Qun Li

PACAP is expressed in sympathoexcitatory bulbospinal C1 neurons of the brain stem and increases sympathetic nerve activity in vivo

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide present in the rat brain stem. The extent of its localization within catecholaminergic groups and bulbospinal sympathoexcitatory neurons is not established. Using immunohistochemistry and in situ hybridization, we determined the extent of any colocalization with catecholaminergic and/or bulbospinal projections from the brain stem was determined. PACAP mRNA was found in tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the C1-C3 cell groups. In the rostral ventrolateral medulla (RVLM), PACAP mRNA was found in 84% of the TH-ir neurons and 82% of bulbospinal TH-ir neurons. The functional significance of these PACAP mRNA positive bulbospinal neurons was tested by intrathecal administration of PACAP-38 in anaesthetized rats. Splanchnic sympathetic nerve activity doubled (110%) and heart rate rose significantly (19%), although blood pressure was unaffected. In addition, as previously reported, PACAP was found in the A1 cell group but not in the A5 cell group or in the locus coeruleus. The RVLM is the primary site responsible for the tonic and reflex control of blood pressure through the activity of bulbospinal presympathetic neurons, the majority of which contain TH. The results indicate 1) that pontomedullary neurons containing both TH and PACAP that project to the intermediolateral cell column originate from C1-C3 and not A5, and 2) intrathecal PACAP-38 causes a prolonged, sympathoexcitatory effect.

Central Command Regulation of Circulatory Function Mediated by Descending Pontine Cholinergic Inputs to Sympathoexcitatory Rostral Ventrolateral Medulla Neurons

Central command is a feedforward neural mechanism that evokes parallel modifications of motor and cardiovascular function during arousal and exercise. The neural circuitry involved has not been elucidated. We have identified a cholinergic neural circuit that, when activated, mimics effects on tonic and reflex control of circulation similar to those evoked at the onset of and during exercise. Central muscarinic cholinergic receptor (mAChR) activation increased splanchnic sympathetic nerve activity (SNA) as well as the range and gain of the sympathetic baroreflex via activation of mAChR in the rostral ventrolateral medulla (RVLM) in anesthetized artificially ventilated Sprague-Dawley rats. RVLM mAChR activation also attenuated and inhibited the peripheral chemoreflex and somatosympathetic reflex, respectively. Cholinergic terminals made close appositions with a subpopulation of sympathoexcitatory RVLM neurons containing either preproenkephalin mRNA or tyrosine hydroxylase immunoreactivity. M2 and M3 receptor mRNA was present postsynaptically in only non-tyrosine hydroxylase neurons. Cholinergic inputs to the RVLM arise only from the pedunculopontine tegmental nucleus. Chemical activation of this region produced increases in muscle activity, SNA, and blood pressure and enhanced the SNA baroreflex; the latter effect was attenuated by mAChR blockade. These findings indicate a novel role for cholinergic input from the pedunculopontine tegmental nucleus to the RVLM in central cardiovascular command. This pathway is likely to be important during exercise where a centrally evoked facilitation of baroreflex control of the circulation is required to maintain blood flow to active muscle.

Cannabinoid receptor activation in the rostral ventrolateral medulla oblongata evokes cardiorespiratory effects in anaesthetised rats

