Regional brain concentrations of neuropeptides in Huntington's chorea and schizophrenia.

To ascertain whether Huntington's chorea and schizophrenia are associated with specific regional alterations in neurotensin, somatostatin, and thyrotropin-releasing hormone, the concentrations of these putative neurotransmitters were measured by radioimmunoassay in postmortem brain samples from patients with Huntington's chorea or schizophrenia. Compared to 50 patients without psychiatric or neurological disease, the patients with Huntington's chorea showed significantly elevated concentrations of all three neuropeptides in the nucleus caudatus. In the nucleus accumbens somatostatin levels were increased threefold, while in the amygdala thyrotropin-releasing hormone levels were elevated. In contrast, the schizophrenics exhibited reduced levels of thyrotropin-releasing hormone in two frontal cortical regions, reduced somatostatin levels in one frontal cortical area, and increased neurotensin levels in one frontal cortical area. None of the differences between the diseased brains and the controls could be accounted for by differences in age, sex, or time between death and autopsy.

The in vitro inhibition of insulin secretion by diphenylhydantoin.

Glucose intolerance has been observed following diphenylhydantoin (DPH) intoxication. Because of this association between DPH and hyperglycemia, the effect of DPH on insulin release in vitro using preparations of isolated islets of Langerhans and pancreatic pieces was examined. In concentrations identical with those considered necessary for adequate anticonvulsant therapy in man, DPH markedly decreases the insulin secretory response of pancreatic pieces to methacholine, 1 mug/ml, tolbutamide, 250 mug/ml, and glucose, 200 mg/100 ml, without any demonstrable alteration in the oxidative conversion of glucose-1-(14)C or glucose-6-(14)C to (14)CO(2) by isolated islets. This DPH-induced inhibition of insulin secretion is not reversed by higher concentrations of glucose (600 mg/100 ml) or by increasing concentrations of extracellular calcium ion (4-6 mmoles/liter). 0.1 mM potassium and 10(-4) M ouabain, however, effectively restore the DPH-induced block of insulin secretion in pancreatic pieces. 60 mM potassium ion, on the other hand, not only restores the insulin secretory response to glucose (200 mg/100 ml) but results in an added stimulation of insulin secretion in the presence of DPH. In the presence of DPH, (22)Na accumulation by isolated islets is decreased by 26-40% as compared with controls. Such evidence is considered to indirectly support the postulate that the electrophysiological properties of DPH on the pancreas are due to a stimulation of the membrane sodium-potassium-magnesium ATPase.

Releasing factors in the circumventricular organs in the rat brain.

With a recently developed microdissection technique, four circumventricular organs were removed from the rat brain, and their contents of LHRH and TRH were measured. The subfornical organ, the organon vasculosum lamia terminalis, the subcommissural organ, the area postrema all contained significant quantities of both releasing factors. The concentration of LHRH in the organon vasculosum (OVLT) was 14 ng/mg protein, 58% of that found in the median eminence. The concentration of LHRH in the remaining circumventricular organs ranged from 4.2 to 10.2 ng/mg protein. The concentration of TRH in these structures, however, was considerably lower, ranging from 0.7 to 1.8 ng/mg protein. The concentrations of LHRH and TRH in the tissue immediately adjacent to the organon vasculosum were nearly 50 times and 4 times less, respectively, than the concentration of these two releasing factors within the OVLT itself.

The effect of bilateral lesions of the ventral noradrenergic bundle on endocrine-induced changes of tyrosine hydroxylase in the rat median eminence.

With the microdissection method of Palkovits, individual hypothalamic nuclei were removed from the brains of adult male rats, and the tyrosine hydroxylase (TH) activity of each nucleus was determined 7 days after gonadectomy or thyroidectomy. Following gonadectomy, TH activity increased significantly in the median eminence with no change in the periventricular (NPV), arcuate (NARC), and dorsomedial nuclei (NDM), or medial forebrain bundle (MF). Following thyroidectomy, TH activity increased significantly in all 5 hypothalamic nuclei examined. Placement of bilateral lesions in the ventral norepinephrine bundles resulted in a greater than an 85% fall in the dopamine-beta-hydroxylase activity of the median eminence, arcuate nucleus, and ventromedial nucleus, but had no effect on tyrosine hydroxylase activity measured in those same areas. Furthermore, placement of such lesions had no effect on the endocrine-induced increases of TH activity found in the median eminence following gonadectomy and thyroidectomy, but did prevent the increase of TH activity found in the NPV, NDM, and MFB following thyroidectomy. Three conclusions appear to be justified: (a) noradrenergic axons which innervate the median eminence, arcuate, and ventromedial nuclei course in the ventral norepinephrine bundles; (b) the TH content of noradrenergic neurons in the median eminence, arcuate nucleus, and ventromedial nuclei is quite small; and (c) the majority, if not all, of the endocrine-responsive catecholaminergic neurons in the median eminence are dopaminergic.

