UVB irradiation induces contralateral changes in galanin, substance P and c-fos immunoreactivity in rat dorsal root ganglia, dorsal horn and lateral spinal nucleus.

The selection of control group is crucial, as the use of an inadequate group may strongly affect the results. In this study we examine the effect on contralateral tissue protein levels, in a model of unilateral UVB irradiation, as the contralateral side is commonly used as a control. Previous studies have shown that UVB irradiation increases immunoreactivity for inflammatory regulated neuropeptides. Unilateral UVB irradiation of rat hind paw was performed and corresponding contralateral spinal cord and dorsal root ganglia (DRG) were collected 2-96 h after and investigated for changes in galanin, substance P and c-fos immunoreactivity. Control tissue was collected from naïve rats. Measurement of skin blood flow from contralateral heel hind paws (Doppler), revealed no change compared to naïve rats. However, UVB irradiation caused a significant reduction in the contralateral proportion of galanin immunopositive DRG neurons, at all-time points, as well as an increase in the contralateral spinal cord dorsal horn, around the central canal and in the lateral spinal nucleus (2-48 h). The contralateral proportion of SP positive DRG neurons and dorsal horn immunoreactivity was unchanged, whereas the lateral spinal nucleus area showed increased immunoreactivity (48 h). UVB irradiation also induced a slight contralateral upregulation of c-fos in the dorsal horn/central canal area (24 and 48 h). In summary, unilateral UVB irradiation induced contralateral changes in inflammatory/nociceptive neuropeptides in spinal cord and afferent pathways involved in pain signaling already within 24 h, a time point when also ipsilateral neurochemical/physiological changes have been reported for rats and humans.

Chronic infusion of lisinopril into hypothalamic paraventricular nucleus modulates cytokines and attenuates oxidative stress in rostral ventrolateral medulla in hypertension.

The hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) play a critical role in the generation and maintenance of sympathetic nerve activity. The renin-angiotensin system (RAS) in the brain is involved in the pathogenesis of hypertension. This study was designed to determine whether inhibition of the angiotensin-converting enzyme (ACE) in the PVN modulates cytokines and attenuates oxidative stress (ROS) in the RVLM, and decreases the blood pressure and sympathetic activity in renovascular hypertensive rats. Renovascular hypertension was induced in male Sprague-Dawley rats by the two-kidney one-clip (2K1C) method. Renovascular hypertensive rats received bilateral PVN infusion with ACE inhibitor lisinopril (LSP, 10µg/h) or vehicle via osmotic minipump for 4weeks. Mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and plasma proinflammatory cytokines (PICs) were significantly increased in renovascular hypertensive rats. The renovascular hypertensive rats also had higher levels of ACE in the PVN, and lower level of interleukin-10 (IL-10) in the RVLM. In addition, the levels of PICs, the chemokine MCP-1, the subunit of NAD(P)H oxidase (gp91(phox)) and ROS in the RVLM were increased in hypertensive rats. PVN treatment with LSP attenuated those changes occurring in renovascular hypertensive rats. Our findings suggest that the beneficial effects of ACE inhibition in the PVN in renovascular hypertension are partly due to modulation cytokines and attenuation oxidative stress in the RVLM.

Aquaporin-4 autoantibody: a neurogenic cause of anorexia and weight loss.

Neuromyelitis optica (NMO) is a severe inflammatory demyelinating disease often associated with a highly specific autoantibody, aquaporin-4 antibody. Although the classic syndrome involves the optic nerves and spinal cord, aquaporin-4 antibody has been important in defining the true spectrum of NMO, which now includes brain lesions in areas of high aquaporin-4 expression. Brainstem involvement, specifically area postrema involvement in the medulla, has been associated with intractable vomiting in some patients with NMO. We describe a 14-year-old female with positive aquaporin-4 antibody whose clinical course was dominated by severe anorexia with associated weight loss (from 68-41kg; body mass index 25.2-15.6). Magnetic resonance imaging showed lesions in the medulla, pons, and thalami. Although she had asymptomatic radiological longitudinally extensive transverse myelitis, she never had symptoms or signs referable to the spinal cord or the optic nerves. We propose that anorexia and weight loss should be considered part of the NMO spectrum, probably related to area postrema involvement.

