Reticularis Rostroventrolateralis Research Articles

Ultrastructural localization and afferent sources of corticotropin-releasing factor in the rat rostral ventrolateral medulla: implications for central cardiovascular regulation.

We investigated the ultrastructural localization, afferent sources, and arterial pressure effects of corticotropin-releasing factor (CRF) in the nucleus reticularis rostroventrolateralis (RVL), a region of the ventrolateral medulla containing C1 adrenergic neurons and sympatho-excitatory reticulospinal afferents to sympathetic preganglionic neurons. A polyclonal antibody to CRF was localized in acrolein-fixed sections through the rat RVL by the peroxidase-antiperoxidase (PAP) method. Light microscopy showed that 1-7 perikarya/30 micron section and numerous varicose processes contained CRF-like immunoreactivity (CRF-LI). By electron microscopy, CRF-LI was most intensely localized to large (80-100 nm) dense-core vesicles within numerous terminals and a few perikarya and large dendrites. Approximately half of the terminals containing CRF-LI were in direct contact with unlabeled perikarya or dendrites; the remainder were in apposition to either unlabeled terminals or astrocytes. Most synaptic specializations were asymmetric synapses on small, unlabeled dendrites. To examine potential extrinsic sources of CRF-containing terminals in the C1 area of the RVL, PAP immunocytochemical localization of CRF was combined with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). In all cases examined, a number of dually labeled neurons were found in the paraventricular nucleus (PVN) of the hypothalamus and a few dually labeled neurons were observed in the nuclei of the solitary tract; these labeled neurons were ipsilateral to the unilateral injection of WGA-HRP into the C1 area. Fewer dually labeled perikarya were detected in the lateral hypothalamic area and the lateral parabrachial nuclei, ipsilateral to the WGA-HRP injection. Additional physiological studies showed that bilateral microinjections of CRF into the C1 area of the RVL of urethane-anesthetized rats elicited a dose-related increase in arterial pressure. The results suggest that within the C1 area of the RVL, CRF released from terminals, arising predominantly from the PVN of the hypothalamus and probably from local neurons as well, may excite sympathoexcitatory reticulospinal neurons.

GABA‐mediated inhibition of medullary vasomotor neurones by area postrema stimulation in rats.

1. The cardiovascular responses, together with the effects on medullary sympathoexcitatory (vasomotor) neurones of the rostral ventrolateral medulla, of area postrema stimulation have been studied in vivo. 2. Electrical (10 Hz) or chemical stimulation using microinjections of L-glutamate of the area postrema produced a vasodepressor response and an inhibition of the medullary sympathoexcitatory neurones in the nucleus reticularis rostroventrolateralis (RVL), while similar stimulation in the adjacent nucleus tractus solitarii (NTS) caused increases in arterial pressure. 3. Single-pulse stimulation of the area postrema revealed at least three influences on the activity of RVL vasomotor neurones, one being excitatory and two inhibitory. 4. The inhibitions evoked in the medullary vasomotor neurones on area postrema stimulation were blocked by ionophoretic application of bicuculline, a GABAA receptor antagonist, without altering the excitatory input to the same neurones. Bilateral microinjections of bicuculline into the RVL in regions where the vasomotor neurones had been identified totally eliminated the vasodepression due to area postrema stimulation. 5. These data support a role for the area postrema in cardiovascular control. It is concluded that the area postrema exerts its action on cardiovascular control in part via GABAergic inhibition of the 'vasomotor' neurones in the nucleus reticularis rostroventrolateralis.

Synthesis, release and receptor binding of acetylcholine in the C1 area of the rostral ventrolateral medulla: contributions in regulating arterial pressure