Identification and immunocytochemical localization of a new thyrotropin-releasing hormone precursor in rat brain.

Antisera were raised to a tridecapeptide, Ser-Asp-Val-Thr-Lys-Arg-Gln-His-Pro-Gly-Arg-Arg-Phe, that was synthesized based on the sequence (residues 166-178) of a proposed cDNA for pro-TRH reported by Lechan et al. With this antiserum, immunostaining of Western blots of rat brain extracts revealed two major proteins with mol wt (Mr = 39,000 and 52,000) considerably larger than that of the largest protein (Mr = 29,000) that could be encoded by the cDNA of Lechan et al. Because these observations suggested the possibility of novel TRH precursors, we studied the immunocytochemical distribution of pro-TRH (39-52K) in rat brain. Our anatomical findings were 4-fold. 1) The distributions of 29K pro-TRH and 39-52K pro-TRH are not identical. 2) TRH is found only in regions containing 29K pro-TRH, 39-52K pro-TRH, or both. 3) There are regions that contain both 29K pro-TRH and 39-52K pro-TRH, but no TRH. 4) Regions containing only 39-52K pro-TRH do not contain 29K pro-TRH mRNA as mapped by Segerson et al. From these electrophoretic and anatomical observations, we postulate the existence of at least one and possibly two additional precursors that can be processed to TRH in rat brain.

Thyrotropin-releasing hormone-like bioactivity in placenta: evidence for the existence of substances other than Pyroglu-His-Pro-NH2 (TRH) capable of stimulating pituitary thyrotropin release.

The purpose of this study was to investigate the chemical nature of the TRH-like bioactivity and immunoreactivity in extracts of human placenta. Methanolic extracts of human placenta contained nearly 36 ng TRH-like bioactivity/g dried tissue. The immunoreactivity of this extract was only 3 ng/g dried tissue. Two molar acetic acid extracts of human placenta yielded 9 ng TRH-like bioactivity/g dried tissue. The immunoreactivity of these acetic acid extracts, however, was 5.5 ng/g dried tissue. When these placental extracts were subjected to gel filtration chromatography, the bioactivity was found to reside in two fractions which were distinct from synthetic TRH. Furthermore, the immunoreactivity present in these placental extracts was also chromatographically distinct from that of synthetic TRH. In conclusion, these experiments confirm the presence of substantial quantities of materials in placenta which possess TRH-like immunoreactivity and bioactivity. These results also argue that the immunoreactive fractions are not bioactive and provide firm evidence that neither the TSH-releasing substances nor the TRH-like immunoreactivity found in human placenta are identical to pyroglu-His-Pro-NH2.

Decreased serum 3,5,3'-triiodothyronine and thyroxine levels accompanying acute and chronic ritalin treatment of developing rats.

Basal serum levels of T3, T4, and TSH and the serum TSH increment after TRH are reported for rats chronically or acutely treated with ritalin. Serum levels of T3 and T4 are significantly reduced in rats chronically treated with ritalin twice daily for 21 days. Female rats appear to be more susceptible than males to the suppressive action of ritalin. Ritalin appears to accelerate the circulatory clearance of serum T3 and T4. Basal serum levels of TSH demonstrate graded elevations after sequentially higher doses of ritalin and, in most cases, hypophyseal responsivity (delta TSH) to TRH challenge appears to be marginally enhanced by chronic ritalin treatment. Thyroidal responsiveness is not adversely affected by chronic ritalin treatment. Serum T3 and T4 levels return to normal levels after chronic ritalin treatment is discontinued; however, the maturational rise of the basal serum level of TSH is permanently depressed in 35 and 100 mg/kg BW ritalin-treated males. Acute administration of ritalin (35 or 100 mg/k BW) results in a significant reduction of the serum levels of T3 and T4 within 5 h and depressed levels persist for 18 days.

