Rostroventrolateral Reticular Nucleus Research Articles

Intrathecal kynurenate but not benextramine blocks hypoxic sympathoexcitation in chemodenervated anesthetized rats

In peripherally chemodenervated anesthetized and ventilated rats, inhalation of 100% N 2 for 20 s rapidly excites reticulospinal vasomotor neurons of the rostroventrolateral reticular nucleus (RVL) and sympathetic nerves. After an initial fall, arterial pressure is also elevated. The sympathoexcitatory response and secondary increases in arterial pressure were abolished by microinjections of muscimol (250 pmol) into the RVL or by intrathecal administration of kynurenate (500 nmol), a broad-spectrum antagonist of excitatory amino acid receptors, but not by benextramine, an α-adrenoceptor antagonist. The results indicate that excitation of pre-ganglionic sympathetic neurons in the spinal cord by hypoxia results from excitation of RVL-spinal vasomotor neurons, probably the glutamatergic neurons.

PHF-1 immunopositive dystrophic neurites are preferentially associated with thioflavine positive β-pleated plaques and not diffuse amyloid plaques : B.J. Cummings, C.J. Pike and C.W. Cotman. IRU in Brain Aging and Dementia, Department of Psychobiology, University of California, Irvine, CA 92717-4550 USA

Recent studies in the rat have demonstrated that at least two populations of sympathoexcitatory reticulospinal neurons reside in the nucleus reticularis rostroventrolateralis. It appears that only one of these populations consists of C1 adrenergic neurons. The present study used both double-labeling (one retrograde tracer and immunohistochemistry) and triple-labeling (two retrograde tracers and immunohistochemistry) to determine if C1 adrenergic neurons, which are immunoreactive for phenylethanolamineN-methyltransferase, exhibit a projection pattern that is sufficiently unique to permit the electrophysiological discrimination between C1 adrenergic and non-adrenergic neurons in the nucleus reticularis rostroventrolateralis. Double-labeling experiments indicated that 71% (range: 53–80) ofphenylethanolamine-N-methyltransferase-immunoreactive neurons in the nucleus reticularis rostroventrolateralis could be retrogradely labeled from the thoracic cord, as were 76% (range: 67–94) following tracer injection in the central tegmental tract at pontine levels. Triple-labeling experiments indicated that 88% (range: 82–93) of nucleus reticularis rostroventrolateralis neurons with projections to both spinal cord and central tegmental tract werephenylethanolamine-N-methyltransferase-immunoreactive.Single-unit recording, in nucleus reticularis rostroventrolateralis, was used to identify antidromic potentials elicted from stimulation sites in the spinal cord and/or central tegmental tract. Since clonidine is known to reduce central adrenaline turnover, sensitivity to this drug was used to identify putative adrenergic neurons. Twenty-six nucleus reticularis rostroventrolateralis neurons with axonal projections to both the ipsilateral spinal cord and the central tegmental tract were recorded in halothane-anesthetized rats. All these cells were barosensitive, pulse-modulated, and 16 of the 16 cells tested exhibited a66 ± 8% reduction in activity upon the intravenous administration of clonidine (20 μg/kg). Most (13 out of 16) exhibited a strong respiratory modulation. The conduction velocity of their spinal collateral was generally low (0.9 ± 0.1m/s) and their firing rate moderate (7.4 ± 1.2spikes/s). Forty-three nucleus reticularis rostroventrolateralis cells with axonal projections exclusively to the thoracic cord were studied for comparison. These cells were strongly barosensitive and pulse-synchronous, had a high discharge rate (25 ± 3spikes/s) and a moderate conduction velocity (3.4 ± 0.3m/s). Only one of the 15 cells tested was inhibited by clonidine and only two to these 15 cells exhibited a detectable respiratory modulation.Thus barosensitive nucleus reticularis rostroventrolateralis neurons with axonal projections to both the spinal cord and the central tegmental tract likely belong the the C1 adrenergic cell group. It is concluded that this subgroup of adrenergic neurons probably subserves a vasomotor function.

