pSer40 tyrosine hydroxylase immunohistochemistry identifies the anatomical location of C1 neurons in rat RVLM that are activated by hypotension.

Identification of neurons, and their phenotype, that are activated in response to specific stimuli is a critical step in understanding how neural networks integrate inputs to produce specific outputs. Here, we developed novel mouse monoclonal antibodies of different IgG isotypes that are specific to tyrosine hydroxylase (TH), and to tyrosine hydroxylase activated at its serine 40 position (pSer40TH), in order to assess changes in the activity of phenotypically identified cardiovascular neurons using fluorescence immunohistochemistry. We find that the proportion of C1 pSer40TH-positive neurons in the central and medial region of the rat rostral ventrolateral medulla (RVLM) increases dramatically following hydralazine treatment, whereas phenylephrine treatment does not significantly change the pSer40TH/TH ratio in these regions compared to control. This finding suggests that there is a mediolateral topology associated with the activation of C1 neurons following baroreceptor loading or unloading. Overall, we conclude first, that our newly characterized monoclonal antibodies are specific, and selective, against TH and pSer40TH. Secondly, that they can be used to label TH and pSer40TH immunoreactive neurons simultaneously, and thirdly that that they can be used to identify the activation state of catecholamine synthetizing neurons after physiological stimuli. Finally, we find that there is basal level of activation of TH neurons in the lateral, central and medial regions (∼ 70%, 30% and 45%, respectively) of the C1 area, but that following unloading of the baroreceptors there is a marked increase in activation of central (∼ 80%) and medial (∼ 90%) C1 neurons in the RVLM.

PACAP causes PAC1/VPAC2 receptor mediated hypertension and sympathoexcitation in normal and hypertensive rats.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide that plays an important role in hypertension and stress responses. PACAP acts at three G protein-coupled receptors [PACAP type 1 receptor (PAC(1)) and vasoactive intestinal peptide receptor types 1 and 2 (VPAC(1) and VPAC(2))] and is localized to sites involved in cardiovascular control, most significantly the rostral ventrolateral medulla (RVLM). The RVLM is crucial for the tonic and reflex control of efferent sympathetic activity. Increases in sympathetic activity are observed in most types of hypertension and heart failure. PACAP delivered intrathecally also causes massive sympathoexcitation. We aimed to determine the presence and abundance of the three PACAP receptors in the RVLM, the role, in vivo, of PACAP in the RVLM on tonic and reflex cardiovascular control, and the contribution of PACAP to hypertension in the spontaneously hypertensive rat (SHR). Data were obtained using quantitative PCR and microinjection of PACAP and its antagonist, PACAP(6-38), into the RVLM of anesthetized artificially ventilated normotensive rats or SHRs. All three receptors were present in the RVLM. PACAP microinjection into the RVLM caused sustained sympathoexcitation and tachycardia with a transient hypertension but did not affect homeostatic reflexes. The responses were partially mediated through PAC(1)/VPAC(2) receptors since the effect of PACAP was attenuated (∼50%) by PACAP(6-38). PACAP was not tonically active in the RVLM in this preparation because PACAP(6-38) on its own had no inhibitory effect. PACAP has long-lasting cardiovascular effects, but altered PACAP signaling within the RVLM is not a cause of hypertension in the SHR.

Intrathecal neurotensin is hypotensive, sympathoinhibitory and enhances the baroreflex in anaesthetized rat.

BACKGROUND AND PURPOSE: The neuromodulatory effects of the gut-neuropeptide neurotensin on sympathetic vasomotor tone, central respiratory drive and adaptive reflexes in the spinal cord, are not known. EXPERIMENTAL APPROACH: Neurotensin (0.5 µM-3 mM) was administered into the intrathecal (i.t.) space at the sixth thoracic spinal cord segment in urethane-anaesthetized, paralysed, vagotomized male Sprague-Dawley rats. Pulsatile arterial pressure, splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, ECG and end-tidal CO(2) were recorded. KEY RESULTS: Neurotensin caused a dose-related hypotension, sympathoinhibition and bradycardia. The maximum effects were observed at 3000 µM, where the decreases in mean arterial pressure (MAP), heart rate (HR) and sSNA reached -25 mmHg, -26 beats min(-1) and -26% from baseline, respectively. The sympathetic baroreflex was enhanced. Changes in central respiratory drive were characterized by a fall in the amplitude of the phrenic nerve activity. Finally, administration of SR 142948A (5 mM), a potent, selective antagonist at neurotensin receptors, caused a potent hypotension (-35 mmHg), bradycardia (-54 beats min(-1) ) and sympathoinhibition (-44%). A reduction in the amplitude and frequency of the phrenic nerve activity was also observed. CONCLUSIONS AND IMPLICATIONS: The data demonstrate that neurotensin plays an important role in the regulation of spinal cardiovascular function, affecting both tone and adaptive reflexes.

