Eugene Nalivaiko

Chronic stress alters the density and morphology of microglia in a subset of stress responsive brain regions

The current study, in parallel experiments, evaluated the impact of chronic psychological stress on physiological and behavioural measures, and on the activation status of microglia in 15 stress-responsive brain regions. Rats were subjected, for 14 days, to two 30 min sessions of restraint per day, applied at random times each day. In one experiment the effects of stress on sucrose preference, weight gain, core body temperature, and struggling behaviour during restraint, were determined. In the second experiment we used immunohistochemistry to investigate stress-induced changes in ionized calcium-binding adaptor molecule-1 (Iba1), a marker constitutively expressed by microglia, and major histocompatibility complex-II (MHC-II), a marker often expressed on activated microglia, in a total of 15 stress-responsive nuclei. We also investigated cellular proliferation in these regions using Ki67 immunolabelling, to check for the possibility of microglial proliferation. Collectively, the results we obtained showed that chronic stress induced a significant increase in anhedonia, a decrease in weight gain across the entire observation period, a significant elevation in core body temperature during restraint, and a progressive decrease in struggling behaviour within and over sessions. With regard to microglial activation, chronic stress induced a significant increase in the density of Iba1 immunolabelling (nine of 15 regions) and the number of Iba1-positive cells (eight of 15 regions). Within the regions that exhibited an increased number of Iba1-positive cells after chronic stress, we found no evidence of a between group difference in the number of MHC-II or Ki67 positive cells. In summary, these results clearly demonstrate that chronic stress selectively increases the number of microglia in certain stress-sensitive brain regions, and also causes a marked transition of microglia from a ramified-resting state to a non-resting state. These findings are consistent with the view that microglial activation could play an important role in controlling and/or adapting to stress.

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.

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 rabbits 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.

Long-term effects of prenatal stress: Changes in adult cardiovascular regulation and sensitivity to stress

Prenatal environment exerts profound influences on the development of an organism and stressful events during pregnancy can bring about long-term physiological/behavioral alterations in the offspring. Epidemiological evidence points to a relationship between intrauterine growth restriction (IUGR), body weight at birth, and adult cardiovascular disease. Experimental research employed different models of IUGR, including altered maternal nutrition, exposure to elevated glucocorticoids, and reduced placental perfusion, all of which can program, when acting during sensitive temporal windows of foetal life, alterations in cardiovascular regulation and stress sensitivity. Original data are presented indicating that prenatal psychological stress (intermittent restraint) does not induce in the rat adult offspring changes of plasma corticosterone levels, cardiac autonomic modulation, and circadian rhythmicity of heart rate (HR), body temperature (T) and physical activity (Act) at rest. However, prenatally stressed rats--when further stimulated in adulthood--exhibit prolonged adrenocortical stress responsivity, disturbed circadian rhythmicity of HR, T, and Act, and increased adrenal weight. This evidence supports the idea that prenatal stress per se does not change dramatically a given structure or function, but it affects resilience and renders the animal more susceptible to pathophysiological outcomes when further insults occur during adulthood.

Tachykinin neurokinin-1 and neurokinin-3 receptor-mediated responses in guinea-pig substantia nigra: an in vitro electrophysiological study

The effects of tachykinin receptor agonists and antagonists were investigated using intra- and extracellular recordings on spontaneously firing nigral neurons in guinea-pig brain slices. In 70 of 76 electrophysiologically identified dopaminergic neurons, a concentration-dependent increase in firing rate was induced by the selective neurokinin-3 tachykinin agonist senktide and by the natural tachykinin agonists neurokinin B and substance P, with EC50 values of 14.7, 31.2 and 12200 nM respectively. These responses were inhibited in a concentration- and time-dependent manner by the selective non-peptide neurokinin-3 receptor antagonist SR 142801 (1-100 nM; n=23), but neither by its S-enantiomer SR 142806 (100 nM; n=4) nor by selective antagonists of neurokinin-1 (SR 140333) or neurokinin-2 (SR 48968) receptors (both at 100 nM; n=3). The selective neurokinin-1 agonist [Sar9,Met(O2)11]substance P (30-100 nM; n=23) and the selective neurokinin-2 agonist [Nle10]neurokinin A(4-10)(30-100 nM; n=13) were without any effect on dopaminergic cells. In 13 of 21 electrophysiologically identified, presumably GABAergic neurons located in the pars compacta of the substantia nigra, excitatory responses were evoked concentration dependently by substance P and [Sar9,Met(O2)11]substance P, with EC50 values of 18.6 and 41.9 nM respectively. These responses were inhibited by SR 140333 (100 nM; n=3), but neither by its R-enantiomer SR 140603 nor by SR 142801 (both at 100 nM; n=3). Senktide and [Nle10]neurokinin A(4-10) (both at 30-100 nM; n=10) were without effect on these presumed GABAergic neurons. A small population (12%) of pars compacta neurons was insensitive to any of the three selective tachykinin agonists. In the nigral pars reticulata, 12 neurons were recorded which had an electrophysiological profile similar to that of presumed GABAergic neurons in the pars compacta. Of these 12 cells, seven did not respond to any of the selective tachykinin agonists tested, while five were excited by senktide in a concentration-dependent manner (EC50=98.5 nM). Although this value was significantly higher than that found for dopaminergic neurons in the pars compacta, senktide-evoked responses were inhibited by SR 142801 (100 nM; n=3). We conclude that, in the guinea-pig substantia nigra, tachykinins evoke excitatory responses in both dopaminergic and non-dopaminergic neurons; however, the sensitivity to tachykinin agonists (neurokinin-1 versus neurokinin-3) depends on both neuronal type and localization.