Absence of luteinizing hormone-releasing hormone (LH-RH) from catecholaminergic neurons.

6-Hydroxy-dopamine was administered into the third ventricle of adult male rats. After 2 successive 250 mug doses of 6-OH-dopamine, the levels of norepinephrine and dopamine in the median eminence and arcuate nucleus fell to 15-20% of the control value. The amount of luteinizing hormone releasing hormone (LH-RH) in these same hypothalamic areas, however, was unchanged. It is concluded that LH-RH containing cells are distinct from catecholaminergic neurons.

Direct evidence that the arcuate nucleus-median eminence tuberoinfundibular system is not of primary importance in the feedback regulation of luteinizing hormone and follicle-stimulating hormone secretion in the castrated rat.

Adult rats which have received monosodium-L-glutamate (MSG, 4 mg/g BW) on alternate days for the first 10 days of life manifest central nervous system lesions largely restricted to the retina and the arcuate nucleus of the hypothalamus in which nearly 90% of the perikarya are destroyed, leaving axons in passage intact. In animals so treated, concentrations of dopamine within the arcuate nucleus and median eminence of the hypothalamus are reduced 52% and 57%, respectively, in males and 45% and 61% in females, whereas concentrations of norepinephrine in these same two areas are normal. Concentrations of both norepinephrine and dopamine in five other hypothalamic nuclei (dorsal septal, medial preoptic, suprachiasmatic, periventricular, and dorsomedial nuclei) are unchanged. Nevertheless, despite the destruction of the arcuate nucleus cell bodies of MSG-treated rats, postcastration levels of serum FSH and LH in males, and FSH in females were not significantly different from FSH and LH values in castrated controls. Serum LH in castrated, MSG-treated females was slightly but significantly lower than in castrated controls. It is concluded that the arcuate nucleus-median eminence tuberoinfundibular neurons are not of primary importance in the tonic, negative feedback regulation of gonadotropin secretion.

Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence.

The bovine median eminence was dissected into eight different subdivisions: rostral, anterior internal, anterior external, middle external medial, middle external lateral, middle internal medial, middle internal lateral, and caudal. Thyrotropin-releasing hormone (TRH) was found in the highest concentrations in the middle external medial and lateral subdivisions; luteinizing hormone-releasing hormone (LHRH) was concentrated in the middle external lateral and anterior internal subdivisions. Among the various neurotransmitters and enzymes assayed, only dopamine and choline acetyltransferase were present in highest concentrations in the same subdivisions of the bovine median eminence found to be rich in TRH and LHRH. The distributions of norepinephrine, dopamine-beta-hydroxylase, serotonin, tryptophan hydroxylase, phenylethanolamine-N-methyltransferase, glutamic acid decarboxylase, and histamine appeared to correlate poorly with the major distributions of TRH and LHRH. These findings suggest that at the level of the median eminence, central neuroendocrine regulation of TRH and LHRH release may involve an interaction only with dopamine and acetylcholine.

The nyctohemeral rhythm of plasma prolactin: effects of ganglionectomy, pinealectomy, constant light, constant darkness or 6-OH-dopamine administration.

