Both Ox1R and Ox2R orexin receptors contribute to the cardiorespiratory response evoked from the perifornical hypothalamus.

Orexin/hypocretin neurons are located in and around the perifornical hypothalamus. Disinhibition of this area in the anaesthetized preparation evokes cardiorespiratory changes that can be reduced to nearly half or more by systemic Almorexant, a dual receptor antagonist of the two known orexin receptors, Ox1R and Ox2R. It is not clear if these reductions result from the blockade of one receptor or both. To determine the contribution of the two receptors, we compared the effects of Almorexant to those of the selective Ox1R antagonist ACT335827 and the selective Ox2R antagonists EMPA and TCS-OX2-29. Bicuculline (20 pmol) was injected in the perifornical hypothalamus of urethane-anaesthetized rats before and after administration of the drugs (all 15 mg/kg, intravenously). The pressor, tachycardic and tachypneic responses to bicuculline were attenuated/reduced by ACT335827 (by 19%, ns; 10%, ns and 24%, P < 0.01, respectively), EMPA (by 35% P < 0.01; 6%, ns; and 26% P < 0.05) and TCS-OX2-29 (by 13%, ns; 10%, ns and 42%, P < 0.001). These reductions represented only a fraction of the reduction after Almorexant (by 43%, P < 0.001; 42%, P < 0.001 and 65% P < 0.001). However, when the selective Ox1R and Ox2R antagonists were given in combination, the reductions were greater and closer to those of Almorexant (ACT335827 + EMPA, by 26%, P < 0.05; 24%, P < 0.05 and 47%, P < 0.001; ACT335827 + TCS-OX2-29, by 40%, P < 0.01; 26%, P < 0.001 and 59%, P < 0.0001). This was particularly clear with the tachypneic response. These results suggest that both orexin receptors contribute to the cardiorespiratory response evoked from the hypothalamus under anaesthesia. They are consistent with our previous study in the conscious animal.

Blockade of the dorsomedial hypothalamus and the perifornical area inhibits respiratory responses to arousing and stressful stimuli.

The dorsomedial hypothalamus (DMH) and the perifornical area (DMH/PeF) is one of the key regions of central autonomic processing. Previous studies have established that this region contains neurons that may be involved in respiratory processing; however, this has never been tested in conscious animals. The aim of our study was to investigate the involvement of the DMH/PeF area in mediating respiratory responses to stressors of various intensities and duration. Adult male Wistar rats (n = 8) received microinjections of GABAA agonist muscimol or saline into the DMH/PeF bilaterally and were subjected to a respiratory recording using whole body plethysmography. Presentation of acoustic stimuli (500-ms white noise) evoked transient responses in respiratory rate, proportional to the stimulus intensity, ranging from +44 ± 27 to +329 ± 31 cycles/min (cpm). Blockade of the DMH/PeF almost completely abolished respiratory rate and tidal volume responses to the 40- to 70-dB stimuli and also significantly attenuated responses to the 80- to 90-dB stimuli. Also, it significantly attenuated respiratory rate during the acclimatization period (novel environment stress). The light stimulus (30-s 2,000 lux) as well as 15-min restraint stress significantly elevated respiratory rate from 95 ± 4.0 to 236 ± 29 cpm and from 117 ± 5.2 to 189 ± 13 cpm, respectively; this response was abolished after the DMH/PeF blockade. We conclude that integrity of the DMH/PeF area is essential for generation of respiratory responses to both stressful and alerting stimuli.

Both Ox1r and Ox2r orexin receptors contribute to the cardiovascular and locomotor components of the novelty stress response in the rat.

