Immunocytochemical detection of acetylcholine in the rat central nervous system.

A specific antibody to acetylcholine was raised and used as a marker for cholinergic neurons in the rat central nervous system. The acetylcholine conjugate was obtained by a two-step immunogen synthesis procedure. An enzyme-linked immunosorbent assay was used to test the specificity and affinity of the antibody in vitro; the results indicated high affinity. A chemical perfusion mixture of allyl alcohol and glutaraldehyde was used to fix the acetylcholine in the nervous tissue. Peroxidase-antiperoxidase immunocytochemistry showed many acetylcholine-immunoreactive cells and fibers in sections from the medial septum region.

Participation of serotonin in thyrotropin release. II. Evidence for the action of serotonin on the phasic release of thyrotropin.

During a 12-h light 12-h dark schedule (lights off at 1900 h), male Sprague-Dawley rats show a circadian rhythm of plasma TSH with a zenith near midday. The participation of serotonin (5HT) in the phasic release of TSH was studied using both pharmacological and surgical-stereotaxical approaches. Animals treated with parachlorophenylalanine methyl ester (pCPA), an inhibitor of 5HT synthesis (one or two injections of 250 mg/kg each) showed a reduction or a disappearance of the diurnal peak of TSH, respectively. Additional treatment by 5-hydroxytryptophan, a precursor of 5HT, completely, restored the diurnal TSH peak. Treatment with 5,6-dihydroxytryptamine creatine sulfate, a neurotoxin which selectively destroys 5HT terminals, also induced alterations of the diurnal peak of TSH. There were no major modifications observed in the low nocturnal levels of TSH in rats treated with pCPA, 5-hydroxytryptophan, or 5,6-dihydroxytryptamine. The major serotoninergic innervation of the hypothalamus originates from the raphe dorsalis or centralis; destruction of these two nuclei caused a quasiabolition of the diurnal TSH peak (only a low amplitude TSH circadian rhythm persisted). Hypothalamic 5HT content was measured in the majority of these experiments; the greatest depletions (near 90%) were observed after two injections of pCPA or in rats bearing raphe lesions. We conclude that the diurnal peak of TSH, observed during the physiological circadian rhythm, is serotoninergic dependent.

One perfusion mixture for immunocytochemical detection of noradrenaline, dopamine, serotonin and acetylcholine in the same rat brain.

A perfusion mixture composed of glutaraldehyde and allyl-alcohol at pH 12 is compatible for the fixation and immunocytochemical detection of noradrenaline, dopamine, serotonin and acetylcholine in the same rodent central nervous system. A technical description of this procedure is provided to facilitate the replication of this immunocytochemical approach with these antibodies.

Serotonin and dopamine afferents to the rat locus coeruleus: a biochemical study after lesioning of the ventral mesencephalic tegmental-A10 region and the raphé dorsalis.

The monoamine levels in the locus coeruleus (LC) were determined by HPLC following specific lesions of the ventral mesencephalic tegmental-A10 regions (VMT-A10) and raphé dorsalis (RD). Only lesions in the VMT-A10 area decreased the dopamine (DA) content, which strongly suggests that the projection from this region to the LC is of dopaminergic nature. Lesions of the RD increased DA metabolism in the LC and provoked significant decreases in the serotonin (5-HT) levels.

Alteration of tyrosine hydroxylase activity in the locus coeruleus after administration of p-chlorophenylalanine.

The time course of the variations in tyrosine hydroxylase activity (THA) and serotonin (5-HT) content were measured in the rat locus coeruleus after parachlorophenylalanine (PCPA) administration. Highly significant decreases in the 5-HT content in LC were found 24-48 h after PCPA treatment (300 mg/kg daily). An increase in THA (in the LC) was found to be significant 4 days after 2 successive injections of PCPA and after 4 successive injections of the drug THA gradually increased, reaching a maximum around 4 days after the last injection. This maximum increase in THA was greatly reduced when 5-HTP was simultaneously administered with PCPA. These results join others which suggest that a serotonin-mediated mechanism could be one of the processes controlling noradrenaline metabolism in the locus coeruleus.