Sex differences in the expression of serotonin-synthesizing enzymes in mouse trigeminal ganglia.

Migraine headaches are more prevalent in women and often occur during the early phases of the menstrual cycle, implying a link between migraine and ovarian steroids. Serotonin (5-HT) and its receptors have been proposed to play a key role in the pathophysiology of migraine. The trigeminal ganglion (TG) has been proposed as a site for 5-HT synthesis based on the expression of the rate limiting enzyme in peripheral 5-HT synthesis, tryptophan hydroxylase 1 (TPH1), in female rodent trigeminal ganglia. Tryptophan hydroxylase levels vary over the estrus cycle, however, the expression and potential regulation of other enzymes involved in 5-HT synthesis has not been reported in this tissue. C57/BL6 mice of both sexes expressed TPH1 and aromatic amino acid decarboxylase (AADC), the key enzymes involved in 5-HT synthesis. Levels of both enzymes were significantly higher in juvenile males compared with females. In naturally cycling females TPH1 and AADC expression was highest during proestrus when compared with the other phases of the cycle, and this regulation was mirrored at the mRNA level. In situ hybridization experiments detected TPH1 and AADC mRNA in presumptive neurons in the trigeminal ganglion. Both key enzymes involved in the synthesis of 5-HT are expressed in mouse trigeminal ganglion and are localized to neurons. The levels of these enzymes are dependent on gender and estrus cycle stage, suggesting that ovarian steroids might play a role in the regulation of sensory neuron 5-HT synthesis.

Intrathecal neuromedin U induces biphasic effects on sympathetic vasomotor tone, increases respiratory drive and attenuates sympathetic reflexes in rat.

BACKGROUND: Neuromedin U (NMU) is a brain-gut peptide that plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow, pain and adrenocortical function via two receptors, the NMU receptor 1 and NMU receptor 2. NMU has several known functions in the periphery, but its role in central cardiorespiratory regulation remains poorly understood. EXPERIMENTAL APPROACH: Experiments were conducted on urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats (n= 42) to determine if NMU modulates sympathetic vasomotor output at the spinal level or modulates baro-, chemo- and somato-sympathetic reflexes. KEY RESULTS: Intrathecal (i.t.) injections of NMU (2.5-20 nmol) caused a dose-dependent biphasic response, initially a brief period of hypertension and sympatho-excitation followed by prolonged hypotension and sympatho-inhibition. Peak excitatory as well as inhibitory responses were observed at 20 nmol. NMU (20 nmol) initially increased mean arterial pressure and splanchnic sympathetic nerve activity by 24 mmHg and 27% and then reduced these by 37 mmHg and 47%, respectively. NMU also dose-dependently increased respiratory drive, as indicated by a rise in phrenic nerve amplitude, an increase in neural minute ventilation and a shortening of the inspiratory period. Both sympatho-excitatory peaks of the somato-sympathetic reflex were abolished by i.t. NMU. Pressor, sympatho-excitatory and tachycardiac responses to chemoreceptor activation (100% N2) were blocked or significantly reduced following i.t. NMU. NMU also reduced barosensitivity. CONCLUSIONS: The data demonstrate that NMU, acting in the spinal cord, differentially contributes to the control of sympathetic tone and adaptive sympathetic reflexes.

Intrathecal orexin A increases sympathetic outflow and respiratory drive, enhances baroreflex sensitivity and blocks the somato-sympathetic reflex.

