateral pontine tegmentum, [(3)H]muscimol binding was saturable, displaced by 80-90% with 10(?5) M GABA or 5.10(?4) M (+) bicuculline, but unchanged in the presence of 10(?4) M nipecotic acid. Analysis of the saturation curve suggested the existence of one population of high affinity binding sites with K(d) = 10.5 nM, B(max) = 2.15 pmol/mg of crude synaptosomal membrane proteins, and K(d) = 14.3 nM, B(max) = 0.36 pmol/mg proteins on frozen tissue sections. Autoradiographic data on (3)H ultrofilms evidenced low densities of sites in this region compared to cerebellum or cortex as stated by previous authors. The highest densities of sites were observed in nuclei locus coeruleus and cuneiform, and in central grey. Dorsal and ventral parabrachial, ventral tegmental, motor trigeminal and superior vestibular nuclei exhibited slightly lower densities of sites, and brachium conjunctivum and medial longitudinal fasciculus presented no binding sites. In addition, light microscopy autoradiographic observations revealed [(3)H]muscimol binding sites between the structures previously quoted. In the locus coeruleus, [(3)H]muscimol binding sites were not strictly superimposed with cell bodies. These data are discussed in relation with the distribution of GABAergic terminals in the various areas of the dorso-lateral pontine tegmentum.

Culture of rat locus coeruleus explants: a model to study transneuronal regulation of tyrosine hydroxylase in central noradrenergic neurons.

A culture method is described to maintain explants of rat locus coeruleus in vitro for several days. Both anatomical and biochemical methods were used to control the evolution of the cultures with time. Explants were first examined 7 days after culture by immunohistochemistry with specific antisera directed against enzymes specific for non-neuronal or neuronal cells. Non-neuronal enolase immunoreactive cells were observed throughout the explants except at their edge where outgrowth zones were observed. Neuronal cells which were separately stained by three markers (neuron-specific enolase, tyrosine hydroxylase and dopamine-?-hydroxylase antisera) share common morphological features: they are grouped in clusters; their soma have similar shape and size and the staining was always seen inside the cytoplasm of the cell bodies and their proximal processes. These results show that noradrenergic neurons are still present in the explants 7 days after culture. Biochemical characteristics of the explants were followed throughout culture. Non neuronal enolase activity doubled during the first week in vitro. In contrast neuron specific enolase activity decreased to about half its initial value. Similarly the amount of 1-[(3)H]noradrenaline taken up in the explants decreased abruptly during the two first days in culture to reach a stable value. Tyrosine hydroxylase activity decreased significantly to about a third its initial value. All biochemical markers became stable after 7 days in culture. The cholinergic control of tyrosine hydroxylase in the noradrenergic cells has been investigated using 1-week old cultures. Tyrosine hydroxylase activity in locus coeruleus explants incubated in the presence of oxotremorine (10(?5) M, during 24 h) is significantly increased (+108%) as compared to non treated tissues. This increase was abolished if atropine sulfate (10(?5) M) was present together with oxotremorine. Immunotitrations of tyrosine hydroxylase in homogenates of the explants treated with the cholinergic agonist revealed that the increased enzyme activity is due to a change in its catalytic properties without variation in the number of enzyme molecules (delayed activation). This experiment indicates that an activation of tyrosine hydroxylase can result from the stimulation of cholinergic receptors located in the locus coeruleus region. The present investigation shows that central noradrenergic neurons of the locus coeruleus region can survive in culture conditions. These neurons retain most of their in situ morphological and biochemical characteristics. This in vitro approach appears to be an interesting means to study tyrosine hydroxylase regulation in central noradrenergic neurons.

Identification of catecholamine cell bodies in the pons and pons-mesencephalon junction of the cat brain, using tyrosine hydroxylase and dopamine-beta-hydroxylase immunohistochemistry.

Using a double labeling immunohistochemical method with antibodies to tyrosine hydroxylase and dopamine-beta-hydroxylase, we reexamined the topographical distribution of dopaminergic (DA) and noradrenergic (NA) cells in the pons-mesencephalon junction of the cat. Besides DA cells of SN and its extensions, DA cells were also observed more caudally in nucleus raphe linearis intermedius and dorsalis, decussation of brachium conjunctivum, fasciculus longitudinalis medialis and periaqueductal gray. In nucleus locus coeruleus, cells exhibited variable levels of DBH, but dopaminergic cells were not evidenced in this structure.

Immunohistochemical study of the catecholaminergic systems in the lower brain stem of the human infant.

The study of catecholaminergic (CA) systems in the lower brain stem was undertaken in the infant. A method of indirect immunohistofluorescence using antibodies to tyrosine-hydroxylase (TH) was applied to five brains from infants whose deaths had no neurological cause; two brains were submitted to fully systematic investigation. A topographical and morphocytological analysis of the CA systems was carried out on horizontal serial sections. A neuronal map is shown presenting four bulbar and four pontine sections. The disposition of the main CA groups (i.e. A1, A2, locus coeruleus) was found to be consistent with the data provided by the literature concerning the human foetus and the adult. Topographic differences and peculiarities are emphasized in the infant. Mapping of phenylethanolamine N methyltransferase (PNMT) positive neurons was performed in one plane of section of mid-medulla. This technique might usefully be applied to the exploration of putative diseases of bulbopontine CA systems, such as the sudden infant death syndrome and central blood pressure disorders.

[Relation between dopamine and serotonin motor effects on isolated rat duodenum. Possible participation of an excitatory dopaminergic receptor]. / Relations entre les effets moteurs de la dopamine et de la sérotonine sur le duodénum isolé de rat. Participation possible d'un récepteur dopaminergique excitant.

The experimental facts presented here allow us to establish a relationship between DA motor effects on isolated rat duodenum, studied in a previous work, (excito-motor between 10(-9) M and 10(-5) M and inhibitor beyond 10(-5) M) and the functional aptitude of this duodenum to answer to 5-HT : 5-HT excito-motor effects are amplified by DA at concentrations less than or equal to 2.6 X 10(-6) M and on the contrary antagonized by DA at concentrations above 5 X 10(-6) M. On the other hand, DA excito-motor effects do not appear on 5-HT-desensitized duodenums. The study of 5-HT dose-response curves in the presence of different concentrations of DA suggests that the amplification of serotonergic effects observed with small DA concentrations can be possibly related to the additional effects of 5-HT on its own receptors and of DA on another receptor. The receptor involved in DA excito-motor action seems to be closely related to the 5-HT receptor since its effects are blocked by the same molecules (methylsergide, cyproheptadine) and since DA excito-motor action is no more evident on 5-HT-desensitized duodenums, but several arguments support the specificity of this receptor for DA (though it is not blocked by haloperidol). Some of its effects are in line with those of DA receptors made conspicuous in other territories and not blocked by haloperidol : central and human colon receptors. On the contrary, DA used at high concentrations (greater than or equal to 5 X 10(-6) M) seems to be able to occupy serotonergic sites, preventing 5-HT access to its receptors until its concentration is high enough to displace DA. From a functional point of view, this antagonistic effect of DA at high concentrations against 5-HT excito-motor effects could facilitate alpha- and beta-mediated inhibitory effects.