The nature of the cardiorespiratory effects mediated by cannabinoids in the hindbrain is poorly understood. In the present study we investigated whether cannabinoid receptor activation in the rostral ventrolateral medulla oblongata (RVLM) affects cardiovascular and/or respiratory function. Initially, we looked for evidence of CB1 receptor gene expression in rostral and caudal sections of the rat ventrolateral medulla (VLM) using reverse transcription–polymerase chain reaction. Second, the potent cannabinoid receptor agonists WIN55,212‐2 (0.05, 0.5 or 5 pmol per 50 nl) and HU‐210 (0.5 pmol per 50 nl) or the CB1 receptor antagonist/inverse agonist AM281 (1 pmol per 100 nl) were microinjected into the RVLM of urethane‐anaesthetised, immobilised and mechanically ventilated male Sprague–Dawley rats (n=22). Changes in splanchnic nerve activity (sSNA), phrenic nerve activity (PNA), mean arterial pressure (MAP) and heart rate (HR) in response to cannabinoid administration were recorded. The CB1 receptor gene was expressed throughout the VLM. Unilateral microinjection of WIN55,212‐2 into the RVLM evoked short‐latency, dose‐dependent increases in sSNA (0.5 pmol; 175±8%, n=5) and MAP (0.5 pmol; 26±3%, n=8) and abolished PNA (0.5 pmol; duration of apnoea: 5.4±0.4 s, n=8), with little change in HR (P<0.005). HU‐210, structurally related to Δ9‐tetrahydrocannabinol (THC), evoked similar effects when microinjected into the RVLM (n=4). Surprisingly, prior microinjection of AM281 produced agonist‐like effects, as well as significantly attenuated the response to subsequent injection of WIN55,212‐2 (0.5 pmol, n=4). The present study reveals CB1 receptor gene expression in the rat VLM and demonstrates sympathoexcitation, hypertension and respiratory inhibition in response to RVLM‐administered cannabinoids. These findings suggest a novel link between CB1 receptors in this region of the hindbrain and the central cardiorespiratory effects of cannabinoids. The extent to which these central effects contribute to the cardiovascular and respiratory outcomes of cannabis use remains to be investigated.

Preprotachykinin A mRNA is colocalized with tyrosine hydroxylase-immunoreactivity in bulbospinal neurons

Previous studies have generated controversy about the extent of co-localization between substance P- and catecholamine-containing neurons that project to the spinal cord. In earlier studies, estimates using immunofluorescence after colchicine have ranged from almost all, to almost none. We sought to resolve this issue by combining in situ hybridization and immunofluorescence. Catecholamine (A1 to A7, C1 to C3; tyrosine hydroxylase immunoreactive) neurons in the rat brainstem were examined to determine their content of mRNA for the preprotachykinin-A gene. In the A1 to A7 and the C1 to C3 cell groups, preprotachykinin-A mRNA was found only in substantial amounts in the C1-C3 cell groups. On average 20.9+/-0.9% (234/1120, n=3) of rostral C1 neurons contained preprotachykinin-A mRNA. Co-localization was also observed in C2 and C3 neurons to a similar extent. Retrograde tract-tracing with cholera toxin B subunit was used to identify bulbospinal neurons and 17.9+/-2.7% (96/529 cells) of the bulbospinal tyrosine hydroxylase-containing neurons of the rostral C1 cell group were found to contain preprotachykinin-A mRNA. In addition a new population of non-catecholaminergic bulbospinal preprotachykinin-A neurons is described in an area corresponding to the recently described caudal pressor area. To confirm that the preprotachykinin-A mRNA observed in cells in the medulla was converted to protein, dual immunofluorescence for fiber labeling at the confocal level was carried out. This confirmed colocalization of substance P and tyrosine hydroxylase in the intermediolateral cell column, but nowhere else, in a small number of cases. The results provide evidence for a much larger population of substance P/neurokinin A containing neurons in the brainstem than was previously suspected. Furthermore, many of these neurons are catecholaminergic and spinally projecting. The specific sympathetic outflow that these neurons influence remains to be determined.

Somatostatin 2A Receptor-Expressing Presympathetic Neurons in the Rostral Ventrolateral Medulla Maintain Blood Pressure

Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are critical for the maintenance of sympathetic vasomotor tone and normal cardiovascular reflex function. So far, selectively eliminating/inhibiting distinct subpopulations of RVLM neurons has not significantly altered arterial pressure. Here we show that RVLM presympathetic neurons that express somatostatin 2A receptors are essential for maintaining and potentially generating sympathetic vasomotor tone. Combined immunocytochemistry and in situ hybridization were used to map the expression of somatostatin receptors 1, 2A, 2B, 3, and 4 (sst1 through 4, respectively) in the rat RVLM. sst1 and sst2B were absent; sst3 and sst4 were sparse. However, sst2A was found postsynaptically and detected in 35±5% of bulbospinal RVLM neurons a population that included 54±4% of catecholaminergic and 30±3% of enkephalinergic neurons. Bilateral microinjection into the RVLM of either somatostatin or the receptor-selective agonist lanreotide evoked dramatic, dose-dependent sympathoinhibition, hypotension, and bradycardia that were blocked by the sst2 receptor antagonist BIM-23627 in anesthetized rats. Bilateral RVLM microinjection of somatostatin also attenuated chemoreceptor and somatosympathetic reflex function. Somatostatin only eliminated the first sympathoexcitatory peak evoked by somatosympathetic reflex activation, whereas muscimol abolished both excitatory peaks providing functional evidence that the activity of only a subpopulation of RVLM presympathetic neurons is inhibited by somatostatin. We suggest that the subpopulation of bulbospinal RVLM neurons that expresses the sst2A receptor sets sympathetic vasomotor output. These neurons are essential for maintaining resting blood pressure under anesthesia and contribute to adaptive reflexes mediated through the RVLM.