Ascending caudal medullary catecholamine pathways drive sickness-induced deficits in exploratory behavior: brain substrates for fatigue?

Immune challenges can lead to marked behavioral changes, including fatigue, reduced social interest, anorexia, and somnolence, but the precise neuronal mechanisms that underlie sickness behavior remain elusive. Part of the neurocircuitry influencing behavior associated with illness likely includes viscerosensory nuclei located in the caudal brainstem, based on findings that inactivation of the dorsal vagal complex (DVC) can prevent social withdrawal. These brainstem nuclei contribute multiple neuronal projections that target different components of autonomic and stress-related neurocircuitry. In particular, catecholaminergic neurons in the ventrolateral medulla (VLM) and DVC target the hypothalamus and drive neuroendocrine responses to immune challenge, but their particular role in sickness behavior is not known. To test whether this catecholamine pathway also mediates sickness behavior, we compared effects of DVC inactivation with targeted lesion of the catecholamine pathway on exploratory behavior, which provides an index of motivation and fatigue, and associated patterns of brain activation assessed by immunohistochemical detection of c-Fos protein. LPS treatment dramatically reduced exploratory behavior, and produced a pattern of increased c-Fos expression in brain regions associated with stress and autonomic adjustments paraventricular hypothalamus (PVN), bed nucleus of the stria terminalis (BST), central amygdala (CEA), whereas activation was reduced in regions involved in exploratory behavior (hippocampus, dorsal striatum, ventral tuberomammillary nucleus, and ventral tegmental area). Both DVC inactivation and catecholamine lesion prevented reductions in exploratory behavior and completely blocked the inhibitory LPS effects on c-Fos expression in the behavior-associated regions. In contrast, LPS-induced activation in the CEA and BST was inhibited by DVC inactivation but not by catecholamine lesion. The findings support the idea that parallel pathways from immune-sensory caudal brainstem sources target distinct populations of forebrain neurons that likely mediate different aspects of sickness. The caudal medullary catecholaminergic projections to the hypothalamus may significantly contribute to brain mechanisms that induce behavioral "fatigue" in the context of physiological stressors.

Immune challenge and satiety-related activation of both distinct and overlapping neuronal populations in the brainstem indicate parallel pathways for viscerosensory signaling.

Caudal brainstem viscerosensory nuclei convey information about the body's internal state to forebrain regions implicated in feeding behavior and responses to immune challenge, and may modulate ingestive behavior following immune activation. Illness-induced appetite loss might be attributed to accentuated "satiety" pathways, activation of a distinct "danger channel" separate from satiety pathways, or both. To evaluate neural substrates that could mediate the effects of illness on ingestive behavior, we analyzed the pattern and phenotypes of medullary neurons responsive to consumption of a preferred food, sweetened milk, and to intraperitoneal lipopolysaccharide challenge that reduced sweetened milk intake. Brainstem sections were stained for c-Fos, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, and glucagon-like peptide-1 (GLP-1) immunoreactivity. Sweetened milk intake activated many neurons throughout the nucleus of the solitary tract (NTS), including A2 noradrenergic neurons in the caudal half of the NTS. LPS challenge activated a similar population of neurons in the NTS, in addition to rostral C2 adrenergic and mid-level A2 noradrenergic neurons in the NTS, many C1 and A1 neurons in the ventrolateral medulla, and in GLP-1 neurons in the dorsal medullary reticular nucleus. Increased numbers of activated GLP-1 neurons in the NTS were only associated with sweetened milk ingestion. Evidence for parallel processing was reflected in the parabrachial nucleus, where sweetened milk intake resulted in activation of the inner external lateral, ventrolateral and central medial portions, whereas LPS challenge induced c-Fos expression in the outer external lateral portions. Thus, signals generated in response to potentially dangerous physiological conditions seem to be propagated via specific populations of catecholaminergic neurons in the NTS and VLM, and likely include a pathway through the external lateral PBN. The data indicate that immune challenge engages multiple ascending neural pathways including both a distinct catecholaminergic "danger" pathway, and a possibly multimodal pathway derived from the NTS.

Organization of immune-responsive medullary projections to the bed nucleus of the stria terminalis, central amygdala, and paraventricular nucleus of the hypothalamus: evidence for parallel viscerosensory pathways in the rat brain.

