Integrity of the dorsolateral periaqueductal grey is essential for the fight-or-flight response, but not the respiratory component of a defense reaction.

Periaqueductal grey is believed to be one of the key structures of the central respiratory stress network. Previous studies established that stimulation of the periaqueductal grey, especially its dorsolateral division (dlPAG), evokes tachypnea as well as increases in other autonomic parameters and motor activity. We investigated the effects of blockade of the dlPAG with GABAA agonist muscimol on respiration during stress and presentation of brief alerting stimuli in conscious unrestrained rats. We found that integrity of the dlPAG is not essential for stress-induced increase in basal/resting respiratory rate or for generation of respiratory responses to brief alerting stimuli. However, blockade of the dlPAG reduced the amount of motor activity and concomitant high-frequency respiratory activity during restraint and the first 5min of novelty stress. We conclude that the integrity of the dlPAG is not essential for generation of respiratory component of the defense reaction, but it mediates expression of the fight-or-flight response including its respiratory component.

Prelimbic prefrontal cortex mediates respiratory responses to mild and potent prolonged, but not brief, stressors.

The prefrontal cortex is one of the key areas of the central mechanism of cardiovascular and respiratory control. Disinhibition of the prelimbic medial prefrontal cortex elicits tachypnoeic responses in anesthetized rats (Hassan et al., J. Physiol. 591: 6069-6088, 2013). The current study examines the effects of inhibition of the prelimbic prefrontal cortex during presentation of stressors of various lengths and intensities in conscious unrestrained rats. 8 Wistar rats were implanted with bilateral guide cannulas targeting the prelimbic prefrontal cortex and received microinjections of either saline of GABAA agonist muscimol prior to recording sessions. Inhibition of the prelimbic prefrontal cortex significantly attenuated respiratory responses to a novel environment stress, 30s light stimulus and restraint stress. It did not affect respiratory responses to 500 ms acoustic stimuli of varying intensities (40-90 dB). We conclude that the prelimbic prefrontal cortex contributes to generation of tachypnoeic responses to prolonged stressors, but does not contribute to respiratory arousal in response to brief stressors.

Ondansetron prevents changes in respiratory pattern provoked by LiCl: a new approach for studying pro-emetic states in rodents?

There are a limited number of biological indices for assessing pro-emetic states in laboratory rodents as they do not possess the vomiting response. In the present study we tested the hypothesis that in rats, pro-emetic intervention would affect the respiratory pattern. To this end, using whole-body plethysmography, in adult male Wistar rats we recorded respiration after i.p. administration of either the emetic agent LiCl or Ringer. Quantification of respiratory signals (from 5 to 35 min post-injection) revealed that post-LiCl, mean respiratory rate was significantly lower (126 ± 9 vs. 178 ± 10 cpm, p < 0.005) and less variable (Kvar 59 ± 8% vs. 73 ± 3%; p<0.05) compared to the post-Ringer condition. Furthermore, while mode values of respiratory rate histograms did not differ between the treatments (indicating that the dominant respiratory frequency remained unchanged), LiCl reduced the fraction of time spent at high respiratory rate (>200 cpm) from 25 ± 3% to 9 ± 2% (p = 0.004). Thus, reduction of the mean respiratory rate by LiCl was predominantly due to reduced contribution of high-frequency breathing that is normally associated with motor activity and/or arousal. Non-linear multifractal analysis of respiratory signals revealed that post-LiCl, respiration becomes less random and more orderly. 5-HT3 antagonist ondansetron prevented respiratory changes elicited by LiCl. We conclude that the observed changes likely reflect effects of LiCl on animals' motion, and that this effect is mediated via 5-HT3 receptors. Providing that the effects observed in our study were quite robust, we suggest that simple and non-invasive respiratory monitoring may be a promising approach for studying emesis in rodents.

Ability of predator odour exposure to elicit conditioned versus sensitised post traumatic stress disorder-like behaviours, and forebrain deltaFosB expression, in rats.