BACKGROUND: Intrathecal (i.t.) injection of orexin A (OX-A) increases blood pressure and heart rate (HR), but the effects of OX-A on sympathetic and phrenic, nerve activity, and the baroreflex(es), somato-sympathetic and hypoxic chemoreflex(es) are unknown. EXPERIMENTAL APPROACH: Urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats were examined in this study. The effects of i.t. OX-A (20 nmol 10 µL⁻¹) on cardiorespiratory parameters, and responses to stimulation of the sciatic nerve (electrical), arterial baroreceptors (phenylephrine hydrochloride, 0.01 mg kg⁻¹ i.v.) and peripheral (hypoxia) chemoreceptors were also investigated. KEY RESULTS: i.t. OX-A caused a prolonged dose-dependent sympathoexcitation, pressor response and tachycardia. The peak effect was observed at 20 nmol with increases in mean arterial pressure, HR and splanchnic sympathetic nerve activity (sSNA) of 32 mmHg, 52 beats per minute and 100% from baseline respectively. OX-A also dose-dependently increased respiratory drive, as indicated by a rise in phrenic nerve amplitude and a fall in phrenic nerve frequency, an increase in neural minute ventilation, a lengthening of the expiratory period, and a shortening of the inspiratory period. All effects of OX-A (20 nmol) were attenuated by the orexin receptor 1 antagonist SB 334867. OX-A significantly reduced both sympathoexcitatory peaks of somato-sympathetic reflex while increasing baroreflex sensitivity. OX-A increased the amplitude of the pressor response and markedly amplified the effect of hypoxia on sSNA. CONCLUSIONS: Thus, activation of OX receptors in rat spinal cord alters cardiorespiratory function and differentially modulates sympathetic reflexes.

Somatostatin selectively ablates post-inspiratory activity after injection into the Bötzinger complex.

Somatostatin (SST) neurons in the ventral respiratory column (VRC) are essential for the generation of normal breathing. Little is known about the neuromodulatory role of SST on ventral respiratory neurons other than that local administration induces apnoea. Here, we describe the cardiorespiratory effects of microinjecting SST into the preBötzinger and Bötzinger complexes which together elaborate a normal inspiratory augmenting and expiratory respiratory pattern, and on spinally projecting respiratory subnuclei (rostral ventral respiratory group; rVRG). Microinjections (20-50 nl) of SST (0.15, 0.45, 1.5 mM) were made into respiratory subnuclei of urethane-anaesthetized, paralysed, vagotomized and artificially ventilated Sprague-Dawley rats (n=46). Unilateral microinjection of SST into the Bötzinger complex converted the augmenting activity of phrenic nerve discharge into a square-wave apneustic pattern associated with a lengthening of inspiratory period and shortening of expiratory time. Following bilateral microinjection the apneusis became pronounced and was associated with a dramatic variability in inspiratory duration. Microinjection of SST into the Bötzinger complex also abolished the post-inspiratory (post-I) motor activity normally observed in vagal and sympathetic nerves. In the preBötzinger complex SST caused bradypnoea and with increasing dose, apnoea. In the rVRG SST reduced phrenic nerve amplitude, eventually causing apnoea. In conclusion, SST powerfully inhibits respiratory neurons throughout the VRC. Of particular interest is the finding that chemical inhibition of the Bötzinger complex with SST ablates the post-I activity that is normally seen in respiratory activity and leads to apneusis. This loss of post-I activity is a unique feature of inhibition with SST and is not seen following inhibition with other agents such as galanin, GABA and endomorphin. The effect seen on post-I activity is similar to the effect of inhibiting the Kölliker-Fuse nucleus in the pons. The mechanism by which SST exerts this effect on Bötzinger neurons remains to be determined.

Pre-protachykinin A mRNA is colocalized with tyrosine hydroxylase-immunoreactivity in bulbospinal neurons.

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

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity identifies a novel subpopulation of sympathetic preganglionic neurons.

Distinct chemical codes are thought to reflect functional specificity in sympathetic preganglionic neurons (SPN). Although a number of chemical candidates have been identified including neurotransmitter-related, calcium-binding and other proteins, signal transduction proteins have been largely neglected. Not only might these chemicals allow discrimination of functionally unique chemical signatures, but they may also identify activated neurons. Immunoreactivity (ir) to phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was differentially located within the thoracic spinal cord depending upon which of three forms of killing was used: the only exception to this was the intermediolateral cell column (IML) which was consistently, densely labeled. The presence or absence of p-ERK1/2 in SPN (n=17,541) within the IML of the thoraco-lumbar spinal cord was determined in seven rats. SPN were identified on the basis of their location, size and that they contained choline acetyltransferase ir. On average, 58% of SPN contained p-ERK1/2, however, more SPN in both the upper (72%; C8-T4) and lower (78%; T11-L3) thoraco-lumbar spinal cord contained p-ERK1/2-ir than the middle thoracic region (47%; T4-T10). p-ERK1/2-ir was also examined in SPN (n=1895) innervating the adrenal medulla (identified by retrograde tracing using cholera toxin B subunit) combined with localization of neuronal nitric oxide synthase (nNOS) in three rats. On average, 64% of adrenal SPN contain p-ERK1/2-ir, and it was confirmed that all adrenal SPN contain nNOS-ir. It appears that p-ERK1/2-ir SPN, described in this study, have tonically activated receptors that are coupled to intracellular signal transduction pathways that lead to the phosphorylation of ERK1/2.

