J. Thomas Cunningham

Induction of c-Fos and ΔFosB Immunoreactivity in Rat Brain by Vagal Nerve Stimulation

Vagus nerve stimulation (VNS) is used as therapy for treatment-resistant depression or epilepsy. This study used immunohistochemistry for biomarkers of short-term (c-Fos) and long-term (ΔFosB) neuronal activation to map regions in brain that are activated by acute (2u2009h) or chronic (3 weeks) VNS in conscious Sprague–Dawley rats. Electrodes (Cyberonics Inc.) were implanted on the left vagus nerve and 1 week after surgery, stimulation began using parameters employed clinically (one burst of 20u2009Hz, 250u2009μs pulse width, 0.25u2009mA stimulation for 30u2009s every 5u2009min). Radio telemetry transmitters were used for monitoring blood pressure, heart rate, activity, and respiratory rate during VNS; neither acute nor chronic VNS significantly affected these parameters. Acute VNS significantly increased c-Fos staining in the nucleus of the solitary tract, paraventricular nucleus of the hypothalamus, parabrachial nucleus, ventral bed nucleus of the stria terminalis, and locus coeruleus but not in the cingulate cortex or dorsal raphe nucleus (DRN). Acute VNS did not affect ΔFosB staining in any region. Chronic VNS significantly increased ΔFosB and c-Fos staining bilaterally in each region affected by acute VNS as well as in the cingulate cortex and DRN. Using these stimulation parameters, VNS was tested for antidepressant-like activity using the forced swim test (FST). Both VNS and desipramine significantly decreased immobility in the FST; whereas desipramine decreased immobility by increasing climbing behavior, VNS did so by increasing swimming behavior. This study, then, identified potential sites in brain where VNS may produce its clinical effects.

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.

High Salt Intake Increases Blood Pressure via BDNF-Mediated Downregulation of KCC2 and Impaired Baroreflex Inhibition of Vasopressin Neurons

The mechanisms by which dietary salt promotes hypertension are unknown. Previous work established that plasma [Na(+)] and osmolality rise in proportion with salt intake and thus promote release of vasopressin (VP) from the neurohypophysis. Although high levels of circulating VP can increase blood pressure, this effect is normally prevented by a potent GABAergic inhibition of VP neurons by aortic baroreceptors. Here we show that chronic high salt intake impairs baroreceptor inhibition of rat VP neurons through a brain-derived neurotrophic factor (BDNF)-dependent activation of TrkB receptors and downregulation of KCC2 expression, which prevents inhibitory GABAergic signaling. We show that high salt intake increases the spontaneous firing rate of VP neurons in vivo and that circulating VP contributes significantly to the elevation of arterial pressure under these conditions. These results provide the first demonstration that dietary salt can affect blood pressure through neurotrophin-induced plasticity in a central homeostatic circuit.

Intra-carotid hyperosmotic stimulation increases Fos staining in forebrain organum vasculosum laminae terminalis neurones that project to the hypothalamic paraventricular nucleus

Body fluid hyperosmolality has long been known to elicit homeostatic responses that range from drinking to inhibition of salt appetite to release of neurohypohyseal hormones (i.e. vasopressin and oxytocin). More recently, it has been recognized that hyperosmolality is capable of also provoking a significant increase of sympathetic nerve activity (SNA). It has been reported that neurones in the forebrain organum vasculosum laminae terminalis (OVLT) and hypothalamic paraventricular nucleus (PVN) each contribute significantly to this response. Here we sought to determine if sympathoexcitatory levels of hyperosmolality activate specifically those OVLT neurones that form a monosynaptic pathway to the PVN. First, we established in anaesthetized rats that graded concentrations of hypertonic NaCl (1.5 and 3.0 osmol kg−1) elicit graded increases of renal SNA (RSNA) when infused at a rate of 0.1 ml min−1 through an internal carotid artery (ICA) – the major vascular supply of the forebrain. Next, infusions were performed in conscious rats in which OVLT neurones projecting to the PVN (OVLT‐PVN) were retrogradely labelled with cholera toxin subunit B (CTB). Immunostaining of the immediate early gene product Fos and CTB was performed to quantify osmotic activation of OVLT‐PVN neurones. ICA infusions of hypertonic NaCl and mannitol each significantly (P < 0.01–0.001) increased the number of Fos immunoreactive (Fos‐ir) neuronal nuclei in the dorsal cap (DC) and lateral margins (LM) of OVLT. In the LM, infusions of 1.5 and 3.0 osmol kg−1 NaCl produced similar increases in the number of Fos‐ir neurones. In the DC, these infusions produced graded increases in Fos expression. Among OVLT neurones with axons projecting directly to the PVN (i.e. CTB‐ir), graded hypertonic NaCl infusions again produced graded increases in Fos expression and this was observed in both the DC and LM. Although the DC and LM contained a similar number of OVLT‐PVN neurones, the proportion of such neurones that expressed Fos‐ir in responses to ICA hypertonic NaCl infusions was greater in the DC (P < 0.001). These findings support the conclusion that PVN‐projecting neurones in the DC and LM of OVLT could participate in behavioural, neuroendocrine, and sympathetic nervous system responses to body fluid hyperosmolality.

