Localization of acetylcholine receptors in central synapses.

The localization of cholinergic receptors in brain synaptosomes and in synapses of the midbrain reticular formation and hypothalamic preoptic nucleus has been demonstrated by means of a horseradish peroxidase-alpha-bungarotoxin (HRP-alpha-Btx) conjugate. Only a small proportion of the total number of synapses was reactive. Axon terminals of reactive synapses contained primarily small clear vesicles, while synapses characterized by large numbers of dense core vesicles were unreactive. Toxin-binding sites were found to occur in a thickened zone of the postsynaptic surface. This procedure can be employed to study the regional distribution and localization of nicotinic receptor sites in the central nervous system.

The regional distribution of somatostatin in the rat brain.

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

Localization of luteinizing hormone-releasing hormone in the preoptic area and hypothalamus of the rat using radioimmunoassay.

To determine the localization of luteinizing hormone-releasing hormone (LHRH), five brains from adult male rats were serially sectioned in a cryostat at - 10 C in either the frontal, horizontal or sagittal planes. Acetic acid-ethanol extracts of each section were assayed for LHRH using radioimmunoassay (RIA) and in some cases using bioassay as well. Approximately 0.2 ng LHRH was concentrated in medial basal preoptic (MB-PO) tissue overlying the rostral portion of the optic chiasm. This LHRH appears to be associated with the organum vasculosum of the lamina terminalis and/or adjacent neural tissue. Uniform, low levels of LHRH were detected in hypothalamic tissue between the preoptic area (POA) and arcuate-median eminence (ARC-ME) region. In the ARC-ME region 2.7 ng of LHRH were concentrated primarily in the median eminence. The lateral distribution of LHRH in the ARC-ME region extended beyond the median eminence into tissue corresponding to the lateral aspect of the ventromedial nucleus. Concomitant bioassay and RIA determinations of LHRH were highly correlated. Of the sections bioassayed, only those sections containing LHRH released FSH. These results confirm the presence of LHRH in the POA and in the rostral hypothalamus of the rat brain. The possible significance of LHRH in the POA for the regulation of LH release is discussed.

Localization of cells containing LHRH-like mRNA in rat forebrain using in situ hybridization.

Using in situ hybridization, we localized cells in the rat forebrain which contain mRNA that hybridizes with a radiolabeled, synthetic oligodeoxyribonucleotide (59-mer) complementary to human LHRH mRNA in the region which includes the coding sequence for the decapeptide. These brain areas have been shown previously to contain immunoreactive LHRH cell bodies.

Luteinizing hormone-releasing hormone neurons in human preoptic/hypothalamus: differential intraneuronal localization of immunoreactive forms.

Luteinizing hormone-releasing hormone (LRH) may be synthesized as part of a larger prohormone, as are several other neuropeptides. In this study, we sought not only to define the distribution and morphological characteristics of LRH neurons within the human preoptic area and hypothalamus, but also to identify sites of initial synthesis, posttranslational conversion to the decapeptide, and storage of LRH in these neurons. Immunoreactive molecular forms were differentiated using a series of antisera with distinct specificities in the peroxidase-antiperoxidase technique. These antisera were capable of detecting the fully processed hormone as well as extended decapeptide sequences. Immunopositive LRH neurons were more abundant in the infundibular area of the hypothalamus than in the preoptic area. Numbers of immunopositive perikarya and subcellular distribution of reaction product varied with binding requirements of the antisera. After treatment with an antiserum that requires the fully processed decapeptide for binding, the reaction product was associated almost entirely with granules in perikarya and processes, while very little was associated with either rough endoplasmic reticulum (RER) or Golgi apparatus. In contrast, with an antiserum capable of detecting extended forms of the decapeptide, the RER and Golgi were labeled in addition to granules. From these data, we infer that in humans, mature decapeptide is present in granules within LRH neuronal perikarya and processes. Furthermore, the molecular forms associated with RER and Golgi may be precursors in which the decapeptide sequence is extended.

