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The authors examined the autopsied brains from three patients who had a fatal hyperthermia syndrome. There was marked hypothalamic noradrenaline depletion in all three patients, severe brain choline acetyltransferase deficiency with nucleus basalis cell loss in two patients, and mild to moderate brain choline acetyltransferase loss in one patient. Striatal dopamine metabolite/dopamine ratio was below normal in two patients and not elevated, as would be expected after short-term neuroleptic administration, in the third. This suggests that reduced capability (aggravated by the cholinergic deficit) of the nigrostriatal dopamine system to respond adequately to stress and/or neuroleptic-induced receptor blockade may be important in the development and course of fatal hyperthermia syndrome.

Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.

A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.

Neuropeptide Y (NPY) messenger RNA was measured by hybridization of mRNA from the cortex, hippocampus, hypothalamus and striatum of rat brains. Adrenalectomized rats showed lowered level of NPY message in the striatum. A similar decline was found in the hypothalamus, while the cortex and hippocampus were unchanged. Levels of NPY message per unit total RNA were about the same for hypothalamus, cortex and striatum and about 50% less for hippocampus. Adrenalectomized rats that received replacement corticosterone had levels of NPY message that had returned to the levels found in rats receiving sham operation. Response elements consistent with our findings are reported in the NPY genomic sequence.

The brains of the chicken, Gallus domesticus, and two amphibians, the anuran Rana ridibunda and the urodele Pleurodeles waltlii, were investigated by means of immunohistochemical techniques with antibodies against dopamine (DA) and tyrosine hydroxylase (TH). As could be expected on the basis of the catecholamine biosynthetic pathway, cell bodies that are immunopositive for the DA antiserum also stain with the TH antibodies. However, a remarkable discrepancy is observed in the hypothalamic periventricular organ where liquor contacting cells exhibit DA- but no TH-immunoreactivity. Since similar results have been obtained in reptiles but not in mammals, it may be concluded that these putative DA-accumulating cells are a primitive brain character of non-mammalian vertebrates.

We recently reported isolation, characterization and synthesis of a novel ovine hypothalamic peptide with 38 residues which stimulates accumulation of cAMP in rat anterior pituitary cell cultures. The peptide was named PACAP38 (pituitary adenylate cyclase-activating polypeptide with 38 residues). The presence of another peptide corresponding to the N-terminal 1-27 residues (PACAP27) was also demonstrated. Both PACAP38 and PACAP27 have an amidated C-terminus. Antisera against synthetic PACAP27 were generated in rabbits. These antisera were tested for titer and specificity in enzyme-linked immunosorbent assay. One of the antisera (no. 88121-3) exhibited a high titer of antibody, which was specific to PACAP27 and PACAP38 with exception of slight cross-reactivity with ovine CRF (oCRF). Therefore, the antibodies against oCRF were removed from the antiserum using a solid phase method. Removal of oCRF antibodies was confirmed by enzyme-linked immunosorbent assay. A dense immunoreactive fiber network was found in both external and internal zones of the median eminence and pituitary stalk. The fibers were demonstrated to be in close contact with the hypophysial portal capillaries. The preabsorption of antiserum with vasoactive intestinal polypeptide or with the mixture containing TRH, LHRH, oCRF, ovine GH-releasing factor, somatostatin, and bovine thyroglobulin did not affect the immunostaining. On the other hand, the preabsorption of antiserum with an excess of PACAP27 or PACAP38 abolished the immunostaining. Therefore, the staining is considered specific for PACAP27 and PACAP38. Stained fibers were also present in the posterior pituitary. A dense fiber network was observed and the lateral hypothalamus the fibers appeared to cling to unstained neuronal cell bodies and their dendrites. In the lateral septum the fibers surrounded some blood vessels. Immunolabeled cell bodies were found in the paraventricular and supraoptic nuclei. These findings support the view that PACAP may play a multifunctional role, including that of a hypophysiotropic hormone, neurotransmitter, neuromodulator, and vasoregulator.

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 mu Ci/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-microns frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.

Hyperphagia and obesity are often associated, and the origins of the biochemical modifications leading to these syndromes might be in the hypothalamus. Indeed, food intake is regulated by numerous neuropeptides in various hypothalamic nuclei, including the paraventricular (PVN), arcuate (ARC), ventromedian (VMN) and suprachiasmatic (SCH) nuclei. Among these peptides, neuropeptide Y (NPY) is the most potent inducer of food intake whereas neurotensin (NT) decreases food intake. We measured these two peptides in microdissected hypothalamic nuclei in obese Zucker rats that ate 30% more food than their lean counterparts. Neuropeptide Y and neurotensin levels varied in opposite directions: In the hyperphagic obese Zucker rats, the NPY concentrations were significantly greater than those in the lean normophagic rats in the ARC (+30%), PVN (+60%) and SCH (+94%) nuclei, whereas the NT levels were significantly lower in the ARC (-40%), PVN (-31%) VMN (-66%) and SCH (-47%) nuclei. Both these variations tend to increase food intake. Feeding periodicity might also be modified because large variations of the two peptides have been measured in the supra-chiasmatic nucleus, which is considered the most important regulator of feeding rhythm. The results reinforce the hypothesis that hyperphagia in obesity is associated with a biochemical modification in the central nervous system because the peripheral status of NT and NPY was not modified in the obese rats. Because levels of other hypothalamic peptides, such as opioid peptides and somatostatin, are also slightly modified, it can be concluded that hyperphagia in obesity is associated with a central peptidergic dysregulation. Research on drugs reacting specifically with the receptor of these peptides might have interesting implications for the treatment of hyperphagia and, therefore, of obesity.