Immune-responsive neurons in the brainstem, primarily in the nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), contribute to a significant drive on forebrain nuclei responsible for brain-mediated host defense responses. The current study investigated the relative contribution of brainstem-derived ascending pathways to forebrain immune-responsive nuclei in the rat by means of retrograde tract tracing and c-Fos immunohistochemistry. Fluorogold was iontophoresed into the bed nucleus of stria terminalis (BST), central nucleus of the amygdala (CEA), paraventricular nucleus of the hypothalamus (PVN), and the pontine lateral parabrachial nucleus (PBL; an important component of ascending viscerosensensory pathways) followed 2 weeks later by intraperitoneal injection of lipopolysaccharide (LPS, 0.1 mg/kg) or saline. The NTS and VLM provide immune-responsive input to all four regions, via direct, predominantly catecholaminergic, projections to the PVN, the lateral BST, and the CEA, and mostly non-catecholaminergic projections to the PBL. The PBL provides a major LPS-activated input to the BST and CEA. The pattern of LPS-activated catecholaminergic projections from the VLM and NTS to the forebrain is characterized by a strong predominance of VLM input to the PVN, whereas the NTS provides a greater contribution to the BST. These findings indicate that direct and indirect pathways originate in the caudal brainstem that propagate immune-related information from the periphery with multiple levels of processing en route to the forebrain nuclei, which may allow for integration of brain responses to infection.

Orcokinin immunoreactivity in the accessory medulla of the cockroach Leucophaea maderae.

The accessory medulla is the master circadian clock in the brain of the cockroach Leucophaea maderae and controls circadian locomotor activity. Previous studies have demonstrated that a variety of neuropeptides are prominent neuromediators in this brain area. Recently, members of the orcokinin family of crustacean neuropeptides have been identified in several insect species and shown to be widely distributed in the brain, including the accessory medulla. To investigate the possible involvement of orcokinins in circadian clock function, we have analyzed the distribution of orcokinin immunostaining in the accessory medulla of L. maderae in detail. The accessory medulla is densely innervated by approximately 30 orcokinin-immunoreactive neurons with cell bodies distributed in five of six established cell groups in the accessory medulla. Immunostaining is particularly prominent in three ventromedian neurons. These neurons have processes in a median layer of the medulla and in the internodular neuropil of the accessory medulla and send axonal fibers via the posterior optic commissure to their contralateral counterparts. Double-labeling experiments have revealed the colocalization of orcokinin immunostaining with immunoreactivity for pigment-dispersing hormone, FMRFamide, Mas-allatotropin, and gamma-aminobutyric acid in two cell groups of the accessory medulla, but not in the ventromedian neurons or in the anterior and posterior optic commissure. Immunostaining in the ventromedian neurons suggests that orcokinin-related peptides play a role in the heterolateral transmission of photic input to the pacemaker and/or in the coupling of the bilateral pacemakers of the cockroach.

Reversible inactivation of the dorsal vagal complex blocks lipopolysaccharide-induced social withdrawal and c-Fos expression in central autonomic nuclei.

Peripheral administration of lipopolysaccharide (LPS), a potent activator of the immune system, induces symptoms of behavioral depression, such as social withdrawal, concommitant with increases in c-Fos expression in central autonomic network nuclei. Previous studies implicated vagal visceral sensory nerves in transduction of immune-related signals relevant to for the induction of social withdrawal, a symptom of behavioral depression. Vagal sensory nerves terminate in the dorsal vagal complex (DVC) of the brainstem, a region that functions to integrate visceral signals and may also play a role in modulating arousal and affect. The objective of the current study was to determine whether the DVC contributes to immunosensory pathways driving symptoms of social withdrawal associated with LPS-induced behavioral depression, using a reversible lesion technique to temporarily inactivate the DVC. To assess the effects of DVC inactivation on LPS-induced social withdrawal and the subsequent changes in brain activation, we used behavioral assessment of social withdrawal, and analyzed c-Fos expression, a marker of neuronal activation, in the central nucleus of the amygdala (CEA), bed nucleus of the stria terminalis (BST), hypothalamic paraventricular nucleus (PVN), and ventromendial preoptic area (VMPO). Two hours following intraperitoneal LPS injection, there was a significant increase in c-Fos immunoreactivity in forebrain regions in animals treated with LPS. DVC inactivation completely blocked LPS-induced social withdrawal and dramatically reduced LPS-induced Fos expression in all four forebrain regions assessed. Collectively, these findings support the idea that the DVC acts as an immune-behavior interface between the peripheral stimuli and brain areas involved in modulating social behavior.

