Parabrachial nucleus induces suppression of baroreflex bradycardia by the release of glutamate in the rostral ventrolateral medulla of the rat

The involvement of glutamatergic neurotransmission in the rostral ventrolateral medulla (RVLM) in the suppression of baroreflex bradycardia by the parabrachial nucleus (PBN) was investigated. Repeated electrical activation of the PBN increased the concentration of glutamate in the dialysate collected from the RVLM. The same stimulation also suppressed baroreflex bradycardia in response to transient hypertension evoked by phenylephrine (5 microg/kg, intravenously). Microinfusion of L-glutamate (10, 50 or 100 microM) via the microdialysis probe into the RVLM dose-dependently elicited a significant inhibition of baroreflex bradycardia that paralleled the concentration and time course of the PBN-elicited elevation in extracellular glutamate in the RVLM. The suppression of baroreflex bradycardia elicited by microinjection of L-glutamate (1 nmol) into the RVLM was appreciably reversed by coinjection of the NMDA receptor antagonist, dizocilpine (500 pmol), or the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (50 pmol). These results suggest that an increase in the extracellular concentration of glutamate and activation of both NMDA and non-NMDA receptors in the RVLM may mediate the suppression of baroreflex bradycardia by activation of the PBN.     

Parvalbumin in respiratory neurons of the ventrolateral medulla of the adult rat

A column of parvalbumin immunoreactive neurons is closely associated with the location of respiratory neurons in the ventrolateral medulla of the rat. The majority (66%) of bulbospinal neurons in the medullary ventral respiratory column (VRC) that were retrogradely labeled by tracer injections in the phrenic nucleus were also positive for parvalbumin. In contrast, only 18.8% of VRC neurons retrogradely labeled after a tracer injection in the VRC, also expressed parvalbumin. The average cross-sectional area of VRC neurons retrogradely labeled after VRC injections was 193.8 μm² ± 6.6 SE. These were significantly smaller than VRC parvalbumin neurons (271.9 μm² ± 12.3 SE). Parvalbumin neurons were found in the Bötzinger Complex, the rostral ventral respiratory group (VRG), and the caudal VRG, areas which all contribute to the bulbospinal projection. In contrast, parvalbumin neurons were sparse or absent in the preBötzinger Complex and in the vicinity of the retrotrapezoid nucleus, areas that have few bulbospinal projections. Parvalbumin was rarely colocalized within Neurokinin-1 receptor positive (NK1R) VRC neurons, which are found in the preBötzinger complex and in the anteroventral part of the rostral VRG. Parvalbumin neurons in the Bötzinger Complex and rostral VRG help define the rostrocaudal extent of these regions. The absence of parvalbumin neurons from the intervening preBötzinger complex also helps establish the boundaries of this region. Regional boundaries described in this manner are in good agreement with earlier physiological and anatomical studies. Taken together, the distributions of parvalbumin, NK1R and bulbospinal neurons suggest that the rostral VRG may be subdivided into distinct, anterodorsal, anteroventral, and posterior subdivisions.     

Immunocytochemical localization of GABA containing neurons in the ventrolateral medulla oblongata of the rat

Summary Localization of -aminobutyric acid (GABA) in the ventrolateral medulla oblongata of the rat was studied, using antisera directed against GABA molecule fixed to bovine serum albumin. Within the rostral portion of the ventrolateral medulla, GABA-like immunoreactive neurons were found in the lateral wing of the raphe magnus and in the region of the paragigantocellular reticular nucleus. In the caudal portion of the ventrolateral medulla, a lesser number of GABA-stained neurons were found in the region around the nucleus reticularis lateralis. GABA-like immunoreactive punctate structures were also found throughout the ventrolateral medulla. These results provide further evidence for the existence of GABAergic neurons in the ventrolateral medulla oblongata of the rat.     

