Musa A. Haxhiu

CNS innervation of airway-related parasympathetic preganglionic neurons: a transneuronal labeling study using pseudorabies virus

The CNS cell groups that innervate the tracheal parasympathetic preganglionic neurons were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the tracheal wall of C8 spinal rats and after 4 days survival, brain tissue sections from these animals were processed for immunohistochemical detection of PRV. Retrogradely labeled parasympathetic preganglionic neurons were seen in three sites in the medulla: the compact portion of the nucleus ambiguus, the area ventral to the nucleus ambiguus, and the rostralmost portion of the medial nucleus tractus solitarius (NTS); this labeling pattern correlated well with the retrograde cell body labeling seen following cholera toxin beta-subunit injections in the tracheal wall. PRV transneuronally labeled neurons were found throughout the CNS with the most abundant labeling concentrated in the ventral medulla oblongata. Labeled neurons were identified along the ventral medullary surface, and in nearby areas including the parapyramidal, retrotrapezoid, gigantocellular and lateral paragigantocellular reticular nuclei as well as the caudal raphe nuclei (raphe pallidus, obscurus, and magnus). Serotonin (5-HT) neurons of the caudal raphe complex (B1-B3 cell groups) and ventromedial medulla were labeled as well as a few C1 adrenergic neurons. The A5 cell group was the major noradrenergic area labeled although a small number of locus coeruleus neurons were also labeled. Several NTS regions contained labeled cells including the commissural, intermediate, medial, central, ventral, and ventrolateral subnuclei. PRV infected neurons were present in the Kölliker-Fuse and Barringtons nuclei. In the rostral mesencephalon, the precommissural nucleus of the dorsal periventricular gray matter was labeled. Labeling was present in the dorsal, lateral and paraventricular hypothalamic nuclei. In summary, the airway parasympathetic preganglionic neurons are innervated predominantly by a network of lower brainstem neurons that lie in the same regions known to be involved in respiratory and cardiovascular regulation. These findings are discussed in relationship to some of the potential CNS mechanisms that may be operative in airway disorders as well as potentially involved in certain fatal respiratory conditions such as Ondines curse and sudden infant death syndrome (SIDS).

Nitric oxide in the sensory function of the carotid body

Recent studies suggest that nitric oxide (NO) may act as a chemical messenger in the nervous system. Since neurotransmitters are considered necessary for the sensory function of the carotid body, and molecular O2 is a co-factor for NO synthesis, we examined whether (a) chemoreceptor tissue also synthesizes NO and if so, (b) does endogenous NO affect chemosensory activity. Experiments were performed on carotid bodies obtained from anesthetized cats (n = 20). Distribution of nitric oxide synthase (NOS), an enzyme that catalyzes the formation of NO was examined using NADPH-diaphorase histochemistry. Many nerve plexuses innervating the chemoreceptor tissue were positive for NADPH-diaphorase, indicating that the nerve fibers are the primary source of NO production in the carotid body. Radiometric analysis of NOS activity of the chemoreceptor tissue averaged 1.94 pmol [3H]citrulline/min/mg protein. NOS activity was significantly less in low pO2 reaction medium than in room air controls. Chemosensory activity in vitro increased in a dose-dependent manner in response to L-omega-nitro arginine (L-NNA), an inhibitor of NOS activity. The effects of NOS inhibitor were enantiomer selective as evidenced by reversal of the responses by L- but not D-arginine. These observations imply that endogenous NO is inhibitory to carotid body sensory activity. cGMP levels of L-NNA-treated carotid bodies were significantly less than untreated controls, suggesting that the actions of NO are coupled to the cGMP second messenger system, as elsewhere in the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

