Targeting the lateral hypothalamus with short hairpin RNAs reduces habitual behaviour following extended instrumental training in rats.

Instrumental actions are initially goal-directed but with repeated performance can become habitual. Habitual actions are adaptive, learned behaviours that are automated in order to reduce cognitive load and to allow for efficient interaction with the environment. Goal-directed and habitual actions are mediated by distinct neurocircuits which centre on the dorsal striatum and involve different cortical and limbic inputs. The lateral hypothalamus (LH) has yet to be considered in this neurocircuitry despite its anatomical connections with these neurocircuits and its established role in motivated behaviour. The aim of the current study was to determine whether the LH has a role in the development of habitual actions in rats by knocking down protein expression in the LH with short hairpin RNAs (shRNA). Two shRNAs were utilised, both of which were shown to reduce the expression of two neuropeptides within the LH, orexin and melanin-concentrating hormone, compared to a saline-vehicle control. This was unexpected given that one shRNA was a control vector (i.e, scrambled sequence), and the other shRNA was supposed to selectively target orexin's precursor protein. Given this lack of specificity and that shRNA's are known to be neurotoxic, the current study examined the impact of non-selective dysfunction of the LH on habitual actions. Adult male Long-Evans rats were trained to press a lever for a food outcome and were tested for goal directed and habitual behaviour following devaluation of the food. The shRNA groups displayed goal-directed actions following moderate instrumental training, but did not develop habitual actions following extended training. That is, control rats developed the expected habitual behaviour where lever-response rates were insensitive to outcome value when tested, whilst the shRNA groups reduced rates of responding on the lever under devalued conditioned and hence remained goal-directed. This failure to demonstrate habitual actions was unlikely to be secondary to changes in motivation or arousal as the shRNA groups did not show altered food consumption, body weight, lever response rates, or motor performance on a rota rod or tapered balance beam. However, locomotor activity was reduced in an open field test, consistent with the proposed role of the LH in spontaneous locomotor activity. Therefore, this study implicates the LH in habitual learning, and adds to the emerging evidence that the LH has a role in associative learning processes. This finding has implications for human conditions where there is dysfunction or neurodegeneration in the LH, as well as altered habitual actions, such as in Parkinson's disease and drug addiction.

Regulatory T Cells and Their Derived Cytokine, Interleukin-35, Reduce Pain in Experimental Autoimmune Encephalomyelitis.

Sensory problems such as neuropathic pain are common and debilitating symptoms in multiple sclerosis (MS), an autoimmune inflammatory disorder of the CNS. Regulatory T (Treg) cells are critical for maintaining immune homeostasis, but their role in MS-associated pain remains unknown. Here, we demonstrate that Treg cell ablation is sufficient to trigger experimental autoimmune encephalomyelitis (EAE) and facial allodynia in immunized female mice. In EAE-induced female mice, adoptive transfer of Treg cells and spinal delivery of the Treg cell cytokine interleukin-35 (IL-35) significantly reduced facial stimulus-evoked pain and spontaneous pain independent of disease severity and increased myelination of the facial nociceptive pathway. The effects of intrathecal IL-35 therapy were Treg-cell dependent and associated with upregulated IL-10 expression in CNS-infiltrating lymphocytes and reduced monocyte infiltration in the trigeminal afferent pathway. We present evidence for a beneficial role of Treg cells and IL-35 in attenuating pain associated with EAE independently of motor symptoms by decreasing neuroinflammation and increasing myelination.SIGNIFICANCE STATEMENT Pain is a highly prevalent symptom affecting the majority of multiple sclerosis (MS) patients and dramatically affects overall health-related quality of life; however, this is a research area that has been largely ignored. Here, we identify for the first time a role for regulatory T (Treg) cells and interleukin-35 (IL-35) in suppressing facial allodynia and facial grimacing in animals with experimental autoimmune encephalomyelitis (EAE). We demonstrate that spinal delivery of Treg cells and IL-35 reduces pain associated with EAE by decreasing neuroinflammation and increasing myelination independently of motor symptoms. These findings increase our understanding of the mechanisms underlying pain in EAE and suggest potential treatment strategies for pain relief in MS.

Effects of chemogenetic excitation or inhibition of the ventrolateral periaqueductal gray on the acquisition and extinction of Pavlovian fear conditioning.