This study sought to determine whether release of acetylcholine (ACh) within the C1 area of nucleus reticularis rostroventrolateralis (RVL) contributes to the tonic maintenance of arterial pressure (AP) in the rat. The activity of choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh, varied 5.5-fold in micropunches of the 6 medullary regions examined. ChAT activity in the C1 area (179 ± 35nmol [ 14C]ACh formed/mg protein/60 min;n = 4) was intermediate between that of the hypoglossal nucleus (249 ± 38; highest) and the pyramids (45 ± 11; lowest) and equivalent to that found in the parietal cortex (147 ± 15). Release of [ 3H]ACh from C1 are micropunches was increased by raising extracellular K + concentrations (5–55 mM) and was entirely Ca 2+-dependent. Muscarinic receptor binding density was assessed using [ 3H]quinuclidinyl benzylate ([ 3H]QNB) as ligand and a recently developed ‘electronic micropunch’ technique which allows measurement of quench-corrected [ 3H]QNB binding within corresponding cylinders of tissue obtained by the mechanical micropunch cannula. [ 3H]QNB binding density varied 2.6-fold: lateral reticular nucleus pars lateralis > C1 area > nucleus ambiguus = hypoglossal nucleus = pyramid = oral spinal trigeminal nucleus. In urethane-anesthetized rats, inhibition of ACh synthesis by hemicholinium-3 (HC-3, 3 nmol/50 nl), or blockade of muscarinic receptors by scopolamine (SCOP, 3 nmol/50 nl), reduced resting mean AP by 18–24 mm Hg following bilateral microinjection into the C1 area. These concentrations of HC-3 and SCOP were sufficient to attenuate by 70–80% the increase in local cholinergic neurotransmission elicited by the cholinesterase inhibitor physostigmine given systemically. High concentrations of SCOP (30–150 nmol/50 nl) lowered AP by 46–60 mm Hg. Similarly, bilateral microinjections of GABA (10 nmol/50 nl) into the C1 area markedly reduced mean AP by51 ± 6mm Hg to levels normally found after transection of the spinal cord. Thus, a substantial portion of tonic sympathetic activity may be driven by activation of muscarinic receptors in the C1 area. In the spontaneously hypertensive rat (SHR), a genetic model of hypertension, neither spontaneous nor K +-evoked release of [ 3H]ACh from the C1 area differed from that of normotensive Wistar-Kyoto rats (WKY). In conclusion: (1) ACh may serve a neurotransmitter role in the C1 area of the RVL since substantial quantities of ACh are synthesized, stored and released from local neurons and axon terminals therein; (2) muscarinic receptors are present in a very high density and are sympathoexcitatory when activated in the C1 area; (3) release of ACh within the rostral C1 area may contribute significantly to the sympathoexcitatory drive that maintains resting AP in normotensive rats; and (4) it is unlikely that enhanced release of ACh in the C1 area alone contributes to either the initiation or the maintenance of hypertension in SHRs.

Anatomical substrates of cholinergic‐autonomic regulation in the rat

Acetylcholine (ACh) plays a major role in central autonomic regulation, including the control of arterial blood pressure (AP). Previously unknown neuroanatomic substrates of cholinergic-autonomic control were mapped in this study. Cholinergic perikarya and bouton-like varicosities were localized by an immunocytochemical method employing a monoclonal antiserum against choline acetyltransferase (ChAT), the enzyme synthesizing ACh. In the forebrain, bouton-like varicosities and/or perikarya were detected in the septum, bed nucleus of the stria terminalis, amygdala (in particular, autonomic projection areas AP1 and AP2 bordering the central subnucleus), hypothalamus (rostrolateral/innominata transitional area, perifornical, dorsal, incertal, caudolateral, posterior [PHN], subparafascicular, supramammillary and mammillary nuclei). Few or no punctate varicosities were labeled in the paraventricular (PVN) or supraoptic (SON) hypothalamic nuclei. In the mid- and hindbrain, immunoreactive cells and processes were present in the nucleus of Edinger-Westphal, periaqueductal gray, parabrachial complex (PBC), a periceruleal zone avoiding the locus ceruleus (LC), pontine micturition field, pontomedullary raphe, paramedian reticular formation and periventricular gray, A5 area, lateral tegmental field, nucleus tractus solitarii (NTS), nucleus commissuralis, nucleus reticularis rostroventrolateralis (RVL), and the ventral medullary surface (VMS). In the PBC, immunoreactive varicosities identified areas previously unexplored for cholinergic autonomic responsivity (superior, internal, dorsal, and central divisions of the lateral subnucleus, nucleus of Koelliker-Fuse and the medial subnucleus). In the NTS, previously undescribed ChAT-immunolabeled cells and processes were concentrated at intermediate and subpostremal levels and distributed viscerotopically in areas receiving primary cardiopulmonary afferents. In the nucleus RVL, cholinergic perikarya were in proximity to the VMS and medial to adrenergic cell bodies of the C1 area. Punctate varicosities of unknown origin and dendrites extending ventrally from the nucleus ambiguus overlapped the C1 area and immediate surround of RVL. 1) Cholinergic perikarya and putative terminal fields, overlap structures that are rich in cholinoreceptors and express autonomic, neuroendocrine, or behavioral responsivity to central cholinergic stimulation (PHN, NTS, RVL). The role of ACh in most immunolabeled areas, however, has yet to be determined. Overall, these data support the concept that cholinergic agents act at multiple sites in the CNS and with topographic specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic regulation of arterial pressure by the C1 area of the rostral ventrolateral medulla