CO2-induced c-fos expression in the CNS catecholaminergic neurons

In these studies we examined c-fos expression in catecholaminergic neurons following exposure of unanesthetized rats to hypercapnic stress. Breathing a gas mixture with elevated CO2 (15% CO2, 21% O2 and 64% N2, or 15% CO2 balance O2) for 60 min, induced activation of the c-fos gene in widespread regions of the CNS, as indicated by the expression of Fos-like immunoreactive protein (Fos). Similar results were obtained in carotid body denervated animals. Colocalization studies of tyrosine hydroxylase (TH) and Fos protein revealed that in the brainstem, 73 to 85% of noradrenaline-containing cells expressed Fos immunoreactivity. Double-labeled neurons were found in the ventrolateral medullary reticular formation (A1 noradrenaline cells), in the dorsal aspect of medulla oblongata (A2 noradrenaline cells), in the ventrolateral pons (A5 noradrenaline cells), and in the locus coeruleus (A6 noradrenaline cells). However, over 90% of TH-immunoreactive neurons in the mesencephalon and diencephalon (dopaminergic cells) did not express Fos-like immunoreactivity in response to CO2. These results indicate that the brainstem noradrenaline-containing neurons are part of the neuronal networks that react to hypercapnic exposure.

Selective antihypertensive action of moxonidine is mediated mainly by I1-imidazoline receptors in the rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM) is the primary region maintaining vasomotor tone, and a site of action for central antihypertensive agents. In vitro [125I]p-iodoclonidine binding studies showed that moxonidine was selective for I1-imidazoline over alpha 2-adrenergic receptors in the RVLM. We identified efaroxan and SK&F 86466 as selective I1- and alpha 2-antagonists, respectively. We tested moxonidines action within the RVLM of spontaneously hypertensive rats (SHRs) on I1-imidazoline or alpha 2-adrenergic receptors, and determined whether the RVLM mediates the action of systemic moxonidine. SHRs were anesthetized, paralyzed, and ventilated and the RVLM was localized by testing for a pressor response to 2 nmol glutamate. To test whether I1 or alpha 2 mediates hypotensive effects of moxonidine, the I1/alpha 2 antagonist efaroxan (4 nmol) or the alpha 2-blocker SK&F 86466 (10 nmol) was administered 15 min before 4 nmol moxonidine. Efaroxan elevated blood pressure and abolished the action of moxonidine, whereas alpha 2-blockade with SK&F 86466 slightly lowered blood pressure and only partially attenuated moxonidines effect. The depressor effect of intravenous moxonidine (40 micrograms/kg) was reversed within 10 min by microinjection of 10 nmol efaroxan into the RVLM. Prior bilateral microinjections of efaroxan (10 nmol in 80 nl/site) into the RVLM prevented the hypotensive action of moxonidine given i.v. (40 micrograms/kg). Pharmacokinetic studies showed that at the peak vasodepressor response (8 min post-injection), [3H]moxonidine spread less than 1 mm from the injection site. Moxonidine is a centrally acting antihypertensive with a selective action on I1-imidazoline receptors in RVLM.

CNS cell groups projecting to pancreatic parasympathetic preganglionic neurons.

The CNS cell groups that project to the pancreatic parasympathetic preganglionic neurons were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the pancreas of C8 spinal rats and after 6 days survival, the animals were perfused and their brains processed for immunohistochemical detection of PRV. Parasympathetic preganglionic neurons of the dorsal vagal nucleus were retrogradely labeled with PRV. Several CNS cell groups consistently contained transneuronally labeled neurons. In the medulla oblongata, labeled neurons were found in the nucleus tractus solitarius, area postrema, paratrigeminal nucleus, lateral paragigantocellular reticular nucleus, raphe pallidus and obscurus nuclei, C3 region and scattered cells in the ventral medullary reticular formation. In the pons, the A5 cell group, Barringtons nucleus and the subcoeruleus region contained labeled neurons. Only an occasional labeled cell was identified in the parabrachial nucleus. In the midbrain, almost no labeling was found except for an occasional neuron in the central gray matter. In the diencephalon, labeling was found in the paraventricular hypothalamic nucleus (PVN) as well as in the lateral hypothalamic nucleus at two levels (one at the level of the PVN and the other at the level of the subthalamic nucleus). In addition, the perifornical and dorsal hypothalamic nuclei contained labeled neurons. A few cells were found in the peripheral part of the dorsomedial hypothalamic nucleus. No labeling was seen in the ventromedial hypothalamic nucleus. In the telencephalon, the central amygdaloid nucleus and the bed nucleus of the stria terminalis were labeled.

