Shirley A. Joseph

The unlabeled antibody method. Contrasting color staining of paired pituitary hormones without antibody removal.

tetrahydrochioride (DAB) to yield a brown reaction product. In the second sequence, antiserum to the second antigen was again followed by anti-immunoglobulin and PAP, and then by hydrogen peroxide and 4-chloro-1-napthol (CN) to yield a blue reaction product. Even though the same anti-immunoglobulin and PAP were used in both sequences, and even though hydrogen peroxide was applied as enzyme substrate twice, no color mixing occurred. Therefore, it has been found unnecessary to remove the immunoreagents of the first staining sequence prior to applying the second sequence. Apparently, the DAB reaction product masked antigen and catalytic sites of the first sequence of immunoreagents and thus prevented interaction with reagents of the second sequence. This conclusion had been derived from attempts at staining the same antigen with the same primary antiserum in the first and second sequence. Only brown reaction product was obtained and no color mixing occurred. However, when either primary antiserum or DAB in the otherwise complete first reaction sequence were progressively diluted, colors became mixed until upon omission of primary antiserum or DAB, standard (control) blue was obtained. Similarly, standard brown was obtained when primary antiserum was omitted in the second sequence. In the pars distalis of the pituitary, separate cells stained brown and blue when pairs of antisera to the following hormones were applied: growth hormone, prolactin, and either ACTH’24 or ovine f1-lipotropin. Separate cells were also visualized when anti-ACTH’24 was followed by anti-LH. When anti-growth hormone was followed by anti-LH, a few cells were mixed in color, even though most cells were either brown or blue. When anti-ACTh’24 was followed by anti-ACTh’3, mixed color staining occurred in cells of the pars intermedia and distalis, and fibers in the pars nervosa were blue.

Distribution of somatostatin in the rat brain: Telencephalon and diencephalon

The distribution of somatostatin (SRIF) was examined using the unlabeled antibody enzyme method of immunocytochemistry on thick 30-50 microns Vibratome sections. The greatest population of SRIF-neurons was observed along the ventricular wall in the preoptic area and anterior hypothalamus. Dense accumulations of fibers were observed in the suprachiasmatic, ventromedial and arcuate nuclei, the internal and external zone of the median eminence, the organum vasculosum of the lamina terminalis and the subfornical organ. Extrahypothalamic sites of SRIF-containing neurons and fibers were also observed throughout the telencephalon. The widespread distribution of SRIF is consistent with radioimmunoassay data and suggests a diverse physiological role for somatostatin.

Systemic nitroglycerin induces Fos immunoreactivity in brainstem and forebrain structures of the rat

Nitroglycerin is a vasodilator which induces vascular relaxation by releasing nitric oxide in the wall of blood vessels. It has been suggested that the cardiovascular inhibitory responses which are induced by this drug are mediated by central structures. In this study, we evaluated the distribution and intensity of Fos immunoreactivity in rat brain nuclei following the systemic administration of nitroglycerin. In the medulla, a significant number of Fos-immunoreactive neurons were observed in the nucleus tractus solitarius, ventrolateral medulla, area postrema and spinal trigeminal nucleus caudalis. A robust staining was seen in the parabrachial nucleus, locus coeruleus and ventrolateral periaqueductal grey. In the hypothalamus, Fos-positive cells were densely packed in the paraventricular and supraoptic nuclei. Other areas where significant staining was observed include the central nucleus of the amygdala and the subfornical organ. These findings demonstrate that the systemic administration of nitroglycerin is capable of activating a spectrum of functionally diverse brain regions. This spectrum includes areas involved in reflex adjustments to nitroglycerin-induced hypotension, areas involved in sensory nociceptive perception and areas associated with integrative regulation of autonomic, behavioral and neuroendocrine functions.

Immunocytochemical localization of ACTH perikarya in nucleus tractus solitarius: evidence for a second opiocortin neuronal system

Immunocytochemical localization of ACTH-opiocortin perikarya was demonstrated in the medulla of colchicine-treated rat. Neuronal cell bodies and fibers containing ACTH-immunoreactivity were abundant in the caudal region of the nucleus tractus solitarius, specifically within pars commisuralis. Location of these opiocortin neurons within the nucleus tractus solitarius provides additional evidence for a role of these peptides in cardiovascular functions.

The distribution and cells of origin of ACTH(1-39)-stained varicosities in the paraventricular and supraoptic nuclei.

ACTH(1-39)-immunoreactive fibers and varicosities were localized using indirect immunofluorescence histochemistry in normal rats, and were found to be distributed in specific parts of the parvocellular division of the paraventricular nucleus, and in regions of the magnocellular division of the paraventricular and supraoptic nuclei in which oxytocinergic cells predominate. A combined retrograde transport-immunohistochemical method was used to confirm that these projections arise from a group of ACTH(1-39)-stained cells in the arcuate nucleus (and in adjacent regions along the base of the hypothalamus), and to describe their distribution within this region.

Arcuate nucleus projections to brainstem regions which modulate nociception.

