The distribution of cholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay

The regional distribution of cholecystokinin (CCK) in the rat brain was determined utilizing a radioimmunoassay which detects both gastrin and CCK. CCK concentration is highest in the caudate nucleus (10–14 ng CCK8 equivalents/mg protein), followed by the cerebral cortex. Within the cerebral cortex, CCK is highest in the cingulate, pyriform, and entorhinal areas. There are substantial CCK concentrations in all other brain regions except pons, medulla and cerebellum. CCK is widely distributed in the hypothalamus, where it is highest in the median eminence and ventromedial nucleus. Considerable CCK-like immunoreactivity is also present in the posterior lobe of the pituitary gland, but is not detectable in anterior and intermediate lobes.Though the antisera used in this study cross-react with gastrin the dominant CCK-like material found in rat brain co-elutes with sulfated CCK8 and separates from gastrin on Sephadex G-25 and HPLC chromatography.     

Identification of the C-terminal flanking peptide of preprocholecystokinin in rat brain by a novel radioimmunoassay

The C-terminal flanking peptide of preprocholecystokinin (preproCCK) has been identified in extracts of rat brain using a novel radioimmunoassay. There is a single form of immunoreactive material on gel filtration, ion exchange and reversed-phase HPLC. The C-terminal preproCCK immunoreactivity had a similar pattern of distribution to CCK8 in different regions of rat brain. This assay should help in studies of neuronal CCK biosynthesis.     

Localization of vasopressin in the rat brain

The distribution of arginine vasopressin (AVP) in the rat brain was studied using a sensitive radioimmunoassay. The highest concentration of AVP was found in the hypothalamus. Individually, the supraoptic, paraventricular and suprachiasmatic nuclei contained in the order of 10% of the total hypothalamic content. Vasopressin was also found in the thalamus, medulla, cerebellum, amygdala, substantia nigra and hippocampus. Much lower levels were detected in the pons, spinal cord, frontal and occipital lobes and caudate putamen. No AVP could be detected in any other regions of the cortex or corpus callosum. Chromatographically the vasopressin found outside the hypothalamus is of a similar nature to that of hypothalamo-hypophysial origin. This study supports previous reports of extrahypothalamic localization of vasopressin by immuno-histochemical methods. It is clear that AVP is not confined to the hypothalamo-hypophysial axis, and the possibilities that this may reflect an involvement in brain function are discussed.     

Regional distribution of [H]imipramine binding in rat brain

[³H]imipramine binding was measured in 23 microdissected areas of the rat brain and compared to published values for the endogenous levels of serotonin, noradrenaline and dopamine in the same areas.

The density of [³H]imipramine binding sites appears to be highly correlated with the distribution of endogenous serotonin especially where the serotonin is located mainly in nerve terminals. A weak but still significant correlation also exists with the distribution of endogenous noradrenaline whereas no such correlation could be detected for endogenous dopamine.     

Distribution of luteinizing hormone-releasing hormone in the nervus terminalis and brain of the mouse detected by immunocytochemistry

Immunoreactive luteinizing hormone-releasing hormone (LHRH) was localized in a relatively large number of ganglion cells and fibers of the nervus terminalis of neonatal and adult mice, indicating that this nerve is a substantial source of LHRH in the mouse brain. Whole-head specimens of neonatal mice, prior to calcification of the cranium, revealed an extensive distribution of LHRH neurons and fine fibers throughout the peripheral, intracranial, and central parts of the nervus terminalis. The most striking difference between the neonatal and adult animals, in the nervus terminalis, was the increase in immunoreactive axons that made up the fiber bundles of this nerve. In the adult mouse, the intracranial and central projections were composed of thick fascicles of immunoreactive axons, ensheathed by glial cells and accompanied by ganglia that contained both LHRH-reactive and nonimmunoreactive neurons. LHRH-immunoreactive cells and axons were seen in a branch of the nervus terminalis that coursed along the medial, posterodorsal aspect of the olfactory bulb and in branches of this nerve that accompany the vomeronasal nerves to the accessory olfactory bulb. A few LHRH neurons and many immunoreactive processes were seen in the accessory and main olfactory bulbs. LHRH-reactive neurons were seen in the hypothalamus and extrahypothalamic structures. Examination of adult mouse brains revealed a pattern of distribution and number of immunoreactive neurons similar to that seen in the neonate. However, many more LHRH-reactive axons were seen in all areas of the brain of the mature animal.     

