Uraemia: an unrecognized driver of central neurohumoral dysfunction in chronic kidney disease?

Chronic kidney disease (CKD) carries a large cardiovascular burden in part due to hypertension and neurohumoral dysfunction - manifesting as sympathetic overactivity, baroreflex dysfunction and chronically elevated circulating vasopressin. Alterations within the central nervous system (CNS) are necessary for the expression of neurohumoral dysfunction in CKD; however, the underlying mechanisms are poorly defined. Uraemic toxins are a diverse group of compounds that accumulate as a direct result of renal disease and drive dysfunction in multiple organs, including the brain. Intensive haemodialysis improves both sympathetic overactivity and cardiac baroreflex sensitivity in renal failure patients, indicating that uraemic toxins participate in the maintenance of autonomic dysfunction in CKD. In rodents exposed to uraemia, immediate early gene expression analysis suggests upregulated activity of not only pre-sympathetic but also vasopressin-secretory nuclei. We outline several potential mechanisms by which uraemia might drive neurohumoral dysfunction in CKD. These include superoxide-dependent effects on neural activity, depletion of nitric oxide and induction of low-grade systemic inflammation. Recent evidence has highlighted superoxide production as an intermediate for the depolarizing effect of some uraemic toxins on neuronal cells. We provide preliminary data indicating augmented superoxide production within the hypothalamic paraventricular nucleus in the Lewis polycystic kidney rat, which might be important for mediating the neurohumoral dysfunction exhibited in this CKD model. We speculate that the uraemic state might serve to sensitize the central actions of other sympathoexcitatory factors, including renal afferent nerve inputs to the CNS and angiotensin II, by way of recruiting convergent superoxide-dependent and pro-inflammatory pathways.

Chronic Methamphetamine Self-Administration Dysregulates Oxytocin Plasma Levels and Oxytocin Receptor Fibre Density in the Nucleus Accumbens Core and Subthalamic Nucleus of the Rat.

The neuropeptide oxytocin attenuates reward and abuse for the psychostimulant methamphetamine (METH). Recent findings have implicated the nucleus accumbens (NAc) core and subthalamic nucleus (STh) in oxytocin modulation of acute METH reward and relapse to METH-seeking behaviour. Surprisingly, the oxytocin receptor (OTR) is only modestly involved in both regions in oxytocin attenuation of METH-primed reinstatement. Coupled with the limited investigation of the role of the OTR in psychostimulant-induced behaviours, we primarily investigated whether there are cellular changes to the OTR in the NAc core and STh, as well as changes to oxytocin plasma levels, after chronic METH i.v. self-administration (IVSA) and after extinction of drug-taking. An additional aim was to examine whether changes to central corticotrophin-releasing factor (CRF) and plasma corticosterone levels were also apparent because of the interaction of oxytocin with stress-regulatory mechanisms. Male Sprague-Dawley rats were trained to lever press for i.v. METH (0.1 mg/kg/infusion) under a fixed-ratio 1 schedule or received yoked saline infusions during 2-h sessions for 20 days. An additional cohort of rats underwent behavioural extinction for 15 days after METH IVSA. Subsequent to the last day of IVSA or extinction, blood plasma was collected for enzyme immunoassay, and immunofluorescence was conducted on NAc core and STh coronal sections. Rats that self-administered METH had higher oxytocin plasma levels, and decreased OTR-immunoreactive (-IR) fibres in the NAc core than yoked controls. In animals that self-administered METH and underwent extinction, oxytocin plasma levels remained elevated, OTR-IR fibre density increased in the STh, and a trend towards normalisation of OTR-IR fibre density was evident in the NAc core. CRF-IR fibre density in both brain regions and corticosterone plasma levels did not change across treatment groups. These findings demonstrate that oxytocin systems, both centrally within the NAc core and STh, as well as peripherally through plasma measures, are dysregulated after METH abuse.

Comparison of noradrenaline, dopamine and serotonin in mediating the tachycardic and thermogenic effects of methamphetamine in the ventral medial prefrontal cortex.

Methamphetamine (METH) is a psychostimulant that disrupts monoaminergic neurotransmission to evoke profound behavioral and physiological effects. Rapidly distributing to forebrain regions to increase synaptic concentrations of three monoamines (dopamine (DA), serotonin (5-HT) and noradrenaline (NA)), the medial prefrontal cortex (mPFC) is important in METH-altered behavioral and psychological profiles. Activation of the ventral mPFC can modify physiological variables, however, METH-evoked autonomic changes from this region are unknown. Therefore, the aim of this study was to characterize the respiratory, metabolic and cardiovascular effects of microinjection of METH, DA, 5-HT and NA into the ventral mPFC in urethane-anesthetized Sprague-Dawley rats. METH and NA microinjection evoked dose-related increases in heart rate, interscapular brown adipose tissue temperature and expired CO2, a pattern of response characteristic of non-shivering thermogenesis. NA and 5-HT microinjection elicited pressor and depressor responses, respectively, with matching baroreflex adjustments in sympathetic nerve activity while METH and DA evoked no change in vasomotor outflow. Low doses of METH and DA may evoke respiratory depression. These data suggest that METH's actions in the ventral mPFC, likely via adrenergic receptors, evoke non-shivering thermogenesis which may contribute to the increased body temperature and tachycardia seen in those that abuse METH.