Genetic studies of daily variations of first-step enzymes of monoamines metabolism in the brain of inbred strains of mice and hybrids. I. Daily variations of tryptophan hydroxylase activity in the nuclei raphe dorsalis, raphe centralis and in the striatum.

A daily variation of tryptophan hydroxylase (TrH) activity was observed in the raphe dorsalis (RD), raphe centralis (RC) and striatum (St) of 3 inbred strains of mice (BALB/c, C57BL/6, C57BR) and of the reciprocal hybrids obtained from Balb/c and C57BL6. Significant differences of the characteristics of these rhythms have been found in the same strain between different structures and for the same structure between different strains. In RD and RC hybridization led to less defined daily variations which, in the striatum, remained well synchronized and could be controlled by a dominant genetic mechanism part. These results help to discuss evidence for selective control mechanisms of regulation responsible for daily variation of TrH in the 5-HT cell bodies and terminals and their relative independence.

Genetic studies of daily variations of first-step enzymes of monoamines metabolism in the brain of inbred strains of mice and hybrids. II. Daily variations of tyrosine hydroxylase activity in the locus coeruleus.

Daily variations of tyrosine hydroxylase (TH) activity in the locus coeruleus of 3 inbred strains of mice (BALB/c; C57BL6; C57Br) and the F1 hybrids obtained from BALB/c and C57BL/6 are discussed. Precise characteristics of the circadian rhythms were observed in each strain. They were found significantly different in two genetically pure parents (BALB/c and C57BL6). In their two F1 hybrids the daily variation of TH activity was similar to that observed in one of the parents (C57BL6). This strongly suggests selective and genetically controlled mechanisms of regulation responsible for the daily variation of TH activity in the locus coeruleus of mice.

Influence of ascending serotonergic pathways on glucose use in the conscious rat brain. I. Effects of electrolytic or neurotoxic lesions of the dorsal and/or median raphé nucleus.

The regional cerebral metabolic effects of manipulations of the central serotonergic pathways are largely unknown. To address this topic, we have examined the consequences of electrolytic lesions of the rostral (median and/or dorsal) raphé nuclei on local cerebral glucose utilization (CMRglu) in the unanaesthetized rat brain. These studies were complemented by comparing control rats to rats that received prior intraventricular administration of the serotonergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). CMRglu was determined in 56 neuroanatomically defined regions of the central nervous system in lightly restrained rats, by the quantitative autoradiographic 2-deoxyglucose technique. In all, 6 groups of rats were studied: sham-lesioned rats, rats with electrolytic lesion of the median, dorsal, or both these raphé nuclei; sham-injected and 5,7-DHT pretreated rats. The efficacy of both electrolytic and neurotoxic lesions was verified, in each animal, by neurochemical microassay of 5-hydroxytryptamine and its metabolite in samples of striatum, hippocampus and prefrontal cortex. Chronic interruption of serotonergic transmission was remarkable for the lack of resultant change in CMRglu. In rats that were subjected to electrolytic lesions of both median and dorsal raphé nuclei, discrete and significant decreases in CMRglu were observed in the red nucleus, substantia nigra and inferior olivary nucleus only. The rats subjected to 5,7-DHT treatment displayed no significant changes in CMRglu in all the brain regions analyzed, despite an 80% decrease in the concentrations of endogenous 5-hydroxytryptamine. Thus, it would appear that a viable serotonergic transmission is not a major determinant of integrated functional activity, even in those brain structures that receive rich raphé projections. Two hypotheses are advanced for this lack of change: firstly, the chronic reduction of 5-hydroxytryptamine levels is accompanied by compensatory changes in this or other neurotransmitter systems; secondly, serotonergic neurones may exert a phasic--rather than tonic--influence on glucose use in the mammalian brain.

GABA-accumulating neurons in the nucleus raphe dorsalis and periaqueductal gray in the rat: a biochemical and radioautographic study.

