Reduced computational modelling of Kölliker-Fuse contributions to breathing patterns in Rett syndrome.

KEY POINTS: Reduced computational models are used to test effects of loss of inhibition to the Kölliker-Fuse nucleus (KFn). Three reduced computational models that simulate eupnoeic and vagotomized respiratory rhythms are considered. All models exhibit the emergence of respiratory perturbations associated with Rett syndrome as inhibition to the KFn is diminished. Simulations suggest that application of 5-HT1A agonists can mitigate the respiratory pathology. The three models can be distinguished and tested based on their predictions about connections and dynamics within the respiratory circuit and about effects of perturbations on certain respiratory neuron populations. ABSTRACT: Rett syndrome (RTT) is a developmental disorder that can lead to respiratory disturbances featuring prolonged apnoeas of variable durations. Determining the mechanisms underlying these effects at the level of respiratory neural circuits would have significant implications for treatment efforts and would also enhance our understanding of respiratory rhythm generation and control. While experimental studies have suggested possible factors contributing to the respiratory patterns of RTT, we take a novel computational approach to the investigation of RTT, which allows for direct manipulation of selected system parameters and testing of specific hypotheses. Specifically, we present three reduced computational models, developed using an established framework, all of which successfully simulate respiratory outputs across eupnoeic and vagotomized conditions. All three models show that loss of inhibition to the Kölliker-Fuse nucleus reproduces the key respiratory alterations associated with RTT and, as suggested experimentally, that effects of 5-HT1A agonists on the respiratory neural circuit suffice to alleviate this respiratory pathology. Each of the models makes distinct predictions regarding the neuronal populations and interactions underlying these effects, suggesting natural directions for future experimental testing.

Deficiency of GABAergic synaptic inhibition in the Kölliker-Fuse area underlies respiratory dysrhythmia in a mouse model of Rett syndrome.

KEY POINTS: Life threatening breathing irregularity and central apnoeas are highly prevalent in children suffering from Rett syndrome. Abnormalities in inhibitory synaptic transmission have been associated with the physiopathology of this syndrome, and may underlie the respiratory disorder. In a mouse model of Rett syndrome, GABAergic terminal projections are markedly reduced in the Kölliker-Fuse nucleus (KF) in the dorsolateral pons, an important centre for control of respiratory rhythm regularity. Administration of a drug that augments endogenous GABA localized to this region of the pons reduced the incidence of apnoea and the respiratory irregularity of Rett female mice. Conversely, the respiratory disorder was recapitulated by blocking GABAergic transmission in the KF area of healthy rats. This study helps us understand the mechanism for generation of respiratory abnormality in Rett syndrome, pinpoints a brain site responsible and provides a clear anatomical target for the development of a translatable drug treatment. Central apnoeas and respiratory irregularity are a common feature in Rett syndrome (RTT), a neurodevelopmental disorder most often caused by mutations in the methyl-CpG-binding protein 2 gene (MECP2). We used a MECP2 deficient mouse model of RTT as a strategy to obtain insights into the neurobiology of the disease and into mechanisms essential for respiratory rhythmicity during normal breathing. Previously, we showed that, systemic administration of a GABA reuptake blocker in MECP2 deficient mice markedly reduced the occurrence of central apnoeas. Further, we found that, during central apnoeas, post-inspiratory drive (adductor motor) to the upper airways was enhanced in amplitude and duration in Mecp2 heterozygous female mice. Since the pontine Kölliker-Fuse area (KF) drives post-inspiration, suppresses inspiration, and can reset the respiratory oscillator phase, we hypothesized that synaptic inhibition in this area is essential for respiratory rhythm regularity. In this study, we found that: (i) Mecp2 heterozygous mice showed deficiency of GABA perisomatic bouton-like puncta and processes in the KF nucleus; (ii) blockade of GABA reuptake in the KF of RTT mice reduced breathing irregularity; (iii) conversely, blockade of GABAA receptors in the KF of healthy rats mimicked the RTT respiratory phenotype of recurrent central apnoeas and prolonged post-inspiratory activity. Our results show that reductions in synaptic inhibition within the KF induce rhythm irregularity whereas boosting GABA transmission reduces respiratory arrhythmia in a murine model of RTT. Our data suggest that manipulation of synaptic inhibition in KF may be a clinically important strategy for alleviating the life threatening respiratory disorders in RTT.

Sympathetic overactivity occurs before hypertension in the two-kidney, one-clip model.

Our knowledge of mechanisms responsible for both the development and the maintenance of hypertension remains incomplete in the Goldblatt (two-kidney, one-clip; 2K1C) model. We tested the hypothesis that elevated sympathetic nerve activity (SNA) occurs before the onset of hypertension in 2K1C rats, considering the time course of the increase in SNA in relationship to the onset of the hypertension. We used a decorticated in situ working heart-brainstem preparation of three groups of male Wistar rats, namely sham-operated animals (SHAM, n = 7) and animals 3 weeks post-2K1C, of which some were hypertensive (2K1C-H, n = 6) and others normotensive (2K1C-N, n = 9), as determined in vivo a priori. Perfusion pressure was higher in both 2K1C groups (2K1C-H, 76 ± 1 mmHg; 2K1C-N, 74 ± 3 mmHg; versus SHAM, 60 ± 2 mmHg, P < 0.05). The SNA was significantly elevated in both 2K1C groups (2K1C-H, 47.7 ± 6.1 µV; 2K1C-N, 32.8 ± 2.8 µV; versus SHAM, 20.5 ± 2.5 µV, P < 0.05) owing to its increased respiratory modulation; the chemoreflex was augmented and baroreflex depressed. Precollicular transection reduced SNA in all groups (2K1C-H, -32.5 ± 7.5%; 2K1C-NH, -48 ± 6.9%; versus SHAM, -13.2 ± 1%, P < 0.05). Subsequent medullary spinal cord transection abolished SNA in both SHAM and 2K1C-N groups, but decreased it by only 57 ± 5.5% in 2K1C-H preparations. Thus, SNA is raised before the onset of hypertension, by the third week after renal artery clipping, and this originates, in part, from its enhanced respiratory modulation. Spinal circuits contribute to the elevation of SNA in the 2K1C model, but only after hypertension has developed.

