John S. Kizer

The projections of the A8, A9 and A10 dopaminergic cell bodies: evidence for a nigral-hypothalamic-median eminence dopaminergic pathway

Using the microdissection method of Palkovits and a sensitive microassay for dopamine (DA) and norepinephrine (NE), the CNS projections of the nigral A8, A9 and A10 dopaminergic cell bodies have been determined in rat brain. Following bilateral electrolytic lesions of the A8-A9-A10 region, DA levels fell significantly in the following areas: dorsal septal nucleus (85%), olfactory tubercle (75%), lateral amygdaloid nucleus (75%), habenula (75%), parietal cortex (75%), cingulate cortex (70%), cerebellum (50%), thalamic paraventricular nucleus (50%), and preoptic suprachiasmatic nucleus (45%). Of particular interest was the finding of a 40% fall in the dopamine levels of the median eminence after such lesions. Concentrations of NE in these same areas following lesions of the A8-A9-A10 region were unchanged. These findings suggest that the nigral A8, A9 and A10 dopaminergic cell bodies, project diffusely to many different areas of cortex, hypothalamus, and brain stem, and, more importantly, provide evidence for a previously unsuspected dopaminergic pathway from substantia nigra to median eminence, an area intimately concerned with neuroendocrine regulation.

Serotonergic innervation of the forebrain: Effect of lesions on serotonin and tryptophan hydroxylase levels

Concentrations of serotonin and tryptophan hydroxylase activity in individual hypothalamic and limbic nuclei, as well as cortical regions, were measured by radioenzymatic microassays after dorsal raphe nucleus lesions and various surgical transections of the brain stem. Fourteen days after lesioning of the dorsal raphe nucleus there were 43-65% decreases in 5-HT concentrations of certain hypothalamic and limbic nuclei. More pronounced decreases were found in the parietal cortex and in the locus coeruleus and substantia nigra. Degenerations of nerve terminals in various regions of the hypothalamus and the limbic system were detected by electron microscopic studies 1-8 days after dorsal raphe nucleus lesions. There was no change in the 5-HT concentration of the spinal cord following dorsal raphe nucleus lesions. Surgical transections at the level of mesencephalic-pontine junction caused no significant changes in 5-HT concentrations or tryptophan hydroxylase levels in the hypothalamus, limbic system, cerebral cortex or spinal cord. Serotonergic fibers ascending from the mesencephalic raphe nuclei constitute a well circumscribed bundle dorsal to the interpeduncular nucleus. The axons enter the hypothalamus between the mammillary body and crus cerebri just ventral to the medial forebrain bundle. The ascending pathway contains all the ascending 5-HT fibers innervating the hypothalamus and the rostral limbic and cortical areas.

DISTRIBUTION OF BIOGENIC AMINES AND RELATED ENZYMES IN THE RAT PITUITARY GLAND

Abstract— Biogenic amines and related enzymes were quantitatively measured in the pituitary gland of the rat. The sensitivity of the assays used allows the determinations to be performed in single pituitary lobes. Relatively high values of histamine and serotonin were found in all three lobes, with higher amounts in the posterior and intermediate lobes. Highest catecholamine concentrations were detected in the posterior lobe, and only very low amounts of dopamine were measured in the anterior lobe. Throughout the gland, norepinephrine concentrations were low, about one‐tenth that of dopamine. Tryptamine could not be detected. High levels of A and B monoamine oxidase were found in all three pituitary lobes. Tyrosine hydroxylase activity was measured in the posterior and intermediate lobes, but was not detected in the anterior lobe. Tryptophan hydroxylase was present in all three pituitary lobes. A relatively low catechol‐O‐methyltransferase activity was found in the anterior lobe, and none was detected in the intermediate and posterior lobe. Choline acetyltransferase, dopamine‐β‐hydroxylase and phenylethanolamine‐N‐methyltransferase activities could not be detected.

Regional Distribution of Neurotensin in Human Brain

Abstract: Neurotensin (NT) is an endogenous neuropeptide that is active in many preclinical screening tests for neuroleptic drugs. Using a radioimmunoassay, we have studied the regional distribution of NT in postmortem human brain and in cerebrospinal fluid. Highest levels were present in the hypothalamus, substantia nigra, and limbic areas, whereas much lower amounts were found in the cortex and striatum. The chromatographic properties of hypothalamic immunoreactivity on ion‐exchange and high pressure liquid chromatography were similar to those of the synthetic tridecapeptide. We conclude that NT is present in human brain with a distribution resembling that seen in other species, such as rat and monkey.