In male rats maintained on a 12 h light-dark schedule (6 AM-6 PM), there is a nyctohemeral cycle of plasma prolactin which consists of a nadir at 11:30 AM and an apogee at approximately 11:30 PM. In rats exposed to constant darkness, this rhythm persists for 7 days. Seven days of constant light, however, reverses this diurnal variation such that plasma prolactin levels peak at 11:30 AM and reach a nadir at approximately 11:30 PM. In animals maintained on a 12 h light-dark cycle, ganglionectomy and lateral ventricular injections of 6-OH-dopamine (250 mug) also appear to reverse the diurnal variation of plasma prolactin, whereas a single injection of 6-OH-dopamine (250 mug) into the third ventricle decreases plasma prolactin values at all times intervals but does not alter the diurnal rhythm. Both sites of 6-OH-dopamine administration markedly deplete hypothalamic dopamine and norepinephrine, but injection of 6-OH-dopamine into the lateral ventricle destroys the catecholaminergic terminals in the pineal, whereas injection of 6-OH-dopamine into the third ventricle does not. Pinealectomy slightly increases the early morning values of plasma prolactin, but otherwise has no effect on the diurnal variation of prolactin. Five conclusions appear to be justified: 1) there is a nyctohemeral rhythm of plasma prolactin, which is reversed by constant light; 2) the pineal gland probably plays no role in the diurnal regulation of plasma prolactin secretion; 3) the diurnal rhythm of plasma prolactin is controlled by sympathetic input into the brain via the superior cervical ganglion; 4) a rhythm of plasma prolactin develops in constant light which is the exact opposite of the normal diurnal variation; 5) there appears to be a noradrenergic pathway in the hypothalamus or brainstem which stimulates release of prolactin.

The regional distribution of somatostatin in the rat brain.

A sensitive and specific radioimmunoassay for somatostatin (SRIF) has been used to determine the regional distribution of SRIF in rat brain. The hypothalamus contained the highest concentration of SRIF. Lower, but significant amounts of SRIF were present outside of the hypothalalmus. Within the hypothalamus, the concentration of SRIF was highest in the median eminence and arcuate nucleus although all of the hypothalmic nuclei contained some fo this material. The implications of this distribution are discussed.

The effect of surgical isolation of the hypothalamus on its luteinizing hormone-releasing hormone content.

The luteinizing hormone-releasing hormone (LHRH) content of the medical basal hypothalamus was determined by radioimmunoassay 10 days after the isolation of this region. Between 70 and 90% of the LHRH disappeared from the hypothalamus after the surgical procedure. Thus, it seems that most of the LHRH present in the medial basal hypothalamus arises from, or is controlled by, cells elsewhere in the brain.

The effect of hypothalamic deafferentation on somatostatin-like activity in the rat brain.

Somatostatin-like activity (SLA) was determined by radioimmunoassay in several brain regions of animals which had either undergone complete deafferentation of the medial basal hypothalamus or a sham-operation. Surgical isolation of the medial basal hypothalamus caused this region to lose most of its SLA, but did not affect activity elsewhere. Similarly, partial (frontal) deafferentation of the medial basal hypothalamus resulted in a substantial decrease in SLA there.

Lack of effect of various endocrine manipulations on tryptophan hydroxylase activity of individual nuclei of the hypothalamus, limbic system, and midbrain of the rat.

The tryptophan hydroxylase activity of individual nuclei of the limbic system, hypothalamus, and midbrain was determined after various endocrine manipulations in an attempt to identify specific endocrine-responsive serotonergic structures in the rat brain. Following adrenalectomy, thyroidectomy, castration, or treatment with pharmacological doses of dexamethasone, testosterone, or thyroxine, no changes in tryptophan hydroxylase activity occurred in any of the areas of brain examined. Furthermore, in large sections of the hypothalamus and midbrain, total tryptophan hydroxylase activity was unaltered, and no changes in Km for substrate or co-factor were found after adrenalectomy. This study, therefore, has failed to detect any hormonally responsive tryptophan hydroxylase activity in the rat brain. These findings suggest that endocrine-induced alterations in serotonin turnover, if they occur, do so without measurable changes in the activity or kinetic properties of tryptophan hydroxylase.

Purification and characterization of a peptidyl glycine monooxygenase from porcine pituitary.

A peptide alpha-amidating enzyme was purified to apparent homogeneity from porcine pituitary. This enzyme is a glycoprotein with a mol wt of 64,000, a metal prosthetic group, and a dependence upon ascorbate and molecular oxygen. The purified enzyme has a strong preference for peptides ending in glycine. It also catalyzes the oxidation of valylglycine bonds more rapidly than prolylglycine bonds, and demonstrates a primary isotope effect greater than 5 when the alpha-hydrogens of glycine are replaced by deuterium. Kinetic analysis is consistent with a ping-pong or double displacement catalytic mechanism in which both the peptide substrate and ascorbate are competitive inhibitors with respect to each other. With respect to its kinetic properties, catalytic mechanism, and cofactor requirements, the purified amidating enzyme is very similar to dopamine beta-hydroxylase, a finding which supports the previous suggestion that the peptide alpha-amidating enzyme be classified as a peptidyl glycine monooxygenase.