At present, exposure of a rodent to the odour of a predator is one of the most common animal models of post traumatic stress disorder (PTSD). Despite this, the model remains incompletely characterized, particularly in regard to within subject assessment of major PTSD-like behaviours. In an attempt to redress this situation, we have extensively characterized the two broad categories of behaviour that are considered to characterize PTSD, that is sensitized behaviours such as social withdrawal and hypervigilance and conditioned behaviours such as avoidance of trauma linked cues. Specifically, we determined the presence and duration of both conditioned and sensitized behaviours, in the same cohort of animals, after three exposures to predator odour. Conditioned fear was assessed on the basis of inhibition of locomotor activity upon return to context 2, 7, 14, 21, and 28 days after the last odour exposure session. To assess the impact on sensitization behaviours, we monitored acoustic startle responses and social interaction behaviour 4, 9, 16, 23, and 30 days after the last exposure session. In addition to examining the behavioural consequences associated with odour exposure, we also determined the key brain regions that were activated using DeltaFosB immunohistochemistry. Our results show that the two groups of behaviours thought to characterize PTSD (conditioned and sensitized) do not travel together in the predator odour model, with clear evidence of enduring changes in conditioned fear but little evidence of changes in social interaction or acoustic startle. With regard to associated patterns of activity in the brain, we observed that odour-exposed animals exhibited significantly higher numbers of FosB-positive nuclei in only the medial prefrontal cortex (mPFC), a finding that can be viewed as being consistent with the observed behavioural changes.

Functional asymmetry in the descending cardiovascular pathways from dorsomedial hypothalamic nucleus.

Neurons in the dorsomedial hypothalamus (DMH) play a key role in mediating tachycardia elicited by emotional stress. DMH activation by microinjections of the GABA(A) antagonist evokes tachycardia and physiological changes typically seen in experimental stress. DMH inhibition abolishes the tachycardia evoked by stress. Based on anatomic evidences for lateralization in the pathways from DMH, we investigated a possible inter-hemispheric difference in DMH-evoked cardiovascular responses. In anesthetized rats we compared changes in heart rate (HR), renal sympathetic activity (RSNA), mesenteric blood flow (MBF) and tail vascular conductance produced by activation of right (R) and left (L) sides of the DMH. We also evaluated the tachycardia produced by air jet stress after inhibition of R or L DMH. There were always greater increases in RSNA when bicuculline was injected ipsilaterally to the side where these parameters were recorded (average DeltaRSNA: L=+50% and R=+26%; P<0.05). Compared to pre-injection values, right DMH activation caused pronounced decrease (0.87+/-0.1% vs. 0.4+/-0.11%/mm Hg; P<0.05), whereas bicuculline methiodide (BMI) into left DMH produced no significant changes (0.95+/-0.09% vs. 1.04+/-0.25%/mm Hg) in tail vascular conductance. R or L DMH disinhibition produced decreases in MBF, but no differences in the range of these changes were observed. Activation of the right DMH caused greater tachycardia compared to the left DMH activation (average DeltaHR: R=+92 bpm; L=+48 bpm; P<0.05). Tachycardia evoked by air jet stress was smallest after right DMH inhibition (average DeltaHR: R=+57 bpm and L=+134 bpm; P<0.05). These results indicate that the descending cardiovascular pathways from DMH are predominantly lateralized and the right DMH might exert a prominent control on heart rate changes during emotional stress.

Blockade of 5-HT2A receptors suppresses hyperthermic but not cardiovascular responses to psychosocial stress in rats.