Innervation of serotonergic medullary raphe neurons from cells of the rostral ventrolateral medulla in rats

The Rostral ventral medulla has been shown to consist of three distinct subrogions: the midline or raphéregion, the lateral paragigantocellular-gigantocellular region and the rostro-ventrolateral reticular nucleus. All three regions have been shown to contribute to central vaso-regulation and to project towards sympathetic preganglionic neurons of the thoracic spinal cord. Therefore it is of particular interest to describe the interconnections between the three regions and to see if local afferents reach cells which have been implicated in the regulation of descending inputs. Following injections of the anterograde tract tracer Phaseolus vulgaris leucoagglutinin into the lateral paragigantocellular nucleus or the rostroventrolateral reticular nucleus, labelled axons were traced into the medullary raphénuclei and the contralateral rostral ventrolateral medulla. Efferents originating from both regions innervated the raphépallidus, raphéobscurus and raphémagnus. However the distribution of terminals originating from the two regions was different in the contralateral ventrolateral medulla oblongata. The data indicate that the connection between the ipsi- and contralateral equivalents of both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus are stronger than the cross-connection between the ipsi- and contralateral parts of the two different regions. In the second part of the study, the existence of direct projections from the rostroventrolateral reticular nucleus and the lateral paragigantocellular-gigantocellular region onto serotonin-immunogold-labelled cells of the ventromedial medulla were investigated. The correlated light and electron microscopic analysis revealed direct synaptic contacts between axons originating from both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus, and serotonin-immunoreactive cells of the raphéobscurus and raphépallidus. The results of the present light microscopic tract-tracing study revealed a different pattern of the intramedullary projection of the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus. These data are in support of the proposed parcellation of the two cytoarchitectonically different areas of the rostral ventrolateral medulla into two functionally distinct subdivisions. Furthermore, the direct anatomical connection revealed in the present study between cells of the rostral ventrolateral and ventromedial medulla oblongata indicates the possibility that vasoregulatory effects of some cells of the rostral ventrolateral medulla oblongata might be executed via direct projections onto serotonin-immunoreactive cells of the medullary raphénuclei.

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.

Monosynaptic projections from the rostral ventrolateral medulla oblongata to identified sympathetic preganglionic neurons

The rostral ventrolateral medulla oblongata plays an important role in the control of arterial blood pressure and it has strong descending projections into the intermediolateral nucleus of the thoracic spinal cord, where the majority of sympathetic preganglionic neurons are located. The purpose of this study was to see whether these projections form synaptic contacts with sympathetic preganglionic neurons in the rat. Projections from both the lateral part of the rostral ventrolateral medulla (rostroventrolateral reticular nucleus) and from the more medial region (lateral paragigantocellular nucleus) were investigated separately in view of their different functional roles in sympatho-regulation and their different chemical composition. Using anterograde tract-tracing of descending medullary pathways with Phaseolus vulgaris leucoagglutinin and retrograde labelling of sympatho-adrenal preganglionic neurons with cholera B chain conjugated to horseradish peroxidase, the existence of monosynaptic connections was sought by electron microscopy. Synaptic inputs from both the lateral and medial aspects of the rostral ventrolateral medulla oblongata were found on identified sympathetic preganglionic neurons. Synaptic specializations were of both the symmetrical and asymmetrical type. The targets of boutons forming asymmetrical synaptic contacts differed according to their origin: boutons originating from neurons in the rostroventrolateral reticular nucleus were mainly in contact with dendrites of sympathetic preganglionic neurons, while those originating from the lateral paragigantocellular nucleus mainly innervated the cell bodies. Our observations provide anatomical support for the view that there are two distinct classes of sympatho-regulatory cells in the rostral ventrolateral medulla, each of which can directly influence the activity of sympathetic preganglionic neurons; they also emphasize the importance of detailed investigation of the subrogions of the ventrolateral medulla with respect to their sympatho-regulatory functions.