Inhibition of protein kinase A activity depresses phrenic drive and glycinergic signalling, but not rhythmogenesis in anaesthetized rat.

The cAMP-protein kinase A (PKA) pathway plays a critical role in regulating neuronal activity. Yet, how PKA signalling shapes the population activity of neurons that regulate respiratory rhythm and motor patterns in vivo is poorly defined. We determined the respiratory effects of focally inhibiting endogenous PKA activity in defined classes of respiratory neurons in the ventrolateral medulla and spinal cord by microinjection of the membrane-permeable PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) in urethane-anaesthetized adult Sprague Dawley rats. Phrenic nerve activity, end-tidal CO2 and arterial pressure were recorded. Rp-cAMPS in the preBötzinger complex (preBötC) caused powerful, dose-dependent depression of phrenic burst amplitude and inspiratory period. Rp-cAMPS powerfully depressed burst amplitude in the phrenic premotor nucleus, but had no effect at the phrenic motor nucleus, suggesting a lack of persistent PKA activity here. Surprisingly, inhibition of PKA activity in the preBötC increased phrenic burst frequency, whereas in the Bötzinger complex phrenic frequency decreased. Pretreating the preBötC with strychnine, but not bicuculline, blocked the Rp-cAMPS-evoked increase in frequency, but not the depression of phrenic burst amplitude. We conclude that endogenous PKA activity in excitatory inspiratory preBötzinger neurons and phrenic premotor neurons, but not motor neurons, regulates network inspiratory drive currents that underpin the intensity of phrenic nerve discharge. We show that inhibition of PKA activity reduces tonic glycinergic transmission that normally restrains the frequency of rhythmic respiratory activity. Finally, we suggest that the maintenance of the respiratory rhythm in vivo is not dependent on endogenous cAMP-PKA signalling.

Distribution and localisation of Gα proteins in the rostral ventrolateral medulla of normotensive and hypertensive rats: focus on catecholaminergic neurons.

About 860 G-protein-coupled receptors (GPCRs) mediate their actions via heterotrimeric G-proteins. Their activation releases Gα from Gßλ subunits. The type of Gα subunit dictates the major signalling proteins involved: adenylyl cyclase, PLC and rhoGEF. The rostral ventrolateral medulla (RVLM), containing the rostral C1 (rC1) cell group, sets and maintains the tonic and reflex control of blood pressure and a plethora of inputs converge onto these neurons. We determined the relative abundance of 10 Gα subunit mRNAs, representing the four major families, within the RVLM, using quantitative RT-PCR. In situ hybridisation (ISH) combined with immunohistochemistry (IHC) was used to quantify and compare this expression in rC1 with that in the A1 and A5 cell groups. The relative abundance of Gα subunit mRNAs and a comparison of gene expression levels were quantitatively determined in normotensive and hypertensive rat strains. All 10 Gα mRNAs were detected in the RVLM of Sprague-Dawley (SD) rats with relative abundance such that Gαs>Gαi2>Gαo>Gαq>GαL>Gα11>Gαi3>Gαi1>Gα12>Gα13. The high abundance of Gα mRNAs signalling via adenylyl cyclase indicates the importance of associated GPCRs. Within the rC1 and A1 groups similar differential Gα mRNA expression profiles were seen with Gαs being found in all rC1 cells, Gα11 absent and Gαi3 rarely expressed. Thus functionally distinct subgroups exist within the rC1 and A1 cell groups as differing distributions of Gα subunits must reflect the array of GPCRs that influence their activity. In contrast, all A5 cells expressed all Gα mRNAs suggesting a functionally homogeneous group. When the 10 Gα mRNAs of the RVLM in spontaneously hypertensive rats (SHR) were compared quantitatively to Wistar-Kyoto (WKY), only Gαs and Gα12 were significantly elevated. However when the expression in normotensive SD and WKY was compared with SHR no significant differences were evident. These findings demonstrate a range of GPCR signalling capabilities in brainstem neurons important for homeostasis and suggest a prominent role for signalling via adenylyl cyclase.