The aim of this study was to determine whether 5-HT2A receptors mediate cardiovascular and thermogenic responses to acute psychological stresses. For this purpose, adult male Wistar hooded rats instrumented for telemetric recordings of either electrocardiogram (ECG) (n=12) or arterial pressure (n=12) were subjected, on different days, to four 15-min episodes of social defeat. Prior to stress, animals received s.c. injection of the selective 5-HT2A receptor antagonist SR-46349B (trans-4-((3Z)3-[(2-dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate) (at doses of 0.3, 1.0 and 3.0 mg/kg) or vehicle. The drug had no effect on basal heart rate or heart rate variability indexes, arterial pressure, and core body temperature. Social defeat elicited significant and substantial tachycardic (347+/-7 to 500+/-7 bpm), pressor (77+/-4 to 97+/-4 mm Hg) and hyperthermic (37.0+/-0.3 to 38.5+/-0.1 degrees C) responses. Blockade of 5-HT2A receptors, at all doses of the antagonist, completely prevented stress-induced hyperthermia. In contrast, stress-induced cardiovascular responses were not affected by the blockade (except small reduction of tachycardia by the highest dose of the drug). We conclude that in rats, 5-HT2A receptors mediate stress-induced hyperthermic responses, but are not involved in the genesis of stress-induced rises in heart rate or arterial pressure, and do not participate in cardiovascular control at rest.

Intra-amygdala injection of GABAA agonist, muscimol, reduces tachycardia and modifies cardiac sympatho-vagal balance during restraint stress in rats.

At present, little is known about the brain origin of stress-induced cardiac sympathetic drive responsible for stress-induced tachycardia. Our aim was to determine the effect of bilateral microinjections of the GABA(A) receptor agonist, muscimol, into the amygdaloid complex on both the heart rate and cardiac autonomic activity during restraint stress. Experiments were performed in male Sprague-Dawley rats (n=9), with pre-implanted electrocardiographic electrodes. Heart rate increased sharply after the onset of the restraint and reached a peak 1-2 min later (from 344+/-6-440+/-20 BPM). Subsequently, heart rate began to fall, and during the next 10-15 min approached the steady-state level of 384+/-11. After vehicle, mean heart rate during each of three 10-min restraint epochs was significantly higher compared with the pre-restraint level. After muscimol, mean heart rate was significantly elevated only during the first 10 min of restraint. There was no difference in the early peak tachycardia between both conditions. Muscimol substantially accelerated the fall of the HR from the peak to the steady-state level, and thus the area under the curve value for muscimol (503+/-162 BPM x min) was significantly smaller than that for vehicle (1221+/-231 BPM x min); P<0.05. After vehicle, the high-frequency spectral power of the heart rate decreased and the low-frequency power increased during the restraint, resulting in a significant rise of the low frequency/high frequency ratio from 1.2+/-0.2-2.8+/-0.6 (n=9, P<0.05). Muscimol suppressed these stress-induced effects. We conclude that inhibition of the amygdala neurons abolishes the sustained component of tachycardia during the restraint, has no effect on the early tachycardic component, and prevents stress-induced alterations in the heart rate variability indices.

The yin and yang of cardiac autonomic control: vago-sympathetic interactions revisited.

We review the pattern of activity in the parasympathetic and sympathetic nerves innervating the heart. Unlike the conventional textbook picture of reciprocal control of cardiac vagal and sympathetic nervous activity, as seen during a baroreceptor reflex, many other reflexes involve simultaneous co-activation of both autonomic limbs. Indeed, even at 'rest', the heart receives tonic drives from both sympathetic and parasympathetic cardiac nerves. Autonomic co-activation occurs during peripheral chemoreceptor, diving, oculocardiac, somatic nociceptor reflex responses as well as being evoked from structures within the brain. It is suggested that simultaneous co-activation may lead to a more efficient cardiac function giving greater cardiac output than activation of the sympathetic limb alone; this permits both a longer time for ventricular filling and a stronger contraction of the myocardium. This may be important when pumping blood into a constricted vascular tree such as is the case during the diving response. We discuss that in some instances, high drive to the heart from both autonomic limbs may also be arrhythmogenic.

CRF1 receptor antagonist CP-154,526 reduces cardiovascular responses during acute psychological stress in rabbits.

We examined the effect of CP-154,526 on cardiovascular changes elicited in conscious rabbits by stressful stimuli (loud sound, cage move, pinprick, formaldehyde vapour and air-jet stress). CP-154,526 substantially reduced pressor and heart rate responses to these stimuli (both vagally and sympathetically mediated), and reduced QT shortening during air-jet stress. Blocking of central CRF1 receptors attenuates cardiovascular responses to environmental stimuli, presumably by affecting brain centres that control cardiovascular functions.