Comparison of the distribution of oxytocin and vasopressin in the rat brain

While immunohistochemistry has been used extensively to map both oxytocin (OT) and vasopressin (VP) pathways in the brain, little information is available concerning the quantitative distribution of these hormones--particularly oxytocin. We have isolated oxytocin from extrahypothalamic regions of the rat brain and shown it to behave identically with standard oxytocin in radioimmunoassay (RIA) and on high-performance liquid chromatography. Using sensitive RIA we have measured and compared levels of both oxytocin and vasopressin in the rat brain. Both hormones are widely distributed, with the largest amounts outside the hypothalamus being found in the locus coeruleus. Considerable quantities of both peptides (but particularly oxytocin) are found in mesencephalic, pontine and medullary nuclei. This distribution is similar to that of the catecholamines, and the possible interaction of oxytocin and vasopressin with catecholaminergic pathways in the central control of various functions is discussed.     

Distribution of immunoreactive melanin-concentrating hormone in the central nervous system of the rat

The distribution of melanin-concentrating hormone-like immunoreactivity (MCH-LI) in 41 microdissected brain and spinal cord regions was determined using radioimmunoassay with antibodies to salmon MCH. The highest concentration of MCH-LI was detected just ventral to the zona incerta (subzona incerta) (2923.2 fmol/mg protein). Very high concentrations of MCH-LI (greater than 1000 fmol/mg protein) were detected also in the nucleus of the diagonal band, medial forebrain bundle, posterior hypothalamic nucleus and medial mammillary nucleus. High concentrations of the peptide (between 500-1000 fmol/mg protein) were measured in 11 brain regions, including bed nucleus of stria terminalis, paraventricular nucleus, anterior hypothalamic nucleus, median eminence, parabrachial nucleus. Moderate concentrations of MCH-LI (between 250-500 fmol/mg protein) were measured in 16 brain regions, such as frontal cortex, central amygdaloid nucleus, medial septum, periventricular nucleus (preoptic) and nucleus of the solitary tract. Low concentrations of MCH-LI (less than 250 fmol/mg protein) were measured in 9 brain regions such as cortical areas, hippocampus, caudate nucleus and substantia nigra. Cervical spinal cord and neurointermediate lobe of the pituitary gland contain low concentrations of the peptide.     

Luteinizing hormone-releasing hormone (LH-RH) production and degradation by rat medial basal hypothalami in vitro

The in vitro production of hypothalamic LH-RH was examined by measuring the differences in tissue content of this neurohormone in incubated versus non-incubated rat medial basal hypothalami. A highly significant increase in hypothalamic LH-RH content was noted which was proportional to the duration of incubation. This observed increase was completely inhibited by incubating tissues at 0 °C, and was not found to occur in similar sized pieces of lateral cortex. A much smaller production of LH-RH was seen by incubation of the preoptic hypothalamic area. In all experiments very little LH-RH was detected in the incubation media. In experiments comparing physiologically different animals, LH-RH production was greater in adult males than in prepubertal males or females, and was higher in intact adult males than in castrates of the same age.Enzymic LH-RH degradation was investigated by adding exogenous synthetic hormone to either the incubating hypothalami or various tissue homogenates and measuring its disappearance as a function of time and/or concentration. These experiments demonstrate a hypothalamic peptidase(s) which degrades LH-RH at a V_max of 1.7 pg/min/μg of protein (24 °C) with a K_m of 2.1 × 10⁻⁸ M/1. Peptidase activity was found in several areas of the brain as well as in the pituitary. Furthermore, there was considerable enzymic activity present in the media from the incubated hypothalami rendering it infeasible to accurately quantitate LH-RH secretion.     

The distribution of glutamine synthetase in subcellular fractions of rat brain

The subcellular localization of glutamine synthetase, an enzyme fundamental to the compartmentation of glutamate hypothesis, was investigated using brain tissue of adult rats. The distribution of this enzyme in relation to the distribution of glucose-6-phosphatase, glutamate dehydrogenase and acetycholine esterase was studied using a fractionation scheme which had been previously extensively characterized in terms of intramitochondrial enzyme complements. Glutamine synthetase was found to be predominantly localized at the nerve terminal and a number of results suggested a possible association with the synaptic membrane.The observations are discussed in relation to the compartmentation of glutamate metabolism. Acetate and ammonia are precursors of the ‘small’ pool of glutamate from which most of the synthesis of glutamine occurs. Since one population of synaptic mitochondria has previously been shown to be enriched in glutamate dehydrogenase and acetyl CoA synthetase and in view of the current observations that synaptosomes are probably in association with a large proportion of brain glutamine synthetase, it is tentatively suggested that the synaptic complex represents at least in part the site of the ‘small’ glutamate pool.     