Anterograde tracing studies were conducted in order to identify efferents from the arcuate nucleus, which contains the hypothalamic opiocortin neuronal pool. Phaseolus vulgaris leucoagglutinin (PHA-L) was stereotaxically iontophoresed into the arcuate nucleus and the terminal fields emanating from the labelled perikarya were identified immunocytochemically. PHA-L-immunoreactive (-ir) fibers were identified in nucleus accumbens, lateral septal nucleus, bed nucleus of the stria terminalis, medial and lateral preoptic areas, anterior hypothalamus, amygdaloid complex, lateral hypothalamus, paraventricular nucleus, zona incerta, dorsal hypothalamus, periventricular gray, medial thalamus and medial habenula. In the brainstem, arcuate terminals were identified in the periaqueductal gray (PAG), dorsal raphe nucleus (DRN), nucleus raphe magnus (NRM), nucleus raphe pallidus, locus coeruleus, parabrachial nucleus, nucleus reticularis gigantocellularis pars alpha, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve. Dual immunostaining was used to identify the neurochemical content of neurons in arcuate terminal fields in the brainstem. Arcuate fiber terminals established putative contacts with serotonergic neurons in the ventrolateral PAG, DRN and NRM and with noradrenergic neurons in periventricular gray, PAG and locus coeruleus. In the PAG, arcuate fibers terminated in areas with neurons immunoreactive to substance P, neurotensin, enkephalin and cholecystokinin (CCK) and putative contacts were identified with CCK-ir cells. This study provides neuroanatomical evidence that putative opiocortin neurons in the arcuate nucleus influence a descending system which modulates nociception.

Immunocytochemical distribution of LHRH neurons and processes in the rat: Hypothalamic and extrahypothalamic locations

SummaryThe distribution of luteinizing hormone-releasing hormone (LHRH)-immunoreactive perikarya and processes was examined, in the untreated rat, with the unlabeled antibody enzyme method of immunocytochemistry on thick 50 μm vibratome sections. LHRH neurons were primarily observed in the preoptico-anterior hypothalamic and septal areas. Projections from these cell bodies to the median eminence form three distinct pathways, one laterally along the course of the optic tracts, one medially through the periventricular stratum of the third ventricle, and one through the tractus infundibularis. In addition, some of these cell bodies project to the organum vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO). LHRH immunoreactive neurons were also noted in the anterior olfactory regions; they project along the medial olfactory tract to the olfactory bulb.

Corticotropin releasing factor: Immunocytochemical localization in rat brain

Using antiserum generated against the synthetic CRF1-41 we have immunocytochemically localized perikarya and processes in rat brain. Areas observed to have particularly dense accumulation of CRF-ir cells were in the extra-hypothalamic areas of nucleus accumbens septi, nucleus of the stria terminalis, the medial preoptic region, and the central amygdaloid nucleus. Within the hypothalamus cell bodies were scattered throughout the anterior hypothalamic region and densely packed in the paraventricular nucleus. Fibers appear most dense in the lateral septal region and throughout the external layer of the median eminence.

THE EFFECTS ON THE CENTRAL NERVOUS SYSTEM OF NITROGLYCERIN-PUTATIVE MECHANISMS AND MEDIATORS

Nitroglycerin is an organic nitrate that has been used as a vasodilator in the treatment of cardiac diseases for over a century. Only recently it has been demonstrated that the vasodilator effect of this drug depends upon the formation of nitric oxide in the blood vessel wall. However, clinical and research data gathered during the last decades have suggested that nitroglycerin possesses, besides its peripheral vasodilator effect, additional, puzzling biological activities. This organic nitrate compound provokes reflex cardiovascular activities via its interaction with the central sympathetic system. Its cerebrovascular effect, on the other hand, is probably mediated by the local release of neuropeptides. The direct application of nitroglycerin onto brain nuclei causes a prompt increase in the neuronal discharge rate. From a neurological point of view, nitroglycerin consistently induces a specific headache attack in patients suffering from migraine. Because of its temporal pattern and clinical characteristics, nitroglycerin-induced headache cannot be solely ascribed to the a drug-induced vasorelaxation. The demonstration that systemic nitroglycerin administration activates a widespread set of vegetative, nociceptive and neuroendocrine structures in the central nervous system seems to further support the occurrence of central mechanisms in the biological activity of nitroglycerin. Double labeling immunocytochemical and neuropharmacological studies have provided information on the putative neurotransmitters and neurochemical mechanisms involved in nitroglycerin-induced neuronal activation.

Nitrous oxide analgesia: partial antagonism by naloxone and total reversal after periaqueductal gray lesions in the rat

Analgesia induced by nitrous oxide was examined using radiant heat tail flick and electrical evoked foot flick tests in rats. Rats exposed to 80 and 60% nitrous oxide expressed statistically significant elevations of percent analgesia (% MPE) compared to air exposed rats. Rats exposed to 30% nitrous oxide showed no significant difference in percent analgesia. Pretreatment with naloxone (10 mg/kg s.c.) produced a significant decrease in %MPE and an increase in variance of response after exposures to 80% nitrous oxide in a double blind study. Kainic acid lesions of the ventral and caudal periaqueductal grey (PAG) reversed analgesia produced by 80% nitrous oxide in a crossover blink study compared to saline lesions. In conclusion, this evidence suggests that the caudal-PAG-raphe mangus-dorsal horn pain inhibition pathway is in part involved in the analgesia induced by nitrous oxide.