Electrocardiographic changes associated with the nasopharyngeal reflex in conscious rabbits: vago-sympathetic co-activation.

Electrocardiographic responses were assessed in conscious rabbits when the nasopharyngeal reflex was elicited by inhalation of formaldehyde vapour. There was a profound fall in heart rate (224+/-5 to 64+/-4 beats per min (bpm)) associated with abnormal or absent P-waves. There were no changes in the QRS complex. The R-T interval (control value 118+/-4 ms) was initially shortened to 107+/-3 ms and then prolonged to 130+/-4 ms. Heart rate and P-wave changes were prevented by muscarinic cholinergic blockade with methylscopolamine. The R-T shortening was reduced by 79+/-4% by beta-adrenergic blockade with propranolol. Methylscopolamine also unmasked small tachycardic responses (5-25 bpm) in 5/7 animals. This tachycardia was prevented by propranolol. Thus both parasympathetic vagal cardiac nerves and sympathetic cardiac nerves are activated during the nasopharyngeal reflex, with increased vagal effects in the sino-atrial node, and increased sympathetic effects in the ventricular myocardium.

CRF1-receptor antagonist CP-154526 reduces alerting-related cutaneous vasoconstriction in conscious rabbits.

Cutaneous vasoconstrictor responses elicited by salient stimuli in conscious rabbits may be a sensitive physiological index of emotional arousal/anxiety. Ear-pinna blood flow was measured by preimplanted laser Doppler probes, and animals were exposed to situations involving different types of potentially salient stimuli before and after i.v. administration of CP-154526 (15 mg/kg) or diazepam (4 mg/kg). At rest, ear-pinna blood flow was stable (coefficient of varition=11+/-2) and remained at high level 93+/-13% of test time. Exposure to novel environment elicited flow fluctuations (coefficient of variation=79+/-8) and reduced amount of time spent at high level to 25+/-6%. Defined unconditioned stimuli caused rapid falls in ear-pinna flow, with nociceptive stimulation producing more vigorous and consistent effects (flow response index 0.66+/-0.02) compared with non-nociceptive (flow response index 0.49+/-0.04). CP-154526 slightly raised mean arterial pressure (from 81+/-2 to 93+/-3 mmHg), increased heart rate (from 198+/-1 to 220+/-4 beats/min) and produced a mild vasoconstriction in the ear-pinna bed (flow fell from 46+/-10 to 25+/-6 cm/s). CP-154526 substantially reduced cutaneous vasoconstrictor responses elicited by the exposure to novel environment and by defined non-nociceptive stimuli, with flow-response index fall from 0.53+/-0.10 to 0.17+/-0.09 and from 0.47+/-0.04 to 0.24+/-0.04, respectively, without affecting responses to nociceptive stimuli. Diazepam reduced only vasoconstrictor responses elicited by the exposure to novel environment, with flow-response index fall from 0.40+/-0.12 to 0.27+/-0.07. Sensitivity of rapid changes in rabbit ear-pinna blood flow to anxiolytic drugs supports the idea that increased cutaneous vascular tone reflects enhanced arousal in rabbits.

Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats.

In urethane-anesthetized rats with body temperature maintained at 39-40 degrees C, electrical stimulation of raphe magnus/pallidus/parapyramidal region within 0.5 mm of the ventral medullary surface reduced arterial blood flow to the tail cutaneous bed (measured with a chronically implanted Doppler ultrasonic flowmeter) from 28+/-5 to 6+/-1 cm/s (P<0.01), without changing mesenteric arterial blood flow, and with only small, variable changes in arterial pressure. Injection of bicuculline (50 pmol in 50 nl) at the same site reduced tail flow from 19+/-2 to 3+/-1 cm/s (P<0.01), again without significantly changing mesenteric flow, but with a moderate increase in arterial pressure. When the rat was cooled to reduce basal tail blood flow, injection of muscimol (1 nmol in 100 nl) or GABA (100 nmol in 100 nl) into the raphe site restored tail blood flow to 93+/-4% of the pre-cooling level. These recordings are the first reported direct measurements of rat tail blood flow changes elicited by alteration of neuronal function in the brainstem. The rostral medullary raphe controls the tail cutaneous vascular bed in a relatively selective manner. Our findings add to evidence that raphe magnus/pallidus/parapyramidal neurons are involved in regulating cutaneous blood flow in response to changes in body temperature in the rat.