Presynaptic delta opioid receptors differentially modulate rhythm and pattern generation in the ventral respiratory group of the rat.

The specific role of the Delta opioid receptor (DOR), in opioid-induced respiratory depression in the ventral respiratory group (VRG) is largely unknown. Here, we sought to determine (1) the relationship between DOR-immunoreactive (ir) boutons, bulbospinal and functionally identified respiratory neurons in the VRG and (2) the effects of microinjection of the selective DOR agonist, D-Pen 2,5-enkephalin (DPDPE), into different subdivisions of the VRG, on phrenic nerve discharge and mean arterial pressure. Following injections of retrograde tracer into the spinal cord or intracellular labelling of respiratory neurons, in Sprague-Dawley rats, brainstem sections were processed for retrograde or intracellular labelling and DOR-ir. Bulbospinal neurons were apposed by DOR-ir boutons regardless of whether they projected to single (cervical or thoracic ventral horn) or multiple (cervical and thoracic ventral horn) targets in the spinal cord. In the VRG, a total of 24 +/- 5% (67 +/- 13/223 +/- 49) of neurons projecting to the cervical ventral horn, and 37 +/- 3% (96 +/- 22/255 +/- 37) of neurons projecting to the thoracic ventral horn, received close appositions from DOR-ir boutons. Furthermore, DOR-ir boutons closely apposed six of seven intracellularly labelled neurons, whilst the remaining neuron itself possessed boutons that were DOR-ir. DPDPE was microinjected (10 mM, 60 nl, unilateral) into regions of respiratory field activity in the VRG of anaesthetised, vagotomised rats, and the effects on phrenic nerve discharge and mean arterial pressure were recorded. DPDPE depressed phrenic nerve amplitude, with little effect on phrenic nerve frequency in the Bötzinger complex, pre-Bötzinger complex and rVRG, the greatest effects occurring in the Bötzinger complex. The results indicate that the DOR is located on afferent inputs to respiratory neurons in the VRG. Activation of the DOR in the VRG is likely to inhibit the release of neurotransmitters from afferent inputs that modulate the pattern of activity of VRG neurons.

Maintenance of sympathetic tone by a nickel chloride-sensitive mechanism in the rostral ventrolateral medulla of the adult rat.

In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection of the divalent cation nickel chloride (Ni(2+); 50 mM, 50 nl) into the rostral ventrolateral medulla elicited a dramatic inhibition of splanchnic sympathetic nerve activity (-44+/-6%) and a marked depressor response (-35+/-7 mmHg). Selective blockade of high-voltage activated Ca(2+) channels with omega-agatoxin IVA (P/Q-type), omega-conotoxin GVIA (N-type) and nifedipine (L-type) did not decrease arterial pressure or splanchnic sympathetic nerve activity when injected separately into the rostral ventrolateral medulla, or combined with kynurenate. Injection of caesium chloride or ZD 7288, a blocker of the hyperpolarization-activated cation current, into the rostral ventrolateral medulla had no effect on arterial pressure or splanchnic sympathetic nerve activity. Bilateral microinjection of nickel chloride into the caudal ventrolateral medulla/pre-Bötzinger complex elicited small increases in splanchnic sympathetic nerve activity (+17+/-13%) and arterial pressure (+12+/-4 mmHg). These were substantially smaller than those evoked by blockade of glutamatergic receptors or high-voltage activated Ca(2+) channels in this area. Injection of kynurenate or high-voltage activated Ca(2+) channel blocker, but not Ni(2+), in this area evoked respiratory termination. The results indicate the existence of a distinct mechanism maintaining the tonic activity of rostral ventrolateral medulla presympathetic neurons that is different from that maintaining the tonic activity in the caudal ventrolateral medulla/pre-Bötzinger region. We conclude that ion channels that are sensitive to Ni(2+), but are insensitive to high-voltage activated (L, P/Q, N) Ca(2+) channel blockers, and are located postsynaptically on the presympathetic rostral ventrolateral medulla neurons are responsible for the tonic activity of the presympathetic neurons in rostral ventrolateral medulla. These channels could well be the low-voltage-activated (or T-type) Ca(2+) channels although other conductances cannot be conclusively excluded.