Expression and distribution of TRPV2 in rat brain

Transient receptor potential (TRP) proteins are non-selective cation channels that mediate sensory transduction. The neuroanatomical localization and the physiological roles of isoform TRPV2 in the rodent brain are largely unknown. We report here the neuroanatomical distribution of TRPV2 in the adult male rat brain focusing on the hypothalamus and hindbrain regions involved in osmoregulation, autonomic function and energy metabolism. For this we utilized immunohistochemistry combined with brightfield microscopy. In the forebrain, the densest immunostaining was seen in both the supraoptic nucleus (SON) and the magnocellular division of the paraventricular nucleus (PVN) of the hypothalamus. TRPV2 immunoreactivity was also seen in the organum vasculosum of the lamina terminalis, the median preoptic nucleus and the subfornical organ, in addition to the arcuate nucleus of the hypothalamus (ARH), the medial forebrain bundle, the cingulate cortex and the globus pallidus to name a few. In the hindbrain, intense staining was seen in the nucleus of the solitary tract, hypoglossal nucleus, nucleus ambiguous, and the rostral division of the ventrolateral medulla (RVLM) and some mild staining in the area prostrema. To ascertain the specificity of the TRPV2 antibody used in this paper, we compared the TRPV2 immunoreactivity of wildtype (WT) and knockout (KO) mouse brain tissue. Double immunostaining with arginine vasopressin (AVP) using confocal microscopy showed a high degree of colocalization of TRPV2 in the magnocellular SON and PVN. Using laser capture microdissection (LCM) we also show that AVP neurons in the SON contain TRPV2 mRNA. TRPV2 was also co-localized with dopamine beta hydroxylase (DBH) in the NTS and the RVLM of the hindbrain. Based on our results, TRPV2 may play an important role in several CNS networks that regulate body fluid homeostasis, autonomic function, and metabolism.

Chronic intermittent hypoxia increases sympathetic control of blood pressure: role of neuronal activity in the hypothalamic paraventricular nucleus

Like humans with sleep apnea, rats exposed to chronic intermittent hypoxia (CIH) experience arterial hypoxemias and develop hypertension characterized by exaggerated sympathetic nerve activity (SNA). To gain insights into the poorly understood mechanisms that initiate sleep apnea/CIH-associated hypertension, experiments were performed in rats exposed to CIH for only 7 days. Compared with sham-treated normoxic control rats, CIH-exposed rats (n = 8 rats/group) had significantly increased hematocrit (P < 0.001) and mean arterial pressure (MAP; P < 0.05). Blockade of ganglionic transmission caused a significantly (P < 0.05) greater reduction of MAP in rats exposed to CIH than control rats (n = 8 rats/group), indicating a greater contribution of SNA in the support of MAP even at this early stage of CIH hypertension. Chemical inhibition of neuronal discharge in the hypothalamic paraventricular nucleus (PVN) (100 pmol muscimol) had no effect on renal SNA but reduced lumbar SNA (P < 0.005) and MAP (P < 0.05) more in CIH-exposed rats (n = 8) than control rats (n = 7), indicating that CIH increased the contribution of PVN neuronal activity in the support of lumbar SNA and MAP. Because CIH activates brain regions controlling body fluid homeostasis, the effects of internal carotid artery injection of hypertonic saline were tested and determined to increase lumbar SNA more (P < 0.05) in CIH-exposed rats than in control rats (n = 9 rats/group). We conclude that neurogenic mechanisms are activated early in the development of CIH hypertension such that elevated MAP relies on increased sympathetic tonus and ongoing PVN neuronal activity. The increased sensitivity of Na(+)/osmosensitive circuitry in CIH-exposed rats suggests that early neuroadaptive responses among body fluid regulatory neurons could contribute to the initiation of CIH hypertension.

FosB expression in the central nervous system following isotonic volume expansion in unanesthetized rats.

We examined c-Fos and FosB staining in the central nervous system 8 and 24 h following acute volume expansion in unanesthetized rats. Male rats were instrumented with a femoral artery catheter for measurement of blood pressure and heart rate (HR), a jugular venous catheter for measurement of central venous pressure (CVP), and a femoral vein catheter for i.v. infusion. After 48 h, rats were volume expanded with isotonic saline (10% of body weight for 10 min i.v.) or given a control infusion (0.01 ml/min for 10 min i.v.). After a period of 8 or 24 h, the rats were deeply anesthetized and perfused transcardially with 4% paraformaldehyde. Separate sets of serial sections of the hypothalamus were processed for either FosB (Santa Cruz) or c-Fos (Oncogene AB-5) immunocytochemistry. The volume expansion protocol significantly increased central venous pressure but did not affect blood pressure or heart rate. Volume expansion produced a significant increase in FosB-positive cells in the paraventricular nucleus (PVN) of the hypothalamus, the supraoptic nucleus (SON), the perinuclear zone (PNZ) of the supraoptic nucleus, the nucleus of the solitary tract (NST), and the caudal ventrolateral medulla (CVL) in both the 8- and 24-h groups. In the area postrema (AP), the number of FosB-positive cells was significantly increased only at 8 h post-infusion. However, c-Fos was not significantly increased above control levels at either time point. The results demonstrate that FosB activation is maintained for at least 24 h following an acute increase in central venous pressure.