The possibility of a GABAergic innervation of the nucleus raphe dorsalis (NRD) has been investigated by using the following approaches: (i) the identification of the principal neuronal groups afferent to the NDR by using horseradish peroxidase retrograde transport, (ii) the determination of glutamate decarboxylase activity (GAD) in the NRD after lesioning these groups or their putative pathways, and (iii) the radioautographic identification of terminals axons and nerve cells accumulating intraventricularly injected [3H]GABA. The hypothesis of a local GABAergic network is supported by the failure to obtain important changes in GAD after lesions of NRD afferents and the presence in this nucleus of terminals, fibers and nerve cell bodies accumulating [3H]GABA. It appears that these GABA-accumulating neurons could represent a portion of aperiventricular GABAergic system in the periaqueductal gray and the pontine ventricular gray.

Fetal noradrenergic transplants into amine-depleted basal forebrain nuclei restore drinking to angiotensin.

6-Hydroxydopamine-induced catecholamine denervations in the organum vasculosum of the lamina terminalis and the median preoptic nucleus attenuate drinking responses to systemic angiotensin II (ANG II) injections. Transplanting catecholamines in these nuclei using fetal noradrenergic (NE) cell suspension restores ANG II-elicited thirst. These results emphasize the functional importance of NE neuronal systems in nuclei of the anteroventral third ventricle (AV3V) in mediating ANG II-induced drinking behaviors.

Alterations in tyrosine hydroxylase activity elicited by raphe nuclei lesions in the rat locus coeruleus: evidence for the involvement of serotonin afferents.

The time course of the variations in tyrosine hydroxylase (TH) activity was measured in the rat locus coeruleus (LC) after lesions of the nucleus raphe dorsalis (NRD), nucleus raphe centralis superior (NRCS) and nucleus raphe pontis (NRP). A certain number of lesions were performed in the raphe magnus (RM), the caudal and rostral NRP and the caudal and rostral NRCS, lateral to raphe nuclei and in adrenalectomized animals. The serotonin (5-HT) content in the LC was also determined after these lesions. Only raphe nuclei producing significant decreases in the 5-HT content in the LC are successful in provoking increases in the TH activity in the LC, thus these results suggest that the noradrenaline (NA) synthesis in the LC may be regulated by 5-HT afferents. Moreover, intraventricular injections of 5,6-dihydroxytryptamine (5,6-DHT) and administration of parachlorophenylalanine (PCPA) also produce significant increases in TH in the LC. After immunotitrations of TH in the LC it was shown that, with exception of a high dose of 5,6-DHT (75 micrograms), all these treatments provoke an increase in the concentration of the enzyme. It therefore seems that one of the functional roles of 5-HT in the LC could be the regulation of the concentration of TH.

Viability of locus coeruleus cultures in the fourth ventricle of the adult rat.

Both tyrosine hydroxylase and dopamine-beta-hydroxylase immunocytochemistry indicated the survival of catecholamine neurons and processes in 7-day-old organotype cultures of the locus coeruleus. Cultures of this age were transplanted in the 4th ventricle of adult rats via the cisterna magna. Two months after transplantation microscopic observations revealed that the transplant developed with a defined neural organization. Following tyrosine hydroxylase immunocytochemistry, immunoreactive cells were identified within the transplants. These results indicate that organotype cultures of the locus coeruleus survive within the 4th ventricle of adult rats.

Correlation between the increase in tyrosine hydroxylase activity and the decrease in serotonin content in the rat locus coeruleus after 5,6-dihydroxytryptamine.

A correlation is demonstrated between the increase in tyrosine hydroxylase activity and the decrease in serotonin concentration in the rat locus coeruleus after various doses of 5,6-dihydroxytryptamine and gradual protection of serotonin neurons by pretreatment with two serotonin uptake inhibitors: fluoxetine or citalopram.

Pharmacological investigation on the role of dopamine in the rat locus coeruleus.

The role of dopamine (DA) in the rat locus coeruleus (LC) was investigated by determining the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), DA and noradrenaline (NA) in the LC after pharmacological treatments by pargyline, haloperidol, 6-hydroxydopamine (6-OHDA) and desmethylimipramine (DMI). The DA, DOPAC and NA contents of the LC were determined by high pressure liquid chromatography. Fifteen days after 6-OHDA, the DOPAC and NA levels were reduced by 60%, but they remained constant after 6-OHDA + DMI. Pargyline provoked highly significant increases in DA and NA but reduced DOPAC to non-measurable amounts. haloperidol caused a 54% decrease in the DOPAC levels. Pargyline and haloperidol administered to rats having received 6-OHDA + DMI 15 days before, caused similar effects on DA, DOPAC and NA levels as those in non-treated rats. It is suggested that DOPAC is mainly located in noradrenergic neurons, thus eliminating the possibility of a significant DA cell body population in the rat LC.