A Novel Pressor Area at the Medullo-Cervical Junction That Is Not Dependent on the RVLM: Efferent Pathways and Chemical Mediators

Chemical stimulation of a region extending from the most caudal ventrolateral medulla into the upper cervical spinal cord evoked large sympathetically mediated pressor responses. These responses were not dependent on the integrity of the rostral ventrolateral medulla (RVLM) and may be mediated by glutamatergic neurons embedded in the white matter that project to the thoracic spinal cord. We term this new region the medullo-cervical pressor area (MCPA). This region is distinct from the caudal pressor area, because blockade of the RVLM with muscimol inhibited this pressor response but not that evoked from the MCPA. This is the first study to provide functional evidence for a cardiovascular role for neurons in the cervical spinal cord white matter that innervate sympathetic preganglionic neurons (Jansen and Loewy, 1997). Using retrograde tracing, in combination with immunohistochemistry and in situ hybridization, we identified two groups of spinally projecting neurons in the region. Approximately 50% of neurons in one group were excitatory because they contained vesicular glutamate transporter 1 (VGluT1)/VGluT2 mRNA, whereas the other contained a mixed population of neurons, some of which contained either VGluT1/VGluT2 or GAD67 (glutamic acid decarboxylase 67) mRNA. Despite the fact that activation of the MCPA causes potent sympathoexcitation, it does not act to restore arterial pressure after chemical lesion of the RVLM so that a role for this novel descending sympathoexcitatory region remains to be elucidated.

Metabotropic neurotransmission and integration of sympathetic nerve activity by the rostral ventrolateral medulla in the rat.

1 Cardiovascular sympathetic nerve activity at rest is grouped into waves, or bursts, that are generally, although not exclusively, related to the heart rate and to respiration. In addition, activity is also generated in response to central commands and to environmental stimuli. 2 Responsibility for the integration of all these different elements of sympathetic activity rests with pre‐motoneurons in the rostral ventrolateral medulla oblongata. These pre‐motoneurons are glutamatergic and spinally projecting where they form synapses with sympathetic preganglionic neurons. 3 Pre‐motoneurons also contain and presumably release, neurotransmitters other than glutamate, including amines and neuropeptides that act on metabotropic receptors with long‐term effects on cell function. 4 Similarly, in the rostral ventrolateral medulla oblongata the pre‐motoneurons are mainly regulated by excitatory influences from glutamate and inhibitory influences from γ‐aminobutyric acid (GABA). Major focuses of recent studies are the interactions between non‐glutamatergic and GABAergic systems and reflexes that regulate the activity of the sympathetic nervous system. 5 The results indicate that neurotransmitters acting at metabotropic receptors selectively affect different reflexes in the rostral ventrolateral medulla. It is suggested that this differential activation or attenuation of reflexes by different neurotransmitters is a mechanism by which the organism can fine‐tune its responses to different homeostatic requirements.

Brainstem galanin-synthesizing neurons are differentially activated by chemoreceptor stimuli and represent a subpopulation of respiratory neurons