Immunocytochemical localization of GABA containing neurons in the ventrolateral medulla oblongata of the rat

Localization of gamma-aminobutyric acid (GABA) in the ventrolateral medulla oblongata of the rat was studied, using antisera directed against GABA molecule fixed to bovine serum albumin. Within the rostral portion of the ventrolateral medulla, GABA-like immunoreactive neurons were found in the lateral wing of the raphe magnus and in the region of the paragigantocellular reticular nucleus. In the caudal portion of the ventrolateral medulla, a lesser number of GABA-stained neurons were found in the region around the nucleus reticularis lateralis. GABA-like immunoreactive punctate structures were also found throughout the ventrolateral medulla. These results provide further evidence for the existence of GABAergic neurons in the ventrolateral medulla oblongata of the rat.     

Pharmacological profile of depressor response elicited by sarthran in rat ventrolateral medulla

Injection of sarthran, an angiotensin receptor antagonist, bilaterally into the rostral ventrolateral medulla (RVLM) of alpha-chloralose-anesthetized rats decreases arterial pressure (AP) to the same extent as total autonomic blockade. This response is not reproduced by selective AT(1) antagonists. To examine the pharmacological profile of the response elicited by [Sar(1), Thr(8)]ANG II (sarthran), the ability of angiotensin analogs to inhibit the effect of sarthran injected into the RVLM was tested. Coinjection of angiotensin II (ANG II) prevented the sarthran-evoked decrease in AP, but this action of ANG II was markedly attenuated by pretreatment of the RVLM with the aminopeptidase inhibitor amastatin. Coinjection of ANG(3-8) or a selective agonist of AT(4) receptors prevented the effect of sarthran injected into the RVLM. ANG(1-7) was also able to prevent the effect of sarthran. None of the angiotensin fragments tested substantially altered blood pressure when injected alone into the RVLM. These results suggest that the depressor action of sarthran injected into the RVLM is not dependent on ANG II receptors, though the nature of the site or sites of action of sarthran within the RVLM remains uncertain.     

Engagement of ubiquitination and de-ubiquitination at rostral ventrolateral medulla in experimental brain death

Background

Whereas brain death is a vitally important clinical phenomenon, our contemporary understanding on its underlying cellular mechanisms remains elusive. This study evaluated whether the ubiquitin-proteasome system (UPS) in the rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is engaged in this fatal process.

Methods

We performed proteomics, Western Blot, real-time PCR, ELISA and pharmacological experiments in conjunction with a clinically relevant experimental endotoxemia model of brain death based on intravenous administration of Escherichia coli lipopolysaccharide in adult male Sprague–Dawley rats.

Results

Proteomics, Western blot and enzyme activity analyses demonstrated that polyubiquitination was preserved and de-ubiquitination by ubiquitin C-terminal hydrolase isozyme-L1 (UCH-L1) was sustained, alongside increased monoubiquitin availability or proteasome activity in RVLM over the course of experimental endotoxemia. However, real-time PCR revealed no significant alteration in proteasome subunit alpha type-1, ubiquitin or UCH-L1 at mRNA level. Functionally, whereas microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) potentiated survival, an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or an UCH-L1 inhibitor exacerbated mortality.

Conclusions

We proposed previously that the progression towards brain death entails a tug-of-war between pro-death and pro-life programs in RVLM. It is conceivable that ubiquitination or de-ubiquitination in RVLM participate in brain death by regulating the degradation of the proteins involved in those programs.     

Baroreflex modulation by angiotensins at the rat rostral and caudal ventrolateral medulla