The midbrain periaqueductal gray (PAG) has been implicated in the generation and transmission of a prediction error signal that instructs amygdala-based fear and extinction learning. However, the PAG also plays a key role in the expression of conditioned fear responses. The evidence for a role of the PAG in fear learning and extinction learning has been obtained almost exclusively using PAG-dependent fear responses. It is less clear whether the PAG regulates fear learning when other measures of learned fear are used. Here we combined a chemogenetic approach, permitting excitation or inhibition of neurons in the ventrolateral PAG (VLPAG), with conditioned suppression as the measure of learned fear to assess the role of VLPAG in the acquisition and extinction of fear learning. We show that chemogenetic excitation of VLPAG (with some encroachment on lateral PAG [LPAG]) impairs acquisition of fear and, conversely, chemogenetic inhibition impairs extinction of fear. These effects on fear and extinction learning were specific to the combination of DREADD expression and injection of CNO because they were observed relative to both eYFP controls injected with CNO as well as DREADD expressing controls injected with vehicle. Taken together, these results show that activity of L/VLPAG neurons regulates both the acquisition and extinction of Pavlovian fear learning.

The respiratory control of carbon dioxide in children and adolescents referred for treatment of psychogenic non-epileptic seizures.

Psychogenic non-epileptic seizures (PNES) are a common problem in paediatric neurology and psychiatry that can best be understood as atypical responses to threat. Threats activate the body for action by mediating increases in arousal, respiration, and motor readiness. In previous studies, a range of cardiac, endocrine, brain-based, attention-bias, and behavioral measures have been used to demonstrate increases in arousal, vigilance, and motor readiness in patients with PNES. The current study uses respiratory measures to assess both the motor readiness of the respiratory system and the respiratory regulation of CO2. Baseline respiratory rates during clinical assessment and arterial CO2 levels during the hyperventilation component of routine video electroencephalogram were documented in 60 children and adolescents referred for treatment of PNES and in 50 controls. Patients showed elevated baseline respiratory rates [t(78) = 3.34, p = .001], with 36/52 (69%) of patients [vs. 11/28 (39%) controls] falling above the 75th percentile (χ 2 = 6.7343; df = 1; p = .009). Twenty-eight (47%) of patients [vs. 4/50 (8%) controls] showed a skewed hyperventilation-challenge profile-baseline PCO2 <36 mmHg, a trough PCO2 ≤ 20 mmHg, or a final PCO2 <36 mmHg after 15 min of recovery-signaling difficulties with CO2 regulation (χ 2 = 19.77; df = 1; p < .001). Children and adolescents with PNES present in a state of readiness-for-action characterized by high arousal coupled with activation of the respiratory motor system, increases in ventilation, and a hyperventilation-challenge profile shifted downward from homeostatic range. Breathing interventions that target arousal, decrease respiratory rate, and normalize ventilation and arterial CO2 may help patients shift brain-body state and avert PNES episodes.

A Split-Luciferase Reporter Recognizing GFP and mCherry Tags to Facilitate Studies of Protein-Protein Interactions.

The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein-protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins. By using nanobodies and performing tests in cell-free expression systems, we accelerated the development of multiple reporters, detecting heterodimers and homodimers or oligomers tagged with GFP or mCherry. We demonstrate that the system can detect interactions at a broad range of concentrations, from low nanomolar up to micromolar.

Long-lasting bradypnea induced by repeated social defeat.

Repeated social defeat in the rat induces long-lasting cardiovascular changes associated with anxiety. In this study, we investigated the effects of repeated social defeat on breathing. Respiratory rate was extracted from the respiratory sinus arrhythmia (RSA) peak frequency of the ECG in rats subjected to social defeat for 4 consecutive days. Respiratory rate was recorded under anesthesia 6 days (D+10) or 26 days (D+30) after social defeat. At D+10, defeated (D) rats spent less time in the open arms of the elevated plus maze test, had heavier adrenal glands, and displayed bradypnea, unlike nondefeated animals. At D+30, all signs of anxiety had disappeared. However, one-half of the rats still displayed bradypnea (DL rats, for low respiratory rate indicated by a lower RSA frequency), whereas those with higher respiratory rate (DH rats) had recovered. Acute blockade of the dorsomedial hypothalamus (DMH) or nucleus tractus solitarii (NTS) 5-HT3 receptors reversed bradypnea in all D rats at D+10 and in DL rats at D+30. Respiratory rate was also recorded in conscious animals implanted with radiotelemetric ECG probes. DH rats recovered between D+10 and D+18, whereas DL rats remained bradypneic until D+30. In conclusion, social stress induces sustained chronic bradypnea mediated by DMH neurons and NTS 5-HT3 receptors. These changes are associated with an anxiety-like state that persists until D+10, followed by recovery. However, bradypnea may persist in one-half of the population up until D+30, despite apparent recovery of the anxiety-like state.

