Glenn M. Toney

Water deprivation activates a glutamatergic projection from the hypothalamic paraventricular nucleus to the rostral ventrolateral medulla.

Elevated sympathetic outflow contributes to the maintenance of blood pressure in water‐deprived rats. The neural circuitry underlying this response may involve activation of a pathway from the hypothalamic paraventricular nucleus (PVH) to the rostral ventrolateral medulla (RVLM). We sought to determine whether the PVH‐RVLM projection activated by water deprivation is glutamatergic and/or contains vasopressin‐ or oxytocin‐neurophysins. Vesicular glutamate transporter 2 (VGLUT2) mRNA was detected by in situ hybridization in the majority of PVH neurons retrogradely labeled from the ipsilateral RVLM with cholera toxin subunit B (CTB; 85% on average, with regional differences). Very few RVLM‐projecting PVH neurons were immunoreactive for oxytocin‐ or vasopressin‐associated neurophysin. Injection of biotinylated dextran amine (BDA) into the PVH produced clusters of BDA‐positive nerve terminals within the ipsilateral RVLM that were immunoreactive (ir) for the VGLUT2 protein. Some of these terminals made close appositions with tyrosine‐hydroxylase‐ir dendrites (presumptive C1 cells). In water‐deprived rats (n = 4), numerous VGLUT2 mRNA‐positive PVH neurons retrogradely labeled from the ipsilateral RVLM with CTB were c‐Fos‐ir (16–40%, depending on PVH region). In marked contrast, few glutamatergic, RVLM‐projecting PVH neurons were c‐Fos‐ir in control rats (n = 3; 0–3%, depending on PVH region). Most (94% ± 4%) RVLM‐projecting PVH neurons activated by water deprivation contained VGLUT2 mRNA. In summary, most PVH neurons that innervate the RVLM are glutamatergic, and this population includes the neurons that are activated by water deprivation. One mechanism by which water deprivation may increase the sympathetic outflow is activation of a glutamatergic pathway from the PVH to the RVLM. J. Comp. Neurol. 494:673–685, 2006.

Sympathoexcitation by PVN-Injected Bicuculline Requires Activation of Excitatory Amino Acid Receptors

Abstract—Acute blockade of &ggr;-aminobutyric acid (GABA)-A receptors in the hypothalamic paraventricular nucleus (PVN) increases mean arterial pressure (MAP), heart rate (HR), and sympathetic nerve activity (SNA). However, the underlying neural mechanisms have not been fully determined. We tested the hypothesis that responses to GABA-A receptor blockade in the PVN require activation of local ionotropic excitatory amino acid (EAA) receptors. MAP, HR, and renal SNA responses to unilateral PVN microinjection of bicuculline methobromide (BIC, 0.1 nmol) were recorded before and after ipsilateral PVN injection of either vehicle (saline), the nonselective ionotropic EAA receptor antagonist kynurenate (KYN), the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5), or the non-NMDA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX). Responses to PVN-injected BIC were unaltered by vehicle injection. In contrast, injection of KYN (7.2 nmol; n=4) nearly abolished ABP and renal SNA responses to BIC (P <0.01) and significantly attenuated (P <0.05) HR responses as well. Similarly, graded doses of AP5 (0.6, 3, and 6 nmol) and NBQX (0.26, 1.3, and 2.6 nmol) reduced responses to PVN-injected BIC in a dose-related manner, with the 3 nmol (n=7) and 1.3 nmol (n=6) doses producing maximal effects (P <0.05). KYN, AP5, and NBQX did not affect baseline parameters. Effects of a cocktail containing AP5 (3 nmol) and NBQX (1.3 nmol) were greater (P <0.01) than either antagonist alone and were not statistically different from KYN. These data indicate that cardiovascular and renal sympathetic responses to acute GABA-A receptor blockade in the PVN require local actions of EAAs at both NMDA and non-NMDA receptors.

Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice

Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressant-like effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.

Median preoptic neurones projecting to the hypothalamic paraventricular nucleus respond to osmotic, circulating Ang II and baroreceptor input in the rat

The present study sought to determine whether individual neurones of the median preoptic nucleus (MnPO) with axonal projections to the hypothalamic paraventricular nucleus (MnPO‐PVN) respond to osmotic, circulating angiotensin II (Ang II), and baroreceptor stimulation. Hypertonic NaCl (0.75 or 1.5 osmol l−1) or Ang II (150 ng) was injected into the internal carotid artery (ICA). Baroreceptor stimulation was performed by i.v. injection of phenylephrine or sodium nitroprusside to increase or decrease arterial blood pressure, respectively. Of 65 MnPO neurones, 50 units were antidromically activated from the PVN with an average onset latency of 11.3 ± 0.7 ms. Only 9.5% of MnPO‐PVN neurones were antidromically activated from the PVN bilaterally. Type I MnPO‐PVN neurones (n= 14) responded to osmotic but not Ang II stimulation. In 79% (11/14) of these type I neurones, the response was an increase in cell discharge. Type II MnPO‐PVN neurones (n= 7) displayed a significant increase in cell discharge in response to ICA injection of Ang II but not hypertonic NaCl. Type III MnPO‐PVN neurones (n= 16) responded to both ICA injection of hypertonic NaCl and Ang II. In 88% (14/16) of type III neurones, osmotic and Ang II stimulation each increased cell discharge. Type IV MnPO‐PVN neurones (n= 13) displayed no change in cell discharge in response to ICA injection of hypertonic NaCl or Ang II. Baroreceptor stimulation altered the discharge in subpopulations of type I, II and III MnPO‐PVN neurones (43–63% depending on neuronal type). Only one MnPO‐PVN neurone responded solely to baroreceptor stimulation (type IV). In addition, a subset of type I, II and III neurones displayed a significant correlation with sympathetic nerve activity and/or the cardiac cycle. These findings suggest that a significant population of MnPO‐PVN neurones respond to osmotic and circulating Ang II stimulation and thereby represents a neural substrate through which neurohumoral inputs are integrated within the forebrain lamina terminalis.

Hypothalamic paraventricular nucleus differentially supports lumbar and renal sympathetic outflow in water‐deprived rats

The present study sought to determine whether the hypothalamic paraventricular nucleus (PVN) contributes in a time‐dependent manner to the differential patterning of lumbar and renal sympathetic nerve activity (SNA) in water‐deprived rats. Mean arterial blood pressure (MAP) and both lumbar SNA (LSNA) and renal SNA (RSNA) were recorded simultaneously in control, 24 and 48 h water‐deprived rats, and the PVN was inhibited bilaterally with microinjection of the GABAA agonist muscimol (100 pmol in 100 nl per side). Inhibition of the PVN significantly decreased RSNA in 48 h water‐deprived rats but not in 24 h water‐deprived or control rats (48 h, −17 ± 4%; 24 h, −2 ± 5%; control, 4 ± 6%; P < 0.05). In addition, injection of muscimol significantly decreased LSNA in 48 and 24 h water‐deprived rats but not in control rats (48 h, −41 ± 4%; 24 h, −14 ± 6%; control, −3 ± 2%; P < 0.05). Interestingly, the decrease in LSNA was significantly greater than the decrease in RSNA of 24 and 48 h water‐deprived rats (P < 0.05). Inhibition of the PVN also significantly decreased MAP to a greater extent in 48 and 24 h water‐deprived rats compared to control rats (48 h, −34 ± 5 mmHg; 24 h, −26 ± 4 mmHg; control, −15 ± 3 mmHg; P < 0.05). When 48 h water‐deprived rats were acutely rehydrated by giving access to tap water 2 h before experiments, inhibition of the PVN with muscimol did not alter LSNA (−12 ± 8%) or RSNA (7 ± 4%) but did produce a small decrease in MAP (−15 ± 4 mmHg) that was not different from control rats. In a parallel set of experiments, acute rehydration of 48 h water‐deprived rats significantly attenuated the increased Fos immunoreactivity in PVN neurones that project to the spinal cord or rostral ventrolateral medulla. Collectively, the present findings suggest that PVN autonomic neurones are synaptically influenced during water deprivation, and that these neurones differentially contribute to LSNA and RSNA in water‐deprived rats.