Effects of Stress on Catecholamines and Tyrosine Hydroxylase Activity of Individual Hypothalamic Nuclei

The effects of acute stress on norepinephrine (NE) and dopamine (DA) concentrations and of repeated stress on tyrosine hydroxylase (TH) activity were measured in individual hypothalamic nuclei and other rat brain regions. A microdissection technique and radioisotopic enzymatic assays enables these studies to be performed. NE and DA concentrations were reduced and TH activity increased selectively in the arcuate nucleus. These results suggest that the arcuate nucleus may be selectively involved in the stress response and support the hypothesis that catecholamines in the medial basal hypothalamus mediate certain of the neuroendocrine changes observed in stress.

The effect of castration, thyroidectomy and haloperidol upon the turnover rates of dopamine and norepinephrine and the kinetic properties of tyrosine hydroxylase in discrete hypothalamic nuclei of the male rat

Adult male rats were either castrated, thyroidectomized, or treated with haloperidol and the rates of turnover of dopamine (DA) and norepinephrine (NE) in the median eminence (ME), the arcuate and dorsomedial nuclei of the hypothalamus were estimated from the rate of decay of DA and NE concentrations as determined by radioenzymatic assay following blockade of catecholamine synthesis by alpha-methyl-p-tyrosine. The ME of animals similarly prepared was also examined for changes in the total activity and kinetic properties of tyrosine hydroxylase (TH). Four days following the administration of haloperidol (400 microgram/kg) or 10 days after castration, there was a significant increase in the rate of turnover of DA but not NE in the ME accompanied by an increase in the Vmax but not Km for the substrate or cofactor of TH. Furthermore, the administration of haloperidol to hypophysectomized rats also significantly increased the TH activity in the ME, indicating that such changes may occur independently of any changes in serum prolactin levels. Ten days after thyroidectomy, or three weeks after treatment with prophylthiouracil, there was a significant increase in the turnover rate of DA in both the ME and dorsomedial nucleus but not in the arcuate nucleus. No changes in the turnover rates of NE in any of the three areas were observed following thyroidectomy. In the ME, the increase in turnover of DA was accompanied by an increase in the total TH activity (Vmax) as welll as a decrease in Km for tetrahydrobiopterin but not tyrosine. From these results 4 conclusions were drawn: (1) following halperidol, castration, and thyroidectomy there are increases in the activity of dopaminergic terminals within the ME; (2) castration, haloperidol and thyroidectomy may influence the activity of dopaminergic terminals within the ME by different mechanisms; (3) changes in tyrosine hydroxylase and turnover of catecholamines within the ME may occur independently of changes in prolactin levels; and (4) local recurrent afferent circuits may exist in the arcuate nucleus region of the hypothalamus.

Models of Neuroendocrine Regulation: Use of Monosodium Glutamate as an Investigational Tool

The administration of monosodium-L-glutamate (MSG) during the neonatal period is known to result in central nervous system lesions in the arcuate nucleus of the hypothalamus and the retina. Rodents so treated exhibit behavioral deficts and endocrinopathies including obesity, hypogonadism, hypothyroidism, pituitary atrophy, tail automutilation and diminished locomotor activity. Assessment of endocrine status revealed normal serum levels of glucagon, thyroid-stimulating hormone and luteinizing hormone, and diminished levels of thyroid hormones and growth hormone in MSG-treated rats. Prolactin levels were elevated in the glutamate-treated male rats. Within the brain hypothalamic levels of thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, and somatostatin were unchanged. Measurement of neurotransmitters and neurotransmitter-related enzymes in individual hypothalamic nuclei derived from MSG-treated rats revealed normal levels of norepinephrine, serotonin and glutamic acid decarboxylase, but reduced levels of choline acetyltransferase and dopamine in the arcuate nucleus and median eminence. Histochemical methods for visualization of dopamine and acetylcholinesterase in the mediobasal hypothalamus confirmed these findings. The MSG-treated animals exhibited a normal diurnal rhythm of pineal serotonin N-acetyltransferase activity. These data indicate that the MSG-induced endocrine deficiency syndrome results at least partly from destruction of cholinergic and dopamingeric tuberoinfundibular systems in the hypothalamus.