Orexin contributes to the expression of the cardiovascular and behavioural response of some forms of stress, including novelty stress. Thus, Almorexant, a dual receptor antagonist that blocks the two known orexin receptors, Ox1R and Ox2R, reduces these responses. However, it is not known if the reduction results from blockade of one receptor only or both. To answer this question, the selective Ox1R antagonist ACT335827 and the selective Ox2R antagonist EMPA were injected intragastrically (300 mg/kg) or intraperitoneally (30 and 100 mg/kg) either alone or as a cocktail and compared to Almorexant in rats exposed to novelty stress. Cardiovascular and locomotor responses were recorded by radio-telemetry. Triple immunolabelling was also conducted to establish the distribution of Ox1R and Ox2R in sympathetic preganglionic neurons and orexin neurons. Intraperitoneal injections of ACT335827 (100 mg/kg) reduced the pressor and tachycardic but not the locomotor response of novelty (by 32% and 48%, respectively). Intraperitoneal injections of EMPA (100 mg/kg) only reduced the pressor response (42%). However when given together, ACT335827 and EMPA reduced all 3 components (65%, 60% and 57% of the tachycardic, pressor and locomotor responses, respectively) as Almorexant (100 mg/kg) did (69%, 87% and 72%, respectively). Triple immunolabelling revealed that sympathetic preganglionic neurons were mainly Ox1R positive only while orexin neurons were both Ox1R and Ox2R positive. This study shows that orexin's contribution to the cardiovascular and locomotor components of the novelty stress response is not mediated by one receptor alone, but by both receptors and at different levels of the neuraxis.

Does exposure to chronic stress influence blood pressure in rats?

The principal aim of this study was to determine whether prolonged chronic footshock stress can evoke sustained changes in blood pressure in rats and to elucidate possible underlying neurochemical mechanisms as mediated by the sympathoadrenal system. Adult male Wistar rats instrumented for telemetric recording of arterial pressure, heart rate and locomotor activity were subjected to six weeks of inescapable unpredictable electrical footshocks (FS+) or were exposed to shock chambers but were not shocked (FS-). Compared to FS- animals, FS+ animals had significantly reduced body weight gain (by 30%), locomotor activity (by 25%) and social interaction time (by 30%)--symptoms commonly induced by chronic stress and depression in humans. These changes were associated with small, but significant increases in systolic blood pressure (by 7%) and pulse pressure (by 11%) in FS+ rats relative to FS- rats. We have also found neurochemical alterations in sympathoadrenal pathways (that lasted for at least one week post-stress) including about 2-3 fold increases in the levels of tyrosine hydroxylase phosphorylation in the sympathetic ganglia and adrenal gland and a 1.8-fold increase in the expression of the Angiotensin II receptor type 1 protein in the adrenal gland of FS+ rats relative to FS- rats. We conclude that uncontrollable and unpredictable footshock stress can lead to elevation in systolic blood pressure when applied for an extended period of time (six weeks) in Wistar rats, and that these changes could be mediated by stress-induced modifications in sympathoadrenal pathways.

Prolonged vasoconstriction of resistance arteries involves vascular smooth muscle actin polymerization leading to inward remodelling.

AIMS: Inward remodelling of the resistance vasculature is predictive of hypertension and life-threatening cardiovascular events. We hypothesize that the contractile mechanisms responsible for maintaining a reduced diameter over time in response to prolonged stimulation with vasoconstrictor agonists are in part responsible for the initial stages of the remodelling process. Here we investigated the role of vascular smooth muscle (VSM) actin polymerization on agonist-induced vasoconstriction and development of inward remodelling. METHODS AND RESULTS: Experiments were conducted in Sprague-Dawley rat resistance vessels isolated from the cremaster and mesentery. Within blood vessels, actin dynamics of VSM were monitored by confocal microscopy after introduction of fluorescent actin monomers through electroporation and by differential centrifugation to probe globular (G) and filamentous (F) actin content. Results indicated that 4 h of agonist-dependent vasoconstriction induced inward remodelling and caused significant actin polymerization, elevating the F-/total-actin ratio. Inhibition of actin polymerization prevented vessels from maintaining prolonged vasoconstriction and developing inward remodelling. Activation of the small GTPases Rho/Rac/Cdc42 also increased the F-/total-actin ratio and induced inward remodelling, while inhibition of Rho kinase or Rac-1 prevented inward remodelling. Disruption of the actin cytoskeleton reversed the inward remodelling caused by prolonged vasoconstriction, but did not affect the passive diameter of freshly isolated vessels. CONCLUSION: These results indicate that vasoconstriction-induced inward remodelling is in part caused by the polymerization of actin within VSM cells through activation of small GTPases.