Vasopressinergic mechanisms in the nucleus reticularis lateralis in blood pressure control

We sought to determine whether arginine vasopressin (AVP) modulates arterial pressure (AP) by a receptor-mediated action in the nucleus reticularis rostroventrolateralis (nRVL). Immunocytochemical labeling with an antiserum against a synthetic AVP conjugate revealed a discrete although modest presumptive neuropeptidergic innervation of the nRVL. Electron microscopic analysis of vasopressinergic processes in the nRVL revealed that AVP-like immunoreactivity (AVP-LI) was primarily in axons and axon terminals. Immunoreactive terminals contained numerous small clear vesicles and large dense core vesicles and formed synapses with unlabeled dendrites. In the nRVL, retrograde transport-immunofluorescence data demonstrated close appositions between vasopressinergic beaded processes and a compact subambigual column of reticulospinal neurons labeled by deposits of cholera toxin β-subunit into the thoracic spinal cord. Similar methods were used to define the origins of the AVP-afferent projection to nRVL. These retrograde transport-immunofluorescence studies demonstrated numerous retrogradely labeled neurons in the hypothalamus, including the paraventricular nucleus (PVN), after injections of a retrograde tracer, Fluoro-Gold into the ventrolateral medulla. However, double-labeled neurons were rare and confirmed a diffuse AVP afferent innervation of the sympathoexcitatory area. Microinjection of AVP into the nRVL in anesthetized rats produced a large dose-related increase in AP different from control at a dose of 1 pmol or higher. AVP injected intravenously elevated AP only at significantly higher doses. Microinjections of AVP into the nucleus tractus solitarii (NTS) had a smaller effect whereas into the caudal ventrolateral medulla exerted no effect on AP. Bilateral microinjections of an AVP antagonist, d(CH 2) 5[Tyr(Me) 2]AVP into the nRVL produced no change in AP but blocked the increase produced by subsequent injections of AVP. An acute hemorrhage produced by withdrawal of 2 ml of blood from the femoral vein did not alter AP. However, bilateral microinjections of the AVP antagonist into the nRVL 5 min after hemorrhage decreased AP. In contrast, the AVP-antagonist injected intravenously after hemorrhage had no effect on AP. Our data suggest that under conditions demanding increased sympathetic drive to maintain AP, such as hemorrhage, a functional AVP receptor mechanism via terminals in the nRVL may be activated to restore normal levels of AP.

Identification of Rat Brainstem Sites with Neuronal Fos Protein Induced by Acoustic Stimulation with Pure Tones

Induction of Fos, a proto-oncogene c-fos protein product, was immunohistochemically examined in the rat brainstem by using pure tone stimuli (0.25 kHz, 4kHz and 8 kHz) at 60 and 80 dB sound pressure levels for 30 min. In comparison with non-stimulated control rats, the brainstem of sound-stimulated rats exhibited distinct Fos-labeled neuronal cell nuclei in i) the ascending auditory nuclei, ii) the brainstem sites which receive afferents from the auditory nuclei, i.e. the pretectum and rostroventrolateral reticular nucleus, iii) the parvocellular part of the ventral lateral geniculate nucleus, lateral reticular nucleus, ventral border region of the spinal trigeminal nucleus, and the medial vestibular and spinal vestibular nuclei, all of which lack established auditory projections, and iv) the brainstem sites where some Fos-labeled neurons were present in controls. Sound stimulation at 80 dB led to widespread Fos-labeling in all these sites. Sound stimulation at 60 dB caused a decrease in number of positive cells in the brainstem sites and yielded a tone-dependent subregional distribution pattern in the dorsal cochlear nucleus and central nucleus of the inferior colliculus.

Distribution of enkephalin and its relation to serotonin in cat and monkey spinal cord and brain stem.