Central losartan attenuates increases in arterial pressure and expression of FosB/ΔFosB along the autonomic axis associated with chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH) increases mean arterial pressure (MAP) and FosB/ΔFosB staining in central autonomic nuclei. To test the role of the brain renin-angiotensin system (RAS) in CIH hypertension, rats were implanted with intracerebroventricular (icv) cannulae delivering losartan (1 μg/h) or vehicle (VEH) via miniosmotic pumps and telemetry devices for arterial pressure recording. A third group was given the same dose of losartan subcutaneously (sc). Two groups of losartan-treated rats served as normoxic controls. Rats were exposed to CIH or normoxia for 7 days and then euthanized for immunohistochemistry. Intracerebroventricular losartan attenuated CIH-induced increases in arterial pressure during CIH exposure (0800-1600 during the light phase) on days 1, 6, and 7 and each day during the normoxic dark phase. FosB/ΔFosB staining in the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus (MnPO), paraventricular nucleus of the hypothalamus (PVN), the rostral ventrolateral medulla (RVLM), and the nucleus of the solitary tract (NTS) was decreased in icv losartan-treated rats. Subcutaneous losartan also reduced CIH hypertension during the last 2 days of CIH and produced bradycardia prior to the effect on blood pressure. Following sc losartan, FosB/ΔFosB staining was reduced only in the OVLT, MnPO, PVN, and NTS. These data indicate that the central and peripheral RAS contribute to CIH-induced hypertension and transcriptional activation of autonomic nuclei and that the contribution of the central RAS is greater during the normoxic dark phase of CIH hypertension.

Neural Control of Blood Pressure in Chronic Intermittent Hypoxia.

Sleep apnea (SA) is increasing in prevalence and is commonly comorbid with hypertension. Chronic intermittent hypoxia is used to model the arterial hypoxemia seen in SA, and through this paradigm, the mechanisms that underlie SA-induced hypertension are becoming clear. Cyclic hypoxic exposure during sleep chronically stimulates the carotid chemoreflexes, inducing sensory long-term facilitation, and drives sympathetic outflow from the hindbrain. The elevated sympathetic tone drives hypertension and renal sympathetic activity to the kidneys resulting in increased plasma renin activity and eventually angiotensin II (Ang II) peripherally. Upon waking, when respiration is normalized, the sympathetic activity does not diminish. This is partially because of adaptations leading to overactivation of the hindbrain regions controlling sympathetic outflow such as the nucleus tractus solitarius (NTS), and rostral ventrolateral medulla (RVLM). The sustained sympathetic activity is also due to enhanced synaptic signaling from the forebrain through the paraventricular nucleus (PVN). During the waking hours, when the chemoreceptors are not exposed to hypoxia, the forebrain circumventricular organs (CVOs) are stimulated by peripherally circulating Ang II from the elevated plasma renin activity. The CVOs and median preoptic nucleus chronically activate the PVN due to the Ang II signaling. All together, this leads to elevated nocturnal mean arterial pressure (MAP) as a response to hypoxemia, as well as inappropriately elevated diurnal MAP in response to maladaptations.

Angiotensin II type 1a receptors in subfornical organ contribute towards chronic intermittent hypoxia-associated sustained increase in mean arterial pressure

Sleep apnea is associated with hypertension. The mechanisms contributing to a sustained increase in mean arterial pressure (MAP) even during normoxic awake-state remain unknown. Rats exposed to chronic intermittent hypoxia for 7 days, a model of the hypoxemia associated with sleep apnea, exhibit sustained increases in MAP even during the normoxic dark phase. Activation of the renin-angiotensin system (RAS) has been implicated in chronic intermittent hypoxia (CIH) hypertension. Since the subfornical organ (SFO) serves as a primary target for the central actions of circulating ANG II, we tested the effects of ANG II type 1a receptor (AT1aR) knockdown in the SFO on the sustained increase in MAP in this CIH model. Adeno-associated virus carrying green fluorescent protein (GFP) and small-hairpin RNA against either AT1aR or a scrambled control sequence (SCM) was stereotaxically injected in the SFO of rats. After recovery, MAP, heart rate, respiratory rate, and activity were continuously recorded using radiotelemetry. In the normoxic groups, the recorded variables did not deviate from the baseline values. Both CIH groups exhibited significant increases in MAP during CIH exposures (P < 0.05). During the normoxic dark phase in the CIH groups, only the SCM-injected group exhibited a sustained increase in MAP (P < 0.05). The AT1aR-CIH group showed significant decreases in FosB/ΔFosB staining in the median preoptic nucleus and the paraventricular nuclei of the hypothalamus compared with the SCM-CIH group. Our data indicate that AT1aRs in the SFO are critical for the sustained elevation in MAP and increased FosB/ΔFosB expression in forebrain autonomic nuclei associated with CIH.