Relation between QT interval variability and cardiac sympathetic activity in hypertension

Elevated QT interval variability is a predictor of malignant ventricular arrhythmia, but the underlying mechanisms are incompletely understood. A recent study in dogs with pacing-induced heart failure suggests that QT variability is linked to cardiac sympathetic nerve activity. The aim of this study was to determine whether increased cardiac sympathetic activity is associated with increased beat-to-beat QT interval variability in patients with essential hypertension. We recorded resting norepinephrine (NE) spillover into the coronary sinus and single-lead, short-term, high-resolution, body-surface ECG in 23 patients with essential hypertension and 9 normotensive control subjects. To assess beat-to-beat QT interval variability, we calculated the overall QT variability (QTVN) as well as the QT variability index (QTVi). Cardiac NE spillover (12.2 ± 6.5 vs. 20.7 ± 14.7, P = 0.03) and QTVi (-1.75 ± 0.36 vs. -1.42 ± 0.50, P = 0.05) were significantly increased in hypertensive patients compared with normotensive subjects. QTVN was significantly correlated with cardiac NE spillover (r(2) = 0.31, P = 0.001), with RR variability (r(2) = 0.20, P = 0.008), and with systolic blood pressure (r(2) = 0.16, P = 0.02). Linear regression analysis identified the former two as independent predictors of QTVN. In conclusion, elevated repolarization lability is directly associated with sympathetic cardiac activation in patients with essential hypertension.

Short-term heart rate variability and cardiac norepinephrine spillover in patients with depression and panic disorder

Changes in measures of heart rate variability (HRV) have been associated with an increased risk for sudden cardiac death. The mechanisms underlying this association are not known. The objective of this study was to assess the relationship between the amount of norepinephrine (NE) released from the cardiac sympathetic terminals and short-term HRV. The study comprised 8 healthy subjects, 12 patients with major depression, and 7 patients with panic disorder. Cardiac NE spillover was determined using direct coronary sinus blood sampling coupled with an NE isotope dilution methodology. Short-term HRV was quantified using detrended fluctuation analysis, symbolic dynamics, sample entropy, and standard time and frequency domain measures. Neither HRV nor cardiac NE spillover was significantly different between the analyzed groups. None of the standard HRV metrics was significantly correlated with cardiac NE spillover, but there was a moderate correlation between two complexity measures of HRV (symbolic dynamics) and cardiac NE spillover (patterns with 2 like variations, r = -0.37 and P = 0.05; and patterns with no variations: r = 0.34 and P = 0.06). In conclusion, there is no correlation between standard HRV measures and cardiac NE spillover in humans. Short-term complexity of heart rate is only moderately affected by sympathetic neural outflow. Therefore, the predictive value of most HRV measures for sudden cardiac death may predominantly result from their capacity to capture vagally mediated heart rate modulations.

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.

QT interval variability and cardiac norepinephrine spillover in patients with depression and panic disorder

Suggestions were made that increased myocardial sympathetic activity is reflected by elevated QT variability (dynamic changes in QT interval duration). However, the relationship between QT variability and the amount of norepinephrine released from the cardiac sympathetic terminals is unknown. We thus attempted to assess this relationship. The study was performed in 17 subjects (12 with major depressive disorder and 5 with panic disorder). Cardiac norepinephrine spillover (measured by direct catheter technique coupled with norepinephrine isotope dilution methodology) was assessed before and 4 mo after treatment with selective serotonin reuptake inhibitor (SSRI) antidepressants. The distribution of the cardiac norepinephrine spillover was bimodal, with the majority of patients having values of < or =10 ng/min. There was a positive correlation between cardiac norepinephrine spillover and corrected QT interval (r = 0.7, P = 0.03) but not with any of the QT variability measures. However, in a subgroup of five patients who had high levels of cardiac norepinephrine spillover (>20 ng/min) a tendency for a strong positive correlation with variance of QT intervals (r = 0.9, P = 0.08) was observed. There were significant correlations between the severity of depression and QT variability indexes normalized to the heart rate [QTVi and QT interval/R-R interval (QT/RR) coherence] and between the severity of anxiety and the QT/RR residual and regression coefficient, respectively. Treatment with SSRI antidepressants substantially reduced depression score but did not affect any of the QT variability indexes. We conclude that in depression/panic disorder patients with near-normal cardiac norepinephrine levels QT variability is not correlated with cardiac norepinephrine spillover and is not affected by treatment with SSRI.

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.