The distribution of enkephalin (ENK)-like immunoreactivity (LI) in spinal cord and medulla oblongata of cat and gray monkey (Macaca fascicularis) was studied by use of immunofluorescence and peroxidase antiperoxidase (PAP) techniques. Possible coexistence between ENK- and 5-hydroxytryptamine (5-HT)-LI was also analyzed with double labeling immunofluorescence. Furthermore, in situ hybridization was used to demonstrate cell bodies in the brain stem expressing mRNA encoding for ENK. ENK-immunoreactive (IR) axonal varicosities and fibers were demonstrated throughout the spinal cord gray matter, with the highest density in the superficial dorsal horn, the area around the central canal, the intermediolateral cell column, the sacral parasympathetic nucleus, and in Onuf's nucleus. In the monkey ventral horn, ENK-IR varicose fibers could in some cases be demonstrated in very close apposition to cell bodies. A low degree of co-localization between ENK- and 5-HT-LI was seen in the spinal cord of both species. Still, fibers containing both compounds could as a rule be demonstrated in every section studied. The highest degree of coexistence was encountered in the motor nucleus of the ventral horn. Six weeks after a low thoracic spinal cord transection a decreased staining for ENK-LI was demonstrated in the ventral horn motor nucleus, whereas other parts of the spinal cord appeared unaffected. In the brain stem of cats after colchicine treatment, ENK-LI was found in a majority of the 5-HT-IR cell bodies in the raphe nuclei (nucleus raphe magnus, pallidus and obscurus) and in the lateral reticular nucleus (rostroventrolateral reticular nucleus). In cat not pretreated with colchicine, a few weakly stained ENK-IR cell bodies could be found in the midline raphe nuclei and in the lateral reticular nucleus with the PAP technique. In the monkey brain stem without colchicine treatment, using the PAP technique, heavily stained ENK-IR cell bodies could be seen in the lateral reticular nucleus whereas, as in the cat, only a few, weakly stained ENK-IR cell bodies could be seen in the midline raphe nuclei. Using in situ hybridization technique, ENK mRNA expressing cells were demonstrated in the lateral reticular nucleus while no convincing mRNA signal could be found over cell bodies in the raphe nuclei. It is concluded that part of the ENKergic innervation of the cord in both species derives from supraspinal or suprasegmental levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Brainstem enkephalinergic projections to spinal autonomic nuclei

The present studies in the rat employed a combined retrograde transport-immunocytochemical technique to determine the origin in the brainstem of enkephalin (Enk) projections to spinal sympathetic nuclei, including the intermediolateralis nucleus, pars principalis (ILp). We found that Enk projections to the ILp nucleus are found in such serotonergic-containing areas as the raphe obscurus; raphe pallidus; gigantocellular reticular nucleus, pars alpha; paragigantocellular lateral nucleus; raphe magnus; and the rostral extension of the raphe magnus nucleus. The adrenergic-containing rostroventrolateral reticular nucleus as well as the noradrenergic-containing areas A5, A7, ventral locus coeruleus, subcoeruleus, and fiber pathway linking the locus coeruleus and A5/A7 send Enk projections to ILp. In the pons, a large contralateral Enk projection to spinal sympathetic nuclei was found medial to the facial nerve and medial to the motor nucleus of the trigeminal nerve. These observations show the existence of a large number of Enk brainstem regions that can influence spinal autonomic centers via descending supraspinal projections.

Morphology of rostral medullary neurons with intrinsic pacemaker activity in the rat

Neurons with regular ongoing activity attributable to intrinsic pacemaker properties were recorded in coronal tissue slices within the nucleus reticularis rostroventrolateralis of the rat medulla oblongata (RVL). The cells were injected with horseradish peroxidase or Lucifer yellow and their dendritic and proximal axonal characteristics were investigated ( n=15). These small-to-medium-sized neurons had a simple dendritic arborization (3–6 primary dendrites branching up to 3 times) apparently confined within the limits of nucleus RVL and with limited extension in the rostrocaudal direction. Their axons originated either from the cell body or from a primary dendrite and coursed in a dorsomedial direction without giving rise to lical arborizations. It is concluded that RVL pacemaker neurons, presumed to represent a non-adrenergic class of sympathoexcitatory premotor neurons, exhibit characteristics reminiscent of the archetypal ‘reticular core’ 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.

Excitation of rostral medullary pacemaker neurons with putative sympathoexcitatory function by cyclic AMP and β-adrenoceptor agonists ‘in vitro’

This study explores the mechanism of action of catecholamines on rostral medullary pacemakers neurons with putative sympathoexcitatory function, in tissue slices. The firing rate of the pacemaker neurons of nucleus reticularis rostroventrolateralis (RVL pacemakers) was reversibly increased by agents which elevate intracellular levels of cAMP (forskolin and 8-br-cAMP). Forskolin dideoxy, an analog without action on adenylate cyclase, was ineffective and adenosine, a potential degradation product of 8-br cAMP produced inhibition exclusively and only in high doses (0.1–1 mM). The firing rate of these cells was uniformly increased by epinephrine and isoproterenol (10 μM) but unaffected by both phenylephrine (100 μM) and clonidine (up to 1 μM). These effects were abolished by pretreatment with the β-adrenoceptor antagonist propranolol (10 μM) but they were unaffected by the α-antagonist phentolamine (100 μM). The indirectly-acting sympathomimetic amine tyramine (0.1–1 mM) activated all the cells tested. The effect of tyramine was antagonized by the β-blocker pindolol and was absent 7 days after microinjection of the neurotoxin 6-hydroxydopamine into the lateral aspect of the RVL. Intracellular recordings indicated that both isoproterenol and tyramine enhanced the rate of depolarization of the pacemaker neurons during the interspike interval and produced a decrease in input resistance. After tetrodotoxin (TTX) pretreatment, isoproterenol produced a depolarization also associated with a reduction in input resistance. Three conclusions are proposed. First, RVL pacemakers have functional β-adrenergic receptors whose activation increases their discharge rate via the intracellular production of cAMP. The effect of cAMP is due at least in part to the activation of an inward current which may be carried by a cation. Secondly, RVL neurons are in close proximity to a releasable pool of catecholamines which is susceptible to destruction by the cytotoxic agent 6-hydroxydopamine (6-OHDA). Finally it is tentatively suggested that the reduction in sympathetic tone produced by centrally acting β-blockers could be due, at least in part, to an action of these agents on RVL pacemaker cells.

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)

Subpopulations of primary sensory neurons in rat are labelled immunohistochemically by an antibody against the 27KD gap junctional protein (CONNEXIN32): Implications for electrical coupli between sensory fibers

We sought to determine whether arginine vasopressin (AVP) modulates arterial pressure (AP) by a receptor-mediated action in the nucleus reticularis rostroventrolateralis (nRVL). Immunocytochemical labeling with an antiserum against a synthetic AVP conjugate revealed a discrete although modest presumptive neuropeptidergic innervation of the nRVL. Electron microscopic analysis of vasopressinergic processes in the nRVL revealed that AVP-like immunoreactivity (AVP-LI) was primarily in axons and axon terminals. Immunoreactive terminals contained numerous small clear vesicles and large dense core vesicles and formed synapses with unlabeled dendrites. In the nRVL, retrograde transport-immunofluorescence data demonstrated close appositions between vasopressinergic beaded processes and a compact subambigual column of reticulospinal neurons labeled by deposits of cholera toxin β-subunit into the thoracic spinal cord. Similar methods were used to define the origins of the AVP-afferent projection to nRVL. These retrograde transport-immunofluorescence studies demonstrated numerous retrogradely labeled neurons in the hypothalamus, including the paraventricular nucleus (PVN), after injections of a retrograde tracer, Fluoro-Gold into the ventrolateral medulla. However, double-labeled neurons were rare and confirmed a diffuse AVP afferent innervation of the sympathoexcitatory area. Microinjection of AVP into the nRVL in anesthetized rats produced a large dose-related increase in AP different from control at a dose of 1 pmol or higher. AVP injected intravenously elevated AP only at significantly higher doses. Microinjections of AVP into the nucleus tractus solitarii (NTS) had a smaller effect whereas into the caudal ventrolateral medulla exerted no effect on AP. Bilateral microinjections of an AVP antagonist, d(CH2)5[Tyr(Me)2]AVP into the nRVL produced no change in AP but blocked the increase produced by subsequent injections of AVP. An acute hemorrhage produced by withdrawal of 2 ml of blood from the femoral vein did not alter AP. However, bilateral microinjections of the AVP antagonist into the nRVL 5 min after hemorrhage decreased AP. In contrast, the AVP-antagonist injected intravenously after hemorrhage had no effect on AP. Our data suggest that under conditions demanding increased sympathetic drive to maintain AP, such as hemorrhage, a functional AVP receptor mechanism via terminals in the nRVL may be activated to restore normal levels of AP.

Diverse afferents converge on the nucleus paragigantocellularis in the rat ventrolateral medulla: Retrograde and anterograde tracing studies

The nucleus paragigantocellularis in the ventrolateral medulla has been implicated in cardiovascular, pain, and analgesic functions; and it has also been found to be a major afferent to the pontine nucleus locus coeruleus. In the present study, afferents to the nucleus paragigantocellularis were identified in the rat by means of the retrograde tracers wheat germ agglutinin-conjugated horseradish peroxidase or Fluoro-Gold. Projections to the nucleus paragigantocellularis arise from a wide variety of nuclei with autonomic, visceral, and sensory-related functions. Major afferents with consistent and robust retrograde labeling include most laminae of the spinal cord, the caudal lateral medulla, the contralateral paragigantocellularis, the nucleus of the solitary tract, the A1 area, the lateral parabrachialis, the Kölliker-Fuse nucleus, the periaqueductal gray, and a preoculomotor nucleus in the ventral central gray, the supraoculomotor nucleus. Other notable afferents, seen only after large caudal injections into the nucleus paragigantocellularis, include the lateral hypothalamus, the paraventricular nucleus of the hypothalamus, and the medial prefrontal cortex. Minor afferents include the gigantocellular nucleus, the area postrema, the caudal raphe groups, the inferior colliculus, the A5 area, and the locus coeruleus. The projection from the supraoculomotor nucleus, not previously reported as an afferent to the ventrolateral medulla, was confirmed with anterograde tracing by means of Phaseolus vulgaris-leucoagglutinin. Iontophoretic deposits of Phaseolus vulgaris-leucoagglutinin into the nucleus of the solitary tract (commissuralis level) or into the periaqueductal gray also yielded terminal fiber labeling in the nucleus paragigantocellularis. Fibers from the supraoculomotor nucleus and the nucleus of the solitary tract were densest in the lateral aspect of the nucleus paragigantocellularis (corresponding to the rostroventrolateral reticular nucleus), while fibers from the periaqueductal gray were more medially located. Previous studies have defined inputs to the rostral ventrolateral medulla from the cochlear nucleus as well as from the colliculi. In the present study, deposits of wheat germ agglutinin-conjugated horseradish peroxidase or Phaseolus vulgaris-leucoagglutinin into the cochlear nucleus or the superior colliculus yielded only sparse anterograde labeling in the nucleus paragigantocellularis, but heavily labeled adjacent areas. The inferior collicular injections yielded strong but restricted anterograde labeling in the rostromedial paragigantocellularis, medial to the facial nucleus. These results indicate that the paragigantocellularis area receives inputs from diverse brain structures. Neurons in the nucleus paragigantocellularis afferent to the locus coeruleus, being distributed throughout this region, may provide a channel where several types of information are integrated and transmitted to the extensive locus coeruleus noradrenergic efferent network...

Effects of vasopressin and other neuropeptides on rostral medullary sympathoexcitatory neurons ‘in vitro’

Neurons with intrinsic pacemaker activity and presumed sympathoexcitatory function were recorded in rat tissue slices within the confines of the rostroventrolateral reticular nucleus (RVL). These cells were excited in dose-dependent fashion by arginine vasopressin (AVP, 10 −8–10 −6 M) but not by oxytocin (up to 10 −7 M). The effect of AVP was mimicked by the V 1-selective agonist [Phe 2, Orn 8]vasotocin (VT) (1 μM) but not by the V 2-agonist [Val 4, d-Arg 8]vasopressin (VP) (1.9 μM). The effect of AVP (10 −7 M) was completely blocked by SKF 101926 (10 −7 M), a non-selective antagonist and by d(CH 2) 5[Tyr(Me) 2]AVP, a V 1-selective antagonist but was unaffected by the V 2-selective antagonist d(CH 2) 5[ d-Ile 2,Ile 4,Ala-NH 2 9]AVP. These cells were also activated by thyrotropin-releasing hormone (TRH) (10 −7–10 −6 M), calcitonin gene-related peptide (CGRP) (4 × 10 −8M), substance P, (10 −6 M), neuropeptide Y (NPY) (10 −8 M) and inhibited by Met-enkephalin (10 −6 M) and morphine (2 mM). Corticotropin-releasing factor (CRF) (10 −7 M) and angiotensin II (10 −6 M) were ineffective. In conclusion, RVL pacemaker neurons have vasopressin receptors reminiscent of the V 1 (vascular and pressor) subtype. Their pacemaking activity is modulated by low doses of several other peptides also known to produce large vasomotor effects after introduction into the cerebroventricular space.

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.

The amygdala directly innervates adrenergic (C1) neurons in the ventrolateral medulla in the rat

The innervation of adrenergic (C1) neurons in the ventrolateral medulla by the central amygdaloid nucleus (Ce) was investigated using immunohistochemical detection of phenylethanolamine N-methyltransferase (PNMT) combined with anterograde tracing through Phaseolus vulgaris leucoagglutinin (PHA-L) transport and lesion-induced axonal degeneration. Injections of PHA-L into the medial Ce labelled axons in close proximity to PNMT-immunoreactive dendrites and somata in the ventrolateral medulla. The PNMT-immunoreactive neurons within the rostral part of the nucleus reticularis rostroventrolateralis were preferentially innervated by the amygdaloid terminals. Degenerating terminals formed synaptic contacts on PNMT-immunoreactive cells of the ventrolateral medulla in animals with lesions of the Ce. The synaptic contacts were mainly found on dendrites and were usually of the symmetrical type. The present findings provide evidence that cells within the amygdala directly innervate presumed adrenergic cells in the ventrolateral medulla. This pathway may be part of the anatomical substrates that are activated during amygdaloid-mediated sympathetic activity.

Electrophysiological characterization of putative C1 adrenergic neurons in the rat

Recent studies in the rat have demonstrated that at least two populations of sympathoexcitatory reticulospinal neurons reside in the nucleus reticularis rostroventrolateralis. It appears that only one of these populations consists of C1 adrenergic neurons. The present study used both double-labeling (one retrograde tracer and immunohistochemistry) and triple-labeling (two retrograde tracers and immunohistochemistry) to determine if C1 adrenergic neurons, which are immunoreactive for phenylethanolamineN-methyltransferase, exhibit a projection pattern that is sufficiently unique to permit the electrophysiological discrimination between C1 adrenergic and non-adrenergic neurons in the nucleus reticularis rostroventrolateralis. Double-labeling experiments indicated that 71% (range: 53–80) ofphenylethanolamine-N-methyltransferase-immunoreactive neurons in the nucleus reticularis rostroventrolateralis could be retrogradely labeled from the thoracic cord, as were 76% (range: 67–94) following tracer injection in the central tegmental tract at pontine levels. Triple-labeling experiments indicated that 88% (range: 82–93) of nucleus reticularis rostroventrolateralis neurons with projections to both spinal cord and central tegmental tract werephenylethanolamine-N-methyltransferase-immunoreactive. Single-unit recording, in nucleus reticularis rostroventrolateralis, was used to identify antidromic potentials elicted from stimulation sites in the spinal cord and/or central tegmental tract. Since clonidine is known to reduce central adrenaline turnover, sensitivity to this drug was used to identify putative adrenergic neurons. Twenty-six nucleus reticularis rostroventrolateralis neurons with axonal projections to both the ipsilateral spinal cord and the central tegmental tract were recorded in halothane-anesthetized rats. All these cells were barosensitive, pulse-modulated, and 16 of the 16 cells tested exhibited a66 ± 8% reduction in activity upon the intravenous administration of clonidine (20 μg/kg). Most (13 out of 16) exhibited a strong respiratory modulation. The conduction velocity of their spinal collateral was generally low (0.9 ± 0.1m/s) and their firing rate moderate (7.4 ± 1.2spikes/s). Forty-three nucleus reticularis rostroventrolateralis cells with axonal projections exclusively to the thoracic cord were studied for comparison. These cells were strongly barosensitive and pulse-synchronous, had a high discharge rate (25 ± 3spikes/s) and a moderate conduction velocity (3.4 ± 0.3m/s). Only one of the 15 cells tested was inhibited by clonidine and only two to these 15 cells exhibited a detectable respiratory modulation. Thus barosensitive nucleus reticularis rostroventrolateralis neurons with axonal projections to both the spinal cord and the central tegmental tract likely belong the the C1 adrenergic cell group. It is concluded that this subgroup of adrenergic neurons probably subserves a vasomotor function.