Role of the medullary raphe nuclei in the respiratory response to CO2

We characterized the role of neurons within the midline of the medulla oblongata on phrenic and hypoglossal nerve responses to hypercapnia during early-development. Studies were performed on decorticate or anesthetized; vagotomized and mechanically ventilated 14-20 day old piglets. Reversible withdrawal of midline neuronal activity was induced by microinjections of lidocaine (2%, 300 nl; n = 10) and lesioning was caused by microinjections of the neurotoxic agent, ibotenic acid (n = 12), at the same sites. At any given end-tidal CO2, peak phrenic and hypoglossal activities after lidocaine were significantly lower than in the control period (P < 0.01). Similarly, 1-2 h after injections of ibotenic acid, both phrenic and hypoglossal nerve responses to CO2 were significantly lower than in the control period (P < 0.01). The results indicate for the first time that the medullary midline neurons are required for full expression of ventilatory responses to hypercapnia and raise the possibility that dysfunction of these nuclei may contribute to respiratory instability during early postnatal life.

CNS innervation of vagal preganglionic neurons controlling peripheral airways : A transneuronal labeling study using pseudorabies virus

The CNS cell groups that project to vagal preganglionic neurons which innervate the most distal part of the airways were identified by the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the lung parenchyma of C8 spinal rats and after 5 days survival, brain tissue sections from these animals were processed for immunohistochemical detection of PRV. Retrogradely labeled parasympathetic preganglionic cells (first-order neurons) were seen mainly in the ventral medulla oblongata: the compact portion of the nucleus ambiguus and the area ventral to it. Occasionally, a few labeled cells were seen within the rostral part of the dorsal vagal nucleus. This labeling pattern correlated well with the retrograde cell body labeling seen following cholera toxin beta-subunit (CT-b) injections in the lung parenchyma. PRV transneuronally labeled neurons (second-order and/or presumed third-order neurons) were found throughout the CNS with the characteristic labeling in the brainstem. Labeled neurons were identified along and just beneath the ventral medullary surface, and in nearby areas: the parapyramidal, retrotrapezoid, gigantocellular and lateral paragigantocellular reticular nuclei, as well as the caudal raphe nuclei (raphe pallidus, obscurus, and magnus). Several nucleus tractus solitarius (nTS) regions contained labeled cells including the commissural, medial, and ventrolateral nTS subnuclei. The A5 cell group and a small number of locus coeruleus neurons were also labeled. PRV-infected neurons were present in the Kölliker-Fuse and Barringtons nuclei. In the mesencephalon, neurons within the ventral periventricular gray matter were labeled. Labeling was present in the dorsal, lateral and paraventricular hypothalamic nuclei, and within the amygdaloid complex. In summary, the parasympathetic preganglionic neurons that innervate the peripheral airways are controlled by networks of lower brainstem and suprapontine neurons that lie in the same regions known to be involved in central regulation of autonomic functions.

The I1-imidazoline-binding site is a functional receptor mediating vasodepression via the ventral medulla

I1-imidazoline-binding sites fulfill all essential criteria for identification as receptors, including specificity of binding, association with physiological functions, appropriate anatomic and cellular and subcellular localization, and specific cell signaling pathways. Moreover, binding affinities correlate with functional drug responses. The evidence linking I1 receptors to vasodepression includes expression in RVLM and consistent correlations between vasodepressor potency in humans and animals and I1 binding affinity. Some I1 agonists are antagonists at α2-adrenergic receptors (α2AR), and these elicit vasodepression in RVLM. Potent α2-agonists with phenylethylamine or guanidine structures are inactive in RVLM, yet highly effective in nucleus of the solitary tract, a region with well-defined α2-mediated vasodepressor responses. Selective I1 agonists are used clinically to lower blood pressure with minimal α2-mediated sedation. Moreover, when microinjected into the RVLM only antagonists active at I1 receptors can block the vasodepressor action of either local or systemic imidazolines. RVLM α2-blockade has no effect. Some reports appear to conflict with the I1 receptor hypothesis; but these reports often make incorrect assumptions regarding drug specificity, overlook systemic effects of α2-antagonists, or inappropriately analyze data. Blockade of γ-aminobutyric acid (GABA) receptors blocks the vasodepressor action of imidazolines, implying a multisynaptic pathway. Thus imidazolines act via I1receptors in RVLM to lower blood pressure, although α2AR are also important, especially in NTS.