Tail artery blood flow measured by chronically implanted Doppler ultrasonic probes in unrestrained conscious rats.

We describe a surgical procedure for chronically implanting a Doppler ultrasonic probe around the tail artery of the rat to measure phasic flow velocity in the tail artery of the unrestrained conscious rat. The phasic tail flow signal is highly correlated with the simultaneously recorded superior mesenteric flow signal (range 0.70-0.89 in seven rats) during vasoconstriction induced by exposure to formaldehyde vapour. In response to two quick alerting taps on the cage, tail flow velocity fell from 20+/-2 to 7+/-1 cm/s (P<0.01) and mesenteric flow fell from 30+/-5 to 25+/-4 cm/s (P<0.05), with the fall in tail flow being significantly greater than the fall in mesenteric flow (P<0.05, n=7 rats). In anesthetized rats, the phasic tail flow signal was highly correlated with phasic arterial pressure (range 0.71-0.83 in seven rats). The ability to reliably measure phasic arterial tail flow in the conscious unrestrained rat should facilitate experimental studies of brain pathways regulating flow to this principally cutaneous vascular bed in different physiological situations.

Raphe region mediates changes in cutaneous vascular tone elicited by stimulation of amygdala and hypothalamus in rabbits.

Raphe pallidus/parapyramidal neurons control cutaneous vasoconstriction induced by noxious stimuli. To determine whether they mediate forebrain-induced cutaneous vasoconstriction, we assessed changes in ear pinna blood flow elicited by electrical stimulation of amygdala and hypothalamus before and after injection of muscimol into the raphe/parapyramidal region. We compared ear flow with simultaneously recorded mesenteric flow. Experiments were performed in rabbits anesthetized with urethane (1.25-1.5 g/kg), paralysed and mechanically ventilated. Amygdala stimulation reduced skin conductance from 0.32+/-0.06 to 0.10+/-0.02 cm/s per mmHg (P<0.05, n=9), without effect on mesenteric conductance. Hypothalamic stimulation caused vasoconstriction in both cutaneous and mesenteric beds (conductances fell from 0.27+/-0.05 to 0.05+/-0.02 cm/s per mmHg and from 0.27+/-0.06 to 0.14+/-0.04 cm/s per mmHg (P<0.05, n=9), respectively). Muscimol microinjection (5 nmol in 100 nl) to raphe/parapyramidal region eliminated amygdala- and hypothalamus-induced skin vasoconstriction (pre-stimulus conductance 0.42+/-0.13 and 0.41+/-0.11 cm/s per mmHg, post-stimulus 0.41+/-0.12 and 0.39+/-0.10 cm/s per mmHg, respectively), but not hypothalamically-induced mesenteric vasoconstriction (pre-stimulus 0.29+/-0.06, post-stimulus 0.16+/-0.03 cm/s per mmHg, P<0.05, n=8). The latter was strongly attenuated by bilateral injection of muscimol to the rostral ventrolateral medulla. Data suggest that descending hypothalamo-spinal and amygdala-spinal pathways constricting the cutaneous vascular bed relay in the raphe/parapyramidal area. A relay in the rostral ventrolateral medulla contributes substantially to mesenteric vasoconstriction elicited from the hypothalamus.

Cutaneous vascular bed is not involved in arterial pressure changes elicited by increasing or decreasing the activity of inhibitory vasomotor neurons in caudal ventrolateral medulla in rabbits.