Neuropeptide coding of sympathetic preganglionic neurons; focus on adrenally projecting populations.

Chemical coding of sympathetic preganglionic neurons (SPN) suggests that the chemical content of subpopulations of SPN can define their function. Since neuropeptides, once synthesized are transported to the axon terminal, most demonstrated chemical coding has been identified using immunoreactive terminals at the target organ. Here, we use a different approach to identify and quantify the subpopulations of SPN that contain the mRNA for pituitary adenylate cyclase activating polypeptide (PACAP) or enkephalin. Using double-labeled immunohistochemistry combined with in situ hybridization (ISH) we firstly identified the distribution of these mRNAs in the spinal cord and determined quantitatively, in Sprague-Dawley rats, that many SPN at the T4-T10 spinal level contain preproPACAP (PPP+, 80 ± 3%, n=3), whereas a very small percentage contain preproenkephalin (PPE+, 4 ± 2%, n=4). A similar neurochemical distribution was found at C8-T3 spinal level. These data suggest that PACAP potentially regulates a large number of functions dictated by SPN whereas enkephalins are involved in few functions. We extended the study to explore those SPN that control adrenal chromaffin cells. We found 97 ± 5% of adrenally projecting SPN (AP-SPN) to be PPP+ (n=4) with only 47 ± 3% that were PPE+ (n=5). These data indicate that adrenally projecting PACAPergic SPN regulate both adrenal adrenaline (Ad) and noradrenaline (NAd) release whereas the enkephalinergic SPN subpopulation must control a (sub) population of chromaffin cells - most likely those that release Ad. The sensory innervation of the adrenal gland was also determined. Of the few adrenally projecting dorsal root ganglia (AP-DRG) observed, 74 ± 12% were PPP+ (n=3), whereas 1 ± 1% were PPE+ (n=3). Therefore, if sensory neurons release peptides to the adrenal medulla, PACAP is most likely involved. Together, these data provide a neurochemical basis for differential control of sympathetic outflow particularly that to the adrenal medulla.

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.

Hypercapnia selectively attenuates the somato-sympathetic reflex.

The effects of hyperoxic hypercapnia (5, 10 or 15% CO2 in O2) on splanchnic sympathetic nerve activity (sSNA) and sympathetic reflexes such as the somato-sympathetic reflex or baroreflex were studied in urethane anaesthetised, paralysed, artificially ventilated and vagotomized Sprague-Dawley rats. Hypercapnia caused a small increase in mean arterial blood pressure (MAP) in the 10% CO2 group and a fall in heart rate (HR) in all three groups. sSNA increased in all three groups. Phrenic frequency and amplitude increased during hypercapnia, with frequency adapting back towards baseline during the CO2 exposure. The somato-sympathetic reflex was attenuated in the 5% CO2 group and abolished in the 10 and 15% CO2 groups, whereas there was little effect on the sSNA baroreflex. Hypercapnia significantly affects phrenic nerve activity (PNA), sSNA and selectively inhibits the somato-sympathetic reflex with little effect on the sSNA baroreflex.

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.

Distribution of calbindin, parvalbumin, and calretinin immunoreactivity in the reticular thalamic nucleus of the marmoset: evidence for a medial leaflet of incertal neurons.

The placement of the reticular thalamic nucleus (RTN) between the dorsal thalamus and the cortex and the inhibitory nature of reticulothalamic projections has led to suggestions that it "gates" the flow of sensory information to the cortex. The New World diurnal monkey, the marmoset, Callithrix jacchus is emerging as an important "model primate" for the study of sensory processing. We have examined the distribution of Nissl-stained somata and calbindin, parvalbumin, and calretinin immunoreactivity in the ventral thalamus for comparison with other species. Cells were labeled using standard immunohistochemistry, ExtraAvidin-HRP, and diaminobenzidine reaction products. The RTN is constituted by a largely homogeneous population of parvalbumin immunoreactive cells with respect to size and orientation. Calbindin and calretinin immunoreactive cells were only found along the medial edge of the RTN adjacent to the external medullary lamina of the dorsal thalamus and laterally near the ventral RTN. These cells were considered to be part of the zona incerta (ZI). The marmoset ZI could be subdivided into dorsal and ventral regions on the basis of its immunoreactivity to calcium binding proteins. Both the ZI and nucleus subthalamicus Luysi contained scattered calbindin and calretinin immunoreactive cells with well-defined dendritic processes. These cells were clearly different to cells in the dorsal thalamus. Parvalbumin immunoreactive cells in RTN, ZI, and subthalamic nucleus were on average larger than neurons positive for the other calcium binding proteins. Future studies reporting the afferent and efferent projections to the RTN must view their results in terms of the close apposition of RTN and ZI somata.