Hypothalamic LHRH in aging rats: effects of ovariectomy and steroid replacement.

Hypothalamic LHRH was measured by RIA in young and middle-aged (MA) female rats in several endocrine conditions. Temporal alterations in LHRH content associated with the steroid induced gonadotropin surge were compared in medial basal hypothalamic and anterior hypothalamic-preoptic area fragments of ovariectomized young and MA subjects. LHRH content was also compared in ovariectomized, untreated subjects from the two age groups. Finally, LHRH content in MA constant estrous females was compared with content in young females on the morning of proestrus. In all conditions, LHRH levels in both brain regions of MA females were similar to, or significantly elevated above levels measured in young females, yet both the steroid induced surge and the castration induced hypersecretion of gonadotropins were markedly attenuated in aging females. Because studies have verified the responsiveness of the pituitary of MA rats to LHRH, the data suggest that adequate amounts of hypothalamic LHRH do not reach the pituitary. Rather, high levels of hypothalamic LHRH measured in MA subjects may represent accumulation of the peptide in LHRH neurons due to an age-related impairment in release.

Neurotransmitter specificity of cells and fibers in the medial preoptic nucleus: an immunohistochemical study in the rat.

The medial preoptic nucleus (MPN) is a sexually dimorphic complex with three major subdivisions. The cell-dense central (MPNc) and medial (MPNm) subdivisions are larger in male rats, while the cell-sparse lateral subdivision (MPNl) occupies a majority of the nucleus in females. In the present study we evaluated the distribution of possible monoaminergic and peptidergic cells and fibers within the MPN, as well as in adjacent regions of the medial preoptic area of the adult male rat. For this, we used an indirect immunohistochemical method with antisera to serotonin (5HT), dopamine beta-hydroxylase (DBH), tyrosine hydroxylase (TH), neuropeptide Y (NPY), cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP), substance P (SP), neurotensin (NT), corticotropin-releasing factor (CRF), luteotropin-releasing hormone (LRH), somatostatin (SS), thyrotropin-releasing hormone (TRH), oxytocin (OXY), vasopressin (VAS), adrenocorticotropic hormone (1-24; ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), leucine-enkephalin (L-ENK), and calcitonin gene-related peptide (CGRP). The results suggest that cell bodies and/or fibers crossreacting with all of these putative neurotransmitters are differentially distributed within the MPN. Within the MPNm, the densest plexuses of fibers were stained with antisera to SP and NPY, while moderate densities of fibers were stained with anti-DBH, SS, CCK, CGRP, ACTH, and alpha-MSH, and only a few fibers were stained with anti-5HT, TH, NT, VAS, and L-ENK. Moderate numbers of SP- and L-ENK-immunoreactive cell bodies, and a few SS-, NT-, CRF-, and TRH-stained cell bodies were also found within the MPNm. The MPNc contained a dense plexus of CCK-immunoreactive fibers, as well as a few CRF-immunoreactive fibers. Both fiber types were localized almost exclusively to this subdivision, while most of the others studied here appeared to avoid it selectively. This suggests that there are relatively few inputs to the MPNc, and that they tend to avoid other parts of the nucleus, although moderate densities of DBH- and NPY-immunoreactive fibers were found in both the MPNm and MPNc. The MPNc contained several CCK-immunoreactive cell bodies as well as a moderate number of TRH-stained cell bodies. Both cell types were nearly completely localized to the MPNc. The major inputs to the MPNl studied here appear to be stained with antisera to 5HT and L-ENK, although moderate numbers of NT- and CRF- immunoreactive fibers were also found in this part of the nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of neuropeptides in the limbic system of the rat: the bed nucleus of the stria terminalis, septum and preoptic area.

