Steve Mifflin

Chronic Hypertension Enhances the Postsynaptic Effect of Baclofen in the Nucleus Tractus Solitarius

Microinjection of the inhibitory neurotransmitter &ggr;-aminobutyric acid B-subtype receptor agonist baclofen into the nucleus tractus solitarius increases arterial blood pressure and sympathetic nerve discharge. The baclofen-induced pressor response is enhanced in chronic hypertension. We hypothesized that a postsynaptic mechanism contributes to the enhanced responses to baclofen in hypertension. We investigated the postsynaptic effect of baclofen on second-order baroreceptor neurons, identified by 1,1′-dilinoleyl-3,3,3′,3′-tetra-methylindocarbocyanine, 4-chlorobenzenesulphonate labeling of the aortic nerve, in nucleus tractus solitarius slices from sham-operated normotensive and unilateral nephrectomized, renal-wrap hypertensive rats. After 4 weeks, arterial blood pressure was 153±7 mm Hg in hypertensive rats (n=9) and 93±3 mm Hg in normotensive rats (n=8; P<0.05). There was no difference in resting membrane potential (54.5±0.7 versus 53.3±0.6 mV) or input resistance (1.07±0.11 versus 1.03±0.11 G&OHgr;) between hypertensive and normotensive neurons (both n=18). Baclofen induced a net outward current in nucleus tractus solitarius neurons in the presence of 1 &mgr;mol/L tetrodotoxin. The EC50 of the baclofen effect was greater in normotensive cells (9.1±3.2 &mgr;mol/L; n=5) than hypertensive cells (3.0±0.5 &mgr;mol/L; n=7; P<0.05), and baclofen (10 &mgr;mol/L) induced a greater decrease in input resistance in hypertensive cells (61±2%; n=6) than in normotensive cells (45±4%; n=9; P<0.05). Both potassium and calcium channels were involved in the baclofen-evoked whole-cell current. The results suggest an enhanced postsynaptic response to activation of inhibitory neurotransmitter &ggr;-aminobutyric acid B-subtype receptors in second-order baroreceptor neurons in the nucleus tractus solitarius in renal-wrap hypertensive rats. This enhanced inhibition could alter baroreflex function in chronic hypertension.

Responses to GABAA receptor activation are altered in NTS neurons isolated from chronic hypoxic rats

The inhibitory amino acid GABA is released within the nucleus of the solitary tract (NTS) during hypoxia and modulates the respiratory response to hypoxia. To determine if responses of NTS neurons to activation of GABA(A) receptors are altered following exposure to chronic hypoxia, GABA(A) receptor-evoked whole cell currents were measured in enzymatically dispersed NTS neurons from normoxic and chronic hypoxic rats. Chronic hypoxic rats were exposed to 10% O(2) for 9-12 days. Membrane capacitance was the same in neurons from normoxic (6.9+/-0.5 pF, n=16) and hypoxic (6.3+/-0.5 pF, n=15) rats. The EC(50) for peak GABA-evoked current density was significantly greater in neurons from hypoxic (21.7+/-2.2 microM) compared to normoxic rats (12.2+/-0.9 microM) (p<0.001). Peak and 5-s adapted GABA currents evoked by 1, 3 and 10 microM were greater in neurons from normoxic compared to hypoxic rats (p<0.05) whereas peak and 5-s adapted responses to 30 and 100 microM GABA were not different comparing normoxic to hypoxic rats. Desensitization of GABA(A)-evoked currents was observed at concentrations greater than 3 microM and, measured as the ratio of the current 5 s after the onset of 100 microM GABA application to the peak GABA current, was the same in neurons from normoxic (0.37+/-0.03) and hypoxic rats (0.33+/-0.04). Reduced sensitivity to GABA(A) receptor-evoked inhibition in chronic hypoxia could influence chemoreceptor afferent integration by NTS neurons.

