Teresa L. Krukoff

Efferent projections from the parabrachial nucleus demonstrated with the anterograde tracer Phaseolus vulgaris leucoagglutinin

Efferent projections from the parabrachial complex (PBN) were studied in the rat using the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). Projections to the hypothalamus (ventromedial, dorsomedial, paraventricular, and supraoptic nuclei) originate primarily in the lateral PBN (1PBN). The amygdalar central nucleus (ACE) receives strong projections from all parts of the PBN although the external 1PBN projects primarily to the lateral ACE. Whereas the projections to the lateral bed nucleus of the stria terminalis, median preoptic nucleus, diagonal band of Broca, and lateral preoptic area originate primarily from the 1PBN, those to the insular cortex arise from the medial PBN (mPBN). The mPBN projects to the ventral posteromedial thalamus and the 1PBN and mPBN project to the zona incerta. Descending projections from the mPBN and Kölliker-Fuse area target the commissural nucleus tractus solitarius (NTS); the mPBN projects to the more rostral NTS. Similarly, the caudal parvicellular reticular formation (RF) receives projections from the mPBN and 1PBN, whereas input to the rostral RF arises from the former. All compartments of the PBN project to the ventrolateral medulla, although the projections arising from the 1PBN are densest. Finally, the raphe nuclei and periaqueductal gray receive some projections from most PBN divisions. These pathways provide a potential means whereby autonomic information can be relayed through the PBN to other structures important in regulating autonomic functions.

Central actions of nitric oxide in regulation of autonomic functions.

The identification of nitric oxide (NO) as a gaseous, nonconventional neurotransmitter in the central nervous system has led to an explosion of studies aimed at learning about the roles of NO, not only at a cellular level, but also in regulating the activity of specific physiological systems that are coordinated by the brain. In the 1980s, publications began to appear which pointed to a role for NO in regulating peripheral autonomic function. In the 1990s, it became apparent that NO also acts centrally to affect autonomic responses. In this review, I will discuss the state of the current knowledge about the central role of NO in physiological functions which are related specifically to the control of sympathetic output. Studies which do not differentiate a central from a peripheral role for NO in these functions have not been included. After a brief discussion about the cellular events in which NO is involved, the distribution of NO-producing neurons in central autonomic areas of the brain will be presented. The more general actions of central NO in regulating sympathetic activity, as assessed with i.c.v. injections of pharmacological agents, will be followed by more specific sites of action achieved with microinjections into discrete brain areas. The review will be concluded with discussions about central NO in two physiological states of sympathetic imbalance, hypertension and stress.

STRESS-INDUCED ACTIVATION OF NITRIC OXIDE-PRODUCING NEURONS IN THE RAT BRAIN

Nitric oxide (NO) is a gaseous neurotransmitter that may mediate a decrease in sympathetic output to the periphery. This implication predicts that NO‐producing neurons in the brain are activated in animals experiencing increased levels of sympathetic activity. To test this prediction, we subjected three groups of experimental rats to differing levels of environmental stimulation for 1 hour: minimal stimulation, moderate stimulation, and restraint stress. NO‐producing neurons were histochemically visualized in sections of the brain, and activation of these neurons was assessed according to the neuronal expression of the immediate early gene c‐fos.

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.

Decreased gene expression of neuronal nitric oxide synthase in hypothalamus and brainstem of rats in heart failure

Nitric oxide may act at autonomic sites in the brain to regulate sympathetic outflow. Our goal was to determine whether gene expression of the neuronal isoform of nitric oxide synthase (nNOS) is altered in discrete autonomic brain regions of rats in the chronic phase of heart failure compared to sham-operated control rats. Experiments were performed in rats 4 to 5 weeks after left coronary artery ligation. Histological data indicated that there was a 39% outer and a 45% inner infarct of the left ventricular myocardium in the heart failure group. The myocardium in sham-operated rats showed no observable damage. Total RNA was purified from microdissected brain tissue blocks containing hypothalamus, dorsal pons, dorsal medulla, rostral ventrolateral medulla, and caudal ventrolateral medulla. Changes in nNOS mRNA were semiquantified in each region using reverse transcription-polymerase chain reactions in which known concentrations of deletion mutant of the gene were coamplified as an internal standard. Compared with controls, significant decreases in nNOS mRNA levels were found in hypothalamus (19%), dorsal pons (43%) and dorsal medulla (34%) of rats with heart failure. There were no statistically significant differences in nNOS mRNA levels in rostral or caudal ventrolateral medulla between the control and heart failure groups. Concomitant with the changes nNOS gene expression in central sites, the plasma concentration of norepinephrine was significantly elevated in rats with heart failure compared to sham-operated control rats. Our results show that heart failure is associated with decreases in nNOS gene expression in at least three regions of the brain and with increased sympathetic outflow to the periphery. The decreased NO production that is likely associated with the decreases in nNOS gene expression may lead to the increased sympathetic drive seen in chronic heart failure.

Expression of c-fos protein in rat brain after electrical stimulation of the aortic depressor nerve

To reveal central nervous system (CNS) structures involved in the baroreceptor reflex we studied the distribution of Fos protein-like immunoreactivity in the rat brain after one hour of electrical stimulation of the aortic depressor nerve (ADN). In 13 male Wistar rats under urethane the ADN was cut on both sides and the central ends were placed on stimulating electrodes. Intermittent (11 s on, 6 s off) electrical stimulation at parameters set to elicit a drop in mean arterial pressure of 15-30 mmHg was applied to one, both or neither ADNs for 1 h. CNS sections were incubated for 48 h in anti-Fos antibody and prepared for visualization of the reaction product using the ABC immunoperoxidase technique. Label was found in several discrete brain nuclei primarily on the side ipsilateral to the side of stimulation. In the medulla labelled nuclei were found in the nucleus tractus solitarius, area postrema, rostral and caudal ventrolateral medulla, nucleus ambiguus and medullary reticular formation. In the pons labelled neurons were found in the lateral and ventrolateral parabrachial nucleus, locus coeruleus, pontine reticular field and A5 region. In the forebrain labelled nuclei were observed in the peri- and paraventricular hypothalamus, supraoptic nucleus, subfornical organ, preoptic area, central nucleus of the amygdala, median preoptic area, horizontal limb of the diagonal band, bed nucleus of the stria terminalis and islands of Calleja. In control animals moderate amounts of label were present in the supraoptic nucleus and periventricular hypothalamus bilaterally. These results define central pathways involved in mediating the baroreceptor reflex.

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.

Activation by hypotension of neurons in the hypothalamic paraventricular nucleus that project to the brainstem

To investigate the involvement of neuronal nitric oxide (NO) in the response of the brain to changes in blood pressure, we studied the activation of putative NO‐producing neurons in the paraventricular nucleus of the hypothalamus (PVN) in rats whose mean arterial pressures (MAPs) were decreased by 40–50% with hemorrhage (HEM) or infusion of sodium nitroprusside (NP). Activation was assessed on the basis of expression of the immediate early gene, c‐fos; putative NO‐producing neurons were identified with the histochemical stain for nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d); and the proportions of neurons projecting to the nucleus of the tractus solitarius (NTS) and/or caudal ventrolateral medulla (CVLM) were determined with retrograde tracing techniques.

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.