The distribution of the neuropeptides vasoactive intestinal polypeptide, cholecystokinin octapeptide, substance P, neurotensin, methionine-enkephalin and somatostatin has been mapped immunocytochemically in the bed nucleus of the stria terminalis, one of the major sites of termination for efferent projections from the amygdala. Immunoreactive fibres and terminals were distributed more or less topographically and largely in accordance with the previously described localization of peptide-containing cell bodies in the amygdala and the amygdaloid projection fields in the bed nucleus as described by neuroanatomical techniques. Thus, vasoactive intestinal polypeptide, which was found in some of the lateral amygdaloid nuclei, had a substantial projection to the lateral bed nucleus. The lateral bed nucleus also contained cholecystokinin-octapeptide, substance P, neurotensin and methionine-enkephalin immunoreactivity which probably derived from the central amygdaloid nucleus, whilst cholecystokinin-octapeptide, and especially substance P-containing fibres, were found in the medial bed nucleus and probably arise from cells in the medial amygdala. Reciprocal amygdalopetal projections were suggested by the presence of substance P- and somatostatin-containing cell bodies in the mediodorsal bed nucleus and vasoactive intestinal polypeptide cells in the lateral bed nucleus, but somatostatin otherwise had a widespread distribution. Numerous local peptidergic connections were also noted both within the bed nucleus and between it and adjacent structures, especially the preoptic area, hypothalamus and the basal ganglia. These data provide further evidence that neuropeptides play a major role in the connectivity of the limbic system and show that the bed nucleus of the stria terminalis is an important relay station, particularly between amygdaloid efferents and other forebrain areas.

A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers.

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.

Quantitative radioimmunohistochemical method using [125I]-protein A to measure the content of methionine enkephalin in discrete rat brain areas.

We report a quantitative radioimmunohistochemical method, using [125I]-protein A in combination with a specific antibody to methionine enkephalin (Met-enk), for determination of the content of this peptide in discrete areas of rat brain. After paraformaldehyde fixation, rat brain sections were incubated with a Met-enk polyclonal antibody, followed by incubation with [125I]-protein A. After autoradiography with 3H-sensitive Ultrofilm, optical densities (OD) were quantified by computerized microdensitometry. The OD obtained were compared to a standard curve, constructed after determination by radioimmunoassay of the Met-enk content in corresponding brain areas from adjacent tissue sections. After comparing 15 different brain areas over a ninetyfold range of concentrations, we found a linear relationship between the content of Met-enk, as determined by radioimmunoassay, and the OD generated by autoradiography. The content of Met-enk in other discrete brain areas can be quantified by interpolation of the OD determined by autoradiography in the standard curve. The method allows, for the first time, precise quantification of peptide concentrations in discrete areas and nuclei from thin sections of rat brain. This technique has a more than 100-fold higher sensitivity than classical radioimmunoassays, with the additional advantage of neuroanatomical localization. It also has the potential for application to the quantification of many other antigens present in brain and other tissues.

Heat acclimation: role of norepinephrine in the anterior hypothalamus.

The hypothesis that anterior hypothalamic (AH) sensitivity to norepinephrine (NE) is altered by chronic exercise in the heat was tested in male Sprague-Dawley rats. Treadmill exercise 6 days/wk for 3 wk at 21 m/min was performed at 23 degrees C (control; C) or at 35 degrees C (heat acclimated; HA), progressing from 20 to 50 min/day in 2 wk. Time for core temperature (Tco) to rise from 39.5 to 40.5 degrees C during a heat-tolerance test after conditioning increased (P less than 0.05) in the HA group. To test for a change in AH sensitivity, the change in Tco to 2-, 5-, 10-, 20-, and 40-micrograms doses of NE injected bilaterally into the AH was determined after conditioning. Dose-response regression lines showed that exercise in the heat increased the slope and shifted the Tco-NE dose relation to the left. In a separate series of experiments on 6 sedentary(s), 10 C, and 10 HA animals, the amounts of NE, dopamine, and 3,4-dihydroxyphenylglycol (DOPEG) were determined by high-pressure liquid chromatography in the AH, median preoptic area (PO), cortex, and cerebellum after 9 wk of conditioning. Results showed that in the PO there was a significant increase in NE and DOPEG in the HA vs. C group and a trend of increasing NE from the S to C to HA groups. The data indicate that exercise in the heat increases NE-induced peripheral heat-dissipating capacity and increases catecholamine storage in the PO.