Beef cows (n = 64) were slaughtered to evaluate effects of dietary energy and calf removal (CR) on hypothalamic and adenohypophysial endocrine characteristics. From d 190 of gestation until parturition, cows received maintenance (ME; n = 32) or low (LE; n = 32) energy diets (ME = 100%, LE = 70% NRC recommendations). After parturition, half (n = 16) of each prepartum diet group received low (LE; n = 32) or high (HE = 130% NRC; n = 32) energy diets. At 30 d postpartum, cows were slaughtered 0 or 48 hr after CR. Hypothalami [preoptic area (POA), hypothalamus (HYP), stalk-median eminence (SME)] and pituitaries were collected. Basal and K(+)-induced release of GnRH from SME, and pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) did not differ among groups (P greater than .05). Hypophyseal LH was correlated (P less than .01) with body condition score (BCS) at parturition and slaughter (r = .36 and .47, respectively). Prepartum LE diet increased (P less than .05) met-enkephalin in POA compared to prepartum ME (.59 +/- .05 vs. .44 +/- .04 pmol/mg) regardless of postpartum diet or suckling status. Concentrations of beta-endorphin in combined HYP + POA were decreased (P less than .05) by 48 hr CR (15.1 +/- 1.1 vs. 18.1 +/- 0.7 fmol/mg).(ABSTRACT TRUNCATED AT 250 WORDS)

By means of the indirect immunohistochemical method distribution of S-100 containing cells has been studied in sections of the mediobasal hypothalamus (astrocytes) and adenohypophysis (follicular-stellate cells) in newborn, 10- and 21-day-old rats under normal development and under protein insufficiency. For this the animals are given the diet containing 6% of protein (control--25% of protein). S-100 containing cells are revealed in the hypothalamus and adenohypophysis in 10- and 21-day-old animals. In the brain of the newborn rats S-100 immunoreactive cells are not revealed. At the ultrastructural level the diaminobenzidine (DAB) reaction products in the immunoreactive cells are revealed diffusely along the whole cytoplasm of the cells, in nuclei the DAB reaction products are absent. Part of S-100 containing cells is essentially lowered, comparing with the control. In the rat adenohypophysis part of S-100 containing cells from the 10th up to the 21st day also decreases. Unlike the hypothalamus, however, content of cells, immunopositive to S-100 exceeds the analogous index in the control rats of the corresponding age groups.

Catecholamine content was studied in hypothalamus of neonatal Wistar female rats treated with 4-hydroxyestradiol-17 (4-OH-E2) in a dose of 10 mg for 1-5 days of life. 4-OH-E2 induced a reliable increase in hypothalamic noradrenaline level in 24 h after the last injection, but not on the 7th, 10th or 12th postnatal days. There was no change in dopamine level. We have postulated a relationship between the increase in hypothalamic noradrenaline content induced by 4-OH-E2 and defeminization effects of 4-OH-E2 on the developing brain of female rats.

Galanin is a widely distributed regulatory peptide which modulates the pituitary secretion of PRL and GH. Estrogen administration strongly stimulates galanin gene expression in the rat anterior pituitary. In adult female Fischer 344 rats, estrogen also induces hyperplasia of lactotropes. We used immunocytochemical analysis to assess the effects of estrogen on galanin-like immunoreactivity (Gal-IR) in the rat pituitary and hypothalamus during sc diethylstilbestrol (DES) implantation and after its removal at 30 days. In the anterior pituitary, DES implantation increased the portion of Gal-IR-containing cells from less than 2% in the control rats to 18.3% after 3 days of DES and 36% after 30 days. These changes paralleled the lactotrope hyperplasia exhibited in response to DES exposure. Ten and 30 days after removal of the DES capsules, the percentage of Gal-IR-containing cells in the anterior pituitary decreased to 6.3% and 1.5%, respectively. Colocalization studies revealed that Gal-IR-containing cells were predominantly lactotropes. Immunoelectron microscopy demonstrated that Gal-IR was concentrated in the Golgi region of these hyperplastic lactotropes and suggests that little of the synthesized galanin is secreted. The distribution of Gal-IR in the hypothalamus, median eminence, and neurohypophysis was unaffected by DES treatment. These data demonstrate that galanin is synthesized by hyperplastic pituitary lactotropes of Fischer 344 rats and that peptide accumulation is dependent on the presence of circulating estrogens. In contrast, neuronal galanin synthesis in the hypothalamus does not appear to be regulated by estrogen.

A battery of antibodies directed against different portions of the precursor to gonadotropin-releasing hormone (GnRH), as well as to the mature decapeptide, were characterized immunocytochemically in two ways. Absorption experiments were used to determine the epitope recognized by each antiserum. Electron microscopic immunocytochemistry was then used to define the subcellular organelles that contained reaction product when tissue was incubated with these reagents. These latter observations helped to determine if the antibody recognized the epitope as part of the intact precursor or only after it had been cleaved from parent protein. Our results demonstrate that the GnRH precursor is routed from the rough endoplasmic reticulum through the Golgi apparatus to the secretory vesicles. Furthermore, we show that initial cleavage and processing of the GnRH precursor begin in the cell soma. These antibodies should be useful in the future in determining changes in processing of precursor in animals that differ in endocrine function.

Effects of the glucocorticoid milieu on the basal and ether stress-induced prolactin (PRL) release and on the immunostaining for hypothalamic vasoactive intestinal peptide (VIP), beta-endorphin (beta-EP), dynorphin-A (DYN-A) and methionine-enkephalin (Met-ENK), were examined in separate groups of male rats. After colchicine treatment in intact rats, VIP-containing cell bodies were observed only in the suprachiasmatic nucleus (SCN). Adrenalectomy (ADX), performed 7 days previously, resulted in the additional appearance of VIP-immunoreactive neurons in the parvocellular subdivision of the paraventricular nucleus (PVN), as well as in significantly higher basal and stressed PRL levels than intact values. Treatment of intact rats with a high dose (500 micrograms/kg body weight (s.c.) daily for 7 days) of dexamethasone (DEX), but not with a low dose (50 micrograms/kg) of DEX, significantly reduced both the basal and stressed PRL release. Administration of either the low or high dose of DEX to ADX rats prevented the appearance of the PVN-VIP neurons. In addition, the ADX-induced high basal and stressed PRL levels were restored to intact values by the low dose of DEX, and completely suppressed by the high dose of DEX. The staining of SCN-VIP-, beta-EP-, DYN-A or Met-ENK neurons was not affected by any treatment employed in this study. These results suggest that the appearance of PVN-VIP immunostaining in ADX rats may, at least in part, be responsible for the enhanced PRL secretion observed in this group. However, SCN-VIP-, beta-EP-, DYN-A- or Met-ENK neurons do not seem to play a pivotal role in the glucocorticoid regulation of PRL secretion.

Mouse monoclonal antibody (mAb) L6 identifies an antigen expressed on the cell surface of many different human carcinomas. While studying the binding activity of mAb L6 to intracerebral tumor xenografts of human lung carcinoma LX-1 cells in nude rats using immunohistological techniques, we observed that L6 can also bind to a cytoplasmic antigen expressed in the magnocellular component of the hypothalamo-neurohypophysial system. Double-labeling experiments with antisera to vasopressin and oxytocin confirmed the localization of L6 immunoreactivity within both peptide-containing cell groups. L6 immunoreactivity in Brattleboro rats (with genetic deletion in the vasopressin gene) was exclusively localized within oxytocin neurons. Oxytocin and vasopressin failed to block L6 staining which suggested that its target epitope resides within the neurophysin sequence, and this explanation was supported by the finding that adsorption of L6 with porcine neurophysin completely eliminated hypothalamic immunoreactivity. Western blot analysis of bovine neurophysin and human pituitary extracts identified L6-immunoreactive bands which corresponded to the position of neurophysin and pro-pressophysin, confirming that L6 immunoreactivity in hypothalamus is related to neurophysin. Thus, monoclonal antibody L6, which is highly reactive with a membrane antigen of human lung cancer cell line LX-1, recognizes a cytoplasmic epitope in hypothalamic neurons identified as neurophysin by immunohistochemistry and Western analysis.

Recent results have demonstrated altered corticotropin-releasing factor (CRF)-41 content of the neurointermediate lobe (NIL) of the pituitary gland in response to various manipulations including osmotic stimulation. This study was undertaken to determine whether changes in CRF-41 content of the NIL are accompanied by changes in intensity of CRF-41-like immunoreactivity (CRF-41-LI) of neurosecretory neurones of the hypothalamus in response to osmotic stimulation. Wistar rats of both sexes given either tap water ad libitum, 2% NaCl solution, or access to tap water was limited to 20 min daily, for 7 days. Subsets of rats from each group were adrenalectomized (ADX) or treated with dexamethasone (DEX). Thirty-six hour before perfusion with fixative consisting of buffered formaldehyde and picric acid, animals received 75 micrograms colchicine i.c.v. Forty micrometer thick vibratome sections were stained for CRF-LI, arginine vasopressin (AVP-LI) and oxytocin (OXY-LI) using the avidin-biotin-peroxidase complex method. In response to both types of osmotic stimulation magnocellular neurones of the paraventricular (PVN) and supraoptic nuclei (SON) showed increased CRF-LI, AVP-LI and OXY-LI, while CRF-LI of parvocellular perikarya of the PVN decreased. The enhanced CRF-LI seemed to appear in a subset of magnocellular neurones with OXY-LI but not AVP-LI. Increased staining intensities were also observed in magnocellular neurones in ADX rats challenged osmotically. In contrast, systemic DEX administration, as well as implantation of DEX in the area on the SON, sharply attenuated CRF-LI but not AVP-LI or OXY-LI of magnocellular neurones in osmotically stimulated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Two antisera (Anti-P7 and Anti-P10) were raised against (-Gln-His-Pro-Gly-) elongated peptides: P7 Gln-His-Pro-Gly-Lys-Arg-Phe) and P10 (Ser-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-Phe). They recognized TRH extended peptides but not TRH. A RIA against P7 and a highly sensitive enzyme immunoassay against P10 were used to identify two major high mol wt forms of 25-35 K and 6-8 K in chromatography fractions of adult and fetal mouse as well as adult rat hypothalami. The existence of the largest form was confirmed by immunoblotting with Anti-P7. During mouse hypothalamus development in vivo and in vitro, the ratio of TRH content vs. P10-associated immunoreactivity increased several times. This suggests that these Pro-TRH peptides are precursors of TRH biosynthesis and indicate an acceleration of TRH processing during development. Double immunostaining with A-TRH and A-P7 of hypothalamic cells taken on the 16th fetal day and cultured for 6, 12, and 18 days in vitro (DIV) revealed three populations of neurons: 1) a very minor population (approximately 2%) of small round cells positive with A-TRH only; 2) a major population of neurons positive with both A-TRH and A-P7. 3) multipolar neurons positive with A-P7 only (up to approximately 45% after 18 DIV). The respective distribution of TRH and P7 along neurites also varied with time in culture. Whatever perikarya staining, TRH was restricted to short neurites and growth cones before synapse formation and, during synapse development, to varicosities and terminal boutons. However even at the latest stage examined some varicosities and terminal boutons were positive with A-P7 only. These results suggest a preferential processing of pro-TRH at a post-Golgi step during axonal transport to growth cones and synaptic boutons.

An interaction between the immune and endocrine systems has been long known. This association is further strengthened by the finding that splenic lymphocytes have the capacity to produce molecules similar to if not the same as classical hormones, including several members of the opiate family, PRL, GH, and neuropeptide Y. Because of such findings and because of information from other laboratories suggesting that LHRH might have direct effects upon the immune system, we hypothesized that immune cells themselves might contain LHRH. Lymphocytes were purified from spleens of intact adult male Sprague-Dawley rats and the cells were lysed with sodium hydroxide. The concentration of immunoreactive LHRH was 403 +/- 184 pg/20 X 10(6) lymphocytes. Increasing amounts of lymphocyte lysate displaced [125-I]LHRH from LHRH antibody in a manner parallel to that produced by synthetic hypothalamic LHRH, suggesting immunologic similarity between lymphocyte and hypothalamic LHRH. Lymphocyte LHRH-like immunoactivity coeluted from Nova-Pak C18 columns with synthetic hypothalamic LHRH. When lymphocyte lysates were applied to rat anterior pituitary cells in monolayer culture, significant stimulation of LHRH secretion was seen, from 2,144 +/- 54 pg LH/ml.4 h to 15,364 +/- 587 pg LH/ml.4 h (P less than 0.001), a finding verified in five additional experiments. In other studies, this LH response evoked by lymphocyte lysates was found to be dose dependent and could be significantly inhibited by an LHRH-antagonist. Furthermore, when lymphocyte lysate and identically treated synthetic LHRH were HPLC fractionated, there was coelution of lysate and hypothalamic LHRH bioactivity. The lysate itself contained no substantial LH immunoreactivity. Thus, lymphocytes from spleens of adult male rats contain an immunoactive and bioactive LHRH, a finding further strengthening an association between the endocrine and immune systems.

Two molecular forms of gonadotropin-releasing hormone (GnRH) were demonstrated in hypothalamic extracts of M. domestica using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. One form eluted in the same position as synthetic mammalian GnRH and was quantified equally by two mammalian GnRH antisera, while the second form coeluted with synthetic chicken GnRH II and was quantified equally with two chicken GnRH II antisera. The finding of chicken GnRH II in a South American species of marsupial, which has previously been reported in some Australian species of marsupial and in species of Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.

A commercial layer breeder flock that was suffering from hysteria was fed a diet containing 5 grams tryptophan/kg for six days. The incidence of episodes of hysteria declined from five times/hour on day 0 to once/hour on day 6 and none on day 8. Feed consumption increased from 107 g to 145 g/hen/day and egg production increased 23% during the six day feeding period. The tryptophan concentration in plasma doubled (from 95.6 to 188.2 mumol/mL). Plasma phenylalanine and tyrosine also increased. Birds that were not in lay, by postmortem examination, had significantly higher plasma valine concentrations (476.4 vs 372.7 mumol/mL). Tryptophan, serotonin and related metabolites increased in both the hypothalamic region and the remainder of the brain following tryptophan feeding, and subsequently declined. High levels of dietary tryptophan may be useful in alleviating hysteria in poultry.

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.

Maharishi-4 (M-4), an ayurvedic food supplement, was tested for anticarcinogenic and anticancer properties against 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. The 6% M-4-supplemented diet protected DMBA-induced carcinogenesis by reducing both tumor incidence and multiplicity during initiation and promotion phases. The control animals who developed tumors when supplemented with M-4 diet for four weeks showed tumor regression in 60% of cases. There was no significant difference in the food intake or weight gain in rats who were on M-4-supplemented diet compared to control group. Possible mechanisms of action of M-4 are discussed.

This study was designed to evaluate profiles of serum concentrations of LH, pituitary content of LH and GnRH receptors, and hypothalamic content of GnRH in undernourished, ovariectomized ewes. In earlier studies, pulsatile secretion of LH diminished as duration of undernutrition progressed in prepubertal or adult ovariectomized ewes. Ewes having similar body condition scores (CS) of 5 to 9 (1 = extremely thin, 5 = moderate, 9 = obese) were fed maintenance or low-energy diets (100% and 60% of NRC requirements, respectively). Blood samples for analysis of LH were collected at 15-min intervals for 4 h at initiation of the project and immediately prior to slaughter. Serum concentrations of LH did not differ (P greater than .05) among groups at the initial sampling period. At slaughter, ewes with CS less than or equal to 2 (n = 7) had lost 26.8 kg (42% of initial weight). Ewes with CS greater than or equal to 3 (n = 12) had lost an average of 13.7 kg (18% of initial weight). Concentrations of LH in ewes with final CS greater than or equal to 3 was similar (P greater than .05) to that observed during the initial sampling period. However, release of LH was reduced (P less than .01) in ewes with CS less than or equal to 2 compared with ewes with CS greater than or equal to 3 (2.6 vs 9.5 and 3.2 vs 10.5 ng/ml for basal and mean concentrations, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The GnRH gene has been cloned in several species, but the location of the promoter and the exact start of transcription have not previously been determined. To characterize the low abundance human GnRH mRNA in the hypothalamus and placenta, we have employed the polymerase chain reaction. The hypothalamus was found to have a 61-base pair first exon, and its transcriptional start site was determined. The human hypothalamic GnRH cDNAs isolated thus far have all contained a short 5' untranslated region which would correspond to this start site. However, all human placental GnRH cDNAs reported to date have a long 5' untranslated region, which extends more than 140-base pairs 5' to this start site in the hypothalamus, suggesting the utilization of an alternative promoter in the placenta. In addition, the human GnRH gene undergoes differential splicing in these tissues. The first intron is removed from the hypothalamic, but retained in the placental, GnRH mRNA. Thus, the placenta has a very long first exon, while the hypothalamus has a comparatively short first exon, followed by a long first intron. This characterization of the human GnRH gene will now allow hormonal regulatory studies to be performed using gene transfer techniques.

The occurrence of prolactin binding sites in some brain structures (telencephalon, ventral hypothalamus, myelencephalon, hypophysis, and choroid plexus) from Xenopus laevis (anuran amphibian) was studied by the in vitro biochemical technique. The higher binding values were obtained at the level of the choroid plexus and above all of the hypothalamus. On the bases of hormonal specificity and high affinity, these binding sites are very similar to those of prolactin receptors of classical target tissues as well as of those described by us in other structures from Xenopus. To our knowledge, the present results provide the first demonstration of the occurrence of prolactin specific binding sites in Xenopus laevis choroid plexus cells.

Several peptidergic PRL-releasing factors (PRFs) have been described; however, none have been proven to be of primary physiological importance in the control of hormone release. Similarly dopamine withdrawal alone cannot completely explain the profiles of PRL secretion observed under a variety of conditions. We describe here the isolation in semipurified form of both a PRF and a PRL-inhibiting factor (PIF) from bovine neurointermediate lobe (NIL) extracts. Acid extracts of bovine NILs stimulated, in a dose-related manner, PRL release from cultured anterior pituitary cells, even after immunoabsorption of endogenous oxytocin from the extract. PIF and PRF activities were semipurified from NIL extracts by Sephadex chromatography and detected by in vitro and in vitro bioassays. The PRF material could be separated from oxytocin by gel sieving and was active in the presence of dopamine in vitro unlike synthetic oxytocin and in cell preparations in which the oxytocin-responsive lactotrophs had been removed by selective cytotoxin cell targeting using an oxytocin-ricin A chain cytotoxic conjugate. The PRF material stimulated PRL secretion in a dose-dependent fashion in conscious male rats after iv injection. The PIF material comigrated on sizing gel chromatography with immunoreactive oxytocin and was active in vitro during dopamine blockade with domperidone and in vivo in the presence of endogenous dopaminergic tone. These data suggest that novel factors present in the NIL might exert physiologically relevant control over lactotroph function and add to the growing literature on the presence of a PRF in the NIL.

Artificial selection of gray rats by the domestic type of behaviour resulted in an increase of the noradrenaline content, but in a decrease of the alpha 2-adrenergic receptors number in the hypothalamus. The data suggest a significant difference in noradrenergic system between domesticated and aggressive animals associated with changes in aggressive behaviour towards man during selection.

We have completely purified, in parallel, a low molecular weight, non-specific, non-lipidic, Na+,K(+)-ATPase inhibitory factor from bovine hypothalamic and pituitary tissues. In the final purification step we obtain, from both tissues, a single, homogeneous peak, with a maximal absorbance at 247 nm. This factor, at physiological concentrations of potassium (5-25 mM), inhibits in a dose-response manner Na+,K(+)-ATPase and displaces ouabain from its receptor at the enzyme structure. The factor isolated from both tissues is identical, being the specific activity per weight of tissue higher in hypophysis. No factor was found in cerebral cortex, used as tissue control.

The physiological roles of chicken LHRH-I and -II (cLHRH-I and -II) in the regulation of gonadotrophin release were investigated in the domestic chicken. Measurements of the neuropeptides, using specific radioimmunoassays, in brain sections cut in three planes or in grossly dissected brain areas, showed that cLHRH-II occurs in low amounts throughout the brain whereas cLHRH-I is most abundant in the diencephalon. Within the diencephalon, the largest amount of cLHRH-I occurred in the median eminence of the hypothalamus. The amount of cLHRH-I in the median eminence was higher (P less than 0.05) in laying than in out-of-lay hens. No cLHRH-II was detected in the median eminence in either reproductive state. The amount of cLHRH-I in the hypothalamus was increased (P less than 0.05) in cockerels at the onset of puberty and in somatically immature birds after castration. There were no correlated changes in the amounts of hypothalamic cLHRH-II measured in the same experimental samples. Active immunization of laying hens against cLHRH-I but not against cLHRH-II resulted in the complete regression of the reproductive system and a depression in the concentration of plasma LH. These observations, taken together, suggest that gonadotrophin secretion in the hen is more likely to be directly regulated by cLHRH-I than by cLHRH-II.

The changes in adrenocorticotropin (ACTH) release before, during and after sympathetic nerve degeneration following superior cervical ganglionectomy (SCGx) were examined in male rats. A 12-fold increase of circulating ACTH was found in both SCGx and sham-operated rats 6 h after surgery. In sham-operated rats, plasma ACTH decreased by about half 16-22 h after surgery, whereas in SCGx rats it remained at a high concentration from 16 to 54 h after surgery, attaining basal values by 120 h post-SCGx. In SCGx rats, MBH corticotropin-releasing hormone (CRH) content decreased significantly from 16 to 54 h after surgery, while in controls it remained unmodified. Significantly smaller arginine vasopressin (AVP) contents were found in MBH of SCGx rats as compared to sham-operated controls, 16-54 h after surgery. In rats exposed to ether or immobilization stress 22 h after SCGx, plasma ACTH levels were significantly higher than in controls; however, since unstressed ACTH levels were about twice as high in SCGx rats, the percent increase of ACTH was smaller in the SCGx group. A decreased response of plasma ACTH to ether or immobilization stress was found in rats 7 days after SCGx. In rats subjected to a simultaneous adrenalectomy (Adx) and SCGx or sham-SCGx, plasma ACTH levels increased to a similar extent in both groups. ACTH increase after Adx was accompanied by decreases in MBH CRH, and absence of significant changes in MBH AVP contents. Rats subjected to pinealectomy (Px) or sham-Px 1 week earlier and killed 22 h earlier exhibited similar responses in plasma ACTH and MBH CRH to SCGx regardless of pineal intactness.(ABSTRACT TRUNCATED AT 250 WORDS)

Galanin gene expression in the anterior pituitary is potently stimulated by estrogen in adult rats. To evaluate the influence of estrogen on galanin during the peripubertal period 30- to 32-day-old female rats were treated with pregnant mare serum gonadotropin (PMSG, 10 IU s.c., 10.00 h). Galanin-like immunoreactivity (galanin-LI) in hypothalamic and pituitary tissues was evaluated 1, 2 or 3 days after PMSG treatment between 17.00 and 19.00 h. The PMSG treatment stimulated 17 beta-estradiol secretion, which induced a midafternoon LH surge 2 days after the PMSG treatment. Concentrations of galanin-LI at the time of this LH surge were elevated 82% in the anterior pituitary and 58% in the hypothalamus (without the median eminence) when compared to saline-treated female rats. On the 3rd day after the PMSG injection, galanin-LI was increased 236% in the anterior pituitary, 88% in the neurointermediate lobe and 39% in the median eminence compared to saline-treated female rats. These changes in galanin-LI were not observed in similarly aged male rats or ovariectomized rats treated with PMSG. In adult male rats, daily injections with 17 beta-estradiol valerate (10 micrograms/daily s.c.) for 1 week increased galanin-LI in the median eminence and neurointermediate lobe to an extent similar to that seen in juvenile female rats following PMSG treatment. In contrast, the high serum levels of 17 beta-estradiol achieved after 17 beta-estradiol valerate treatment increased galanin-LI in the anterior pituitary 65-fold. These studies indicate that galanin-LI is influenced by estrogen in peripubertal and adult rats.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate possible sex differences in the feedback regulation of growth hormone (GH) secretion, concentrations of immunoreactive GH-releasing hormone (GRF) and somatostatin (SS) were measured in the median eminence (ME) and the hypothalamus of male and female rats bearing the MtTW15 tumor, which secretes high amounts of GH and prolactin (PRL). Four weeks after tumor implantation in male rats, the GRF concentration in the whole hypothalamus, including the ME, was decreased by 37% (0.29 +/- 0.02 vs. 0.46 +/- 0.02 ng/mg protein in intact male controls; p less than 0.001) and the concentration of SS was increased by 40% (11.5 +/- 0.7 vs. 8.1 +/- 0.3 ng/mg protein in male controls; p less than 0.01). In female rats, the presence of tumor for 4 weeks caused a smaller (18%) reduction in GRF concentrations (0.27 +/- 0.02 vs. 0.33 +/- 0.03 ng/mg protein in intact female controls; p less than 0.05) and no significant change in SS concentrations (10.2 +/- 0.08 vs. 9.7 +/- 0.8 ng/mg protein in female controls). Tumor-related changes in GRF and SS concentrations were also more pronounced in male rats than in females, when determined separately in the microdissected ME and in the remaining hypothalamus. These differences occurred despite similar increases in serum GH, PRL and insulin-like growth factor I concentrations in male and female tumor-bearing rats. To assess which hormone (GH or PRL) was responsible for these changes, intact male rats were treated for 10 days with 2 daily s.c. injections of rat GH (rGH; 100 and 250 micrograms/day), rat PRL (100 and 250 micrograms/day) or vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)

It is known that in adult rats, GH by itself and by promoting secretion of the somatomedins acts at the level of the hypothalamus to trigger release of somatostatin and decrease output of GH-releasing hormone (GHRH), thereby inhibiting further secretion of GH. To assess whether these mechanisms are already operative in the early postnatal period, we have evaluated the effect of short-term administration of GH in 10-day-old rats. Twice-daily s.c. administration of 25 micrograms human GH/rat, from days 5 to 9 of life, significantly reduced pituitary content of GH, decreased hypothalamic levels of GHRH mRNA and abolished the in-vivo GH response to a challenge dose of GHRH (20 ng/100 g body weight, s.c.). GHRH (20 ng/100 g body weight, twice daily, s.c.) given concomitantly with the GH treatment, completely counteracted the inhibitory effect of the latter on pituitary content of GH and restored to normal the in-vivo GH response to the GHRH challenge. These data indicate that impaired secretion of GHRH is involved in the inhibitory effect elicited by GH treatment in infant rats. However, concomitant involvement of hypothalamic somatostatin as a result of GH treatment cannot be ruled out. In fact, pituitaries from rats pretreated with GH responded in the same manner as pituitaries from control rats to the GHRH challenge in vitro.

The effect of the biogenic amine depleting drug, reserpine, on the concentration of type II corticosteroid receptors (i.e., glucocorticoid receptors) in neuronal (hippocampus, frontal cortex, hypothalamus), lymphoid (circulating lymphocytes, spleen, thymus) and pituitary tissues as well as hippocampal type I (i.e., mineralocorticoid) receptors was examined in adrenal-intact and adrenalectomized (ADX) rats. Reserpine (2 mg/kg) or vehicle was administered to adrenal-intact rats for 2 consecutive days. Following the second injection rats were ADX and sacrificed 24 h later. Reserpine significantly decreased type I and II hippocampal receptors as well as type II receptors in frontal cortex, hypothalamus, lymphocytes and spleen. Since the reserpine-induced decreases in receptor content could be due to reserpine-induced elevations in circulating corticosterone levels, reserpine (2 mg/kg) or vehicle was administered to 1-day ADX rats which were then sacrificed 2 days later (i.e., 3 days post ADX). A 1-day ADX control group was also included. The 3-day ADX regimen produced significant or nearly significant increases in type II receptors in hippocampus, frontal cortex, hypothalamus, lymphocytes and spleen in vehicle-treated rats. Reserpine attenuated the ADX-induced upregulation of type II receptors in hippocampus, frontal cortex, lymphocytes and spleen, but had no effect on the ADX-induced upregulation of type II receptors in the hypothalamus. The ADX-induced increase in hippocampal type I receptors was not affected by reserpine treatment. In a final experiment, reserpine (2 mg/kg) or vehicle was administered immediately after ADX and rats were sacrificed 24 h later in order to assess the effect of reserpine on basal (i.e., nonupregulated) corticosteroid receptor levels in the absence of circulating corticosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to evaluate the effects of environmental lighting on the hypothalamic secretion of luteinizing hormone-releasing hormone (LHRH), intact and castrated male Wistar rats were kept either in periodic (LD) or in constant light (LL) for 1 week, and the hypothalamic LHRH concentration was measured by radioimmunoassay. In the intact rats the LHRH concentration was lower in constant than in periodic light (30 +/- 3 vs 61 +/- 3 pg mg-1, P less than 0.05), whereas in the castrated rats it was higher (16 +/- 2 vs 5 +/- 1 pg mg-1, P less than 0.05). The intact and castrated rats received melatonin (50 micrograms) or saline injections daily at either 09.00 h or 16.00 h in LD and LL for 1 week. In intact or castrated rats the melatonin injections were not able to prevent the change of hypothalamic LHRH in constant light. In castrated rats testosterone (125 or 250 micrograms) had no effect on the LHRH concentration under either lighting condition. The present results suggest that constant light reduces the synthesis of hypothalamic LHRH in intact rats but increases it in castrated rats. These effects of constant light do not appear to be related to melatonin.

The neuroanatomical distribution and morphology of neurons that produce gonadotropin-releasing hormone (GnRH) in the brain of the postpubertal male Djungarian hamster was studied using light microscopic immunocytochemistry. Analysis of every section from the rostral olfactory tubercle to the medial basal hypothalamus indicate 356 +/- 37 immunoreactive GnRH perikarya per brain (mean +/- SE; n = 4 brains). Over 90% of GnRH cell bodies were found in 6 brain regions; the largest number of somata were located in the medial preoptic area followed by the diagonal band, lateral hypothalamus, lateral preoptic area, lateral septum and anterior hypothalamus. Morphologically, two predominant types of GnRH neurons were identified: unipolar GnRH cells with an ovoid soma and only a single distinct process (about 40% of all GnRH neurons), and bipolar cells with a fusiform-shaped perikaryon. Overall and in most brain regions, the ratio of unipolar to bipolar GnRH perikarya was 2:3 or greater. A significant proportion of GnRH neurons had an unusually "thick" process(es) that exited the soma and tapered gradually. GnRH fibers were evident in most sections, forming dense plexuses in the arcuate nucleus-median eminence, the periventricular region of the third ventricle and organum vasculosum of the lamina terminalis. These findings indicate that the Djungarian hamster is similar to other rodent species, especially the white-footed mouse, in the neuroanatomical distribution of GnRH neurons. The present report provides a working atlas for the rostral ventral forebrain of the postpubertal Djungarian hamster.(ABSTRACT TRUNCATED AT 250 WORDS)

The vasopressin gene is highly transcribed in magnocellular neurons of the supraoptic (SON) and paraventricular nucleus (PVN) in the rat hypothalamus. In order to identify cis-acting elements involved in the expression of the vasopressin gene, approximately 1 kb upstream of the transcription start site has been sequenced. Several putative regulatory elements have been detected, including a glucocorticoid response element (CRE), a cAMP response element (CRE), and four AP2 binding sites. In gel shift assays performed with a labelled DNA fragment corresponding to nucleotide residues -214 to -36 and nuclear proteins extracted from SON-derived tissue enriched in magnocellular neurons, three specific protein-DNA complexes have been detected. Complex formation is effectively competed by addition of an excess of unlabelled fragment.

The present study investigated the effect of repeated treatment with the antidepressant drugs imipramine, amitryptyline, citalopram and mianserin (10 mg/kg PO, twice daily for 14 days) on levels of thyrotropin-releasing hormone (TRH) in several brain structures (cerebral cortex, amygdala + pyriform cortex, hippocampus, nucleus accumbens, striatum and hypothalamus) of the rat. Amitriptyline caused a marked increase in the TRH content in the striatum and nucleus accumbens. Citalopram and mianserin produced a smaller but significant increase in the TRH content in the striatum only, while imipramine did not significantly affect the TRH concentrations in any of the brain structures. None of the antidepressant drugs administered acutely significantly affected the TRH concentrations in the nucleus accumbens or the striatum. These results indicate that changes in brain TRH induced by antidepressant drugs are not related to their therapeutic activity.

Lead exposure alters cardiovascular function and has been implicated in the etiology of hypertension. Therefore it was of interest to study the short term effect of lead treatment on atrial natriuretic factor (ANF), a hormone which produces vascular smooth muscle relaxation and natriuresis. Male Sprague Dawley rats were randomly divided into 5 groups containing 4 animals each and injected intraperitoneally with normal saline (control), 0.01, 0.1, 0.5 or 1.0 mg/kg of body weight with lead acetate solution twice a day for 7 days, and then maintained for a period of 30 days. During this period water consumption and urine volume were measured daily. At the end of the 30 day period, immunoreactive levels of ANF in hypothalamus, atria and plasma were measured by radioimmunoassay. Lead treatment did not alter water consumption, but significantly decreased urine output. At all doses, lead produced a decrease in hypothalamic content of ANF and slightly increased atrial levels. The content of ANF in plasma was decreased. The changes in ANF content indicate that lead interacts with the hormonal regulation of the cardiovascular system and these observations may relate to the cardiovascular toxicity of this heavy metal.

Progesterone and 5 alpha-pregnane-3,20-dione (5 alpha-DHP) concentrations were measured in seven brain areas and in plasma during "anesthesia" induced by progesterone (1-2 mg IV) in female rats. The highest levels of progesterone were detected in the striatum and hypothalamus (23.3 +/- 5.27 and 22.7 +/- 4.30 micrograms/g +/- SEM, respectively); these concentrations were approximately 1000 times higher than those during the post-ovulatory phase. Highest levels of 5 alpha-DHP were detected in the striatum and hippocampus (11.5 +/- 1.74 and 10.4 +/- 3.15 micrograms/g +/- SEM, respectively). The ratio of 5 alpha-DHP to progesterone was approximately 100 times higher in brain tissue than in plasma. We conclude that a conversion of progesterone to 5 alpha-DHP occurs in the brain during the course of progesterone-induced "anesthesia". This metabolic step may be an important contributory factor to the anesthetic potency of progesterone.

Vasogenic brain edema was produced by transplantation of rat glioma C6 cells into rat brain. Using this model, we measured neurotransmitter concentrations and water content of regional brain tissue. In the tumor-implanted controls, monoamine neurotransmitters in the hypothalamus, cortex, and striatum significantly decreased. When treated with DEX, these monoamines tended to return to the levels of the sham-operated controls. Additionally, DEX markedly lowered tumoral monoamines. In the nonsurgical animals, DEX significantly increased norepinephrine but not DA. Regardless of treating with DEX or not, water content showed no changes in the hypothalamus and striatum in the tumor-implanted animals. However, the increase in water content in the cortex was significant, and this increase could be reduced by DEX to the levels of controls. Water content of tumor tissue could also be markedly reduced by DEX. In the nonsurgical animals, there were no changes in water content between DEX-treated and nontreated animals. In conclusion, brain edema produced by the brain tumor may reduce noradrenergic and dopaminergic activities. This is more likely due to compression anoxia caused by the tumor mass, glial hydrops, and edema fluid. It is presumed that the effect of DEX is due to reduction of water content of the tumor and peritumoral white matter as well as by increasing noradrenergic activity.

GnRH-associated peptide (GAP)-like immunonreactive elements located in the human hypothalamus were investigated by PAP immunocytochemistry using specific antiserum against [pro-GnRH (14-69) OH]. Immunoreactive neuronal perikarya were distributed in the MPOA, PVN and infundibular nucleus, with the largest numbers of GAP-like immunoreactive perikarya found in the infundibular nucleus. We also detected the coexistence of GAP-like and GnRH-like immunoreactivities in the same neuronal perikarya in the MPOA by using a double immunolabelling procedure. In addition to the above regions immunoreactive neuronal perikarya were present in the region dorsal to the medial mammillary nucleus. GAP-like immunoreactive fibers were distributed in same areas that immunoreactive perikarya were observed. Many immunoreactive terminals were found adjacent to capillaries in the infundibulum. Immunoreactive dots, presumably terminals, were observed in the posterior pituitary and these were particularly evident along the margin adjacent to the anterior pituitary. The distribution pattern and density of GAP-like immunoreactive neuronal elements are compared with those of other mammalian species. We also compared GAP-like immunoreactive elements with that of GnRH as has been previously observed in the human hypothalamus.

Experimental alloxan- or streptozotocin-produced diabetes in rats was accompanied by an increase in the levels of norepinephrine, dopamine, and serotonin, whereas the contents of metabolites, i.e., 5-hydroxyindoleacetic acid and homovanillic acid, in the whole brain gradually decreased with the duration of diabetes. Among the striatum, thalamus, and hypothalamus of alloxan diabetic rats, monoamine alterations were observed only in the hypothalamus; after 1 week an increase of norepinephrine content and after 13 weeks an increase of norepinephrine and dopamine contents were found. Tissues of 11 brain regions of 10 diabetic and 12 control patients post mortem were investigated for monoamine concentrations. Patients were all male, of similar age and interval between death and autopsy. Diabetic patients had an increase in the content of serotonin in the medial and lateral hypothalamus. The content of dopamine increased in the medial hypothalamus, putamen, and medial and lateral pallidus. In diabetic patients, the content of norepinephrine increased in the lateral pallidus and decreased in the nucleus accumbens and claustrum. Thus, it seems that diabetes mellitus in rats, as well as in humans is associated with regionally specific changes in brain monoamines.

The aim of this work was to study L-DOPA-containing neuronal structures of the rat posterior and dorsal hypothalamus by means of immunohistochemistry using antiserum against glutaraldehyde conjugated L-DOPA. Aspects and distribution of L-DOPA immunoreaction among cells of the supramammillary nucleus and the A11, A13c and A13 cell groups are described and compared to dopamine immunoreactivity, mainly through a double colored labelling procedure employing a color modification of the DAB reaction by metallic ions. Differences between L-DOPA and dopamine stainings within cell groups as the presence of cells with predominant or exclusive L-DOPA coloration are tentatively explained under the light of previous findings using immunohistochemistry of catecholamines synthesizing enzymes and catecholamines histofluorescence.

Sprague-Dawley dams were fed either a protein-calorie deficient or control diet from day 5 to day 21 after parturition. The concentrations of seven amino acids (aspartate, glutamate, gamma-aminobutyric acid, glycine, glutamine, serine, and taurine) were determined in brain regions from 17-day-old undernourished offspring and from 35-day-old rehabilitated rats. The brain regions examined were the cortex, cerebellum, corpus striatum, hippocampus, hypothalamus, brainstem, and midbrain. At 17 days of age, taurine was the amino acid with the highest concentration, whereas at 35 days glutamate had the highest concentration. This change was due to the fact that the concentration of taurine decreased significantly in all brain regions between 17 and 35 days, whereas the concentration of glutamate remained high or increased somewhat in all brain regions except the hypothalamus and brainstem. When the age-matched offspring of control and undernourished rats were compared, several interesting and significant differences were found. The concentrations of glutamate and aspartate were significantly lower (decreased 16-34%) in the cerebellum, brainstem, cortex, and midbrain in 17-day-old undernourished rats. The aspartate level was also significantly decreased in the corpus striatum and hypothalamus in 17-day-old offspring. However, the deficiencies of aspartate and glutamate were transient and reversible. In contrast, the concentration of taurine was increased in the hypothalamus (31%) and hippocampus (12-33%) at both 17 and 35 days of age and in the midbrain (17%) at 17 days. Other transient abnormalities in amino acid levels were found in undernourished offspring. The results of these experiments suggest that undernutrition during lactation causes delayed CNS development, which is manifested in altered concentrations of the neurotransmitters aspartate, glutamate, and taurine.

A gas chromatographic method is described for the concomitant separation and analysis of catecholamines, catecholamine or 3,4-dihydroxyphenylethylamine condensation products (tetrahydroisoquinolines), and their isomeric mono-O-methyl (phenolic) metabolites which may be present in neuronal tissues, utilizing short glass capillary columns and electron-capture detection. Isomeric phenolic amines that were not generally separable with conventional-packed gas chromatographic columns were rapidly resolved on the capillary system, and with their catecholamine or catechol isoquinoline precursors, quantitated with high sensitivity (0.25-7.0 pg) and reproducibility. Key steps in the approach with tissues include initial amine isolation with a weak cation-exchange resin (BioRex-70), fluoracyl derivative formation, and brief washing of the derivatives with ammonium phosphate buffer (pH 5.8) just prior to capillary analysis; overall recoveries of amines or alkaloids added to rat brain homogenates ranged from 79% to 89%. Application of the method is demonstrated in an assay of endogenous dopamine in rat corpus striatum and hypothalamus. This new procedure should complement and in some instances may be preferred over liquid chromatographic assays for catecholic and phenolic amines and isoquinolines, and ought to be applicable to mass spectrometric detectors as well.

A highly sensitive and specific radioimmunoassay for LRF was applied to the measurement of endogenous LRF in various hypothalamic extracts. Specific antiserum was obtained by injecting LRF conjugated to human serum albumin with glutaraldehyde. Thyrotropin-releasing hormone, lysine vasopressin, oxytocin, noradrenaline, LH, FSH and cortical extracts did not appear to affect the assay, and the maximum cross-reaction observed with the LRF analogs tested was 8.5 p. 100 with LRF 2-10. The best detection limit (0.4 pg/tube) was usually obtained when the labelled LRF had been purified by polyacrylamide gel electrophoresis. Within and between-assay coefficients of variation were 8.0 and 12.6 p. 100, respectively (from B/Bo = 20 to 80 p. 100). Synthetic LRF administered to rams by intravenous injection was readily detectable in the peripheral plasma. However, the direct measurement of plasma endogenous LRF may give misleading results due to non-specific interference by plasma factors. No endogenous LRF could be detected in plasma methanol or acetone extracts obtained from rats and rams in various physiological conditions. The inhibition curves parallel to the synthetic LRF curve were obtained by diluting the crude hypothalamic extracts of rams and rats, and a good correlation (r = 0.997) with the Ramirez-McCann bioassay resulted, indicating that using radioimmunoassay to determine hypothalamic LRF content may be fruitful in studying hypothalamo-pituitary gonad interactions. The LRF content of rat and ovine hypothalami ranged from 2-8 to 20-80 ng of LRF, respectively.

Four peptides having SRIF-like bioactivity when assayed by a rat anterior pituitary cell culture system have been isolated from chicken hypothalamic extracts. These four peptides have been purified from 2,000-5,000 chicken hypothalami in a yield of 2.2 nmol to 16 nmol. The isolated SRIFs have been characterized by amino acid analysis. One consists of 28 amino acids which are identical with mammalian SRIF-28 except that one Met residue is replaced by a Leu residue. Three other SRIF-like substances with low mol wt have also been isolated in a pure state. One is identical with mammalian SRIF-14, as judged by chromatographic comparison on HPLC and its amino acid composition as well as the specific bioactivity on rat pituitary cells. The amino acid composition and specific bioactivity of another one is identical with that of mammalian SRIF-14, but it is different from mammalian SRIF-14 in its elution position on HPLC. The third SRIF-like substance consists of 16 amino acids and is less potent than mammalian SRIF-14. It is concluded that several SRIF-like peptides exist and that SRIF-28, with one amino acid substitution from Met to Leu, and SRIF-14, identical with the mammalian one, are the major components in chicken hypothalamus.