In anesthetized, paralyzed rats intravenous administration of the acetylcholinesterase inhibitor physostigmine (PHY) (100 micrograms/kg) evoked a dose-related rise in arterial pressure (AP) and heart rate (HR) and an associated increase in sympathetic nerve activity (SNA). The responses to PHY were blocked by electrolytic lesions of, or microinjection of kainic acid into, a specific site in the rostral ventrolateral medulla containing a cluster of neurons immunoreactive for the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase and corresponding to the C1 area of the nucleus reticularis rostroventrolateralis (RVL). The C1 area and its surround contain a heretofore unrecognized network of varicose neuronal processes and perikarya labeled immunocytochemically with a monoclonal antibody to the ACh-synthesizing enzyme, choline acetyltransferase (CAT). PHY increased, by over 3-fold, the spontaneous activity of reticulospinal cardiovascular neurons in the C1 area which excite preganglionic sympathetic neurons in the intermediolateral spinal column. The effects of PHY on AP, SNA, and the discharge of reticulospinal neurons were blocked by systemic administration of the muscarinic antagonist scopolamine. Microinjections within the C1 area of the RVL of scopolamine, the M2-selective muscarinic receptor antagonist AF-DX 116, or the high-affinity choline uptake inhibitor hemicholinium-3 blocked the pressor effects of PHY. The nicotinic antagonist hexamethonium and the M1-selective muscarinic receptor antagonist pirenzepine were without effect. We conclude that (1) the increases in AP, HR, and SNA elicited by the systemic administration of PHY result from the augmented action of ACh released from cholinergic terminals within the C1 area of the RVL; (2) the locally released ACh acts through muscarinic receptors of the M2 subtype within the C1 area to produce excitation of intrinsic reticulospinal sympathoexcitatory neurons, thereby increasing the activity of sympathetic preganglionic neurons and consequently elevating AP and HR; and (3) while the specific function of the cholinergic innervation of the C1 area in cardiovascular regulation is unknown, it may contribute to the tonic regulation of AP.

Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla

Projections from the nucleus tractus solitarii (NTS) to autonomic control regions of the ventrolateral medulla, particularly the nucleus reticularis rostroventrolateralis (RVL), which serves as a tonic vasomotor center, were analyzed in rat by anterograde, retrograde, and combined axonal transport techniques. Autonomic portions of the NTS, including its commissural, dorsal, intermediate, interstitial, ventral, and ventrolateral subnuclei directly project to RVL as well as to other regions of the ventrolateral medulla. The projections are organized topographically. Rostrally, a small cluster of neurons in the intermediate third of NTS, the subnucleus centralis, and neurons in proximity to the solitary tract selectively innervate neurons in the retrofacial nucleus and nucleus ambiguus. Neurons generally located in more caudal and lateral sites in the NTS innervate the caudal ventrolateral medulla (CVL). The RVL, CVL, and nucleus retroambiguus are interconnected. A combined retrograde and anterograde transport technique was developed so as to prove that projections from the NTS to the ventrolateral medulla specifically innervate the region of RVL containing neurons projecting to the thoracic spinal cord or the region of the nucleus containing vagal preganglionic neurons. When the retrograde tracer, fast blue, was injected into the thoracic spinal cord, and wheat germ agglutinin-conjugate horseradish peroxidase (HRP) was injected into the NTS, anterogradely labeled terminals from the NTS surrounded the retrogradely labeled neurons in the RVL and in the nucleus retroambiguus in the caudal medulla. Among the bulbospinal neurons in the RVL innervated by the NTS were adrenaline-synthesizing neurons of the C1 group. When fast blue was applied to the cervical vagus, and HRP was injected into the NTS, anterogradely labeled terminals from the NTS surrounded retrogradely labeled neurons in the rostral dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, the retrofacial nucleus, and the dorsal portion of the RVL, a region previously shown to contain cardiac vagal preganglionic neurons. This combined anterograde and retrograde transport technique provides a useful method for tracing disynaptic connections in the brain. These data suggest that the RVL is part of a complex of visceral output regions in the ventrolateral medulla, all of which receive afferent projections from autonomic portions of the NTS. Bulbospinal neurons in the RVL, in particular the C1 adrenaline neurons, may provide a portion of the anatomic substrate of the baroreceptor and other visceral reflexes.

Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons.

Anterograde, retrograde, and combined axonal transport methods were used to describe the descending efferent projections of a region of rostral ventrolateral medullary reticular formation important in cardiovascular control. We have termed this region, which contains C1 adrenaline-synthesizing neurons, the nucleus reticularis rostroventrolateralis (RVL). Efferent projections from the RVL innervate all segmental levels of the thoracic intermediolateral and intermediomedial columns as shown using retrograde transport of lectin-conjugated horseradish peroxidase (HRP) or fast blue dye, and anterograde transport of either HRP or labeled amino acids. The projection is highly specific in that there are no projections to thoracic dorsal or ventral horns. This innervation corresponds to the distribution of preganglionic sympathetic neurons in the intermediolateral column. In particular, terminals surround neurons projecting to the adrenal medulla, as demonstrated by combined anterograde and retrograde transport methods at the light level. Terminals containing phenylethanolamine-N-methyl transferase (PNMT) were mapped using immunocytochemical techniques. PNMT-labeled terminals were present at all levels of thoracic intermediolateral column, in a distribution similar to that of the descending projections from the RVL. We have previously shown using double label techniques (Ross et al., '81-'83), that many of the spinal projections of the RVL originate from C1 neurons. These data support our suggestion that certain bulbospinal neurons within the RVL, in particular the C1 neurons, are crucial for tonic vasomotor control.

Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin

We have studied the responses to electrical and chemical stimulation of the ventrolateral medulla in the chloralose-anesthetized, paralyzed, artificially ventilated rat. Locations of most active pressor responses were compared to regions containing neurons labeled immunocytochemically for phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing the synthesis of adrenaline. Elevations of arterial pressure (+81.6 +/- 2.5 mm Hg) and cardioacceleration (+73 +/- 13.6 bpm) were elicited with low current (5 times threshold of 9.5 +/- 1.1 microA) electrical stimulation in a region of rostral ventrolateral medullary reticular formation we have termed the nucleus reticularis rostroventrolateralis (RVL). Electrical stimulation of the RVL increased plasma catecholamines (16.8-fold for adrenaline, 5.3-fold for noradrenaline, and 1.9-fold for dopamine) and vasopressin (1.7-fold before spinal transection, 4.7-fold after). The location of the most active pressor region in the ventrolateral medulla corresponded closely with the location of C1 adrenaline-synthesizing (PNMT-containing) neurons. In addition, the location of the most active pressor region in the dorsomedial medulla corresponded with the location of a bundle of PNMT-containing axons. Unilateral injections into the RVL of the excitatory amino acid monosodium L-glutamate (50 pmol to 10 nmol), but not saline, caused transient dose-dependent and topographically specific elevations (maximum +71.6 +/- 4.9 mm Hg) of arterial blood pressure and tachycardia. Injections of the rigid structural analogue of glutamate, kainic acid, caused large, prolonged (at least 15 min) pressor responses and tachycardia. Unilateral injections of the inhibitory amino acid gamma-aminobutyric acid (GABA) into the RVL caused transient dose-dependent hypotension (maximum -40.8 +/- 6.6 mm Hg) and bradycardia, whereas the specific GABA antagonist bicuculline caused prolonged (10 to 20 min) elevations (+64.2 +/- 6.8 mm Hg) of arterial pressure and tachycardia. By contrast, injections of the glycine antagonist strychnine had no significant effect. Bilateral injections of the neurotoxin, tetrodotoxin, dropped arterial pressure to low levels (51.7 +/- 4.7) not changed by subsequent spinal cord transection at the first cervical segment (52.5 +/- 6.2). We propose the following. (1) Neurons within the RVL, most probably C1 adrenaline-synthesizing neurons, exert an excitatory influence on sympathetic vasomotor fibers, the adrenal medulla, and the posterior pituitary. (2) These neurons are tonically active and under tonic inhibitory control, in part via GABAergic mechanisms--perhaps via the nucleus of the solitary tract (NTS).(ABSTRACT TRUNCATED AT 400 WORDS)