Spontaneously hypertensive rats have more orexin neurons in their medial hypothalamus than normotensive rats.

NEW FINDINGS: What is the central question of this study? Blockade of orexin receptors reduces blood pressure in spontaneously hypertensive rats (SHRs) but not in normotensive Wistar-Kyoto (WKY) rats, suggesting that upregulation of orexin signalling underlies the hypertensive phenotype of the SHR. However, it is not known what causes this upregulation. What is the main finding and its importance? Using orexin immunolabelling, we show that SHRs have 20% more orexin neurons than normotensive WKY and Wistar rats in the medial hypothalamus, which is a good match to their blood pressure phenotype. In contrast, there is no such match for the orexin neurons of the lateral hypothalamus. Essential hypertension may be linked to an increase in orexin neurons in the medial hypothalamus. The neuropeptide orexin contributes to the regulation of blood pressure as part of its role in the control of arousal during wakefulness and motivated behaviour (including responses to psychological stress). Recent work shows that pharmacological blockade of orexin receptors reduces blood pressure in spontaneously hypertensive rats (SHRs) but not in normotensive Wistar-Kyoto (WKY) rats. It is not clear why orexin signalling is upregulated in the SHR, but one possibility is that these animals have more orexin neurons than their normotensive WKY and Wistar relatives. To test this possibility, SHRs, WKY and Wistar male rats (6-16 weeks old) were killed, perfused and their brains sectioned and immunolabelled for orexin A. Labelled neurons were plotted and counted in the six best labelled hemisections (120 µm apart) of each brain. There were significantly more orexin neurons (+20%) in the medial hypothalamus (medial to fornix) of SHRs compared with WKY and Wistar rats (126 ± 4 versus 106 ± 5 and 104 ± 5 per hemisection, respectively, P < 0.05), which matches well the blood pressure phenotypes. In contrast, counts in the lateral hypothalamus did not match the blood pressure phenotypes (69 ± 2 versus 50 ± 3 and 76 ± 4, respectively). The results support the idea that orexin signalling is upregulated in the SHR and suggest that this is due, at least in part, to a greater number of orexin neurons in the medial hypothalamus. These medial orexin neurons, which are also involved in hyperarousal and stress responses, may contribute to the development of essential hypertension.

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 orexin receptors with Almorexant reduces cardiorespiratory responses evoked from the hypothalamus but not baro- or chemoreceptor reflex responses.

Orexin neurons form a restricted group in the dorsal hypothalamus. The group is centered on the perifornical area within the classic hypothalamic defense area, an area which when activated produces marked cardiovascular and respiratory effects. Central administration of orexin can produce cardiorespiratory effects, but the extent to which orexin contributes to such responses evoked from the perifornical hypothalamus is not clear. To determine this, we used the dual orexin receptor antagonist Almorexant to challenge the cardiorespiratory effects evoked by disinhibition of the perifornical hypothalamus. Bicuculline (10 and 20 pmol) was microinjected in the perifornical area before and after administration of Almorexant (15 mg/kg iv) or vehicle in urethane-anesthetized rats. Almorexant significantly reduced the pressor, tachycardic, renal sympathoexcitatory, and tachypneic responses to bicuculline (10 pmol, by 55%, 53%, 28%, 77%; 20 pmol, by 54%, 27%, 51%, 72%, respectively). Reductions of similar magnitude were observed with bicuculline microinjections centered on more caudal sites just peripheral to the orexin neuron group, which would likely have activated fewer orexin neurons. In contrast, Almorexant had no effect on the cardiorespiratory response of the chemoreflex (sodium cyanide injection) or the sympathetic component of the baroreflex. Thus orexin makes a major contribution to the cardiorespiratory response evoked from the perifornical area even though orexin neurons represent only a fraction of the output of this area. Orexin neurons may also mediate cardiorespiratory responses from non-orexin neurons in the caudal hypothalamus. However, under resting conditions, blockade of orexin receptors does not affect the chemo- and baroreflexes.

