Caloric intake and hypothalamic neurotransmitters in Zucker rats made acutely diabetic with streptozocin.

Zucker rats, lean and obese, treated with low dose intraperitoneal injections of streptozocin become hyperglycemic within 24h. Insulin levels fall, although the obese animal remains hyperinsulinemic. Associated with these changes in glucose and insulin there are transient decreases in caloric intake. Macronutrient selection studies show that protein consumption decreases. There is a trend for fat intake to decrease. The levels of hypothalamic neurotransmitters in the lean animals are not altered by streptozocin. The levels of 5-hydroxyindoleacetic acid increases in the streptozocin-treated obese animal in the paraventricular region, ventromedial region and the raphe. Serotonin is also significantly increased in the paraventricular region of the obese rat. These results suggest that acutely, treatment with streptozocin injures pancreatic islets, causing, in turn, decreases in insulin levels so that hyperglycemia ensues in both phenotypes. Associated with these perturbations are decreases in caloric intake. The magnitude of change in insulin levels is much greater in the obese rat. It is hypothesized that in the obese Zucker rat decrements in food intake are mediated by increase in serotonin turnover in the hypothalamus and these changes are related to changes of insulin levels. These data support the concept that circulating insulin affects hypothalamic neurotransmitters.

Fos induction in selective hypothalamic neuroendocrine and medullary nuclei by intravenous injection of urocortin and corticotropin-releasing factor in rats.

CRF and urocortin, administrated systemically, exert peripheral biological actions which may be mediated by brain pathways. We identified brain neuronal activation induced by intravenous (i.v.) injection of CRF and urocortin in conscious rats by monitoring Fos expression 60 min later. Both peptides (850 pmol/kg, i.v.) increased the number of Fos immunoreactive cells in the paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala, nucleus tractus solitarius and area postrema compared with vehicle injection. Urocortin induced a 4-fold increase in the number of Fos-positive cells in the supraoptic nucleus and a 3.4-fold increase in the lateral magnocellular part of the paraventricular nucleus compared with CRF. Urocortin also elicited Fos expression in the accessory hypothalamic neurosecretory nuclei, ependyma lining the ventricles and choroid plexus which was not observed after CRF. The intensity and pattern of the Fos response were dose-related (85, 255 and 850 pmol/kg, i.v.) and urocortin was more potent than CRF. Neither CRF nor urocortin induced Fos expression in the lateral septal nucleus, Edinger-Westphal nucleus, dorsal raphe nucleus, locus coeruleus, or hypoglossal nucleus. These results show that urocortin, and less potently CRF, injected into the circulation at picomolar doses activate selective brain nuclei involved in the modulation of autonomic/endocrine function; in addition, urocortin induces a distinct activation of hypothalamic neuroendocrine neurons.

(+) 3,4-methylenedioxymethamphetamine ('ecstasy') transiently increases striatal 5-HT1B binding sites without altering 5-HT1B mRNA in rat brain.

(+) 3,4-Methylenedioxymethamphetamine (MDMA) is a psychedelic drug of abuse that causes selective degeneration of serotonergic fibers of dorsal raphe neurons that project throughout the forebrain. Previous studies using pharmacological and behavioral approaches suggested that MDMA treatment leads to desensitization of 5-HT1B receptors. We proposed to test whether this occurs by downregulation of 5-HT1B messenger RNA in dorsal raphe, striatum or CA1 hippocampal neurons and/or 5-HT1B binding site density in hippocampus and basal ganglia. In Experiment I, rats were treated with MDMA using several dosing protocols (2.5 or 10 mg kg-1 day-1 s.c. given a single time or twice daily for 4 days). The animals were killed 24 h after the last dose. [3H]-citalopram binding to serotonin transporters in hippocampus was reduced in the high dose protocol, indicating degeneration of forebrain serotonergic fibers. Despite the extensive reduction in serotonergic content, 5-HT1B mRNA did not change from control levels in any region when measured by in situ hybridization. [125I]-Iodocyanopindolol binding to 5-HT1B sites in hippocampus was also not changed. In Experiment II, high dose MDMA had no effect on 5-HT1B mRNA in any brain region either 1 or 14 days after treatment. However, [125I]-iodocyanopindolol binding more than doubled in striatum 1 day after MDMA treatment but returned to control levels by 14 days. This may have been a transient compensation to early neuronal damage caused by MDMA exposure. These results suggest that previously described changes in 5-HT1B function following MDMA treatment involve only posttranscriptional changes in receptor regulation and do not alter 5-HT1B mRNA levels.