Integration of Aortic Nerve Inputs in Hypertensive Rats

The integration of arterial baroreceptor afferent inputs was studied in renal wrap hypertensive (HT) and normotensive (NT) rats. In anesthetized and paralyzed rats, aortic nerve (AN)-evoked depressor responses were reduced in HT compared with NT rats (P<0.05). We tested the hypothesis that the attenuated baroreflex was associated with altered integration of baroreceptor inputs within the nucleus of the solitary tract. Based on onset latency and the ability of monosynaptic neurons (MSNs) to respond to each of 2 AN stimuli separated by 5 ms, cells in HT and NT rats were divided into 3 groups: short-latency MSNs (SLMSNs), long-latency MSNs (LLMSNs), and polysynaptic neurons (PSNs). A higher percentage of PSNs (73% versus 61%) and a lower percentage of SLMSNs (20% versus 27%) or LLMSNs (7% versus 12%) were found in HT rats (P<0.05). In addition, in HT compared with NT rats, the AN onset latency was greater in PSNs (29. 9+/-1.1 versus 26.7+/-0.8 ms) but not in SLMSNs (5.0+/-0.5 versus 5. 0+/-0.3 ms) or LLMSNs (22.9+/-1.2 versus 24.1+/-0.7 ms) (P<0.05). Finally, in HT compared with NT rats, the number of PSNs responding to a single AN stimulus with multiple action potentials was increased (40% versus 19%) (P<0.05). This was not observed in SLMSNs (26% versus 13%) or LLMSNs (12% versus 18%). The results indicate that renal wrap hypertension is associated with reduced AN-evoked depressor responses. There also were alterations in the integration of AN afferent inputs within the nucleus of the solitary tract, and these alterations were most marked in the PSN population.

Responses to GABAA Receptor Activation Are Altered in NTS Neurons Isolated From Renal-Wrap Hypertensive Rats

Abstract—The inhibitory amino acid GABA is a potent modulator of the spontaneous discharge and the responses to afferent inputs of neurons in the nucleus of the solitary tract (NTS). To determine if responses to activation of GABAA receptors are altered in hypertension, GABAA receptor–evoked whole cell currents were measured in enzymatically dispersed NTS neurons from 33 normotensive (NT, 109±4 mm Hg, n=7) and 24 hypertensive (HT, 167±5 mm Hg, n=24) rats. GABAA receptor–evoked currents reversed at the calculated equilibrium potential for chloride and were blocked by bicuculline (n=6). Membrane capacitance was the same in neurons from NT (7.5±0.6 pF, n=62) and HT (6.8±0.6 pF, n=51) rats. The EC50 for peak GABA-evoked currents cells was significantly greater in neurons from HT (21.0±2.6 &mgr;mol/L, n=16) compared with NT rats (13.0±1.8 &mgr;mol/L, n=14, P =0.01). The EC50 of neurons exhibiting DiA labeling of presumptive aortic nerve terminals was no different than that observed in the nonlabeled cells (19.0±4.9 &mgr;mol/L, n=4). The time constant for desensitization of GABAA-evoked currents was the same in neurons from HT (4.5±0.3 seconds, n=17) and NT rats (3.8±0.3 seconds, n=17, P >0.05). Repetitive pulse application of GABA revealed a more rapid decline in the evoked current in neurons from HT compared with NT rats. The amplitude of the 5th pulse of GABA (5-second duration, 2-second interval) was 21±2% the amplitude of the 1st pulse in NT rats (n=10) and 14±2% in HT rats (n=11, P <0.05). These alterations in GABAA-receptor evoked currents could render the neurons less sensitive to GABAA receptor inhibition and influence afferent integration by NTS neurons in HT.

Chronic Hypertension Enhances Presynaptic Inhibition by Baclofen in the Nucleus of the Solitary Tract

The selective &ggr;-aminobutyric acid B-subtype receptor agonist baclofen activates both presynaptic and postsynaptic receptors in the brain. Microinjection of baclofen into the nucleus of the solitary tract increases arterial pressure, heart rate, and sympathetic nerve discharge consistent with inhibition of the arterial baroreflex. The magnitude of these responses is enhanced in hypertension and is associated with increased postsynaptic GABAB receptor function. We tested whether a presynaptic mechanism contributes to the enhanced baclofen inhibition in hypertension. Whole-cell recordings of second-order baroreceptor neurons, identified by 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide labeling of aortic nerve, were obtained in brainstem slices from normotensive control and renal-wrap hypertensive rats. After 4 weeks, arterial blood pressure was 162±9 mm Hg in hypertensive (n=6) and 107±3 mm Hg in control rats (n=6/11; P<0.001). Baclofen reduced the amplitude of excitatory postsynaptic currents evoked by solitary tract stimulation and the EC50 of this inhibition was greater in control (1.5±0.5 &mgr;mol/L; n=6) than in hypertensive cells (0.6±0.1 &mgr;mol/L; n=9; P<0.05). Baclofen (1 &mgr;mol/L) elicited greater inhibition on evoked response in hypertensive (58±6%; n=9) than in control cells (40±6%; n=8; P<0.05). Another index of presynaptic inhibition, the paired-pulse ratio (ratio of second to first evoked response amplitudes at stimulus intervals of 40 ms), was greater in hypertensive (0.60±0.08; n=8) than in control cells (0.48±0.06; n=5; P<0.05). The results suggest that in renal-wrap hypertensive rats, baclofen causes an enhanced presynaptic inhibition of glutamate release from baroreceptor afferent terminals to second-order neurons in the nucleus of the solitary tract. This enhanced presynaptic inhibition could contribute to altered baroreflex function in hypertension.