Activation of mu-opioid receptors in rat ventrolateral medulla selectively blocks baroreceptor reflexes while activation of delta opioid receptors blocks somato-sympathetic reflexes.

The effects of activation of mu and delta-opioid receptors in the rostral ventrolateral medulla (RVLM) on somato-sympathetic, baroreceptor and chemoreceptor reflexes, as well as respiratory rhythmicity in sympathetic nerves, were examined in urethane anaesthetized (1.1-1.2 g/kg) and artificially ventilated Sprague-Dawley rats. Microinjection of the delta-opioid receptor agonist [D-Pen(2,5)]-enkephalin (DPDPE; 8 mM, 50 nl) bilaterally into the RVLM potently inhibited the post-inspiratory-related burst discharges of lumbar sympathetic nerve activity (LSNA) but had only limited effects on splanchnic sympathetic nerve activity (SSNA) and phrenic nerve discharge. Injection of DPDPE into the RVLM strongly attenuated the somato-sympathetic reflex (approximately 50-80%) evoked in the lumbar sympathetic nerve and splanchnic sympathetic nerve by tibial nerve stimulation but had no effect on baroreceptor reflexes and chemoreceptor reflexes evoked by aortic nerve stimulation and brief hypoxia, respectively. Injection of the mu-opioid receptor agonist, [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO; 4 mM, 50 nl), also elicited a greater inhibition of LSNA than SSNA accompanied by an abolition of phrenic nerve discharge. Injection of DAMGO inhibited the baroreceptor reflex without significant effect on either the somato-sympathetic or the chemoreceptor reflexes. We propose that opioid peptides diminish specific excitatory and inhibitory inputs to the presympathetic neurons in RVLM via distinct presynaptic receptor subclasses.

NK1 receptor and the ventral medulla of the rat: bulbospinal and catecholaminergic neurons.

Bulbospinal C1 neurons are sympathoexcitatory and excited by substance P. However the substance P receptor (NK1 receptor), has been reported to be absent from the somata of C1 neurons. In this study, using double and triple labelling immunofluorescence and retrograde tracing, we provide evidence that the NK1 receptor is present on 5.3% of C1 neurons, and that 4.7% of C1 neurons receive close oppositions from NK1 receptor immunoreactive terminals, indicating a pre-synaptic and post-synaptic site for the action of substance P. These results provide support for the sympathoexcitatory actions of substance P on C1 neurons. We also demonstrate the NK1 receptor on bulbospinal neurons of the ventral respiratory group, in a region overlapping the pre-Bötzinger Complex.

Differential role of kinases in brain stem of hypertensive and normotensive rats.

Spontaneously hypertensive rats (SHR) are characterized by extreme elevations of blood pressure. The genetic factors underlying this are yet to be identified. Here we demonstrate, in vivo, that in SHR and normotensive Wistar-Kyoto rats (WKY), injection of the mitogen-activated protein kinase inhibitor PD 098,059 bilaterally into the rostral ventrolateral medulla (RVLM) dramatically lowers arterial pressure. PD 098,059 does not alter the responses evoked by microinjection of glutamate into the RVLM or brief apnea. Wortmannin (phosphatidylinositol-3 kinase inhibitor) bilaterally into the RVLM causes a 35+/-4% fall in arterial pressure in SHR but has no effect in WKY. Furthermore, wortmannin reduces the pressor response evoked by microinjection of angiotensin (Ang) II in the RVLM of SHR compared with WKY. The response to Ang II microinjection into the RVLM of WKY was unaffected by wortmannin. Simultaneous bilateral injections of PD 098,059 and wortmannin into the RVLM abolished the response to exogenous Ang II in the RVLM but did not affect the response evoked by glutamate in either SHR or WKY. Thus, it appears that PD 098,059- and/or wortmannin-sensitive mechanisms are not involved in the responses evoked by glutamate in the RVLM and that these kinase inhibitors are not neurotoxic. We conclude that a PD 098,059-sensitive pathway in the RVLM of SHR and WKY tonically regulates arterial pressure and that a wortmannin-sensitive pathway in the RVLM is important in the maintenance of hypertension in SHR. This may be related to a phosphatidylinositol-3 kinase-dependent mechanism involved in the action of Ang II on the Ang II type 1 receptor.