We determined whether caudal ventrolateral medulla (CVLM) vasodepressor neurons tonically inhibit vasomotor tone in the ear in anesthetized rabbits. Injection of L-glutamate (10 nmol in 100 nl) into the CVLM decreased arterial pressure and increased superior mesenteric conductance. Ear conductance decreased (0.43+/-0.06 to 0. 33+/-0.05 cm s(-1) per mmHg, n=15 injections, 12 rabbits, P<0.01). Conversely, bilateral injection of gamma-aminobutyric acid (100 nmol in 100 nl) increased arterial pressure and decreased superior mesenteric conductance. At the same time ear conductance increased (0.39+/-09 to 0.48+/-0.27 cm s(-1) per mmHg, n=8 injections, eight rabbits, P<0.05). Results suggest that ear vessels are not tonically inhibited by the CVLM vasodepressor neurons. Presympathetic motoneurons regulating cutaneous flow may be excited, rather than inhibited, by the CVLM neurons.

Regional blood flow and nociceptive stimuli in rabbits: patterning by medullary raphe, not ventrolateral medulla.

1. Regional blood flow was measured with Doppler ultrasonic probes in anaesthetized rabbits. We used focal microinjections of pharmacological agents to investigate medullary pathways mediating ear pinna vasoconstriction elicited by electrical stimulation of the spinal tract of the trigeminal nerve or by pinching the lip, and pathways mediating mesenteric vasoconstriction elicited by electrical stimulation of the afferent abdominal vagus nerve. 2. Bilateral injection of kynurenate into the rostral ventrolateral medulla reduced arterial pressure and prevented the mesenteric vasoconstriction and the rise in arterial pressure elicited by abdominal vagal stimulation. However, kynurenate did not prevent ear pinna vasoconstriction or the fall in pressure elicited by trigeminal tract stimulation. Similar injections of muscimol also failed to prevent the trigeminally elicited cardiovascular changes. 3. Injections of kynurenate into the raphe-parapyramidal area did not diminish trigeminally elicited ear vasoconstriction or the depressor response. However, injections of muscimol substantially reduced or abolished the trigeminally elicited ear vasoconstriction, without affecting the depressor response. Raphe-parapyramidal muscimol injections also entirely abolished ear vasoconstriction elicited by pinching the rabbit's lip. 4. The trigeminal depressor response does not depend on either the rostral ventrolateral medulla or the raphe-parapyramidal region. 5. Mesenteric vasoconstriction elicited by stimulation of the afferent abdominal vagus nerve is mediated via the rostral ventrolateral medulla, but ear vasoconstriction elicited by lip pinch or by stimulation of the trigeminal tract is mediated by the raphe-parapyramidal region. Our study is the first to suggest a brainstem pathway mediating cutaneous vasoconstriction elicited by nociceptive stimulation.

Synchronous changes in ear and tail blood flow following salient and noxious stimuli in rabbits.

Simultaneous recordings were made of ear and tail blood flow during alerting responses to salient environmental stimuli in conscious rabbits, and during electrical stimulation of the spinal trigeminal tract and raphe pallidus in anesthetized rabbits. Blood flow fell in a highly correlated manner (Pearson coefficient ranging from 0.52 to 0.95) in these experimental situations. Salient stimuli in conscious rabbits, and noxious stimuli in anesthetized rabbits appear to cause a generalized vasoconstriction in cutaneous beds.

Raphe pallidus and parapyramidal neurons regulate ear pinna vascular conductance in the rabbit.

We have determined whether alteration of neuronal function in raphe pallidus and the parapyramidal region alters ear blood flow, measured by an implanted Doppler ultrasonic probe, in anesthetized rabbits. Injection of GABA (5 nmol in 50 nl) increased ear flow from 6.0 +/- 1.0 to 31 +/- 10 kHz, without changing arterial pressure or heart rate. Focal electrical stimulation of raphe pallidus at low current amplitude caused ear pinna blood flow to fall from 41 +/- 6 to 9 +/- 3 kHz, again with little or no change in arterial pressure. These excitatory and inhibitory stimuli did not affect superior mesenteric blood flow. The fall in ear flow in response to electrical stimulation of raphe pallidus was not prevented by tetrodotoxin-mediated inhibition of the rostral ventrolateral medulla. Thus raphe pallidus and parapyramidal region may regulate ear pinna vascular conductance via a direct spinal projection.