The ventrolateral medulla oblongata (VLM) of the brainstem contains neurochemically heterogeneous neurons that have a critical role in cardiovascular and respiratory regulation. Previous anatomical studies have shown the existence of galanin immunoreactivity in the medulla oblongata, but a detailed characterization is lacking. In this study, we demonstrate three populations of preprogalanin mRNA (PPG)‐expressing neurons in the VLM of the adult, male Sprague‐Dawley rat: a retrotrapezoid nucleus (RTN) group, a group in the rostral ventral respiratory group (rVRG), and a subpopulation of A1 neurons. PPG+ neurons express tyrosine hydroxylase (TH) only in the A1 region of the VLM, where approximately 56% of PPG+ neurons contain TH (79 ± 14; n = 4). PPG+ neurons do not express vesicular acetylcholine transporter (vAChT) in the VLM (n = 3). However, 33% of PPG+ neurons contain neurokinin‐1 receptor (NK1R) in the rVRG (126 ± 12; n = 12), accounting for ∼28% of all NK1R+ neurons in the region. Retrogradely transported cholera toxin B injected into the thoracic spinal cord (T1) revealed that bulbospinal PPG+ neurons are present in the rVRG (n = 3; ∼26% of PPG+ neurons). PPG+ neurons in the RTN and locus coeruleus are selectively activated (Fos) following 2 hours of exposure to hypercapnia, but not by hypoxia. Neurons in the A1, nucleus of the solitary tract, and dorsomedial hypothalamus are activated by both chemoreceptor stimuli. The results suggest that PPG+ neurons represent a population of brainstem neurons that play a critical and differential role in the chemoreflex responses to hypoxia and hypercapnia. J. Comp. Neurol., 2012.

Effect of haemorrhage on the expression of neurotransmitter-related genes in rat ventrolateral medulla: a quantitative real-time RT-PCR study.

The ventrolateral medulla (VLM) has three functionally defined regions that contain catecholamine-synthesising neurons (rostral C1, caudal C1 and A1 regions). Many neuromessengers can alter cardiovascular functions in the VLM. The aims of this study were, first to validate the utility of real-time RT-PCR SYBR Green assay for quantitation of mRNA expression levels of neuromessengers in small site-specific neuronal populations in the VLM, and second to compare the basal mRNA levels of the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT), neuropeptide Y (NPY) and preproenkephalin (ENK) in the three regions and third to examine the effects of haemorrhage on the expression of these three genes. Rats were anaesthetised with sodium pentobarbital and divided into three groups: perfused, sham-operated and haemorrhaged. A 15% haemorrhage was carried out on the haemorrhaged group. It was found that there are regional differences in the level of mRNA expression for all the three genes: with, in general, decreases from the rostral to caudal regions of VLM. A 15% haemorrhage significantly induced expression of PNMT in the rostral C1 region and NPY in the caudal C1 and A1 regions but had no effect on ENK at any sites, suggesting a differential regulation on the expression of these three genes in the VLM. Our results also demonstrate that real-time RT-PCR is a sensitive and accurate method for quantitative studies on neurotransmitter gene expressions in restricted brain regions.

An aldosterone-related system in the ventrolateral medulla oblongata of spontaneously hypertensive and Wistar-Kyoto rats

1 The actions of aldosterone include mediation of vasoconstriction, vascular fibrosis, endothelial dysfunction and sodium retention. These actions can contribute to hypertension. Recent studies implicate an abnormal aldosterone hormonal system in the brain in hypertension. However, the study of central aldosterone actions is still in its infancy, as the exact location and abundance of its components in the brain are uncertain. 2 We aimed to detect components of the aldosterone cascade in the regions of the ventrolateral medulla oblongata (VLM)‐containing neurons that regulate blood pressure and to see whether there are quantitative differences in these components between the spontaneously hypertensive rat (SHR) and normotensive Wistar‐Kyoto (WKY) rat models. Tissues from four regions of the brainstem, namely, the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively), rostral pressor area and caudal pressor area, were examined. We measured mRNA expression of aldosterone synthase, mineralocorticoid receptor (MR1), 12‐lipoxygenase (12‐LO), serum‐ and glucocorticoid‐ inducible kinase and K‐ras in male rats. Gene expression levels were measured using real‐time reverse transcription–polymerase chain reaction. 3 We detected all aldosterone components in all regions of the VLM. The K‐ras levels were not significantly different in any of the regions. Expression of MR1 mRNA was lower in the RVLM of SHR (n = 5) compared with WKY rats (n = 5; t = 4.590; P = 0.002) and 12‐LO mRNA levels were lower in the CVLM in SHR (n = 6) compared with WKY rats (n = 7; P = 0.04). Thus, we have shown for the first time that components of the aldosterone cascade are present in the VLM. Our results suggest that there may be a differential gene expression profile in the brainstem for genetic hypertension.