Identification of Active Central Nervous System Sites in Renal Wrap Hypertensive Rats

To identify central neurons participating in cardiovascular regulation in hypertension, we studied Fos staining, a marker for synaptically activated neurons, in adult male normotensive and hypertensive (HT) rats. At 1 and 4 weeks after induction of unilateral nephrectomy, renal wrap hypertension mean arterial pressure was 138±4 mm Hg (n=6) in 1-week HT rats and 159±6 mm Hg (n=6) in 4-week HT rats. Mean arterial pressure was 103±2 mm Hg (n=6) in sham-operated, normotensive rats. Mean arterial pressure was greater in both HT groups compared with normotensive rats, and the mean arterial pressure in 4-week HT rats was greater than that in 1-week HT rats. Rats were anesthetized and perfused, brains sectioned and processed using a Fos antibody, and the number of Fos immunoreactive neurons counted in sections through various brain regions. Hypertension of 1 or 4 weeks did not alter the number of Fos immunoreactive neurons in the area postrema, the supraoptic nucleus, and the median preoptic nucleus. The number of Fos immunoreactive neurons was increased after 1 and 4 weeks in the nucleus of the solitary tract, both the caudal and ventral lateral medulla, and the organum vasculosum of the lamina terminalis. In addition, after 4 weeks of HT, the number of Fos immunoreactive neurons was increased in the parabrachial nucleus and the paraventricular nucleus of the hypothalamus. The results indicate central regions active in acute and chronic HT rats and suggest certain areas that may be differentially activated depending on the duration of the hypertension.

Voltage-Dependent Calcium Currents Are Enhanced in Nucleus of the Solitary Tract Neurons Isolated From Renal Wrap Hypertensive Rats

The nucleus of the solitary tract (NTS) is the central site of termination of baroreceptor afferents. We hypothesize that changes occur in voltage-gated calcium channels (VGCCs) within NTS neurons as a consequence of hypertension. Whole-cell patch-clamp recordings were obtained from adult normotensive (109±2 mm Hg; n=6 from 6 sham-operated and 31 nonsurgically treated) and hypertensive (158±6 mm Hg; n=24) rats. In some experiments, 4-(4-[dihexadecylamino]styryl)-N-methylpyridinium iodide was applied to the aortic nerve to visualize NTS neurons receiving baroreceptor synaptic contacts. Ba2+ currents (500 ms; −80 mV prepotential; 500 ms voltage steps in 5-mV increments to +15mV) peaked between −20 and −10 mV and were blocked by 100 &mgr;m of Cd2+. Peak VGCCs were not different comparing non-4-(4-[dihexadecylamino]styryl)-N-methylpyridinium iodide-labeled and 4-(4- [dihexadecylamino]styryl)-N-methylpyridinium iodide-labeled NTS neurons in hypertensive and normotensive rats. The peak VGCC was significantly greater in cells from hypertensive compared with normotensive rats for both non–DiA-labeled (P=0.02) and DiA-labeled (P=0.04) neurons. To separate high-voltage activated (HVA) and low-voltage activated (LVA) components of VGCCs, voltage ramps (−110 mV to +30 mV over 50 ms) were applied from a holding potential of −60 mV (LVA channels inactivated) and a holding potential of −100 mV (both LVA and HVA currents activated). HVA currents were subtracted from HVA+LVA currents to yield the LVA current. Peak LVA currents were not different between hypertensive (8.9±0.8 pA/pF) and normotensive (7.8±0.6 pA/pF) groups of NTS neurons (P=0.27). These results demonstrate that 4 weeks of renal wrap hypertension induce an increase in Ca2+ influx through HVA VGCCs in NTS neurons receiving arterial baroreceptor inputs.

Chronic hypoxia abolishes expiratory prolongation following carotid sinus nerve stimulation in the anesthetized rat

In anesthetized rats, increases in phrenic nerve (PN) amplitude and frequency during brief periods of hypoxia or electrical stimulation of the carotid sinus nerve (CSN) are followed by an increase in expiratory duration. We investigated the effects of chronic exposure to hypoxia on PN responses to CSN stimulation. In Inactin anesthetized (100 mg/kg) Sprague-Dawley rats PN discharge and arterial pressure responses to 10-120 s of CSN stimulation (20 Hz, 0.2 ms duration pulses) were recorded after 7-10 days exposure to hypoxia (10 +/- .5% O2). In normoxic rats, the degree of CSN-evoked expiratory prolongation was dependent upon the duration of CSN stimulation. CSN-evoked increases in PN burst amplitude were not different comparing chronic hypoxic rats to rats maintained at normoxia while CSN-evoked increases in PN burst frequency were greater in chronic hypoxic rats (p<.05). CSN-evoked expiratory prolongation was abolished in chronic hypoxic rats. Following chronic hypoxia, changes occur within the central processing of arterial chemoreceptor inputs so that CSN stimulation evokes an enhanced PN frequency response and no expiratory prolongation.