Adult age destruction versus neonatal age destruction of the serotonin system: two models to investigate the survival of serotonin neurons transplanted in adult rats.

Mesencephalic raphe nuclei were transplanted via the cisterna magna in the IVth ventricle of adult rats having received either a neonatal or an adult age destruction of the serotonin system. In neonatally treated rats both serotonin (5-HT) and glial fibrillary acidic protein (GFA) immunocytochemistry showed an absence of survival of 5-HT neurons in the transplants. However, many 5-HT immunoreactive cell bodies and processes were detected in 10-month-old transplants in adult treated animals. These results suggest that recognition processes between the target tissue and the implant may be a prerequisite for the survival of grafted neurons.

The viability of transplants of mesencephalic raphe nuclei in the IVth ventricle of the adult rat.

Mesencephalic raphe nuclei were transplanted via the cisterna magna near the locus coeruleus. The transplants were observed at different time intervals ranging from 24 h to 4 months. Microscopic observations revealed that from 3 weeks to 4 months the transplant developed with a well-defined neural organization. Radioautographic results showed that some of the neurons in the implant at 3 weeks captured and retained [3H] serotonin. These results indicate that the intact IVth ventricle seems to be a likely culturing floor for mesencephalic raphe nuclei.

Fetal serotoninergic transplants in the fourth ventricle of adult rodents reverse sleep disturbances induced by neonatal administration of 5,7-dihydroxytryptamine.

Rats received a neonatal (at 4 days of age) intracisternal injection of 5,7-dihydroxytryptamine which eliminates serotonin (5-HT) throughout adulthood. Sleep recordings, performed on these rats at adult age, demonstrated significant decreases in paradoxical sleep. Dissociated fetal 5-HT cell suspensions transplanted in the IVth ventricle of these rats restored paradoxical sleep. However, fetal neuronal noradrenergic or cholinergic transplants did not restore paradoxical sleep. These results suggest that paradoxical sleep is directly or indirectly mediated through serotoninergic mechanisms.

In vivo voltammetric measurement of extracellular 5-hydroxyindoleacetic acid in the denervated striatum after transplantation of mesencephalic raphe neurons.

In vivo differential pulse voltammetry with carbon fiber electrodes was used to monitor extracellular 5-hydroxyindoleacetic acid (5-HIAA) levels in a serotonergic denervated target area, the striatum, before and after transplantation of mesencephalic raphé nuclei into the lateral ventricle in the rat. The amplitude of the electrochemical signal recorded in the striatum on the transplanted side was found to be comparable to that measured in control striatum whereas the signal measured from the contralateral nontransplanted striatum was negligible. These findings suggest that mesencephalic raphé nuclei transplants establish connections with the host tissue.

Forebrain and brainstem afferents to the arcuate nucleus in the rat: potential pathways for the modulation of hypophyseal secretions.

Doxorubicin, an anti-oncogenic agent, was used as a retrograde marker to identify arcuate nucleus afferent projections. Injections of this tracer into the arcuate nucleus indicated that the subfornical organ, the organum vasculosum of the lamina terminalis, the nucleus raphe dorsalis and median raphe send projections to the arcuate nucleus. Immunocytochemical procedures were used to demonstrate that the raphe projections to the arcuate nucleus are serotoninergic. This anatomical investigation provides evidence that neural pathways exist between forebrain body fluid and mineral nuclei, mesencephalic serotonin nuclei and the arcuate nuclei.

Implantable microencapsulated dopamine (DA): a new approach for slow-release DA delivery into brain tissue.

Biodegradable controlled-release systems constitute an exciting new technology for drug delivery to the central nervous system (CNS). The present study describes functional and histochemical observations, indicating that implantation of DA microcapsules into striatal tissue assures a prolonged release of the transmitter in situ. This technology has considerable potential for basic and possibly also clinical research.