Behavioural and cardiovascular effects of orexin-A infused into the central amygdala under basal and fear conditions in rats.

The neuropeptide orexin-A (OX-A) has diverse functions, including maintaining arousal, autonomic control, motor activity and stress responses. These functions are regulated at different terminal regions where OX-A is released. The current study examined the physiological and behavioural effects of OX-A microinjections into the central amygdala (CeA) under basal and stressed conditions in rats. When OX-A was microinjected into the CeA and the animals returned to the home-cage, heart rate and mean arterial pressure were increased compared to vehicle-injected controls. General activity of the animal was also increased, indicating that OX-A activity in CeA contributes to increased arousal. This outcome is similar to the effects of central intracerebroventricular infusions of OX-A, as well as the cardiovascular effects previously demonstrated at many of OX's efferent hypothalamic and brainstem structures. In a second study, animals were fear-conditioned to a context by delivery of electric footshocks and then animals were re-exposed to the conditioned context at test. When OX-A was microinjected at test, freezing behaviour was reduced and there was a corresponding increase in the animal's activity but no impact on the pressor and cardiac responses (i.e, blood pressure and heart rate were unchanged). This reduction in freezing suggests that OX-A activates amygdala neurons that inhibit freezing, which is similar to the actions of other neuropeptides in the CeA that modulate the appropriate defence response to fearful stimuli. Overall, these data indicate that the CeA is an important site of OX-A modulation of cardiovascular and motor activity, as well as conditioned freezing responses.

Social defeat: Vagal reduction and vulnerability to ventricular arrhythmias.

Previously, a sub-population of defeated anesthetized rats (Dlow) was characterized by persistent low blood levels of brain-derived neurotrophic factor (BDNF) at day 29 and autonomic alteration at day 30 after social challenge, while the other population (Dhigh) was similar to non-defeated (ND) animals. The aims of this study were to determine the time-course of autonomic dysfunction in awake animals, and whether Dhigh and/or Dlow were vulnerable to cardiac events. Defeated animals were exposed to four daily episodes of social defeats from day 1 to day 4. At day 30, anesthetized Dlow displayed decreased experimental and spontaneous reflex responses reflecting lower parasympathetic efficiency. In addition, Dlow but not Dhigh were characterized by left ventricular hypertrophy at day 30. Telemetric recordings revealed that Dlow had increased low frequency-to-high frequency ratio (LF/HF) and diastolic (DBP) and systolic (SBP) blood pressure, associated with decreased HF and spontaneous baroreflex responses (BRS) from day 3 to day 29. LF/HF, DBP and SBP recovered at day 5, and HF and BRS recovered at day 15 in Dhigh. Ventricular premature beats (VPBs) occurred in Dlow and Dhigh animals from day 5. Time course of VBP fluctuations in Dhigh mirrored that of HF and BRS, but not that of LF/HF, DBP and SBP. These results suggest that a psychosocial stress associated to low serum BDNF levels can lead to vulnerability to persistent autonomic dysfunction, cardiac hypertrophy and ventricular ectopic beats. The parasympathetic recovery seen in Dhigh may provide protection against cardiac events in this population.

Mitochondrial uncoupler BAM15 reverses diet-induced obesity and insulin resistance in mice.

Obesity is a health problem affecting more than 40% of US adults and 13% of the global population. Anti-obesity treatments including diet, exercise, surgery and pharmacotherapies have so far failed to reverse obesity incidence. Herein, we target obesity with a pharmacotherapeutic approach that decreases caloric efficiency by mitochondrial uncoupling. We show that a recently identified mitochondrial uncoupler BAM15 is orally bioavailable, increases nutrient oxidation, and decreases body fat mass without altering food intake, lean body mass, body temperature, or biochemical and haematological markers of toxicity. BAM15 decreases hepatic fat, decreases inflammatory lipids, and has strong antioxidant effects. Hyperinsulinemic-euglycemic clamp studies show that BAM15 improves insulin sensitivity in multiple tissue types. Collectively, these data demonstrate that pharmacologic mitochondrial uncoupling with BAM15 has powerful anti-obesity and insulin sensitizing effects without compromising lean mass or affecting food intake.