We determined the effect of microinjection of ANG-(1-7) and ANG II into two key regions of the medulla that control the circulation [rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively)] on baroreflex control of heart rate (HR) in anesthetized rats. Reflex bradycardia and tachycardia were induced by increases and decreases in mean arterial pressure produced by intravenous phenylephrine and sodium nitroprusside, respectively. The pressor effects of ANG-(1-7) and ANG II (25 pmol) after RVLM microinjection (11 +/- 0.8 and 10 +/- 2 mmHg, respectively) were not accompanied by consistent changes in HR. In addition, RVLM microinjection of these angiotensin peptides did not alter the bradycardic or tachycardic component of the baroreflex. CVLM microinjections of ANG-(1-7) and ANG II produced hypotension (-11 +/- 1.5 and -11 +/- 1.9 mmHg, respectively) that was similarly not accompanied by significant changes in HR. However, CVLM microinjections of angiotensins induced differential changes in the baroreflex control of HR. ANG-(1-7) attenuated the baroreflex bradycardia (0.26 +/- 0.06 ms/mmHg vs. 0.42 +/- 0.08 ms/mmHg before treatment) and facilitated the baroreflex tachycardia (0.86 +/- 0.19 ms/mmHg vs. 0.42 +/- 0.10 ms/mmHg before treatment); ANG II produced the opposite effect, attenuating baroreflex tachycardia (0.09 +/- 0.06 ms/mmHg vs. 0.31 +/- 0.07 ms/mmHg before treatment) and facilitating the baroreflex bradycardia (0.67 +/- 0.16 ms/mmHg vs. 0.41 +/- 0.05 ms/mmHg before treatment). The modulatory effect of ANG II and ANG-(1-7) on baroreflex sensitivity was completely abolished by peripheral administration of methylatropine. These results suggest that ANG II and ANG-(1-7) at the CVLM produce a differential modulation of the baroreflex control of HR, probably through distinct effects on the parasympathetic drive to the heart.     

Neurokinin-1 receptor-expressing cells regulate depressor region of rat ventrolateral medulla

Intraparenchymal injection of the saporin conjugate [Sar9, Met (O2)11] substance P-saporin (SSP-SAP) into the ventrolateral medulla (VLM) destroys neurokinin-1 receptor-immunoreactivity (NK1R-ir) neurons selectively. This treatment attenuates the hypotension caused by injection of DL-homocysteic acid (DLH) into the caudal VLM (CVLM). Here we ask whether SSP-SAP creates this deficit by destroying the CVLM GABAergic interneurons that mediate the sympathetic baroreflex (baroactivated depressor neurons) or by destroying other VLM neurons. Two weeks after unilateral SSP-SAP treatment (97% loss of VLM NK1R-ir neurons) DLH-induced hypotension and sympathetic tone inhibition were blunted on the lesioned side. Unlesioned or unilaterally lesioned rats received phenylephrine (PE) while awake to identify CVLM baroactivated depressor neurons by the presence of Fos-ir nuclei. Although CVLM Fos-ir cells were not NK1R-ir, their number was reduced approximately 60-70% on the SSP-SAP-injected side. SSP-SAP spared VLM neurons devoid of NK1R-ir, such as the catecholaminergic cells and the precerebellar glutamatergic neurons. In the pre-B?tzinger region of the VLM the toxin killed glutamatergic neurons while sparing glycinergic and GABAergic inhibitory neurons. In the CVLM region approximately 26% of the inhibitory cells were destroyed. In conclusion, the baroactivated depressor neurons of the CVLM do not appear to express NK1Rs but their activity is probably modulated by a population of excitatory NK1R-ir cells located in the VLM. The results also suggest that a region located below the CVLM (subCVLM) may contain an unrelated population of GABAergic depressor neurons that are NK1R-ir but are either not barosensitive or do not express Fos during baroreceptor stimulation.     

Sex differences in angiotensin signaling in bulbospinal neurons in the rat rostral ventrolateral medulla

Sex differences may play a significant role in determining the risk of hypertension. Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are involved in the tonic regulation of arterial pressure and participate in the central mechanisms of hypertension. Angiotensin II (ANG II) acting on angiotensin type 1 (AT(1)) receptors in RVLM neurons is implicated in the development of hypertension by activating NADPH oxidase and producing reactive oxygen species (ROS). Therefore, we analyzed RVLM bulbospinal neurons to determine whether there are sex differences in: 1) immunolabeling for AT(1) receptors and the key NADPH oxidase subunit p47 using dual-label immunoelectron microscopy, and 2) the effects of ANG II on ROS production and Ca(2+) currents using, respectively, hydroethidine fluoromicrography and patch-clamping. In tyrosine hydroxylase-positive RVLM neurons, female rats displayed significantly more AT(1) receptor immunoreactivity and less p47 immunoreactivity than male rats (P < 0.05). Although ANG II (100 nM) induced comparable ROS production in dissociated RVLM bulbospinal neurons of female and male rats (P > 0.05), an effect mediated by AT(1) receptors and NADPH oxidase, it triggered significantly larger dihydropyridine-sensitive long-lasting (L-type) Ca(2+) currents in female RVLM neurons (P < 0.05). These observations suggest that an increase in AT(1) receptors in female RVLM neurons is counterbalanced by a reduction in p47 levels, such that ANG II-induced ROS production does not differ between females and males. Since the Ca(2+) current activator Bay K 8644 induced larger Ca(2+) currents in females than in male RVLM neurons, increased ANG II-induced L-type Ca(2+) currents in females may result from sex differences in calcium channel densities or dynamics.     