Regional distribution of a novel pituitary protein (7B2) in the rat brain

The regional distribution of a novel pituitary protein (7B2) in the rat brain was studied using a specific and sensitive radioimmunoassay. Immunoreactive (IR)-7B2 was distributed throughout the brain, with the highest concentrations in the pituitary, hypothalamus and basal ganglia. Immunoreactive 7B2 from the brain and other tissues had an apparent molecular weight of around 20,000 as estimated by SDS-polyacrylamide gel electrophoresis as observed with other tissues. In homozygous Brattleboro rats which do not synthesize vasopressin or its associated neurophysin, IR-7B2 levels in the brain and pituitary gland were shown to be similar to those of control animals. Furthermore, the molecular weight of 7B2 in the brain and pituitary gland of homozygous Brattleboro rats was similar to that of control animals.     

The luteinizing hormone-releasing hormone (LH-RH) system in neonatally estradiol-feminized male gray short-tailed opossums (Monodelphis domestica)

In this study, the luteinizing hormone-releasing hormone (LH-RH) system was examined in adult gray short-tailed opossums that had been treated with estradiol benzoate (EB) on days 1 and 3 of postnatal life, a treatment which results in complete block of testicular development. The finding that the organization of the LH-RH system in neonatally EB-treated males did not differ from that of neonatally untreated animals indicates that the LH-RH system can develop normally despite the absence of gonads throughout perinatal life.     

The luteinizing hormone-releasing hormone (LHRH) system in normal and estrogenized neonatal rats

LHRH perikarya and processes were compared in 6, 9, and 11 day old normal and estrogenized male and female rats. Estradiol benzoate was administered SC in 1 or 1000 μg amounts when the animals were 2 days old. Control animals received either an equivalent amount of the oil vehicle or no injection. After perfusion with Bouin's solution, brains were embedded in paraffin and sectioned 6 μ coronally through the entire brain. The peroxidase-antiperoxidase immunocytochemical technique was used to assess the development of LHRH cells and fibers. The primary antiserum to LHRH was Arimura's antiserum #743. LHRH immunopositive perikarya were present in the septal-preoptic but not in the arcuate-median eminence regions. No immunopositive reaction product was present following treatment with antiserum #743 absorbed with LHRH. At each of the three ages, the amount of detectable LHRH cell bodies was considerably greater in normal males than in females. The high dose of estrogen reduced the quantity of LHRH perikarya localized in the male and increased it in the female. LHRH processes formed a “rostral” pathway in proximity with the organum vasculosm of the lamina terminalis (OVLT) in the vicinity of the rostral preoptic area and a “caudal” one extending through the arcuate-median eminence region. Many more LHRH immunopositive processes were visualized in normal males than in female siblings on days 9 and 11. The high dosage of estrogen also reduced the amount of LHRH immunoreactive material in 9 and 11 day old males but not in equivalent age females. The data are consistent with the findings that the hypothalamic-pituitary-gonadal inhibitory system functions earlier in the male than in the female and that the LHRH system can be modified by estrogen circulating during the period of sexual differentiation.     

Targeted cytotoxic analogue of luteinizing hormone-releasing hormone (LH-RH) only transiently decreases the gene expression of pituitary receptors for LH-RH

A cytotoxic analogue of LH-RH, AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier [D-Lys6]LH-RH, was developed for targeted therapy of cancers expressing LH-RH-receptors. To determine its possible side-effects on the pituitary gland, we investigated the gene expression of pituitary LH-RH-receptors and LH secretion in ovariectomized female and normal male rats after treatment with the maximum tolerated dose of AN-207. The effect of AN-207 on the gene expression of the pituitary GH-RH-receptors and GH secretion was also assessed in male rats. Five hours after a single i.v. injection of AN-207 at 175 nmol/kg, there was a 39-51% decrease in mRNA expression for the pituitary LH-RH-receptors in male and female rats. The carrier, at an equimolar dose, caused a similar reduction (37-39%), whereas the cytotoxic radical AN-201, at an equitoxic dose (110 nmol/kg), produced only a 12-24% decrease (NS) in the mRNA expression of LH-RH-receptors. AN-207 and the carrier analogue induced a comparable 90-100-fold increase in serum LH concentrations in male rats, and the same 12-fold elevation in OVX rats at 5 h. Seven days after treatment with AN-207, the mRNA levels for the LH-RH receptors and the serum LH concentration were back to normal in both sexes. AN-207, the carrier, and AN-201 had no significant effect on the expression of mRNA for GH-RH-receptors in the pituitary. In vitro, a continuous perfusion of pituitary cells with 10 nM AN-207 did not affect the hormone-releasing function of the targeted LH cells or the nontargeted GH cells. Our results demonstrate that cytotoxic LH-RH analogue AN-207, at the maximum tolerated dose causes only a transient decrease in the gene expression of the pituitary LH-RH receptors, and the levels of mRNA for LH-RH receptor fully recover within 7 days. Moreover, the carrier hormone moiety, and not the cytotoxic radical in AN-207 is responsible for this transient suppression. Our findings suggest that the therapy with cytotoxic LH-RH analogues will not inflict permanent damage to pituitary function.     