Hypocretin/orexin contributes to the expression of some but not all forms of stress and arousal.

Hypocretin/orexin has a well-established role in wakefulness and in the maintenance of arousal. Because stress is associated with arousal, it has been proposed that hypocretin is also involved in stress. However, it is not clear if this is true for all forms of stress. To clarify this issue, we compared four conditions combining high arousal with no or low stress (wakefulness and exploration) or high stress (contextual fear and restraint) in the rat. We looked at Fos expression in hypocretin neurons, hypocretin-1 levels in cerebrospinal fluid and cardiovascular and behavioural changes after pharmacological blockade with the dual hypocretin receptor antagonist, almorexant. Fos expression in hypocretin neurons was highest with wakefulness and exploration, also high with fear but not significant with restraint. Hypocretin-1 levels were consistent with this pattern, although the differences were not as marked. Hypocretin receptor blockade with almorexant reduced the pressor, tachycardic and locomotor responses of wakefulness and exploration as well as the pressor and sympathetic component of the tachycardic response of fear. In contrast, almorexant did not reduce the pressor and tachycardic responses of restraint and nor did it reduce the pressor, tachycardic and locomotor responses of another stressor, i.e. cold exposure. Thus, hypocretin is not involved in all forms of stress. Comparison of the different conditions suggests that, regardless of stress, hypocretin involvement occurs when the arousal associated with the response includes increased attention to environmental cues. When it does, hypocretin will at least contribute to the cardiovascular response. The findings are of clinical relevance to some forms of psychological stress.

Inhibition of the cardiovascular response to stress by systemic 5-HT1A activation: sympathoinhibition or anxiolysis?

5-HT(1A) agonists given systemically are known to produce anxiolytic effects. In addition, a growing body of research is showing that those compounds also have central sympathoinhibitory properties. Since emotional arousal gives rise to sympathetic activation, it is not clear whether systemic treatment with a 5-HT(1A) agonist reduces the sympathetic response to emotional stress primarily by a direct action on sympathetic-related sites in the brain or indirectly through reducing anxiety. To test this, we compared the effect of intraperitoneal injections of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.05 and 0.25 mg/kg), a preferential 5-HT(1A) agonist, or vehicle on the cardiovascular responses to four stressors known to produce sympathetic activation, three being emotional stressors, and one physiological. In conscious rats, 30-min exposure to either a neutral context, a fear-conditioned context, or to restraint stress led to increases in heart rate and blood pressure, which were attenuated by 8-OH-DPAT. In contrast, the same treatment did not reduce the cardiovascular response to 30-min cold exposure (4 degrees C). The results suggest that 8-OH-DPAT acts preferentially on limbic, rather than central, autonomic sites. Hence, doses of 5-HT(1A) agonists, which are just sufficient to produce anxiolysis, are not enough to cause true sympathoinhibition.

Local response to cold in rat tail after spinal cord transection.

Subjects with severe chronic spinal cord injury (SCI) are prone to hypothermia when they are exposed to relatively low environmental temperatures that are normally well tolerated by healthy individuals. This impaired thermoregulation is presumably due to disconnection of territories below the SCI from supraspinal thermoregulatory centers. However, it is not known how these territories respond to low temperatures. Using a complete transection at T(11) in rats, we examined the responses of the tail to cold (6-9 degrees C) by measuring changes in tail blood flow and skin temperature weekly for 8 wk after SCI. Despite no significant change in baseline mean flow or temperature in the tail, the transection effectively removed the sympathetically mediated supraspinal control of the tail vasculature, since the amplitude of the pulse flow was markedly increased and the natural variations of the mean flow were almost abolished. As expected, the cold challenge before SCI caused a marked drop in mean flow, pulse amplitude, and temperature of the tail. Surprisingly, the drops in mean blood flow and temperature were observed after SCI, although the decrease in flow was slower and the pulse amplitude was not reduced. The results suggest that the cutaneous vasculature of the tail is sensitive to cold and will constrict, despite disconnection from supraspinal centers. This local effect is slow but may be sufficient to maintain some level of thermoregulation to cold. Without this vascular reaction, the effects of SCI on temperature regulation to cold would probably be much worse.