NO and CO have got to GO for enhanced chemoreceptor sympathoexcitation in heart failure

arterial chemoreceptors located in the carotid body are an important defense mechanism against systemic hypoxemia. Activation of arterial chemoreceptors increases alveolar ventilation and sympathetic outflow to most vascular beds. During acute exposures to hypoxia, the sympathoexcitatory responses

Obesity and the central nervous system

The past decade has witnessed an explosion of information regarding the role of the central nervous system in the development of obesity and the influence of peripheral, hormonal signals that modulate CNS regulation of energy homeostasis. A Journal of Physiology Symposium held in Washington, DC in association with the 2007 Experimental Biology meeting focused on recent work defining central neural mechanisms that mediate ingestive behaviour and the central effects of peripheral hormonal signals that modulate feeding. A crowd estimated at 500 was treated to a series of talks by leaders in the field who discussed state-of-the-art data and models regarding the brain as an initiator of obesity and as a target organ of peripheral feedback signals that regulate feeding behaviour. n nThe opening speaker, Dr Barry Levin, provided a very informative and entertaining overview of his work that some obesity-prone individuals have an inborn reduction in their ability to sense and respond to inhibitory signals from adipose stores and other organs which ordinarily limit their intake of energy when in excess of metabolic needs (see Levin, 2007). Furthermore, the physiological processes which drive all of us to seek and ingest food and limit energy expenditure during periods of negative energy balance provide an irresistible drive to regain lost adipose stores in weight-reduced obese individuals. This provides a potential basis for the well-recognized difficulty of maintaining weight loss. For this reason, prevention of obesity and the identification of factors that promote the development of neural pathways which enhance the sensitivity to negative feedback signals from the periphery should be a major focus of research. n nDr Mary Dallman spoke next and presented a very interesting and compelling case for peripheral signals, specifically glucocorticoids and insulin, as modulators of the central pathways that regulate ingestive behaviour (see Dallman et al. 2007). Glucocorticoids act primarily in a feed-forward fashion on brain to activate CNS pathways that implement wanting appropriate to physiological needs. Thus, depending on the available conditions, elevated glucocorticoids may augment the behavioural desire to run, fight or feed. Although glucocorticoids stimulate intake of chow, fat and sucrose, insulin appears to sculpt calorie-associated desires toward foods high in fat, acting through hepatic branch afferents of the vagus nerve. Conditions of reduced food allowance and chronic stress excite glucocorticoid-augmented central neural networks that may ultimately lead toward ultimate abdominal obesity. This provides a potential link between stress and obesity. n nDr Gregory Morton presented a creative use of gene targeting and viral gene therapy to rescue leptin-receptors in the arcuate nucleus of Koletsky rats that lack leptin receptor protein (see Morton, 2007). Growing evidence suggests that hypothalamic areas that respond to a variety of peripheral signals regulate food intake, energy expenditure and endogenous glucose production. Therefore, in response to a reduction in energy stores or circulating nutrients, the brain initiates responses in order to promote positive energy balance to restore and maintain energy and glucose homeostasis. In contrast, in times of nutrient abundance and excess energy storage, key hypothalamic areas activate responses to promote negative energy balance (i.e. reduced food intake and increased energy expenditure) and decreased nutrient availability (reduced endogenous glucose production). Accordingly, impaired responses or ‘resistance’ to afferent input from these hormonal or nutrient-related signals would be predicted to favour weight gain and insulin resistance and may contribute to the development of obesity and type 2 diabetes. n nFinally, Dr Steven Heymsfield reviewed the development program for MK-0557, a highly selective and potent antagonist of the NPY5 receptor by the Merck Research Laboratories. About one decade ago it became clear that the neuropeptide Y-5 receptor is involved in food intake regulation, with agonism increasing intake. Merck launched an intensive programme leading to the discovery of MK-0557 (an NPY5R antagonist). The successful preclinical work was followed by PET and pharmacokinetic studies in humans that supported safety and receptor occupancy. A short-term positive proof of concept weight loss study then led to a large long-term series of clinical trials which ultimately demonstrated that the antagonist resulted in minimal weight loss. Many lessons in neurobiology and drug development can be learned from this progression of studies. n nThe overall goal is that the understanding of how and why certain individuals gain weight and the central mechanisms that make it difficult to sustain weight loss might lead to therapeutic potential to prevent the process, rather than deal with its consequences in already obese individuals. Insights into the central neural networks that regulate food intake and how these networks are modulated provide a very promising avenue towards achieving these goals.