Altered serotonin innervation patterns in the forebrain of monkeys treated with (+/-)3,4-methylenedioxymethamphetamine seven years previously: factors influencing abnormal recovery.

The recreational drug (+/-)3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") is a potent and selective brain serotonin (5-HT) neurotoxin in animals and, possibly, in humans. The purpose of the present study was to determine whether brain 5-HT deficits persist in squirrel monkeys beyond the 18-month period studied previously and to identify factors that influence recovery of injured 5-HT axons. Seven years after treatment, abnormal brain 5-HT innervation patterns were still evident in MDMA-treated monkeys, although 5-HT deficits in some regions were less severe than those observed at 18 months. No loss of 5-HT nerve cell bodies in the rostral raphe nuclei was found, indicating that abnormal innervation patterns in MDMA-treated monkeys are not the result of loss of a particular 5-HT nerve cell group. Factors that influence recovery of 5-HT axons after MDMA injury are (1) the distance of the affected axon terminal field from the rostral raphe nuclei, (2) the degree of initial 5-HT axonal injury, and possibly (3) the proximity of damaged 5-HT axons to myelinated fiber tracts. Additional studies are needed to better understand these and other factors that influence the response of primate 5-HT neurons to MDMA injury and to determine whether the present findings generalize to humans who use MDMA for recreational purposes.

Serotonin transporter (SERT) mRNA and binding site densities in male rat brain affected by sex steroids.

Estrogen increases serotonin transporter (SERT) mRNA and binding sites in female rat brain. In order to determine whether changes in SERT are gender- and steroid-specific we have now carried out studies on adult male Wistar rats which were either intact or castrated (under halothane anesthesia) and injected with arachis oil, estradiol benzoate (EB), testosterone propionate (TP) or the non-aromatizable androgen, 5alpha-dihydrotestosterone (5alpha-DHT). The number of SERT mRNA-expressing cells in the dorsal raphe (DR) nucleus was decreased by castration and increased by treatment (for approximately 32 h) with EB or TP, but not 5alpha-DHT. Sex steroids had no effect on the number of SERT mRNA-expressing cells in the median raphe nucleus. The density of SERT sites, assessed by autoradiography of [3H]paroxetine binding, was significantly reduced in arcuate nucleus and median raphe after castration, and increased in arcuate, basolateral amygdala and ventromedial hypothalamic nucleus by treatment with EB or TP, but not 5alpha-DHT. Estradiol, but not testosterone or 5alpha-DHT reduced the density of SERT sites in midbrain central grey. These data show that testosterone as well as estrogen affects SERT expression in male brain, and that the action of testosterone probably depends upon its enzymatic conversion, by aromatase, to estradiol. Our findings may have implications for sex steroid control of mood and behavior, and the action of neurotoxic derivatives of amphetamine, such as 3, 4-methylenedioxymethamphetamine, in the human.

Ovarian steroid action on tryptophan hydroxylase protein and serotonin compared to localization of ovarian steroid receptors in midbrain of guinea pigs.

The effect of estrogen (E) and progesterone (P) on the protein expression of the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase (TPH), and the level of serotonin in the hypothalamic terminal field was examined in guinea pigs. In addition, we questioned whether serotonin neurons of guinea pigs contain ovarian steroid receptors (estrogen receptoralpha[ERalpha], estrogen receptor beta[ERbeta], progestin receptors [PRs]) that could directly mediate the actions of E or P. Western blot and densitometric analysis for TPH were used on raphe extracts from untreated-ovariectomized (OVX), OVX-E-treated (28 d), and OVX-E+P-treated (14 d E+14 d E+P) guinea pigs. The medial basal hypothalami from the same animals were extracted and subjected to high-performance liquid chromatography analysis for serotonin, dopamine, 5-hydroxyindole acetic acid, and homovanillic acid. The brains from other animals treated in an identical manner were perfusion fixed and examined for the colocalization of ERalpha plus serotonin and PR plus serotonin with double immunohistochemistry or for expression of ERbeta mRNA with in situ hybridization. E and E+P treatment significantly increased TPH protein levels compared to the untreated control group (p < 0.05), but TPH levels were similar in the E and E+P-treated groups. By contrast, serotonin (nanogram/milligram of protein) in the hypothalamus was significantly increased by E+P treatment, but not by E alone. Neither ERalpha nor PR proteins were detected within serotonin neurons of the guinea pig raphe nucleus. However, ERbeta mRNA was expressed in the dorsal raphe. In summary, E alone increased TPH protein expression and the addition of P had no further effect, whereas E+P increased hypothalamic serotonin and E alone had no effect. The localization of ERbeta, but not ERalpha or PR, in the dorsal raphe nucleus suggests that E acting via ERbeta within serotonin neurons increases expression of TPH, but that P acting via other neurons and transsynaptic stimulation may effect changes in TPH enzymatic activity, which in turn, would lead to an increase in serotonin synthesis.

Immunocytochemical localization of serotonin1A receptors in the rat central nervous system.

Specific anti-rat 5-hydroxytryptamine1A (serotonin1A) receptor antibodies raised in a rabbit injected with a synthetic peptide corresponding to a highly selective portion of the third intracellular loop of the receptor protein (El Mestikawy et al. [1990] Neurosci. Lett. 118:189-192) were used for immunohistochemical mapping of serotonin1A receptors in the brain and spinal cord of adult rats. The highest density of immunostaining was found in limbic areas (lateral septum, CA1 area of Ammon's horn and dentate gyrus in the hippocampus, and frontal and entorhinal cortices), in the anterior raphe nuclei, and in the interpeduncular nucleus, in agreement with previous autoradiographic studies with selective radioligands showing the enrichment of these regions in serotonin1A receptor binding sites. Serotonin1A receptor-like immunoreactivity was also present, but at a moderate level, in the neocortex, in some thalamic and hypothalamic nuclei, in the nucleus of the solitary tract, in the dorsal tegmentum, in the nucleus of the spinal tract of the trigeminal nerve, and in the superficial layers of the dorsal horn in the spinal cord. In contrast, extrapyramidal areas, including the caudate putamen, the globus pallidus, and the substantia nigra as well as the cerebellum, exhibited very low to no immunostaining by antiserotonin1A receptor antibodies. At the cellular level, both the plasma membrane of neuronal perikarya and fine neuronal processes probably corresponding to dendritic fields were found to bind antiserotonin1A receptor antibodies. Regional differences were noted regarding these two types of immunostaining, because only dendrites bound antibodies within the hippocampus and the lateral septum, whereas both dendrites and neuronal cell bodies were immunoreactive in the medial septum, in the diagonal band of Broca, and in the dorsal and median raphe nuclei. Therefore, differential addressing of serotonin1A receptors could occur from one neuron to another. In general, the distribution and density of serotonin1A receptor-like immunoreactivity in the whole brain and in spinal cord were consistent with the mapping of serotonin1A receptor binding sites and serotonin1A receptor mRNA previously established by immunoautoradiographic and in situ hybridization procedures.

Changes in serotonin-immunoreactivity in the dorsal and median raphe nuclei of rats exposed to 2,4-dichlorophenoxyacetic acid through lactation.

Comparison of serotonin-immunoreactive (SER-IR) neurons in nucleus raphe dorsalis (NRD) and median raphe nucleus (MRN) of 25-d-old rat pups exposed to 70 mg/kg/d 2,4-dichloro-phenoxyacetic acid through mothers milk and control pups was made using an immunohistochemical analysis. Significant 2,4-D-treatment-related increase in size and density of SER-IR neuronal somata as well as in fiber length were observed. We postulate that exposure to 2,4-dichlorophenoxyacetic acid on the first day of life would modify the synthesis of 5-HT or the maturation of the brain serotonergic system.

Dietary carbohydrates: effects on self-selection, plasma glucose and insulin, and brain indoleaminergic systems in rat.

The aim of the study was to investigate the effect of different dietary carbohydrates such as corn starch, sucrose, fructose and glucose on carbohydrate and protein self-selection and on arterial and venous concentrations of glucose and insulin, and brain indoleamines in rats. Fructose and sucrose feeding induced the lowest food intakes which were due respectively to a lower carbohydrate and protein selection. The present data showed that feeding with dietary glucose as the main carbohydrate source gave the highest glycemic response, the lowest one being found with fructose and corn starch, and an intermediate one with sucrose feeding. The insulin response to the dietary carbohydrates followed a somewhat different pattern with the highest insulin secretion observed after fructose feeding whereas highly variable and inconsistent results were obtained following corn starch, sucrose and glucose feeding. Feeding chemically different sugars was also characterized by decreased serotonin synthesis in the raphe nuclei, brainstem and thalamus, and increased 5-HT synthesis in the hypothalamus of rats fed fructose when compared to glucose fed animals. The present results highlight the importance of considering the nature of dietary carbohydrates in the regulation of feeding.

The mouse 5-hydroxytryptamine1B receptor is localized predominantly on axon terminals.

The 5-hydroxytryptamine1B receptor is a serotonin receptor subtype which is expressed predominantly in the basal ganglia. It has been suggested to play a role in movement and appetite control as well as in certain pathological states such as migraine. The recent cloning of the 5-hydroxytryptamine1B gene as well as the discovery of a radioligand that labels in rodents 5-hydroxytryptamine1B and possibly 5-hydroxytryptamine1D alpha receptors (S-CM-G[125I]TNH2) allowed us to compare the distribution of the messenger RNA and of the protein in mouse brain sections. A high 5-hydroxytryptamine1B messenger RNA level is found in the caudate-putamen in medium spiny neurons that project to the globus pallidus and the substantia nigra. In contrast, no messenger RNA is expressed in the globus pallidus and substantia nigra although these structures reveal the highest level of 5-hydroxytryptamine1B binding sites. In the hippocampus, 5-hydroxytryptamine1B messenger RNA is localized in the cell bodies of pyramidal cells of the CA1 field while the protein is found predominantly in the dorsal subiculum, a projection zone for the CA1 pyramidal neurons. In the cerebellum, 5-hydroxytryptamine1B messenger RNA is expressed in the Purkinje cells, which display no receptor binding sites. Conversely, moderate binding is found in the deep nuclei of the cerebellum, the main projection zone of the Purkinje cells. 5-Hydroxytryptamine1B sites are also detected in the superficial gray layer of the superior colliculus and the lateral geniculate nucleus, brain regions containing the terminals of retinal ganglion cells. The soma of these ganglion cells express high levels of 5-hydroxytryptamine1B messenger RNA while no 5-hydroxytryptamine1B binding sites were found in the retina. This study demonstrates that the main brain regions, expressing 5-hydroxytrypamine1B messenger RNA contain low densities of 5-hydroxytryptamine1B binding sites. Conversely, the major projection areas of these anatomical structures do not express detectable levels of 5-hydroxytryptamine1B messenger RNA, but present a high density of binding sites. In addition, our data suggest that the distribution of the 5-hydroxytryptamine1D alpha binding sites is different from that of the 5-hydroxytryptamine1D alpha messenger RNA. These results together with previous lesion studies, indicate that the 5-hydroxytryptamine1B and possibly the 5-hydroxytryptamine1D alpha receptors are localized predominantly on axon terminals, while their expression is low or absent at the somatodendritic level. The 5-hydroxytryptamine1D alpha proteins might therefore contain an addressing signal allowing their transport toward nerve endings.(ABSTRACT TRUNCATED AT 400 WORDS)

Central nervous system innervation of the penis as revealed by the transneuronal transport of pseudorabies virus.

Transneuronal tracing techniques were used in order to identify putative spinal interneurons and brainstem sites involved in the control of penile function. Pseudorabies virus was injected into the corpus cavernosus tissue of the penis in rats. After a four day survival period, rats were perfused with fixative and virus-labelled neurons were identified by immunohistochemistry. Postganglionic neurons were retrogradely labelled in the major pelvic ganglia. In the spinal cord, sympathetic and parasympathetic preganglionic neurons were labelled transneuronally. Presumptive interneurons were also labelled in the lower thoracic and lumbosacral spinal cord in locations consistent with what is currently known about such interneurons. In the brainstem, transneuronally labelled neurons were found in the medulla, pons and hypothalamus. Regions consistently labelled included the nucleus paragigantocellularis, parapyramidal reticular formation of the medulla, raphe pallidus, raphe magnus, A5 noradrenergic cell group, Barrington's nucleus and the paraventricular nucleus of the hypothalamus. This study confirmed previous studies from our lab and others concerning the preganglionic and postganglionic neurons innervating the penis. The number, morphology and location of these neurons were consistent with labelling seen following injection of conventional tracers into the penis. The brainstem nuclei labelled in this study were also consistent with what is currently known about the brainstem control of penile function. The labelling appeared to be highly specific, in that descending systems involved in other functions were not labelled. These results provide further evidence that the pseudorabies virus transneuronal tracing technique is a valuable method for identifying neural circuits mediating specific functions.

Expression of c-fos protein in serotonergic neurons of rat brainstem following electro-acupuncture.

The c-fos proto-oncogene encodes a nuclear phosphoprotein, Fos which has been proposed to be a "third messenger" coupling short term extracellular signals to long term alteration in cell function. Using double labeling immunocytochemistry, the present work demonstrated the co-localization of Fos protein and serotonin in the nucleus raphe dorsalis, nucleus raphe centralis superior and rostral ventromedial medulla. The results pose an interesting problem, the possible relation of Fos protein to the biosynthesis of serotonin, awaiting further investigation.

Regional distribution of extracellular 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the brain of freely moving rats.

The extracellular concentrations of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) have been determined in six brain areas of awake rats (frontal cortex, striatum, hypothalamus, hippocampus, inferior colliculus, and raphe nuclei) using intracerebral microdialysis. The extracellular levels of 5-HT showed no significant differences among the brain regions studied. The tissue levels of 5-HT and 5-HIAA as well as the extracellular concentration of 5-HIAA were significantly higher in raphe nuclei. The regional distribution of tissue and extracellular 5-HIAA were very similar, suggesting that extracellular 5-HIAA depends mainly on the output from the intracellular compartment. On the other hand, extracellular 5-HT and tissue 5-HT showed a different distribution pattern. The tissue/extracellular ratio for 5-HT ranged from 739 in frontal cortex to 2,882 in raphe, whereas it only amounted to 1.8-3.6 for 5-HIAA. The relationship between the present results and the density of 5-HT uptake sites in these areas is discussed.

Detection of the release of 5-hydroxyindole compounds in the hypothalamus and the n. raphe dorsalis throughout the sleep-waking cycle and during stressful situations in the rat: a polygraphic and voltammetric approach.

In the present work, voltammetric method combined with polygraphic recordings were used in animals under long-term chronic conditions; the extracellular concentrations of 5-hydroxyindole compounds (5-OHles) and in particular 5-hydroxyindoleacetic acid (5-HIAA) were measured in the hypothalamus and in the nucleus Raphe Dorsalis (n.RD). The hypothesis that extracellular detection of 5-HIAA, in animals under physiological conditions, might reflect serotonin (5-HT) release is suggested by the following observations: serotoninergic neurons are reported to contain only monoamine oxidase type B (MAO-B);--an inhibitor of such an enzyme, MDL 72145 (1 mg/kg), fails to decrease the extracellular 5-HIAA peak 3 height:--MAO type A is contained in non-5-HT cells or neurons;--only the inhibitor of this last type of enzyme (Clorgyline 2.5 mg/kg) induces a complete disappearance of the voltammetric signal. The 5-HIAA measured in the extracellular space thus comes from the 5-HT released and metabolized outside the 5-HT neurons. Throughout the sleep-waking cycle, 5-OHles release occurs following two different modes: 1--during sleep, in the vicinity of the 5-HT cellular bodies in the n.RD; this release might come from dendrites and be responsible for the 5-HT neuronal inhibition occurring during sleep; 2--during waking, at the level of the axonal nerve endings impinging on the hypothalamus; this release might be related to the synthesis of "hypnogenic factors". Finally, we have observed that in the hypothalamus, 30 min. of immobilization-stress (IS) induces a larger increase of the voltammetric signal (+80%) than a painful stimulation of the same duration (+30%); the possible link between the 5-OHles release occurring in this area during an IS and the subsequent paradoxical sleep rebound is discussed.

The metabolism of exogenous L-dopa in the brain: an immunohistochemical study of its conversion to dopamine in non-catecholaminergic cells of the rat brain.

The characterization and localization of non-catecholaminergic cells producing dopamine after L-Dopa load have been investigated in the normal rat brain by a direct immunohistochemical labelling of amines using specific antibodies. The detection of dopamine-containing non-catecholaminergic cells has been achieved in rats given a commonly used mixture of L-Dopa plus peripheral decarboxylase inhibitor, and compared to controls. Results indicate that serotoninergic neurons tend toward a switch of their metabolism into dopamine production after L-Dopa load in a dose-dependent manner. In addition small non-aminergic cells, identified as aromatic amino-acid decarboxylase-containing cells, were observed to produce dopamine after exogenous L-Dopa load. Possible implications of such results concerning the mode of action of L-Dopa in the brain are discussed.