Voluntary exercise does not affect stress-induced tachycardia, but improves resistance to cardiac arrhythmias in rats.

1. It is currently unknown whether long-term voluntary exercise has enduring cardioprotective effects in animal models. 2. The present study was conducted in three groups of rats: (i) sedentary controls (n = 6); (ii) 24 h runners (n = 8; unlimited access to running wheels); and (iii) 2 h runners (n = 8; access to running wheels limited to 2 h daily). After termination of the 6 week exercise protocol, all rats were implanted with the telemetric electrocardiogram transmitters and were studied 1 week later. 3. Resting heart rate (HR) values in the control rats, 24 h runners and 2 h runners were 372 ± 7, 361 ± 9 and 298 ± 5 b.p.m., respectively (P < 0.05 for 2 h runners vs controls). The high-frequency spectral power in the control rats, 24 h runners and 2 h runners was 3.9 ± 0.2, 4.3 ± 0.3 and 5.3 ± 0.3 s², respectively (P < 0.05 for 2 h runners vs controls), whereas intrinsic HR was 383 ± 3, 377 ± 2 and 346 ± 3 b.p.m., respectively (P < 0.001 for 2 h runners vs controls). Restraint stress provoked tachycardia of similar magnitude in all groups. 4. After completion of telemetric studies, haemodynamic indices and susceptibility to cardiac arrhythmias were assessed in anaesthetized animals, there were no major between-group differences in HR, arterial pressure, contractility indices or sensitivity to ß-adrenoceptor stimulation (dobutamine) or blockade (atenolol). The effective refractory period in the control rats, 24 h runners and 2 h runners was 49 ± 2, 55 ± 2 and 60 ± 4 ms, respectively (P = 0.054 for 2 h runners vs controls). A significantly higher dose of aconitine was required to provoke ventricular arrhythmias in the 24 h and 2 h running groups compared with controls (489 ± 76, 505 ± 88 and 173 ± 33 µg, respectively; P < 0.05). 5. We conclude that, in rats, long-term voluntary exercise has enduring cardioprotective effects mediated at the level of both the central nervous system and the heart.

Respiratory pattern in awake rats: effects of motor activity and of alerting stimuli.

Our aim was to assess the impact of motor activity and of arousing stimuli on respiratory rate in the awake rats. The study was performed in male adult Sprague-Dawley (SD, n=5) and Hooded Wistar (HW, n=5) rats instrumented for ECG telemetry. Respiratory rate was recorded using whole-body plethysmograph, with a piezoelectric sensor attached for the simultaneous assessment of motor activity. All motor activity was found to be associated with an immediate increase in respiratory rate that remained elevated for the whole duration of movement; this was reflected by: i) bimodal distribution of respiratory intervals (modes for slow peak: 336+/-19 and 532+/-80 ms for HW and SD, p<0.05; modes for fast peak 128+/-6 and 132+/-7 ms for HW and SD, NS); and ii) a tight correlation between total movement time and total time of tachypnoea, with an R(2) ranging 0.96-0.99 (n=10, p<0001). The extent of motor-related tachypnoea was significantly correlated with the intensity of associated movement. Mild alerting stimuli produced stereotyped tachypnoeic responses, without affecting heart rate: tapping the chamber raised respiratory rate from 117+/-7 to 430+/-15 cpm; sudden side move--from 134+/-13 to 487+/-16 cpm, and turning on lights--from 136+/-12 to 507+/-14 cpm (n=10; p<0.01 for all; no inter-strain differences). We conclude that: i) sniffing is an integral part of the generalized arousal response and does not depend on the modality of sensory stimuli; ii) tachypnoea is a sensitive index of arousal; and iii) respiratory rate is tightly correlated with motor activity.