Hyperosmotic activation of CNS sympathetic drive: implications for cardiovascular disease

Evidence now indicates that exaggerated sympathetic nerve activity (SNA) significantly contributes to salt‐sensitive cardiovascular diseases. Although CNS mechanisms that support the elevation of SNA in various cardiovascular disease models have been intensively studied, many mechanistic details remain unknown. In recent years, studies have shown that SNA can rise as a result of both acute and chronic increases of body fluid osmolality. These findings have raised the possibility that salt‐sensitive cardiovascular diseases could result, at least in part, from direct osmosensory activation of CNS sympathetic drive. In this brief review we emphasize recent findings from several laboratories, including our own, which demonstrate that neurons of the forebrain organum vasculosum laminae terminalis (OVLT) play a pivotal role in triggering hyperosmotic activation of SNA by recruiting neurons in specific regions of the hypothalamus, brainstem and spinal cord. Although OVLT neurons are intrinsically osmosensitive and shrink when exposed to extracellular hypertonicity, it is not yet clear if these processes are functionally linked. Whereas acute hypertonic activation of OVLT neurons critically depends on TRPV1 channels, studies in TRPV1−/− mice suggest that acute and long‐term osmoregulatory responses remain largely intact. Therefore, acute and chronic osmosensory transduction by OVLT neurons may be mediated by distinct mechanisms. We speculate that organic osmolytes such as taurine and possibly novel processes such as extracellular acidification could contribute to long‐term osmosensory transduction by OVLT neurons and might therefore participate in the elevation of SNA in salt‐sensitive cardiovascular diseases.

Functional role of brain AT1 and AT2 receptors in the central angiotensin II pressor response

Intracerebroventricular (i.c.v.) angiotensin II (ANG II) increases vascular resistance and elicits a pressor response characterized by sympathetic nervous system activation (SNS component) and increased vasopressin (VP) secretion (VP component). This study examines the role of brain AT1 and AT2 ANG II receptors in mediating the pressor and renal hemodynamic effects of i.c.v. ANG II in conscious Sprague-Dawley rats. Mean arterial pressure, heart rate and renal vascular resistance responses to i.c.v. ANG II (100 ng in 5 microliters) were determined 10 min after i.c.v. injection of either the AT1 receptor antagonist, DuP 753 (1.0, 2.5, 5.0, 10.0 micrograms), the AT2 receptor ligand, PD 123319 (3.5 x [10(-6), 10(-4), 10(-2), 10(0)] micrograms), or both. In control rats, i.c.v. DuP 753 prevented the pressor response and the increase in renal vascular resistance that occurred following i.c.v. ANG II in a dose-dependent manner (P < 0.05), while i.c.v. PD 123319 was without affect. When the VP- and SNS components were studied individually, by preventing the SNS component with intravenous (i.v.) chlorisondamine or the VP component with a V1 receptor antagonist (i.v.) similar results were obtained; DuP 753 prevented the SNS component and significantly reduced the VP component. These results indicate that both central ANG II pressor components are mediated primarily by brain AT1 receptors. However, doses of DuP 753 were more effective when combined with 3.5 micrograms of PD 123319 than when given alone (P < 0.05), suggesting that the pressor effects of i.c.v. ANG II may involve activation of multiple ANG II receptor subtypes.

Chronic intermittent hypoxia increases blood pressure and expression of FosB/ΔFosB in central autonomic regions

Chronic intermittent hypoxia (CIH) models repetitive bouts of arterial hypoxemia that occur in humans suffering from obstructive sleep apnea. CIH has been linked to persistent activation of arterial chemoreceptors and the renin-angiotensin system, which have been linked to chronic elevations of sympathetic nerve activity (SNA) and mean arterial pressure (MAP). Because Fos and FosB are transcription factors involved in activator protein (AP)-1 driven central nervous system neuronal adaptations, this study determined if CIH causes increased Fos or FosB staining in brain regions that regulate SNA and autonomic function. Male Sprague Dawley rats were instrumented with telemetry transmitters for continuous recording of MAP and heart rate (HR). Rats were exposed to continuous normoxia (CON) or to CIH for 8 h/day for 7 days. CIH increased MAP by 7-10 mmHg without persistently affecting HR. A separate group of rats was killed 1 day after 7 days of CIH for immunohistochemistry. CIH did not increase Fos staining in any brain region examined. Staining for FosB/ΔFosB was increased in the organum vasculosum of the lamina terminalis (CON: 9 ± 1; CIH: 34 ± 3 cells/section), subfornical organ (CON: 7 ± 2; CIH: 31 ± 3), median preoptic nucleus (CON 15 ± 1; CIH: 38 ± 3), nucleus of the solitary tract (CON: 9 ± 2; CIH: 28 ± 4), A5 (CON: 3 ± 1; CIH: 10 ± 1), and rostral ventrolateral medulla (CON: 5 ± 1; CIH: 17 ± 2). In the paraventricular nucleus, FosB/ΔFosB staining was located mainly in the dorsal and medial parvocellular subnuclei. CIH did not increase FosB/ΔFosB staining in caudal ventrolateral medulla or supraoptic nucleus. These data indicate that CIH induces an increase in FosB/ΔFosB in autonomic nuclei and suggest that AP-1 transcriptional regulation may contribute to stable adaptive changes that support chronically elevated SNA.