Adaptation of Respiratory-Related Brain Regions to Long-Term Hypercapnia: Focus on Neuropeptides in the RTN.

Long-term hypercapnia is associated with respiratory conditions including obstructive sleep apnea, chronic obstructive pulmonary disease and obesity hypoventilation syndrome. Animal studies have demonstrated an initial (within hours) increase in ventilatory drive followed by a decrease in this response over the long-term (days-weeks) in response hypercapnia. Little is known about whether changes in the central respiratory chemoreflex are involved. Here we investigated whether central respiratory chemoreceptor neurons of the retrotrapezoid nucleus (RTN), which project to the respiratory pattern generator within the ventral respiratory column (VRC) have a role in the mechanism of neuroplasticity associated with long-term hypercapnia. Adult male C57BL/6 mice (n = 5/group) were used. Our aims were (1) to determine if galanin, neuromedin B and gastrin-releasing peptide gene expression is altered in the RTN after long-term hypercapnia. This was achieved using qPCR to measure mRNA expression changes of neuropeptides in the RTN after short-term hypercapnia (6 or 8 h, 5 or 8% CO2) or long-term hypercapnia exposure (10 day, 5 or 8% CO2), (2) in the mouse brainstem, to determine the distribution of preprogalanin in chemoreceptors, and the co-occurrence of the galanin receptor 1 (GalR1:Gi-coupled receptor) with inhibitory GlyT2 ventral respiratory column neurons using in situ hybridization (ISH) to better characterize galaninergic RTN-VRC circuitry, (3) to investigate whether long-term hypercapnia causes changes to recruitment (detected by cFos immunohistochemistry) of respiratory related neural populations including the RTN neurons and their galaninergic subset, in vivo. Collectively, we found that hypercapnia decreases neuropeptide expression in the RTN in the short-term and has the opposite effect over the long-term. Following long term hypercapnia, the number of RTN galanin neurons remains unchanged, and their responsiveness to acute chemoreflex is sustained; in contrast, we identified multiple respiratory related sites that exhibit blunted chemoreflex activation. GalR1 was distributed in 11% of preBötC and 30% of BötC glycinergic neurons. Our working hypothesis is that during long-term hypercapnia, galanin co-release from RTN neurons may counterbalance glutamatergic inputs to respiratory centers to downscale energetically wasteful hyperventilation, thereby having a role in neuroplasticity by contributing to a decrease in ventilation, through the inhibitory effects of galanin.

Vulnerability to stress consequences induced by repeated social defeat in rats: Contribution of the angiotensin II type 1 receptor in cardiovascular alterations associated to low brain derived neurotrophic factor.

After social stress, rats become vulnerable to depression, and this state is characterized by persistent low blood levels of brain-derived neurotrophic factor (BDNF). The aim of this study was to determine whether low BDNF levels are associated with long term autonomic changes. Defeated animals were subjected to four daily episodes of social defeats. Twenty five days later, defeated rats with low BDNF levels (Dlow) still displayed elevated sympathetic tone (as indicated by an elevated low frequency to high frequency ratio (LF/HF) in heart rate) and elevated blood pressure, as well as reduced baroreflex sensitivity (BRS). In contrast, those with higher BDNF levels (Dhigh) similar to controls, did not. Dlow animals persistent cardiovascular changes were abolished by acute inhibition of the dorsomedial nucleus of the hypothalamus (DMH). These cardiovascular changes were also prevented by chronic sub-cutaneous osmotic infusion of losartan, an angiotensin II type 1 receptor (AT1) receptor antagonist, started immediately after social defeat. In conclusion, the results show that greater vulnerability to stress consequences following a traumatic event is associated with an elevated LF/HF ratio, a persistent high blood pressure and a low BRS, all due to an AT1 receptor activation.

Premotor sympathetic neurons of conditioned fear in the rat.