Evidence for involvement of the neural pathway containing the peripheral vagus nerve, medullary visceral zone and central amygdaloid nucleus in neuroimmunomodulation.

It is now evident that a bidirectional communication network exists between the central nervous system (CNS) and immune system (IS). However, the way in which the IS passes inform to the brain is not quite clear.In the present study, one of the neural pathways involved in the cytokine-to-brain communication was investigated in the rat. This pathway starts at the vagal nerve projecting to the medullary visceral zone (MVZ), an arc-shape band from the dorsomedial to ventrolateral area in the middle-caudal segment of the medulla oblongata, and terminates at the central amygdaloid nucleus (Ce) which receives projections from large catecholaminergic neurons in the MVZ. Animals were randomly divided into two experimental groups. Triple-labeling was used in Group I animals to combine wheat germ aggulutinin-conjugated horseradish peroxidase (WGA-HRP) retrograde tracing with anti-Fos and anti-tyrosine hydroxylase (TH) immunostaining. WGA-RP was stereotaxically injected into the unilateral Ce of the animals and, after a survival period of 48 h, intraperitoneal (IP) injection of lipopolysaccharide (LPS) was performed. Seven kinds of labeled neurons were observed in the MVZ, namely, HRP-, Fos- or TH-singly-labeled neurons; Fos/HRP-, Fos/TH- or HRP/TH-doubly-labeled neurons; and Fos/HRP/TH-triply-labeled neurons. As for Group II animals, bilateral subdiaphragmatic vagotomy (SDV) or sham operation was performed, followed 4 weeks later by IP injection of LPS. The number of Fos-positive neurons within the Ce and MVZ was significantly lower (P<0.01) in rats having SDV when compared with those receiving sham operation. Our results suggest that part of the peripheral immune information can be conveyed through the vagus to the catecholaminergic neurons in the MVZ, where it is transported to the Ce. The MVZ is a neural relay station in the immune-to-brain communication and might play a significant role in neuroimmuno-modulation via the vagus-MVZ-Ce pathway.

Regional distribution of immunoreactive prolactin-releasing peptide in the human brain.

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.

Perfluoro-15-crown-5-ether labelled macrophages in adoptive transfer experimental allergic encephalomyelitis.

In this serial in vivo study, macrophages labelled with perfluoro-15-crown-5-ether (15C5) were monitored in rats after inducing adoptive transfer experimental allergic encephalomyelitis (AT-EAE). AT-EAE is an animal model of multiple sclerosis and is characterized by inflammatory infiltrates in the central nervous system (CNS) and breakdown of the blood-brain-barrier. A particular feature of AT-EAE are macrophage infiltrates. Purpose of this study was to monitor the invasive and evasive phase of the macrophages in AT-EAE by using 3-dimensional 19F magnetic resonance imaging (3D 19F-MRI). In the early stage of the disease, a much stronger 19F-signal intensity was observed in AT-EAE-rats than in healthy control rats in the tissue adjacent to CNS regions severely affected by inflammatory infiltrates, and thereafter the 19F-signal intensity was decreasing over the time. However, no 19F-signal could be observed in the CNS itself neither in AT-EAE-rats nor in control rats. According to these findings it is assumed that we monitored the evasion of the macrophages from the region of inflammation.

Pituitary adenylate cyclase activating polypeptide immunoreactivity in the rat spinal cord and medulla: implication of sensory and autonomic functions.