Experimental studies on the ultrastructural localization of acetylcholinesterase in the mediobasal hypothalamus of the rat.

Acetylcholinesterase (AChE) activity has been demonstrated ultrastructurally in neurons of the arcuate nucleus and associated with fibers in the arcuate nucleus neuropil and the median eminence (ME) of the rat. In addition, the effects of neonatal monosodium glutamate (MSG) treatment and Halász deafferentation on the AChE staining and localization have been studied. Neonatal MSG-treatment resulted in loss of the majority of AChE-positive neurons in the arcuate nucleus while leaving neuropil staining intact. Halász deafferentation caused a loss of arcuate neuropil activity while leaving the neuronal staining unaltered. These observations are consistent with previous biochemical results suggesting the existence of a cholinergic tuberoinfundibular system with nerve cells in the arcuate nucleus and terminals in the median eminence. In addition, the deafferentation experiments indicated that extra-hypothalamic cholinergic fibers may innervate the arcuate nucleus. Supporting evidence from other biochemical studies and the curious paucity of histochemical and biochemical AChE activity in the ME are also discussed.

Neuropeptides in Alzheimer's disease: a postmortem study.

The concentration of 5 neuropeptides, neurotensin (NT), somatostatin (SRIF), corticotropin-releasing factor (CRF), bombesin and thyrotropin-releasing hormone (TRH) was measured in 3 cerebrocortical areas and several subcortical regions in post-mortem brains obtained from patients with histologically verified Alzheimer's disease and from controls without neurological or psychiatric disorders using sensitive and specific radioimmunoassay procedures. In Alzheimer's disease, reductions in the concentration of SRIF and CRF were observed in frontal and temporal cortex. In addition, in Alzheimer's disease, SRIF was also reduced in concentration in the hypothalamus, whereas CRF concentrations were reduced in the caudate nucleus. Neurotensin was reduced in concentration in the amygdala in Alzheimer's disease. No alterations in TRH or bombesin/gastrin-releasing peptide were found. These findings provide further evidence for the pathological involvement of certain neuropeptide-containing neurons in Alzheimer's disease.

A microassay for simultaneous measurement of in vivo rates of tryptophan hydroxylation and levels of serotonin in discrete brain nuclei.

A sensitive procedure for simultaneous determination of in vivo rates of tryptophan hydroxylation and levels of serotonin (5-HT) in discrete rat brain nuclei is described. Rates of tryptophan hydroxylation are estimated by 5-hydroxytryptophan (5-HTP) accumulation following l-aromatic amino acid decarboxylase inhibition by R04-4602/1. 5-HTP is separated from 5-HT by liquid cation exchange after which both 5-HTP and 5-HT are measured by a sensitive radioenzymatic assay. Following decarboxylase inhibition, 5-HTP accumulates over 30 min in 6 brain nuclei examined, with negligible levels of 5-HTP being measured in the absence of decarboxylase inhibition. 5-HT levels do not change significantly up to 45 min after decarboxylase inhibition. Comparison of rates of tryptophan hydroxylation determined in 12 different microdissected rat brain areas reveals a greater rate of 5-HT biosynthetic activity in raphe nuclei containing 5-HT cell bodies than in nuclei containing 5-HT terminals. Pretreatment with para-chlorophenylalanine markedly reduces both 5-HTP accumulation and 5-HT levels in the nucleus raphe dorsalis. With this procedure quantities as little as 10 pg of both 5-HTP and 5-HT can be measured, allowing estimation of in vivo serotonin biosynthesis in microgram quantities of brain tissue.

A reliable method for the quantification of thyrotropin-releasing hormone (TRH) in tissue and biological fluids.

A reliable technique for purification of crude tissue extracts and analysis for TRH by HPLC, TLC, radioimmunoassay and bioassay is presented. We conclude that authentic TRH is present throughout the mammalian brain and accounts for much of the TRH-like immunoreactivity in several peripheral organs, but that it is not present in urine, placenta, or pineal, and its concentration in blood is some 250-fold less than a value obtained from a direct radioimmunoassay of a sample of blood.