Subretrofacial Nucleus Research Articles

Nitric oxide synthase (NOS) coexists with activated neurons by skeletal muscle contraction in the brainstem of cats

Contraction of skeletal muscle evokes increases in arterial blood pressure and heart rate. Some regions of the brainstem have been implicated for expression of the cardiovascular responses to muscle contraction. Previous studies have reported that static muscle contraction induced c-Fos protein in the nucleus of tractus solitarii (NTS), lateral reticular nucleus (LRN), lateral tegmental field (FTL), subretrofacial nucleus (SRF), A1 region and periaqueductal gray (PAG) of the brainstem. Furthermore, neuronal NADPH-diaphorase (NADPH-d), which is considered as a marker of neuronal nitric oxide synthase (nNOS), has been localized in those same regions. In this study, static muscle contraction was induced by electrical stimulation of the L7 and S1 ventral roots in anaesthetized cats. Distribution of c-Fos protein within neurons containing nNOS was evaluated by double labeling methods in order to determine if nNOS containing neurons in the brainstem were activated during muscle contraction. The results indicate that c-Fos protein colocalized with NADPH-d positive staining within the neurons of the SRF and PAG, but not within the NTS neurons. Distinct number of neurons with c-Fos protein was in close proximity to NADPH-d positive staining in the NTS, SRF, and PAG. Coexisting of c-Fos protein and NADPH-d positive staining was not observed in the LRN, FTL and A1 region. These findings demonstrate that nNOS containing neurons were activated by muscle contraction in the selective regions of the brainstem, and nNOS positive staining had close anatomic contacts with the neurons activated by contraction. This result provides neuroanatomic evidence suggesting that nitric oxide modulates the cardiovascular responses to muscle contraction within the NTS, SRF and PAG of the brainstem.

Effect of nitric oxide on release of glutamate in the subretrofacial nucleus (SRF) during the exercise pressor reflex in cats

The subretrofacial nucleus (SRF) has been known to play a crucial role in the expression of the exercise pressor reflex. Previously, we have reported that the release of glutamate (Glu) in the SRF was increased during muscle contraction in anesthetized cats. In this study, static muscle contraction of the triceps surae for 4 min was induced by electrical stimulation of L7 and S1 ventral roots. Endogenous release of Glu and citrulline (Cit) from the SRF was recovered by microdialysis and measured by HPLC. The microdialysis probes were also used to deliver l-arginine and l-NAME to test the effect of nitric oxide (NO) on release of Glu in the SRF and on the cardiovascular responses during muscle contraction. During control, muscle contraction significantly increased mean arterial pressure (MAP) from 98±8 to 151±9 mmHg, and the extracellular concentration of Glu from 610±120 to 1280±290 nM. Dialyzing 2 mM l-arginine into the SRF increased basal Cit concentration from 260±50 to 760±210 nM ( P<0.05). During contraction after l-arginine, the increases in MAP and Glu concentration were significantly attenuated (86±3–124±6 mmHg and 300±60–460±100 nM, respectively). Dialysis of 0.5 mM l-NAME into the SRF decreased Cit concentration from 340±40 to 180±20 nM ( P<0.05). During contraction after dialyzing l-NAME, the increases in MAP and Glu concentration were significantly potentiated (93±6–154±9 mmHg and 520±80–1290±380 nM, respectively). These results suggest that endogenous NO modulates the cardiovascular responses to static muscle contraction by affecting the release of Glu in the SRF.

Analysis of firing correlations between sympathetic premotor neuron pairs in anesthetized cats.

The activity of sympathetic premotor neurons in the rostral ventrolateral medulla (subretrofacial nucleus) supports sympathetic vasomotor tone, but the factors that drive these premotor neurons' activity have not been determined. This study examines whether either direct interconnections between subretrofacial neurons or synchronizing common inputs to them are important for generating their tonic activity. Simultaneous extracellular single-unit recordings were made from 32 pairs of sympathetic premotor neurons in the subretrofacial nucleus of chloralose-anesthetized cats. Paired spike trains were either separated by spike shape from a single-electrode recording (14 pairs) or recorded from two electrodes less than 250 microm apart (18 pairs). All neurons were inhibited by carotid baroreceptor stimulation and most had a spinal axon proven by antidromic stimulation from the spinal cord. Autocorrelation, inter-spike interval, and cardiac cycle-triggered histograms were constructed from the spontaneous activity of each neuron, and cross-correlation histograms covering several time scales were generated for each neuron pair. No significant peaks or troughs were found in short-term cross-correlation histograms (2 ms bins, +/-100 ms range), providing no support for important local synaptic interactions. On an intermediate time scale (20 ms bins, +/-1 s range), cross-correlation revealed two patterns indicating shared, synchronizing inputs. Repeating peaks and troughs (19/32 pairs) were due to the two neurons' common cardiac rhythmicity, of presumed baroreceptor origin. Single, zero time-spanning peaks of 40--180 ms width were seen in 5/32 cases. Calculations based on the prevalence and strength of these synchronizing inputs indicate that most of the ensemble spike activity of the subretrofacial neuron population is derived from asynchronous sources (be they intrinsic or extrinsic). If synchronizing sources such as neuronal oscillators were responsible for more than a minor part of the drive, they would be multiple, dispersed, and weak.

Activation of skeletal muscle afferents evokes release of glutamate in the subretrofacial nucleus (SRF) of cats

The subretrofacial nucleus (SRF) is a region of the rostral ventrolateral medulla known to play a crucial role in sympathoexcitation. SRF neurons send direct projections to the intermediolateral cell columns of the spinal cord where they form synaptic contact with preganglionic sympathetic motor neurons. Activation of this neural pathway increases sympathetic outflow to the heart and blood vessels affecting cardiac function and vasomotor tone. Previous studies utilizing electrophysiological recording techniques and c-Fos expression have established that the activity of SRF neurons is increased during skeletal muscle contraction. However, the excitatory neurotransmitter mediating this increased activity remains in question. In the present study, static contraction of the triceps surae was induced by electrical stimulation of L7 and S1 ventral roots in anesthetized cats ( n=12). Endogenous release of glutamate (Glu) from the SRF was recovered by microdialysis and measured by HPLC. Static muscle contraction for 4 min increased mean arterial pressure (MAP) 38±4 mmHg from a control level of 102±12 mmHg ( P<0.05). During muscle contraction the extracellular concentration of Glu recovered from the SRF increased from 623±117 to 1078±187 nM ( P<0.05). To determine the effect of muscle contraction on Glu release in the absence of synaptic input from other reflexogenic areas, contraction was repeated following acute sinoaortic denervation and vagotomy. Following this denervation, muscle contraction increased MAP 41±4 mmHg ( P<0.05) and Glu concentration from 635±246 to 1106±389 nM ( P<0.05). Muscle paralysis prevented the increases in MAP and Glu concentration during ventral root stimulation. These results suggest that: (i) Glu is released in the SRF during activation of contraction-sensitive skeletal muscle afferent fibers in the cat; and (ii) synaptic input from other reflexogenic areas appears to be ineffective in modulating the release of Glu in the SRF during static muscle contraction.

Expression of c-Fos-like immunoreactivity in the feline brainstem in response to isometric muscle contraction and baroreceptor reflex changes in arterial pressure

This study compared whether activation of muscle ergoreceptor afferents caused by isometric muscle contraction, activation of baroreceptor afferents induced by i.v. infusion of phenylephrine, or baroreceptor afferent inactivation, caused by carotid artery occlusion, elicit similar patterns of c-Fos induction in brainstem areas. Adult cats were anesthetized with α-chloralose, and in each case, the experimental intervention caused an increase in the arterial blood pressure. There were two sets of control experiments: in both, animals underwent the same surgical procedures but then either remained at rest for the entire study, or the tibial nerve was stimulated, as in the contraction group, following muscle paralysis with tubocurarine. Following the procedures, animals rested for 90 min to allow neuronal expression of c-Fos. Control cats showed very little c-Fos immunoreactivity (c-Fos-ir) in the brainstem. Muscle contraction induced c-Fos-ir expression mainly in the nucleus tractus solitarius, lateral reticular nucleus, lateral tegmental field, vestibular nucleus, subretrofacial nucleus, spinal trigeminal tract and in a lateral region of the periaqueductal grey (P 0.5–1.0). The majority of the c-Fos-ir was found in brainstem areas contralateral to the contracted muscle. In addition, muscle contraction induced c-Fos-ir in the dorsal horns of spinal segments L 6–S 1 on the ipsilateral side of the spinal cord. Phenylephrine infusion caused c-Fos-ir expression in the nucleus tractus solitarius, spinal trigeminal tract, solitary tract, and dorsal motor nucleus of the vagus. No c-Fos-ir was apparent in the periaqueductal grey. Carotid occlusions induced c-Fos-ir expression in the area postrema, nucleus tractus solitarius, solitary tract, and spinal trigeminal tract. Expression was bilateral. Areas that exhibited c-Fos-ir correspond to sites previously reported to release various neuropeptides in response to muscle contraction or carotid occlusions. These results indicate that the exercise pressor reflex and baroreflex activate similar, but not completely identical, sites in the brainstem.

Modulation of sympathetic nerve activity by microinjection of the 5-HT 1A receptor agonist 8-OH-DPAT into the rostroventrolateral medulla

In the present study, renal sympathetic nerve activity was recorded simultaneously with sympathetic nerve activity to skeletal muscle vasculature to determine if the sympatho-inhibition evoked by microinjection of the 5-HT 1A receptor agonist 8-hydroxy-2-(di- n-propylamino)teralin (8-OH-DPAT) into the rostroventrolateral medulla (RVLM) was uniform or regional. Three patterns of sympatho-inhibition were observed in these sympathetic outflows and the type of response depended upon location of microinjection within the subretrofacial nucleus (SRF). Inhibition of renal nerve activity only was elicited by microinjections at rostral sites at the caudal pole of the facial nucleus. In contrast, inhibition of muscle sympathetic nerve activity was evoked from more caudal injections at the rostral pole of the inferior olives. Microinjection in the area between these two regions produced inhibition of both sympathetic outflows. This study demonstrates that differential inhibition of regional sympathetic outflows can be elicited by microinjection of the 5-HT 1A receptor agonist 8-OH-DPAT into the RVLM. These data suggests that this modulation is due to differences in anatomical arrangement of the medullary neurons rather than differences in neuron sensitivity to the serotonergic agonist.

Cardiac inotropic, chronotropic, and dromotropic actions of subretrofacial neurons of cat RVLM.

The cardiac actions of microinjecting sodium glutamate (0.5-2 nmol) among sympathetic premotor neurons of the subretrofacial nucleus in the rostral ventrolateral medulla (RVLM) were studied in chloralose-anesthetized cats after bilateral vagotomy, sinoaortic denervation, adrenalectomy, and alpha1-receptor blockade. Glutamate microinjections increased heart rate by 25.9 +/- 1.8 beats/min (17. 5%), systolic rate of rise in left ventricular pressure (LVdP/dt) by 1,443 +/- 110 mmHg/s (119%), and arterial blood pressure by 26.9 +/- 1.7 mmHg (50%), whereas they shortened the electrocardiogram P-R interval in 85 of 103 cases by 7.5 +/- 1.2 ms (11.4%), triggering junctional rhythms on five occasions. The increase in LVdP/dt usually led the rise in blood pressure, and its magnitude greatly exceeded any increase attributable to changes in heart rate, diastolic filling, or afterload. Right-sided microinjections caused significantly greater tachycardias than did left-sided microinjections, but only left-sided microinjections triggered junctional rhythms (5 of 52 vs. 0 of 51; P < 0.05), whereas microinjections on either side raised LVdP/dt equally. Subretrofacial neurons thus drive positive chronotropic, inotropic, and dromotropic actions via the cardiac sympathetic nerves, whereas subsets among them preferentially control different aspects of cardiac function.

Effect of barodenervation on c-Fos expression in the medulla induced by static muscle contraction in cats.

A previous study has shown increased Fos-like immunoreactivity (FLI), a marker of neural activation, in the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM) after static muscle contraction elicited by electrical stimulation of L7 and S1 ventral roots of the spinal cord in anesthetized, baroreceptor-intact cats. Because the electrically induced static muscle contraction reflexly increased arterial blood pressure, the concomitant activation of the arterial baroreceptor reflex during static muscle contraction may have resulted in some of the FLI labeling that was observed in the medulla. The purpose of this study was to determine regions in the medulla that are activated by muscle contraction in the absence of arterial baroreceptor input. Electrical stimulation of L7 and S1 ventral roots of the spinal cord was used to elicit static muscle contraction, and FLI in the medulla was determined in barointact and barodenervated cats. In barointact contraction cats, FLI was observed in the lateral reticular nucleus (LRN), NTS, lateral tegmental field (FTL), subretrofacial nucleus (SRF), and A1 region of the medulla. In barodenervated contraction cats, FLI increased in the same regions; however, the number of FLI-labeled cells in the NTS, FTL, and A1 region was significantly less than in barointact contraction animals. No significant difference in the number of FLI-labeled cells was found in the LRN and SRF between the two groups. These results clearly demonstrate that cardiovascular regions in the medulla are activated by input from afferent activity originating in skeletal muscle independently of concomitant arterial baroreceptor reflex activation.

The Supply of Vasomotor Drive to Individual Classes of Sympathetic Neuron

The vasoconstrictor supplies to different tissues show distinct patterns of ongoing and reflex activity, indicating that they are driven by distinct central pathways. Vasomotor tone depends heavily on connections from the brainstem, so class-specific vasomotor drives have been sought amongst the sympathetic premotor neurons which provide those connections. Premotor neurons of the rostral ventrolateral medulla (subretrofacial nucleus) provide most descending vasomotor drive. Together, they drive the sympathetic supplies to heart, blood vessels and adrenal, but not ‘non-cardiovascular’ sympathetic responses (sweating, pupil dilatation, piloerection, etc.). Individually, they provide preferential or selective drives to particular classes of ‘cardiovascular’ sympathetic outflow. Subretrofacial neurons are arranged topographically, forming a neural map of the functional class (target tissue), not the body region, of the driven outflows. It is still unknown whether other premotor cell groups are organised this way. Nor are the premotor pathways to ‘non-cardiovascula’ sympathetic nerves yet well-defined.

C-Fos expression in the medulla induced by static muscle contraction in cats.

In this study, we examined Fos-like immunoreactivity (FLI) in the medulla after static muscle contraction induced by stimulation of L7 and S1 ventral roots of the spinal cord in anesthetized cats. The results show that FLI increases in the lateral reticular nucleus, nucleus of the solitary tract, lateral tegmental field, vestibular nucleus, subretrofacial nucleus, and A1 region of the medulla in comparison with these same areas in sham-operated animals (P < 0.05 in each region). In the rostral ventrolateral medulla, FLI distribution in neurons containing phenylethanolamine-N-methyltransferase (PNMT, the synthetic enzyme for epinephrine) was also observed utilizing double-labeling methods. The majority of neurons with PNMT also expressed FLI (66 +/- 4%). These data are in contrast to the results from sham-operated animals showing that 24 +/- 3% of the neurons costained with PNMT (P < 0.05). Our findings indicate that expression of FLI can be used to identify neurons activated during static muscle contraction and support previous studies implicating the ventrolateral medulla as a critical region for expression of the exercise pressor reflex. Furthermore, neurons in the rostral ventrolateral medulla containing PNMT were activated during static muscle contraction.

Renal responses to increases in renal sympathetic nerve activity induced by brainstem stimulation in rabbits

We have stimulated the rostral ventrolateral medulla of the central nervous system to increase renal sympathetic nerve activity (RSNA), and measured the effect on renal blood flow, glomerular filtration rate, and urinary excretion. Increases in RSNA were produced by infusion of 0.02 M glutamate at a rate of 30–50 nl/min into the subretrofacial nucleus for 40 min, in 10 urethane anaesthetized rabbits. Changes in RSNA were quantified as the mean nerve activity per 1 s period and as the frequency and amplitude of individual discharges (reflecting the number of activated nerve fibres). Glutamate infusion increased RSNA 59 ± 1 1 % over control levels. This was predominantly due to a 65 ± 15% increase in the frequency of discharges (3.0 ± 0.35 to 4.6 ± 0.4 Hz), rather than the amplitude of the discharges (+9 ± 3% over control). The effects of these changes on the kidney were made against data collected in the last 20 min of the infusion and the 40 min pre- and post-stimulation periods, when arterial pressure and heart rate were unchanged from control levels. Renal blood flow fell significantly from 31.3 ± 4.5 to 17.7 ± 5.1 ml/min (47% decrease) and filtration fraction significantly increased from 12.7 ± 1.1 to 15.7 ± 2.1% (24% increase) during glutamate infusion. Each of these variables returned to their pre-stimulus levels after ceasing the central stimulation. Fluid, sodium and potassium excretion were not changed by this stimulus. In conclusion, the results in this study suggest that a selective increase in sympathetic nerve activity to the kidney without change in renal perfusion pressure can cause constriction of the renal vasculature without alteration in sodium and water excretion.

Renal responses to increases in renal sympathetic nerve activity induced by brainstem stimulation in rabbits

We have stimulated the rostral ventrolateral medulla of the central nervous system to increase renal sympathetic nerve activity (RSNA), and measured the effect on renal blood flow, glomerular filtration rate, and urinary excretion. Increases in RSNA were produced by infusion of 0.02 M glutamate at a rate of 30–50 nl/min into the subretrofacial nucleus for 40 min, in 10 urethane anaesthetized rabbits. Changes in RSNA were quantified as the mean nerve activity per 1 s period and as the frequency and amplitude of individual discharges (reflecting the number of activated nerve fibres). Glutamate infusion increased RSNA 59 ± 1 1 % over control levels. This was predominantly due to a 65 ± 15% increase in the frequency of discharges (3.0 ± 0.35 to 4.6 ± 0.4 Hz), rather than the amplitude of the discharges (+9 ± 3% over control). The effects of these changes on the kidney were made against data collected in the last 20 min of the infusion and the 40 min pre- and post-stimulation periods, when arterial pressure and heart rate were unchanged from control levels. Renal blood flow fell significantly from 31.3 ± 4.5 to 17.7 ± 5.1 ml/min (47% decrease) and filtration fraction significantly increased from 12.7 ± 1.1 to 15.7 ± 2.1% (24% increase) during glutamate infusion. Each of these variables returned to their pre-stimulus levels after ceasing the central stimulation. Fluid, sodium and potassium excretion were not changed by this stimulus. In conclusion, the results in this study suggest that a selective increase in sympathetic nerve activity to the kidney without change in renal perfusion pressure can cause constriction of the renal vasculature without alteration in sodium and water excretion.

FOS-LIKE IMMUNOREACTIVITY (FLI) IN THE MEDULLA OF CATS INDUCED BY STATIC MUSCLE CONTRACTION 1051

Induced static muscle contraction reflexly increases both blood pressure and heart rate. A number of sites in the medulla have been implicated for the complete expression of this pressor response. In order to identify which sites may be involved in the cardiovascular responses to static muscle contraction, we stimulated the L7 and S1 ventral roots in anesthesized cats and measured FLI in multiple regions within the medulla. The results show a distinct distribution of FLI in the following areas: lateral reticular nucleus, lateral tegmental field, nucleus of solitary tract, A1 region, vestibular nucleus, and subretrofacial nucleus (SRF). Numbers of FLI labeling neurons induced by static muscle contraction increased 65%, 46%, 63%, 67%, 47% and 67%, respectively, when compared with the same stereotaxic level of those areas in control animals (no electrical stimulation delivered to the ventral roots, p<0.05 in each region). Double labeling showed that 66% of the neurons which stained with phenylethanolamine-N-methyltransferase (PNMT) in the SRF also were labeled with FLI. By contrast, only 24% PNMT neurons in the SRF were FLI positive in control animals (p<0.01). No distinct FLI double labeling with PNMT was observed in the other areas of the medulla. The results support the hypothesis that the ventrolateral medulla is an important region of integration for the cardiovascular responses induced during static muscle contraction. Further, adrenergic neurons in the SRF are likely involved in these responses.

Brainstem neurones and postganglionic sympathetic nerves: does correlation mean connection?

Short-term correlations in activity have been widely used as evidence to connect brainstem units with postganglionic sympathetic nerves. These may be detected by spike-triggered averaging, cross correlation or coherence analysis. The specificity of this type of evidence has been investigated by cross-correlating the activity of identified cutaneous vasoconstrictor postganglionic fibres with that of medullary premotor neurones of like and of unlike functional type, as determined by physiological testing (preoptic warming), in anaesthetised cats. Single medullary premotor neurones of both types were recorded from the subretrofacial nucleus: they were identified by their barosensitivity and, in most cases, their spinally projecting axons. By the test criteria chosen, the correlation method gave both false-positive and false-negative results as commonly as it gave correct ones. We conclude that it is not a reliable way to determine brainstem-postganglionic connectivity.

Effects of preoptic warming on subretrofacial and cutaneous vasoconstrictor neurons in anaesthetized cats.

1. Sympathetic and subretrofacial neuron responses to preoptic warming were studied in chloralose- or Saffan-anaesthetized, paralysed cats. 2. Warming a thermode in the preoptic region inhibited the activity of cutaneous vasoconstrictor fibres supplying hairy skin. Muscle vasoconstrictor fibre activity recorded at the same time was either unaffected or raised. 3. Small injections of sodium glutamate (5 nl, 0.1 M) were made into the region of the subretrofacial nucleus in the ventrolateral medulla. The part of that region where glutamate injections evoked brisk increases in cutaneous vasoconstrictor fibre activity was chosen for further study. 4. Extracellular single unit recordings were made in that area from seventy-seven subretrofacial neurons, which were identified by their barosensitivity (inhibition by carotid blind sac inflation). Forty-seven of them were antidromically activated by stimulation in the spinal cord. 5. The activity of twenty subretrofacial neurons (twelve proven bulbospinal) was significantly reduced by periods of preoptic warming. Cutaneous vasoconstrictor activity recorded at the same time also fell. Forty-nine subretrofacial neurons (thirty-five proven bulbospinal) were unaffected or excited by periods of preoptic warming that inhibited cutaneous vasoconstrictor fibres. The response of eight neurons was unclear. 6. No difference in either mean firing rate or axonal conduction velocity was found between neurons inhibited by preoptic warming and other subretrofacial neurons. 7. The subretrofacial neurons inhibited by warming were found intermingled with those unaffected or excited. Marked recording sites of warm-inhibited neurons were clustered around the ventromedial border of the subretrofacial nucleus. 8. In two cats, bilateral inhibition of subretrofacial neurons by surface application of 1 M glycine reduced cutaneous vasoconstrictor fibre activity to 32 and 44% of control levels. 9. The results suggest that specific cutaneous vasoconstrictor premotor neurons exist in the subretrofacial nucleus. These apparently provide most of the background excitatory drive to cutaneous vasomotor neurons. Central warming stimuli may act, at least in part, by withdrawing that drive.

Differential drives from rostral ventrolateral medullary neurons to three identified sympathetic outflows

Simultaneous recordings were made in chloralose-anesthetized cats from muscle vasoconstrictor (MVC), visceral vasoconstrictor (VVC), and renal (RSN) sympathetic fibers. Their barosensitivity was demonstrated, after which all baroreceptor nerves were cut. Multiple microinjections of sodium glutamate (5 nl, 0.1 M) were made in a grid pattern covering the rostral ventrolateral medulla (RVLM) pressor area on seven sides of four animals. Injections increased blood pressure by up to 87 +/- 22 mmHg and MVC, VVC, and RSN activities by up to 169 +/- 55, 62 +/- 23, and 67 +/- 36%, respectively. The relative responses between nerves (taken in pairs) showed significant inhomogeneity across injections into 5/7, 6/7, and 1/7 RVLMs for MVC vs. VVC, MVC vs. RSN, and VVC vs. RSN, respectively. Calculation showed that overall, less than half the response spikes due to RVLM glutamate injections could be accounted for by a hypothetical population of neurons that excited MVC, VVC, and RSN outflows in fixed proportion. Sites that exclusively drove MVC, VVC, or RSN outflows were found in 7/7, 3/7, and 5/7 RVLMs, respectively. Sites that selectively drove either nerve of a given pair were ubiquitous. The RVLM territories from which the three outflows could be activated overlapped but showed clear differences. Their shapes and sizes differed between animals and even between sides. They covered the subretrofacial (SRF) nucleus plus a variable rostromedial extension. The data support the view that the RVLM neurons driving these three sympathetic outflows are largely, perhaps entirely, separate populations.

Emetic reflex arc revealed by expression of the immediate-early gene c- fos in the cat

The organization of the central neuronal circuitry that produces vomiting was explored by mapping the distribution of c-fos protein (Fos)-like immunoreactivity (FLI) as a monitor of functional activity. The brainstem and spinal cord were examined in cats administered multiple emetic drugs (cisplatin, lobeline, protoveratrine, naloxone, apomorphine) or control saline injections. Some animals were decerebrated, paralyzed, and artificially ventilated to avoid possible Fos expression induced by sensory feedback or fluid depletion during vomiting. Fictive vomiting was identified in these animals by a characteristic pattern of respiratory muscle nerve (phrenic and abdominal) coactivation. Tissues were immunoprocessed using an antibody raised against amino acids 1-131 of Fos and the avidin-biotin peroxidase complex method. Enhanced nuclear FLI was observed in experimental animals along portions of the sensorimotor emetic reflex arc, including the nodose ganglia, area postrema, nuclei of the solitary tract (especially medial and subpostrema subnuclei), intermediate reticular zone of the lateral tegmental field, nucleus retroambiguus, C2 inspiratory propriospinal cell region, and dorsal vagal and phrenic motor nuclei. Enhanced FLI was also detected in the raphe magnus, subretrofacial nucleus, and spinal dorsal horn. Regions showing no recognizable differences in FLI between experimental and control animals included the vestibular, cochlear, spinal trigeminal, subtrigeminal, and lateral reticular nuclei. Only minor differences were observed in the distributions of FLI between intact and decerebrate animals. No unique, well-defined group of labeled neurons that might function as a "vomiting center" could be identified. Instead, the pattern of c-fos expression suggests that neurons involved in coordinating the emetic response may radiate from the area postrema and nucleus of the solitary tract to an arc in the lateral tegmental field implicated in somato-autonomic integration.

Projections from inspiratory neurons of the ventral respiratory group to the subretroficial nucleus of the cat

Arterial blood pressure and the activity of many sympathetic nerves are known to be affected by changes in central respiratory activity. The central neurons responsible for this respiratory modulation are unknown. In the present study we have labelled inspiratory neurons ( n = 24) in the rostral ventral respiratory group and Bo¨tzinger complex in the medulla oblongata of the cat using intracellular injection of biocytin. The filled neurons were examined to see if they had axonal projections to the subretrofacial nucleus, an important brainstem nucleus in the tonic and reflex control of blood pressure. The subretrofacial nucleus was identified histologically as a cluster of neurons in the rostral ventrolateral medulla, some of which are tyrosine hydroxylase immunoreactive. Varicose axons arising from labelled inspiratory neurons were mostly found dorsal to this cluster, within the area corresponding to the Bo¨tzinger complex. A small number of axon varicosities were seen in the subretrofacial nucleus. The results suggest that a part of the respiratory modulation of sympathetic nerve activity may be due to a direct synaptic input from inspiratory neurons of the ventral respiratory group to neurons of the subretrofacial nucleus.

Monosynaptic excitation of preganglionic vasomotor neurons by subretrofacial neurons of the rostral ventrolateral medulla

Extracellular single unit recordings were made from barosensitive neurons in the subretrofacial nucleus (SRF) of the rostral ventrolateral medulla in chloralose-anaesthetised cats. At the same time, single preganglionic neuron activity was recorded from filaments of the cervical sympathetic trunk (CST); barosensitive units were selected for study. Evidence for monosynaptic connections between the two neuron groups was sought by cross-correlation analysis of their ongoing activity. Cross-correlograms of 16/16 SRF/CST neuron pairs showed a broad peak (100–200ms wide), reflecting the synchronising action of arterial baroreceptors on both neurons' activity. Two of the 16 cross-correlograms additionally showed a robust, statistically significant, narrow peak of a single 2 ms bin width, providing the first physiological demonstration that ventrolateral medullary neurons monosynaptically excite preganglionic sympathetic neurons. Deductions are made about the strength, convergence and divergence of the connection.

Respiratory responses to microinjection of excitatory amino acid agonists in ventrolateral regions of the lateral parabrachial nucleus in the cat

Sites of tachypneic and hyperpneic responses to localized pressure injection of glutamate agonist were located in the small restricted regions in the ventrolateral regions of the lateral parabrachial nucleus (vlPbl) in the rostral and intermediate levels of the Pbl. The respiration-related neurons in the vlPbl showed different sensitivity to different types of glutamate agonists. They are most sensitive, in descending order, to quisqualate, kainate, N-methyl- d-aspartate (NMDA), and glutamat. The respiratory response to kainate and NMDA was characterized by being slow and long-lasting, while the response to quisqualate and glutamate was rapid and brief. An immunocytochemical study suggested that the respiration-related neurons in the vlPbl receive glutamatergic projections from the solitary tract nucleus, nucleus retroambigualis, nucleus para-ambigualis, para-retrofacial nucleus, subretrofacial nucleus and subfacial cell group in the medulla, Pbl in the contralateral pons, paraventricular nucleus in the hypothalamus and central amygdaloid nucleus in the limbic system. These findings indicate that the respiration-related neurons in the vlPbl play an important role in the modulation of respiration.