Neonatal Monosodium Glutamate

Neonatal administration of monosodium glutamate (MSG) produces neurotoxic degeneration of the retina and medial-basal hypothalamus, including the arcuate nucleus. Since this hypothalamic area contains the only neuronal cell bodies in brain which contain adrenocorticotrophic hormone (ACTH) and beta-lipotropin (beta-LPH) and beta-endorphin, destruction of these cells by MSG may interfere with pain responses mediated by nerve fibers arising from these perikarya. The present study examined whether MSG-treated rats, as compared to littermate controls, exhibited concomitant changes in the immunocytochemical distribution of ACTH and beta-LPH, and their reactivity to several analgesia-inducing manipulations. Although MSG-treated rats did not differ from control rats in their baseline reactivity to electric shock, they displayed an inability to exhibit analgesia following acute exposure to cold-water swim stress. In addition, MSG-treated rats showed an attenuated analgesic response following morphine administration. However, the analgesia elicited by either abrupt food deprivation, or the glucoprivic stress of 2-deoxy-D-glucose, was unaffected by neonatal MSG treatment. Concomitant with these selective analgesic deficits, MSG-treated rats displayed a marked immunocytochemical reduction in ACTH/beta-LPH perikarya and terminals in brain, but not pituitary. These data indicate that multiple pain-inhibitory systems exist, and that some rely upon an intact medial-basal hypothalamus to produce analgesia.

Prolactin in cerebrospinal fluid: A probable site of prolactin autoregulation

The purpose of this study was to examine the thesis that increasing concentrations of prolactin within the cerebrospinal fluid (CSF) increase the activity of dopaminergic terminals within the median eminence and that this increased dopaminergic activity is temporally associated with a suppression of endogenous prolactin secretion. To avoid difficulties encountered in performing catecholamine turnovers in the undisturbed rat, the measurement of tyrosine hydroxylase was validated as an index of dopaminergic activity within the median eminence. In the median eminence, but not the medial preoptic area, parallel increases in the activity of tyrosine hydroxylase and the turnover of dopamine (but not norepinephrine) occurred following hyperprolactinemia. Twenty-six hours but not 2.5 h after the subcutaneous administration of ovine prolactin, the activity of tyrosine hydroxylase was increased in the median eminence, and endogenous prolactin secretion was inhibited. During a 26 h continuous intracerebroventricular (icv) infusion (88 ng/h) of rat prolactin, there was a complete suppression of endogenous prolactin secretion. Twenty-six but not 2.5 h after the initiation of the icv infusion of prolactin, there was an increase in tyrosine hydroxylase activity in the median eminence. The results of these studies suggest that: (1) measurement of tyrosine hydroxylase activity within the median eminence is a useful index of the activity of dopaminergic terminals; (2) increasing concentrations of prolactin within the CSF suppressed prolactin secretion by the anterior pituitary; (3) this suppression of prolactin is accompanied by an increased activity of dopaminergic terminals within the median eminence; (4) those neural structures concerned with the regulation of prolactin secretion respond directly to prolactin itself; (5) the autoregulation by prolactin of its own secretion manifests a certain latency more characteristic of a tonic rather than a phasic inhibitory control; and finally, (6) dopaminergic terminals in the median eminence but not the preoptic area appear uniquely sensitive to prolactin.

Serotonin synthesis and release in brain slices: Independence of tryptophan

The purpose of the present study was to examine the role of substrate availability in the regulation of the release and synthesis of serotonin by brain slices. Electrical field depolarization of the brain slices stimulated the synthesis and release of serotonin in the absence of changes in intracellular tryptophan concentration, in the absence of tryptophan in the incubation bath, and in the absence of changes in total tryptophan uptake. Furthermore, electrical stimulation decreased the apparent Km for tryptophan required for synthesis of serotonin by the slices. Several conclusions were drawn. (1) Rates of serotonin release and synthesis in brain slices may increase independently of the tissue tryptophan concentration or tryptophan uptake. (2) There is little difference in the synthetic rate of serotonin in tissues exposed to pargyline or not at all. (3) Blockade of monoamine oxidase by pargyline appears to give estimates of the synthetic rates of serotonin which are comparable to estimates derived from [3H]-tryptophan incorporation. (4) Newly synthesized serotinin is preferentially released. (5) Only in tissue pretreated with pargyline do increasing concentrations of tryptophan increase the releasable pool of 5-HT. (6) Electrical depolarization may allosterically activate tryptophan hydroxylase; an effect which may be mimicked by homogenization of the tissue.