CNS monoamine cell groups projecting to pancreatic vagal motor neurons: a transneuronal labeling study using pseudorabies virus

The CNS monoamine cell groups that project to the pancreatic parasympathetic preganglionic neurons were identified with the use of the viral retrograde transneuronal labeling method. Pseudorabies virus (PRV) was injected into the pancreas of C8 spinal rats and subsequently, transneuronally-labelled central monoamine neurons were mapped in brain tissue sections that had been stained by an immunohistochemical procedure that allowed for the visualization of PRV products and biogenic amine neurotransmitter enzymes or serotonin (5-HT) in the same neuron. The enzymes studied were tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT), and histidine decarboxylase. Pancreatic vagal motor neurons originate exclusively from the dorsal vagal motor nucleus and some of these may be dopamine neurons because they were TH immunopositive, but DBH and PNMT immunonegative. Transneuronally labeled aminergic neurons were found throughout the medulla oblongata. The adrenergic inputs arose from the C1, C2, and C3 cell groups. Noradrenergic inputs originated predominantly from the A5 cell group, with lesser contributions from the A1 and A2 cell groups as well as from the area postrema. None of the other CNS catecholamine cells were labeled, except for some weakly staining TH-immunoreactive neurons, presumably dopaminergic, in the paraventricular hypothalamic nucleus (PVN). The greatest number of 5-HT neurons that innervate the pancreatic vagal motor neurons come from the gigantocellular reticular nucleus, pars alpha with lesser inputs from the raphe magnus, obscurus, and pallidus nuclei. None of the CNS histaminergic cell groups were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide and ventilatory response to hypoxia

It is believed that hypoxia results in the release of neurotransmitters in the central nervous system, which can excite or inhibit breathing. Recent evidence indicates that nitric oxide (NO) is a physiological messenger molecule that may serve as a neurotransmitter in the CNS. In this study we examined (1) the localization of nitric oxide synthase (NOS) within the nucleus tractus solitarius, and (2) the role of the NO-cGMP pathway in the respiratory response to oxygen deprivation. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was used to determine the distribution of neurons that express NOS, an enzyme involved in NO formation. The NOS inhibitor N omega-nitro-L-arginine was used as tool to assess the NOS activity in the medulla, and to define the role of NO in the respiratory response to acute oxygen deprivation. In the rat and the cat brainstem, histochemical studies showed the presence of NADPH-diaphorase reactive neurons within subnuclei of the nucleus tractus solitarius which receive peripheral chemoreceptor inputs. Chronic pretreatment of rats with N omega-nitro-L-arginine (75 mg/kg, ip, twice daily for 7 days) caused a significant decrease in cGMP, and attenuated the ventilatory response to hypoxia. In anesthetized, paralyzed, vagotomized and artificially ventilated cats with intact carotid sinus nerves (n = 8), administration of N omega-nitro-L-arginine (30-100 mg/kg) attenuated the response to hypoxia, and caused the hypoxia induced roll-off of phrenic nerve activity to occur significantly earlier than when NOS activity was not inhibited. In sinoaortic denervated cats (n=9) blockage of NOS potentiated the decline of the phrenic nerve output. The data suggest that oxygen deprivation leads to activation of NO-cGMP pathway in the central nervous system, which contributes to the induction and maintenance of hypoxia-induced increase in respiratory output. In addition, these findings indicate that NO may inhibit inhibitory synaptic transmission that is triggered by CNS hypoxia, and this is not directly related to peripheral chemoreceptor inputs.