An apparent genetic polymorphism for a protein present in the hypothalamus of Sprague-Dawley rats.

Using two-dimensional gel electrophoresis, an apparent genetic polymorphism was detected in the hypothalamus of a group of inbred Sprague-Dawley rats. The proteins involved in this polymorphism have a molecular weight of 57,000 daltons and isoelectric points ranging from 6.1 to 6.3. These proteins met four criteria that should be met before a positional shift on two-dimension gels can be attributed to a genetic polymorphism. This is the first report of the existence of a genetic polymorphism in the brains of a group of inbred Sprague-Dawley rats. The functional significance of this polymorphism is currently under investigation.

Regional distribution of substance P in the brain of the rat.

Using a sensitive radioimmunoassay we have studied the regional distribution of substance P. The level of substance P is higher in the mesencephalon, hypothalamus and preoptic area than in other regions of the brain. Substance P is found in especially high concentrations in the reticular part of the substantia nigra and the interpeduncular nucleus. It is present in large amounts in several septal, preoptic and hypothalamic nuclei as well.

Acetylcholine rhythm in the preoptic area of the rat hypothalamus is synchronized with the estrous cycle.

Acetylcholine levels in the preoptic area of rat hypothalamus were found to change rhythmically, in synchronization with the estrous cycle. During the proestrous critical period, these levels demonstrated a drop followed by a sharp rise. The absence of this phenomena in males indicates sexual dimorphism. The close association between this newly detected hypothalamic acetylcholine rhythm and the estrous cycle suggests the possible involvement of acetylcholine in regulation of the estrous cycle.

Nuclear and cytosolic androgen receptor levels in the limbic brain of neonatal male and female rats.

Nuclear and cytosolic androgen receptors in the limbic brain were measured in neonatal male and female rat pups. There were no sex differences in cytosolic receptor concentrations during the neonatal period in any of the regions studied (hypothalamus, amygdala, preoptic area and septum). Receptor concentrations in all 4 regions increase gradually over the first 10 days of life, with no change in the affinity for 5-alpha-dihydrotestosterone. Nuclear receptor levels in intact pups, measured using an exchange assay, are highest between days 4 and 8 of life. In general, nuclear receptor levels are higher in males than in females; however, this sex difference is most consistently seen in the amygdala. These results are discussed in relation to sex differences in circulating testosterone levels and with respect to the contribution of androgens to the sexual differentiation of behavior.

Glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in discrete nuclei of hypothalamus and substantia nigra.

The distribution of L-glutamate decarboxylase activity (GAD) and gamma-aminobutyric acid (GABA) was investigated in the hypothalamic nuclei and in parts of the substantia nigra in the rat. GAD varied markedly among these areas. The reticular part of the nigra showed the highest activity two-fold higher than any other nucleus. Among the hypothalamic nuclei, a 5-fold difference was found between the poorest and richest nuclei. High GAD was measured in the preoptic, anterior and dorsomedial nuclei. Low activity was found in arcuate and supraoptic nuclei. The lowest GAD activity was measured in the median eminence with only half the activity of the whole brain homogenate. This suggests that GABAergic neurones might not be involved in neuroendocrine regulation at the median eminence level. GABA was determined using the sensitive cycling microassay. The rats were killed by microwaves, procedure which was found to inactivate enzymatic processes within two sec without affecting the level or the distribution of GABA. Postmortem increments during the first 3 min following decapitation ranged up to 5 times the endogenous levels, and were proportional to the GAD activity of the corresponding nucleus. This confirms that GAD is the limiting factor in GABA synthesis and suggests that GABA turnover might be rapid. Endogenous GABA showed a uniform distribution within hypothalamic nuclei and nigra. No relationship appeared between endogenous GABA levels and GAD activities in the various nuclei. These results suggest that while GABA synthesis is likely to occur in non-evenly distributed nerve cells, most GABA may be stored in surrounding cells, presumably glia cells.

Release of thyrotropin releasing hormone into hypophysial portal blood is high relative to other neuropeptides and may be related to prolactin secretion.

The amount of immunoreactive TRH released into hypophysial portal blood of female rats was about 2 orders of magnitude greater than gonadotropin releasing hormone and somatostatin. The turnover of TRH, as high as 80% of the total hypothalamic content per hour, was also much greater than that of any other known peptide. TRH release increased during the expected proestrous surge of prolactin and also in some animals during suckling.

Development of a radioimmunoassay for Pro-Leu-Gly-NH2 (PLG or MIF-I): evidence that PLG is not present in rat brain.

Pro-Leu-Gly-NH2 (PLG), which is the C-terminal tripeptide tail of oxytocin, has been reported to possess melanocyte-stimulating hormone (MSH)-release-inhibiting activity. Although it has been isolated from bovine hypothalamus, little is known about the CNS distribution of this peptide in other species. In this report, we describe the development of a radioimmunoassay which can be used to measure both PLG and oxytocin following chromatographic separation by high pressure liquid chromatography (HPLC). Using this method, we are unable to demonstrate the presence of any endogenous PLG in rat hypothalamus, preoptic area, pituitary, or eye tissue. However, synthetic PLG, which is added to tissue homogenates as an internal standard, is consistently recovered from all areas. We conclude that the PLG tripeptide is not present in the rat brain and thus cannot be the physiological regulator of MSH secretion.

Noradrenergic regulation of alpha 1-receptors during the postnatal development of the guinea pig.

In guinea pig brain, alpha 1-noradrenergic receptor concentrations undergo region-specific fluctuations during the first weeks of postnatal life. However, the factors involved in the regulation of these receptors have yet to be identified. In this study, the ontogeny of one possible regulatory factor, norepinephrine, was examined in relation to postnatal changes in alpha 1-receptor levels in several different regions of guinea pig brain. Results from these studies showed that while the activity of the noradrenergic system increased throughout the first weeks of postnatal development in each brain area examined, the concentration of alpha 1-receptors decreased in preoptic area and hypothalamus and increased in cortex. In subsequent experiments, the effects of noradrenergic lesions with 6-hydroxydopamine on alpha 1-receptor levels were assessed to examine the possibility that alpha 1-receptors are differentially sensitive to noradrenergic stimulation in cortex and preoptic area/hypothalamus in immature guinea pigs. Noradrenergic lesions which reduced norepinephrine levels by 87-94% resulted in significant elevations in alpha 1-receptors in all regions examined. These results are discussed with reference to the anatomical distribution of alpha 1-receptors and their regulation by norepinephrine.

Androgen receptor levels in hypothalamic and vocal control nuclei in the male zebra finch.

Using a synthetic, non-metabolizable ligand, R1881 (methyltrienolone), an in vitro binding assay was developed to quantify levels of cytosol androgen receptors in microdissected nuclei from the finch brain. Saturable, high affinity binding in the nanomolar range was demonstrated in the brain areas examined, and receptor levels were unaffected by freezing the tissue samples. The assay was specific for androgen receptors when 10 microM triamcinolone acetonide was added to inhibit the binding of R1881 to glucocorticoid and progestin receptors. Levels of cytosol androgen receptors were quantified in 3 hypothalamic and 3 vocal control nuclei presumed to contain high concentrations of androgen receptors on the basis of previous autoradiography. All nuclei examined showed significant levels of androgen receptors ranging from 5.8 to 35.8 fmol/mg protein. Hypothalamic nuclei had higher concentrations than vocal control nuclei.