Age dependence of somatostatin levels and somatostatin binding in the rat brain

1. Somatostatin-like immunoreactivity (SLI) and 125I-Tyrl-somatostatin binding were measured from the brains of rats aged 1, 8 and 18 months. 2. Somatostatin binding was reduced in the striatum, frontal cortex, hypothalamus and hippocampus of the 8-month-old rats compared to the 1-month-old group. 3. Somatostatin binding was reduced in the striatum, frontal cortex and hippocampus of the 18-month-old rats compared to the 1-month-old group. 4. The reduction (40%) was most striking in the frontal cortex. 5. In no area of the brain did changes in SLI differ significantly between the different age groups.     

Effect of moxonidine on putative sympathetic neurons in the rostral ventrolateral medulla of the rat

We used an intracellular recording technique in vitro to investigate the effects of moxonidine on neurons in the rostral ventrolateral medulla (RVLM) with electrophysiological properties similar to premotor sympathetic neurons in vivo. These neurons were classified as firing regularly and irregularly, according to previous reports. Moxonidine is a sympathoinhibitory and antihypertensive agent that is thought to be a ligand of alpha(2)-adrenergic receptors and imidazoline type-1 receptors in the RVLM. Moxonidine (2-10 microM) was applied to the perfusate on 4 irregularly firing neurons, and 2 regularly firing neurons. Moxonidine (2 microM) produced only minor depolarization in 2 of these neurons. However, on 4 tested neurons, moxonidine (10 microM) elicited a profound inhibitory effect with hyperpolarization (near -20 mV); these neurons practically ceased firing. These changes were partially reversible. The results would indicate that neurons in the RVLM, recorded in vitro and with similar electrophysiological characteristics to a group of neurons previously identified in vivo in the same bulbar region as barosensitive premotor sympathetic neurons, can be modulated by imidazoline-derivative adrenergic agonists. These results could help to understand the hypotensive effects of moxonidine.     

Hypoxic excitation in neurons cultured from the rostral ventrolateral medulla of the neonatal rat.

Neurons within cardiorespiratory regions of the rostral ventrolateral medulla (RVLM) have been shown to be excited by local hypoxia. To determine the electrophysiological properties of these excitatory responses to hypoxia, we developed a primary dissociated cell culture system to examine the intrinsic response of RVLM neurons to hypoxia. Neonatal rat neurons plated on medullary astrocyte monolayers were studied using the whole cell perforated patch-clamp technique. Sodium cyanide (NaCN, 0.5-10 mM) was used, and membrane potential (V(m)), firing frequency, and input resistance were examined. In 11 of 19 neurons, NaCN produced a V(m) depolarization, an increase in firing frequency, and a decrease in input resistance, suggesting the opening of a cation channel. The hypoxic depolarization had a linear dose response and was dependent on baseline V(m), with a greater response at more hyperpolarized V(m). In 8 of 19 neurons, NaCN produced a V(m) hyperpolarization, decrease in firing frequency, and variable changes in input resistance. The V(m) hyperpolarization exhibited an all-or-none dose response and was independent of baseline V(m). These differential responses to NaCN were retained after synaptic blockade with low Ca(2+)-high Mg(2+) or TTX. Thus hypoxic excitation 1) is maintained in cell culture, 2) is an intrinsic response, and 3) is likely due to the increase in a cation current. These hypoxia-excited neurons are likely candidates to function as central oxygen sensors.     