Immunoreactivity to pituitary adenylate cyclase activating polypeptide-38 was detected in numerous nerve fibres in layers I and II of the dorsal horn of the rat and some of these fibres extended into the deeper layers of all segments of the spinal cord. Immunoreactivity was also detected in the lateral funiculus projecting into the intermediolateral cell column of the lower cervical and thoracic segments and in the lateral pathway terminating in the intermediate gray area of the lower lumbar and sacral segments. Neurons in the lateral horn area were not immunoreactive nor were the ventral horn motoneurons. In the medulla, numerous immunoreactive fibres were observed in the spinal trigeminal tract and superficial layers of the caudal spinal trigeminal nucleus but few in the interpolar spinal trigeminal nucleus. A prominent immunoreactive nerve bundle emanated from the caudal spinal trigeminal nucleus and projected into the solitary tract. A dense network of immunoreactive neurons and fibres was present in the nucleus raphe obscurus, lateral reticular nucleus and parvocellular lateral reticular nucleus. Immunoreactive fibres could also be detected in the solitary tract and area postrema. Labelled somata were occasionally noted in various subnuclei of the nucleus of the solitary tract and nucleus raphe pallidus. In addition, a small number of positive neurons were detected in an area between the lateral reticular nucleus and inferior olive and near the ventral surface of the medulla (parapyramidal region). A few weakly-labelled cells were occasionally seen in the dorsal motor nucleus of vagus. A population of neurons in the trigeminal, nodose and dorsal root ganglia from all segments of the spinal cord displayed low to intense immunoreactivity. The presence of immunoreactivity in nodose and dorsal root ganglia, dorsal horn, spinal autonomic nuclei, solitary tract and in certain areas of the medulla suggests that this peptide may participate in a variety of sensory and autonomic functions.

Tau immunoreactivity associated with aluminum maltolate-induced neurofibrillary degeneration in rabbits.

Intracisternal administration of aluminum maltolate to rabbits produces a marked argyrophilic neurofibrillary degeneration (NFD) which is also immunoreactive for both phosphorylated and non-phosphorylated microtubule associated protein tau. Using tissue fixation in PBF, the monoclonal antibodies Tau-2 and AT8 stain the NFD. Dephosphorylation markedly reduces the positivity of AT8. Using PLP-fixed tissue, monoclonal antibody Tau-1 also immunostains aluminum-induced NFD.

Anatomic patterns of Fos immunostaining in rat brain following systemic endotoxin administration.

To identify brain neurons that participate in the acute phase response, rat brains were examined immunocytochemically for Fos protein following the intravenous administration of bacterial endotoxin (lipopolysaccharide, LPS). Two to three hours after the injection of LPS, 150 micrograms/kg body weight, to adult male Long-Evans rats, a consistent anatomic pattern of Fos immunostained cell nuclei is seen. In the brain stem, prominant Fos immunostaining is induced in tyrosine hydroxylase immunoreactive neurons of the caudal ventral-lateral medulla (the A1 cell group), in both tyrosine hydroxylase positive and negative neurons of nu. tractus solitarius, in the parabrachial nu., and in a few neurons of the locus ceruleus. In the hypothalamus, endotoxin induces Fos expression in magnocellular neurons of the paraventricular and supraoptic nuclei and internuclear cell groups. A higher percentage of oxytocin-immunoreactive cells is double labeled for Fos nuclear immunostaining than vasopressin-immunoreactive cells. A minority of somatostatin immunoreactive periventricular hypothalamic neurons are Fos positive. Other hypothalamic nuclei that contain endotoxin-induced Fos nuclear immunostaining include the parvocellular neurons of the paraventricular nu., the dorsomedial and arcuate nuclei, the lateral hypothalamus, the dorsal hypothalamic area (zona incerta), and the median nucleus of the preoptic area. LPS induces numerous Fos-positive neurons in regions known to respond to a variety of stressful stimuli; these regions include the preoptic area, bed nucleus of the stria terminalis, lateral septum, and the central and medial nuclei of the amygdala. Moreover, Fos nuclear immunostaining is seen in neurons of circumventricular organs: the organum vasculosum of the lamina terminalis, the subfornical organ, and the area postrema. The maximum intensity of Fos nuclear immunostaining occurs 2-3 h after endotoxin administration and declines thereafter. It is attenuated by pretreatment with indomethacin, 25 mg/kg body weight Sc, or dexamethasone, 1 mg/kg IP. These observations are consistent with the participation of a variety of brain neuronal systems in the acute phase response and elucidate the functional neuroanatomy of that response at the cellular level.

Target regulation of neuronal differentiation in a temperature-sensitive cell line derived from medullary raphe.

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen, a clonal cell line, RN33B, was isolated by serial dilution. At 33 degrees C, RN33B cells divide with a doubling time of 48 h and show T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. RN33B cells are immortal, but not transformed, as they will not grow in soft agar. At non-permissive temperature (38.5 degrees C), T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33 degrees C re-express T antigen, but do not de-differentiate or begin dividing. Co-culture with embryonic hippocampus and cerebral cortex, but not medullary raphe or spinal cord, resulted in significantly greater survival, more complex neuronal morphology, and enhanced expression of neuronal-specific antigens. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. These data suggest that the observed target regulation of RN33B cell neuronal differentiation in co-culture may be mediated by neurotrophin(s).

Activation of brainstem catecholaminergic neurons by conditioned and unconditioned aversive stimuli as revealed by c-Fos immunoreactivity.

In an attempt to define areas of the brain that respond to stressors and influence immune function, we have previously identified stress-induced, c-Fos-immunoreactive areas of the diencephalon. We found that c-Fos was strongly expressed in cells of the paraventricular nuclei (some of which contain corticotropin-releasing hormone (CRH)) and other hypothalamic areas directly associated with autonomic function. To further characterize the presumptive pathways mediating stress-induced immune alterations, including the assessment of brainstem catecholaminergic neuron involvement, the induction of c-Fos immunoreactivity was examined in the brainstem of rats exposed to conditioned and unconditioned, immunomodulating stimuli. In response to electric footshock (the unconditioned stimulus (US)), c-Fos immunoreactivity was strongly induced in the noradrenergic neurons of the locus ceruleus (A6), the nucleus of the solitary tract (A2/C2), the ventral lateral medulla (A1/C1), A5, and A7, as well as in unidentified neurons of the dorsal and ventral subdivisions of the periaqueductal gray (PAG), and in the serotonergic neurons of the dorsal raphe nuclei. Conditioned animals re-exposed to the conditioned stimulus showed c-Fos induction in these same areas but to a lesser degree. Control animals exposed only to the conditioning stimulus (CS) (electronic tone) in the absence of the US, expressed very little, if any, c-Fos activity in the above loci except for a small degree of baseline expression in the PAG. These results further confirm the role of autonomic and endocrine pathways as mediators of the stress response and will help to more fully characterize the pathways of stress-induced immune alteration.

Neurons in the rat medulla oblongata containing neuropeptide Y-, angiotensin II-, or galanin-like immunoreactivity project to the parabrachial nucleus.

Projections from the medulla to the parabrachial complex of the rat were examined for their content of neuropeptide Y-, angiotensin II- or galanin-like immunoreactivity using combined retrograde tracing and immunohistochemical techniques. Rhodamine-labelled latex microspheres were stereotaxically injected into discrete nuclei of the parabrachial complex. After survival of two to five days, colchicine (100 micrograms in 10 microliters saline) was injected into the cisterna magna. One day later, rats were perfused and the brainstems were prepared for visualization of the retrograde tracer and immunoreactivity of one of the three peptides. Retrograde labelling verified that the area postrema, nucleus of the tractus solitarius, caudal spinal nucleus of the trigeminal nerve, parvocellular reticular nucleus, and ventrolateral medulla including the rostral ventrolateral medulla and nucleus paragigantocellularis project to the lateral parabrachial and Kölliker-Fuse nuclei. While most projections were primarily ipsilateral, a small proportion of the projections from the ventrolateral medulla was bilateral. Neurons containing neuropeptide Y-like immunoreactivity were found in the caudal and intermediate nucleus of the tractus solitarius, dorsal to the lateral reticular nucleus and in the nucleus paragigantocellularis. After bilateral microsphere injections into the lateral parabrachial and Kölliker-Fuse nuclei, double-labelled neurons were found dorsal to the lateral reticular nucleus of caudal and intermediate medullary levels, at the ventral surface of the medulla at intermediate levels and in the nucleus paragigantocellularis at rostral levels. Neurons with angiotensin II-like immunoreactivity were observed at the dorsomedial border of the caudal and intermediate nucleus of the tractus solitarius, in the area postrema and in the lateral reticular nucleus and nucleus paragigantocellularis. Of these neurons, small numbers in the nucleus of the tractus solitarius and ventrolateral medulla also projected to the lateral parabrachial and Kölliker-Fuse nuclei. Neurons containing galanin-like immunoreactivity were found in the caudal nucleus of the tractus solitarius, the area postrema, the spinal trigeminal nucleus, the raphe nuclei (pallidus and obscurus), the nucleus paragigantocellularis and dorsal to the lateral reticular nucleus. Of these cells, double-labelled neurons were found in the commissural and medial subdivisions of the caudal nucleus of the tractus solitarius and in the rostral ventrolateral medulla including the ventral surface and the nucleus paragigantocellularis. The results suggest that neuropeptide Y, angiotensin II and galanin may serve as neurochemical messengers in pathways from the medulla to the parabrachial complex. The location of double-labelled neurons suggests that the information relayed by these neurons is related to autonomic activity.

Immunohistochemical localization of calretinin in the rat hindbrain.

The localization of calretinin in the rat hindbrain was examined immunohistochemically with antiserum against calretinin purified from the guinea pig brain. Calretinin immunoreactivity was found within neuronal elements. The distribution of calretinin-immunoreactive cell bodies and fibers is presented in schematic drawings and summarized in a table. Major calretinin-immunoreactive neurons were found in the lateral and medial geniculate nuclei, substantia nigra, ventral tegmental area, interpeduncular nucleus, periaqueductal gray, mesencephalic trigeminal nucleus, superior and inferior colliculi, pontine nuclei, parabrachial nucleus, dorsal and laterodorsal tegmental nuclei, cochlear nuclei, vestibular nuclei, medullary reticular nuclei, nucleus of the solitary tract, area postrema, substantia gelatinosa of the spinal trigeminal nucleus, and cerebellum. These results show that distinct calretinin-immunoreactive neurons are widely distributed in the rat hindbrain.

Gonadotropin hormone-releasing hormone (GnRH) immunoreactivity in the mesencephalon of sharks and rays.

Other than association with the terminal nerve (TN), little is known concerning the distribution of gonadotropin hormone-releasing hormone (GnRH) in elasmobranchs. The purpose of this study was to identify GnRH immunoreactivity in the brains of three elasmobranch species with special regard to the mesencephalon. The round stingray (Urolophus halleri), thornback guitarfish (Platyrhinoidis triseriata), and leopard shark (Triakis semifasciatus) were used and immunocytochemistry was performed with antisera to both salmon and mammalian GnRH. A large GnRH-immunoreactive (ir) nucleus extends rostrocaudally for approximately 1.5 mm along and adjacent to the midline of the midbrain near the area of the oculomotor nerve. GnRH-ir fibers surround the nucleus and are found diffusely throughout the mesencephalon; some of the fibers may contact the ventricle. The medulla and spinal cord contain ir fibers that most likely originate from the midbrain nucleus. Mesencephalic GnRH-ir cell groups have been reported in representatives of all vertebrate classes with the exception of agnathans and mammals. Such a well-developed cell group in elasmobranchs may aid in understanding the evolution of GnRH systems with regard to the mesencephalon as well as providing insight to the functional significance of this cell group. Possible homologies to mesencephalic GnRH systems reported in other vertebrates is discussed as well.

Medullary and mesencephalic neuronal groups reacting to antibodies against VIP, somatostatin and bombesin in adult Gallus gallus domesticus.

The caudo-cranially intermediate one-third of medullary dorsal region, the periaqueductal grey and the rostro-ventral portion of the midbrain tegmentum of adult chickens were studied in detail by means of the PAP-DAB procedure, to define further the main morphological features of the neuronal populations that in previous studies had shown VIP (Vasoactive Intestinal Polypeptide),-Somatostatin (SRIF)-, and Bombesin-like immunoreactivities. In the medulla, VIP-like immunoreactivity was detected within neuronal bodies and processes and extended down to the cervical spinal cord. SRIF-like immunoreactivity was seen only within nerve cell processes, at least a part of which could be sensitive fibre terminals. Bombesin-like immunoreactivity was observed only within neuronal processes. In the periaqueductal grey, all 3 immunoreactivities were detected within perikarya and neuronal processes, with a higher density cranially. In the rostro-ventral portion of the midbrain tegmentum, VIP-like and Bombesin-like immunoreactivities were detected (the latter being located somewhat more cranially) both in neuronal bodies and in processes. SRIF-like immunoreactivity was found in this region only in long neuronal processes.