Conditioned fear to context, a pure form of psychological stress, is associated with sympathetically mediated changes including a marked hypertension. To identify the possible premotor sympathetic neurons mediating these changes, we conducted double-immunolabelling experiments combining fear-induced Fos with retrograde tracing from the thoracic cord (T2-L1). Presympathetic groups showing the greatest increase in the proportion of spinally projecting cells double-labelled with Fos compared with resting controls were the perifornical area (PeF; 22.7% vs. 0.4%) and paraventricular nucleus (Pa; 10.5% vs. 0.2%) in the hypothalamus, and the A5 noradrenergic group (33.6% vs. 0.2%) in the pons. In contrast, there was only a small increase in the presympathetic groups of the rostral ventral medulla, including the lateral paragigantocellular group (LPGi; 4.3% vs. 0.5%), raphe magnus and pallidus (1.1% vs. 0.6% and 1.8% vs. 0.5%), and the vasopressor group of the rostral ventrolateral medulla (RVLM; 1.9% vs. 0.8%). PeF, Pa, A5 and LPGi accounted for 21, 15, 16 and 6% of all the double-labelled cells, respectively, and RVLM for only 1%. Double-immunolabelling of Fos and tyrosine hydroxylase confirmed that many A5 neurons were activated (19%) and that practically no C1 neurons in RVLM were (1.3%). The results suggest that the main premotor sympathetic drive of the fear response comes from hypothalamic (PeF and Pa) and A5 neurons that project directly to the thoracic cord and bypass medullary presympathetic groups, and that the vasopressor premotor sympathetic neurons of the RVLM are unlikely to mediate the hypertensive pressure response of contextual fear.

Psychogenic non-epileptic seizures in children and adolescents: Part I - Diagnostic formulations.

Psychogenic non-epileptic seizures (PNES) are a nonspecific, umbrella category that is used to collect together a range of atypical neurophysiological responses to emotional distress, physiological stressors and danger. Because PNES mimic epileptic seizures, children and adolescents with PNES usually present to neurologists or to epilepsy monitoring units. After a comprehensive neurological evaluation and a diagnosis of PNES, the patient is referred to mental health services for treatment. This study documents the diagnostic formulations - the clinical formulations about the probable neurophysiological mechanisms - that were constructed for 60 consecutive children and adolescents with PNES who were referred to our Mind-Body Rehabilitation Programme for treatment. As a heuristic framework, we used a contemporary reworking of Janet's dissociation model: PNES occur in the context of a destabilized neural system and reflect a release of prewired motor programmes following a functional failure in cognitive-emotional executive control circuitry. Using this framework, we clustered the 60 patients into six different subgroups: (1) dissociative PNES (23/60; 38%), (2) dissociative PNES triggered by hyperventilation (32/60; 53%), (3) innate defence responses presenting as PNES (6/60; 10%), (4) PNES triggered by vocal cord adduction (1/60; 2%), (5) PNES triggered by activation of the valsalva manoeuvre (1/60; 1.5%) and (6) PNES triggered by reflex activation of the vagus (2/60; 3%). As described in the companion article, these diagnostic formulations were used, in turn, both to inform the explanations of PNES that we gave to families and to design clinical interventions for helping the children and adolescents gain control of their PNES.

Psychogenic non-epileptic seizures in children and adolescents: Part II - explanations to families, treatment, and group outcomes.

Psychogenic non-epileptic seizures (PNES) - time-limited disturbances of consciousness and motor-sensory control, not accompanied by ictal activity on electroencephalogram (EEG) - are best conceptualized as atypical neurophysiological responses to emotional distress, physiological stressors and danger. Patients and families find the diagnosis of PNES difficult to understand; the transition from neurology (where the diagnosis is made) to mental health services (to which patients are referred for treatment) can be a bumpy one. This study reports how diagnostic formulations constructed for 60 consecutive children and adolescents with PNES were used to inform both the explanations about PNES that were given to them and their families and the clinical interventions that were used to help patients gain control over PNES. Families were able to accept the diagnosis of PNES and engage in treatment when it was explained how emotional distress, illness and states of high arousal could activate atypical defence responses in the body and brain - with PNES being an unwanted by-product of this process. Patients and their families made good use of therapeutic interventions. A total of 75% of children/adolescents (45/60) regained normal function and attained full-time return to school. Global Assessment of Functioning scores increased from 41 to 67 ( t(54) = 10.09; p < .001). Outcomes were less favourable in children/adolescents who presented with chronic PNES and in those with a chronic, comorbid mental health disorder that failed to resolve with treatment. The study highlights that prompt diagnosis, followed by prompt multidisciplinary assessment, engagement, and treatment, achieves improved outcomes in children/adolescents with PNES.