Differential expression of catecholamine synthetic enzymes in the caudal ventral pons.

The analysis of colocalization of multiple catecholamine biosynthetic enzymes within the ventrolateral part of the medulla oblongata of the rat revealed distinct subpopulations of neurons within the C1 region (Phillips et al., J Comp Neurol 2001, 432:20-34). In extending this study to include the caudal pons, it was shown for the first time that the A5 cell group could be distinguished by the presence of immunoreactivity to tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and dopamine beta hydroxylase (DBH). A novel cell group was also identified. The cells within this new group were immunoreactive to DBH but not TH, AADC, or phenylethanolamine N-methyltransferase (PNMT) and will be referred to as the TH-, DBH+ cell group. The TH-, DBH+ neurons were not immunoreactive for either the dopamine or noradrenaline transporters, suggesting that these neurons do not take up these transmitters. A5 neurons were immunoreactive for the noradrenaline transporter but not the dopamine transporter (as previously shown). Retrograde tracing with cholera toxin B revealed that the TH-, DBH+ neurons do not project to the thoracic spinal cord or to the rostral ventrolateral medulla, but A5 neurons do. A calbindin immunoreactive cell group is located in a region overlapping TH-, DBH+ cell group. However, only a few neurons were immunoreactive for both markers. The physiological role of the TH-, DBH+ cell group remains to be determined.

Firing patterns of pre-Bötzinger and Bötzinger neurons during hypocapnia in the adult rat.

Controversy exists about how a coordinated respiratory rhythm is generated in the brainstem. Some authors suggest that neurons in the pre-Bötzinger complex are key to initiation of all types of breathing. While, on the other hand, it has been reported that some pre-Bötzinger neurons fail to maintain a rhythmic discharge in phase with phrenic nerve discharge during mechanical hyperventilation. Extracellular recordings were made from respiratory units in the pre-Bötzinger and Bötzinger complexes of 13 anaesthetised, paralysed and vagotomised rats. Central respiratory activity was monitored from the C5 phrenic nerve. During mechanical hyperventilation, several changes were observed in the phrenic neurogram. Firstly, the frequency and amplitude of integrated phrenic nerve discharge were reduced and reversibly stopped. Secondly, the patterned discharges changed from an augmenting to a variety of non-augmenting patterns in 53 of 60 cases. In some cases (n=9) we observed that the pattern appeared to have two components, an early short duration discharge followed by a longer duration discharge. Respiratory units also started to show different firing patterns during mechanical hyperventilation. In general, they were divided into those units that fired tonically (n=28) and units that became silent (n=32), before phrenic nerve discharge ceased coincidently with complete apnoea. Of particular interest were those expiratory-inspiratory units in the pre-Bötzinger complex (n=8) that narrowed their firing period towards late expiration and early inspiration during mechanical hyperventilation. Given their firing features, it is possible that these expiratory-inspiratory units may participate in generation of the early inspiratory component of phrenic nerve discharge.

Rostral ventral medulla 5-HT1A receptors selectively inhibit the somatosympathetic reflex.

The role of the 5-hydroxytryptamine (5-HT1A) receptors in the rostral ventrolateral medulla (RVLM) on somatosympathetic, baroreceptor, and chemoreceptor reflexes was examined in anesthetized rats. Microinjection of the selective 5-HT1A agonist 8-hydroxy-di-n-propylamino tetralin (8-OH-DPAT) decreased arterial blood pressure and splanchnic sympathetic nerve activity (SNA). Electrical stimulation of the hindlimb evoked early and late excitatory sympathetic responses. Bilateral microinjection in the RVLM of 8-OH-DPAT markedly attenuated both the early and late responses. This potent inhibition of the somatosympathetic reflex persisted even after SNA and arterial blood pressure returned to preinjection levels. Preinjection of the selective 5-HT1A antagonist NAN-190 in the RVLM blocked the sympathoinhibitory effect of 8-OH-DPAT and attenuated the inhibitory effect on the somatosympathetic reflex. 8-OH-DPAT injected in the RVLM did not affect baroreceptor or chemoreceptor reflexes. Our findings suggest that activation of 5-HT1A receptors in the RVLM exerts a potent, selective inhibition on the somatosympathetic reflex.

Differential expression of catecholamine biosynthetic enzymes in the rat ventrolateral medulla.

Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine beta-hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis.