Theodor Petrov

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat.

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.

The expression of the low affinity nerve growth factor receptor in long-term denervated Schwann cells

Schwann cells in the distal stump of injured peripheral nerves synthesize the low affinity nerve growth factor receptor (p75). In this study we used short‐term (1 week) and long‐term (1‐12 months) transected distal sciatic nerves of rats to determine the variations of p75 expression by using immunocytochemistry and in situ hybridization.

Chemically defined collateral projections from the pons to the central nucleus of the amygdala and hypothalamic paraventricular nucleus in the rat

Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline-acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine-and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.

Parabrachial nucleus projection to the amygdala in the rat: Electrophysiological and anatomical observations

The amygdala, an important limbic forebrain centre, is the recipient of projections from a number of autonomic brainstem nuclei including the pontine parabrachial nucleus. This study examined the influence of electrical stimulation of the parabrachial nucleus on the excitability of amygdala neurons and their response to two cardiovascular stimuli, namely baroreceptor activation and the administration of systemic angiotensin II. We also defined the chemical identity of some amygdala neurons that receive parabrachial nucleus projections by combining the transport of the anterograde tracer Phaseolus vulgaris leucoagglutinin injected into the parabrachial nucleus with immunocytochemical labelling of neurotensin and galanin profiles within the amygdala. In urethane-anesthetized rats, stimulation of parabrachial nucleus evoked four basic types of synaptic responses in amygdala cells: (1) a short duration (< 100 ms) excitation in 75 of 167 neurons, (2) a longer duration (> 100 ms) excitatory response in 36 neurons, (3) an inhibitory response in 32 cells, and (4) more complex responses consisting of excitation-inhibition or inhibition-excitation sequences in the remainder of the cells. Thirty-seven of 72 amygdala neurons activated synaptically by parabrachial nucleus stimulation also responded to baroreceptor activation or intravenous angiotensin II. Anatomical data revealed the presence of Phaseolus vulgaris leucoagglutinin labelled terminals predominantly within the lateral, medial, and capsular subdivisions of the central nucleus of amygdala. Phaseolus vulgaris leucoagglutinin varicosities and boutons were observed apposed to the neurotensin and galanin neuronal perikarya within the central nucleus of amygdala. The electrophysiological results provide a framework whereby parabrachial nucleus efferents influence the activity of amygdala neurons that are responsive to cardiovascular stimuli. Furthermore, the anatomical data indicate that a portion of the parabrachial nucleus input is directed toward galanin and neurotensin neurons within the central nucleus of amygdala.

Distribution of the tight junction-associated protein ZO-1 in circumventricular organs of the CNS.

The immunofluorescent distribution of ZO-1, a tight junction-associated protein, was studied in murine circumventricular organs. These regions generally express a less restrictive blood-brain barrier than is found in other areas of the CNS. In the remaining brain parenchyma, where a characteristic blood-brain barrier exists, ZO-1 was localized in discrete, continuous lines along blood vessels, presumably in association with endothelial cell tight junctions. The ependymal cells in the ventricular walls displayed a more punctate pattern of ZO-1 distribution, indicative of discontinuous tight junctions. In two of the circumventricular organs examined, the median eminence and the subfornical organ, many capillaries lacked detectable ZO-1 immunoreactivity while the apical aspects of the specialized ependymal cells (tanycytes) revealed an unbroken ZO-1 distribution. Scant labelling of ZO-1 in blood vessels was found in the area postrema, and only weak and discontinuous ZO-1 labelling was present in the ventricular wall. Capillaries of the organum vasculosum laminae terminalis expressed ZO-1 immunoreactivity which was comparable to the pattern observed in CNS regions with typical blood-brain barrier. The subcommissural organ, known to contain a blood-brain barrier, also displayed continuous ZO-1 staining in blood vessels. Unbroken ZO-1 distribution was observed in the specialized ependymal cells adjacent to both the organum vasculosum laminae terminalis and subcommissural organ. These immunocytochemical data demonstrate a distribution of ZO-1 in CNS parenchyma outside the circumventricular organs that is consistent with an organization of tight junctions which prevent free paracellular exchange of substances between blood and neuropil but which allow for continuity between CSF and the neuronal environment. The ZO-1 staining pattern in blood vessels and ventricular walls of the circumventricular organs is heterogeneous despite the prevalent absence of a functional blood-brain barrier.

Characterization of the parabrachial nucleus input to the hypothalamic paraventricular nucleus in the rat.

The brainstem parabrachial nucleus (PBN) is viewed as an increasingly important site for the transfer of autonomic‐related information to more rostral structures in the forebrain including the hypothalamus. In this study, we examined electrophysiologically in vivo and anatomically the nature of PBN input to the hypothalamic paraventricular nucleus (PVN) and particularly to the vasopressin‐and oxytocin‐secreting magnocellular neurosecretory cells within this nucleus.

Hypotension induces fos immunoreactivity in NADPH-diaphorase positive neurons in the paraventricular and supraoptic hypothalamic nuclei of the rat

Double staining for Fos and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) was used to study the distribution of activated neurons that synthesize nitric oxide in the paraventricular (PVN) and supraoptic nuclei (SON) following hypotensive stimulation in conscious rats. Fos was detected in many magno- and parvocellular NADPH-D positive neurons in response to haemorrhage or drug-evoked hypotension using i.v. infusions of sodium nitroprusside. However, quantitative analysis did not reveal any differences in the number of Fos positive PVN neurons following either mode of stimulation. These results suggest that a subpopulation of hypothalamic NADPH-D positive neurons is activated following hypotensive challenge. This activation of NADPH-D neurons may occur indirectly through other CNS structures that influence the excitability of hypothalamic SON and PVN. Furthermore, the lack of a difference in activated neurons within the PVN following either haemorrhage or nitroprusside infusion suggests that while a drop in blood pressure causes activation of neurons that produce nitric oxide, a decrease in blood volume, which accompanies haemorrhage, does not.

Activation of nitric oxide-synthesizing neurones during precipitated morphine withdrawal

ACTIVATION of nitric oxide (NO)-synthesizing neurones in the hypothalamus and brain stem was studied during naloxone-precipitated morphine withdrawal in rats. In animals that underwent behavioural changes consistent with withdrawal, Fos and nicotin-amide dinucleotide phosphate-diaphorase (NADPH-D)-positive neurones were identified within the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei as well as the brain stem nucleus tractus solitarius (NTS). A larger proportion of NADPH-D-positive neurones were activated in the PVN than in the NTS. While NO has been implicated in the genesis of opioid withdrawal, the present data provide evidence for the activation of select populations of NO-synthesizing neurones during the opiate withdrawal syndrome.

Activation of neuropeptide FF neurons in the brainstem nucleus tractus solitarius following cardiovascular challenge and opiate withdrawal

Neuropeptide FF (NPFF), a morphine modulatory peptide, is localized within discrete autonomic regions including the brainstem nucleus tractus solitarius (NTS) and the parabrachial nucleus (PBN). We investigated the activation of NPFF neurons in the NTS of rats induced by cardiovascular challenge and centrally generated opiate withdrawal. For hypotensive stimulation, we used systemic infusions of sodium nitroprusside (NP) or hemorrhage (HEM), and hypertension was achieved by intravenous phenylephrine (PHENYL) or angiotensin II (AII). In rats that received continuous intracerebroventricular injections of morphine, intraperitoneal injections of naloxone precipitated behavioural signs of opioid withdrawal. Activated NTS neurons were identified by using a combined immunohistochemistry for Fos and NPFF, and neurons projecting to the PBN were determined with a retrograde tracer. HEM, administration of vasoactive drugs, and opiate withdrawal produced a very robust activation of NTS neurons. In NP and HEM groups, 25.6 ± 3.2% and 7.6 ± 1.3 % of NPFF neurons were activated, respectively. Lesser numbers of NPFF neurons were activated in the PHENYL (4.6 ± 1.6%) and AII (2.4 ± 0.8%) groups. However, following opiate withdrawal, virtually no Fos expression was observed in NPFF neurons. NPFF neurons activated during NP infusion constituted the largest number of cells projecting to the PBN. This study shows that NPFF neurons in NTS that project to the PBN respond selectively to NP as opposed to other cardiovascular challenges or opiate withdrawal. These data support an emerging and important role for NPFF in the context of central cardiovascular regulation. J. Comp. Neurol. 402:210–221, 1998.

Characterization of peptidergic efferents from the lateral parabrachial nucleus to identified neurons in the rat dorsal raphe nucleus

The peptidergic content of the lateral parabrachial nucleus (LPB) efferents to the dorsal raphe nucleus (DRN) was studied by combining visualization of the anterogradely transported tracer Phaseolus vulgaris leucoagglutinin within fibers that were immunocytochemically stained for neurotensin (NT), calcitonin gene-related peptide (CGRP) or galanin (GAL). The identity of DRN target neurons was determined with simultaneous immunocytochemical labelling for serotonin, the major transmitter within the nucleus. Within the DRN, we estimated that about two-thirds of the anterogradely labelled fibers arising from the LPB also showed peptidergic immunoreactivity. NT was the most commonly observed neuropeptide in LPB neuronal efferents directed to the DRN, followed by CGRP and GAL. The peptidergic afferents in the DRN were oriented preferentially in the dorsoventral plane. Peptidergic fibers from the LPB possessed varicosities (diameters not exceeding 3 microns) and were apposed on serotoninergic neuronal somata. Some of the anterogradely labelled peptidergic fibers were not associated with cells showing immunoreactivity for serotonin. The present results suggest that NT-ergic, CGRP-ergic and GAL-ergic neurons within the LPB are in contact with serotoninergic and non-serotoninergic neurons within the DRN. Since the DRN is known to project to the LPB, it is likely that bi-directional interconnections between these nuclei exist. Such linkages may provide anatomical substrates for coordinated autonomic responses.