Transport mechanisms governing serotonin clearance in vivo revealed by high-speed chronoamperometry.

High-speed chronoamperometry was used to determine the kinetics of clearance of exogenously applied serotonin (5-HT) in the dorsal raphe nucleus (DRN), dentate gyrus, CA3 region of the hippocampus or corpus callosum of anesthetized rats. Maximal velocity (Vmax) for 5-HT clearance was greatest in the DRN > dentate gyrus > CA3 > corpus callosum. Apparent affinity (K(T)) of the serotonin transporter (5-HTT) was similar in DRN and CA3 but greater in dentate gyrus and corpus callosum. A 90% loss of norepinephrine transporters (NET) produced by 6-hydroxydopamine pretreatment, resulted in a two-fold reduction in Vmax and a 30% decrease in K(T) in the dentate gyrus, but no change in kinetic parameters in the CA3 region. Pretreatment with 5,7-dihydroxytryptamine that resulted in a 90% reduction in 5-HTT density, modestly reduced Vmax in dentate gyrus but not in CA3. The same treatment had no effect on K(T) in the dentate gyrus but increased K(T) two-fold in the CA3. Neurotoxin treatments had no effect on 5-HT clearance in the corpus callosum. In hippocampal regions of intact rats, local application of the selective serotonin reuptake inhibitor, fluvoxamine, inhibited 5-HT clearance most robustly when the extracellular concentration of 5-HT was less than the K(T) value. By contrast, the NET antagonist, desipramine, significantly inhibited 5-HT clearance when extracellular concentrations of 5-HT were greater than the K(T) value. These data indicate that hippocampal uptake of 5-HT may be mediated by two processes, one with high affinity but low capacity (i.e. the 5-HTT) and the other with low affinity but a high capacity (i.e. the NET). These data show for the first time in the whole animal that 5-HT clearance in brain is regionally distinct with regard to rate and affinity.

Does enhanced respiratory–sympathetic coupling contribute to peripheral neural mechanisms of angiotensin II–salt hypertension?

Hypertension caused by chronic infusion of angiotensin II (Ang II) in experimental animals is likely to be mediated, at least in part, by an elevation of ongoing sympathetic nerve activity (SNA). However, the contribution of SNA relative to non‐neural mechanisms in mediating Ang II‐induced hypertension is an area of intense debate and remains unresolved. We hypothesize that sympathoexcitatory actions of Ang II are directly related to the level of dietary salt intake. To test this hypothesis, chronically instrumented rats were placed on a 0.1 (low), 0.4 (normal) or 2.0% NaCl diet (high) and, following a control period, administered Ang II (150 ng kg−1 min−1, s.c.) for 10–14 days. The hypertensive response to Ang II was greatest in rats on the high‐salt diet (Ang II–salt hypertension), which was associated with increased ‘whole body’ sympathetic activity as measured by noradrenaline spillover and ganglionic blockade. Indirect and direct measures of organ‐specific SNA revealed a distinct ‘sympathetic signature’ in Ang II–salt rats characterized by increased SNA to the splanchnic vascular bed, transiently reduced renal SNA and no change in SNA to the hindlimbs. Electrophysiological experiments indicate that increased sympathetic outflow in Ang II–salt rats is unlikely to involve activation of rostral ventrolateral medulla (RVLM) vasomotor neurons with barosensitive cardiac rhythmic discharge. Instead, another set of RVLM neurons that discharge in discrete bursts have exaggerated spontaneous activity in rats with Ang II–salt hypertension. Although their discharge is not cardiac rhythmic at resting levels of arterial pressure, it nevertheless appears to be barosensitive. Therefore, these burst‐firing RVLM neurons presumably serve a vasomotor function, consistent with their having axonal projections to the spinal cord. Bursting discharge of these neurons is respiratory rhythmic and driven by the respiratory network. Given that splanchnic SNA is strongly coupled to respiration, we hypothesize that enhanced central respiratory–vasomotor neuron coupling in the RVLM could be an important mechanism that contributes to exaggerated splanchnic sympathetic outflow in Ang II–salt hypertension. This hypothesis remains to be tested directly in future investigations.