Naloxone application to the ventrolateral medulla enhances the respiratory response to inspired carbon dioxide

Previous studies have shown that systemic administration of the opiate antagonist naloxone potentiates the ventilatory response to inspired carbon dioxide. The present study was designed to localize the site of action of naloxone for increasing the respiratory chemosensitivity to inhaled carbon dioxide (CO2) in cats. Naloxone applied topically to the caudal chemosensitive area on the ventral medullary surface (VMS) during hypercapnic breathing produced a 75% greater increase in minute ventilation than hypercapnic breathing alone. Furthermore, hypercapnic breathing produced a 200% increase in neuronal activity of VMS chemosensitive cells; this was further increased 120% by naloxone. It is concluded that naloxone increases the sensitivity of neurons in the caudal respiratory chemosensitive area of cats to hypercapnia, and that endogenous opiates may act as modulators at VMS chemosensitive sites during hypercapnic breathing.     

Inspiratory neuron activity in the ventrolateral medulla of the dog

The purpose of this study was to describe the distribution and activity pattern of respiratory neurons located in the ventrolateral medulla (VLM) of the dog. Spike activity of 129 respiratory neurons was recorded in 23 ketamine-anesthetized spontaneously breathing dogs. Pontamine blue dye was used to mark the location of each neuron. Most VLM neurons displaying respiratory related spike patterns were located in a column related closely to ambigual and retroambigual nuclei. Both inspiratory and expiratory neurons were present with inspiratory units being grouped more rostrally. The predominant inspiratory neuron firing pattern was "late" inspiratory, although eight "early" types were located. All expiratory firing patterns were the late expiratory variety. Each neuron burst pattern was characterized by determining burst duration (BD), spikes per burst (S/B), peak frequency (PF), time to peak frequency (TPF), rate of rise to peak frequency (PF/TPF), and mean frequency. CO2-induced minute ventilation increases were associated with decreases in BD and TPF and increases in PF, S/B, and PF/TPF. In 11 experiments the relative influences of vagotomy and tracheal occlusion on late inspiratory units were compared. Tracheal occlusion increased late inspiratory BD and S/B but did not alter PF/TPF. Vagotomy increased BD and S/B beyond those obtained by tracheal occlusion and, in some neurons, decreased the PF/TPF. We conclude that the location of respiratory units in the VLM of the dog is similar to that in other species, the discharge pattern of VLM respiratory units is similar to those in cat VLM, and vagotomy and tracheal occlusion affect discharge patterns differently.     

Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat

We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration ofEscherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0–0.8 Hz) in systemic arterial pressure signals. LPS also elicited progressive hypotension, and death ensued within 4 h. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochromec reductase (Complexes I + III) and cytochromec oxidase (Complex IV) in the RVLM during all three phases of endotoxemia. On the other hand, the activity of succinate cytochromec reductase (Complexes II + III) remained unaltered. We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat.     

Acute tolerance to ethanol inhibition of NMDA-induced responses in rat rostral ventrolateral medulla neurons

The present study was performed to examine the effects of acute ethanol exposure on N-methyl-D-aspartate (NMDA)-induced responses and the development of acute tolerance in rat rostral ventrolateral medulla (RVLM) in vivo and in vitro. Repeated microinjections of NMDA (0.14 nmol) into the RVLM every 30 min caused reproducible increases in mean arterial pressure in urethane-anesthetized rats weighing 325–350 g. Intravenous injections of ethanol (0.16 or 0.32 g, 1 ml) inhibited NMDA-induced pressor effects in a blood-concentration-dependent and reversible manner. The inhibitory effect of ethanol was reduced over time during continuous infusion of ethanol or on the second injection 3.5 h after prior injection of a higher dose of ethanol (0.32 g). A high dose of ethanol (0.32 g) had no significant effects on-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid,-aminobutyric acid and glycine-induced changes in blood pressure. In vitro studies showed that ethanol (10–100 mM) dose-dependently inhibited inward currents elicited by pressure ejection of NMDA (10 mM) in RVLM neurons of neonatal brainstem slice preparations. When the superfusion time of ethanol (100 mM) was increased to 50 min, its inhibitory effect decreased gradually after 30–40 min in 60% of RVLM neurons examined. These data suggested that ethanol inhibition and subsequent tolerance development is associated with changed sensitivity to NMDA in the RVLM, which may play important roles in the ethanol regulation of cardiovascular function.