The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. I. Intra- and extra-hypothalamic projections.

In the guinea pig brain, LH-RH-containing cell bodies are located not only within the classical hypophysiotrophic area but also in the medial preoptic area, septum and olfactory tubercle. LH-RH fiber tracts project not only to the primary portal plexus in the median eminence but also throughout the limbic forebrain and limbic midbrain regions. Using radiofrequency lesions in different brain regions, the projections of LH-RH cell bodies were determined. Cells in the medial preoptic area project ot the organum vasculosum of the lamina terminalis (OVLT), the suprachiasmatic nucleus, the mammillary body complex and the ventral tegmental area. LH-RH neurons in both the medial septal nucleus and medial preoptic area project via the stria medullaris to the medial habenular nucleus and from there via the fasciculus retroflexus to the interpeduncular nucleus of the midbrain. Other LH-RH neurons in the medial septal nucleus, nucleus of the diagonal band of Broca and olfactory tubercle are congregated in small clusters around large blood vessels which penetrate into this area, and they do not appear to send axons outside their immediate vicinity. The types of LH-RH axonal terminations and the roles of these peptide-containing neurons are discussed.     

A non-equilibrium 24-hour vasopressin radioimmunoassay: development and basal levels in the rat brain

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

Differences in regional brain concentrations of neuropeptide Y in spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats

Regional brain concentrations of neuropeptide Y immunoreactivity (NPY) were measured in age-matched Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats using a sensitive and specific radioimmunoassay developed within our laboratory. In 5 of the 9 brain regions examined the SH rats had significantly lower NPY levels compared to the WKY strain. The largest differences occurred within the cortex (−43%), and cervical (−30%) and thoracic spinal cord (−30%), whilst smaller differences were observed in the midbrain (−11%) and medulla oblongata-pons (−18%). The concentration of NPY in the hypothalamus and hippocampus did not vary between the strains. The SH rats contained significantly greater (+18%) NPY levels in the striatum compared to the WKY rats.     

Distribution of cholecystokinin (CCK) in the rat lower brain stem nuclei

The cholecystokinin (CCK) concentration in individual brain stem nuclei of rat was determined using the Palkovits punch method¹⁹ and the CCK RIA³. CCK has a unique distribution in the brain stem, unlike other neuropeptides and biogenic amines^8,19. In general, the CCK levels in the brain stem are 5–20% of rat cerebral cortex. The colliculi, midbrain central gray, nucleus of the solitary tract, and the interpeduncular nucleus had the highest CCK content (2.7-1 ng CCK mg protein).     

Luteinizing hormone-releasing hormone (LH-RH) cells and their projections in the forebrain of the batMyotis lucifugus lucifugus

Luteinizing hormone-releasing hormone (LH-RH) neurons and their projections were studied by immunocytochemistry in the brains of little brown bats (Myotis lucifugus lucifugus: Chiroptera: Vespertilionidae ) as a first step in the study of relationships between these neurons and the seasonal reproductive events characteristic of this species. The majority of immunoreactive neurons in adult male, adult female, and fetal bats were ovoid bipolar cells with one thin and one thicker process, both of which gave rise to fine varicose fibers. LH-RH-immunoreactive perikarya were concentrated in the region of the arcuate nuclei in all bats examined. Perikarya were also consistently found dispersed in the mammillary region, anterior hypothalamus, preoptic areas, septum, diagonal band of Broca, and olfactory tracts; they were occasionally observed in the dorsal hypothalamus, organum vasculosum of the lamina terminalis (OVLT), habenula, amygdala, and cingulate gyrus. LH-RH-immunoreactive fibers projected heavily to the median eminence, infundibular stalk, and posterior pituitary. In extrahypothalamic areas, these fibers were especially abundant in the stria medullaris/habenula and stria terminalis/amygdala, but also contributed to the diagonal band of Broca and the olfactory tracts. Immunoreactive fibers that may be components of many different pathways clustered in the rostral septum and permeated the medial hypothalamus. LH-RH-containing fibers frequently entered the subfornical organ, but were observed less often in the OVLT and only occasionally in the pineal. The organization of the LH-RH system in the little brown bat resembles that of primates, but differs considerably from that in the rat. Anatomical characteristics of the LH-RH system in bats thus suggest that this animal may be a particularly suitable species for further study of neuroendocrine control of reproductive function as it may relate to primates, including humans.