Defining inhibitory neurone function in respiratory circuits: opportunities with optogenetics?

Pharmacological and mathematical modelling studies support the view that synaptic inhibition in mammalian brainstem respiratory circuits is essential for generating normal and stable breathing movements. GABAergic and glycinergic neurones are known components of these circuits but their precise functional roles have not been established, especially within key microcircuits of the respiratory pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes involved in phasic control of respiratory pump and airway muscles. Here, we review briefly current concepts of relevant complexities of inhibitory synapses and the importance of synaptic inhibition in the operation of these microcircuits. We highlight results and limitations of classical pharmacological studies that have suggested critical functions of synaptic inhibition. We then explore the potential opportunities for optogenetic strategies that represent a promising new approach for interrogating function of inhibitory circuits, including a hypothetical wish list for optogenetic approaches to allow expedient application of this technology. We conclude that recent technical advances in optogenetics should provide a means to understand the role of functionally select and regionally confined subsets of inhibitory neurones in key respiratory circuits such as those in the pre-BötC and BötC.

Impaired CO2 sensitivity of astrocytes in a mouse model of Rett syndrome.

Rett syndrome, a prototypical neurological disorder caused by loss of function of the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2) gene, is associated with a severely disordered breathing pattern and reduced ventilatory CO2 sensitivity. In a mouse model of Rett syndrome (MeCP2 knockout), re-introduction of the MeCP2 gene selectively in astrocytes rescues normal respiratory phenotype. In the present study we determined whether the metabolic and/or signalling functions of astrocytes are affected by testing the hypotheses that in conditions of MeCP2 deficiency, medullary astrocytes are unable to produce/release appropriate amounts of lactate or detect changes in PCO2/[H(+) ], or both. No differences in tonic or hypoxia-induced release of lactate from the ventral surface of the medulla oblongata or cerebral cortex in brain slices of MeCP2-knockout and wild-type mice were found. In brainstem slices of wild-type mice, respiratory acidosis triggered robust elevations in [Ca(2+) ]i in astrocytes residing near the ventral surface of the medulla oblongata. The magnitude of CO2 -induced [Ca(2+) ]i responses in medullary astrocytes was markedly reduced in conditions of MeCP2 deficiency, whereas [Ca(2+) ]i responses to ATP were unaffected. These data suggest that (i) metabolic function of astrocytes in releasing lactate into the extracellular space is not affected by MeCP2 deficiency, and (ii) MeCP2 deficiency impairs the ability of medullary astrocytes to sense changes in PCO2/[H(+) ]. Taken together with the evidence of severely blunted ventilatory sensitivity to CO2 in mice with conditional MeCP2 deletion in astroglia, these data support the hypothesis of an important role played by astrocytes in central respiratory CO2 /pH chemosensitivity.

P2X3 receptor antagonism reduces the occurrence of apnoeas in newborn rats.

Hyperreflexia of the peripheral chemoreceptors is a potential contributor of apnoeas of prematurity (AoP). Recently, it was shown that elevated P2X3 receptor expression was associated with elevated carotid body afferent sensitivity. Therefore, we tested whether P2X3 receptor antagonism would reduce AoP known to occur in newborn rats. Unrestrained whole-body plethysmography was used to record breathing and from this the frequency of apnoeas at baseline and following administration of either a P2X3 receptor antagonist - AF-454 (5 mg/kg or 10 mg/kg s.c.) or vehicle was derived. In a separate group, we tested the effects of AF-454 (10 mg/kg) on the hypoxic ventilatory response (10 % FiO2). Ten but not 5 mg/kg AF-454 reduced the frequency of AoP and improved breathing regularity significantly compared to vehicle. Neither AF-454 (both 5 and 10 mg/kg) nor vehicle affected baseline respiration. However, P2X3 receptor antagonism (10 mg/kg) powerfully blunted hypoxic ventilatory response to 10 % FiO2. These data suggest that P2X3 receptors contribute to AoP and the hypoxic ventilatory response in newborn rats but play no role in the drive to breathe at rest.

Methamidophos, an Organophosphorus Insecticide, Induces Pro-aggressive Behaviour in Mice.

Although evidence indicates that exposure to organophosphorus (OP) pesticides induces neurobehavioral disorders, little is known about the effects of OP on aggressive behaviour. Our study investigated the effects of repeated exposure to an OP pesticide, methamidophos, on the isolation-induced aggressive behaviour in mice. Forty seven male mice were individually housed for a month. Socially isolated animals were then confronted with a standard non-isolated opponent for 15 min (pre-treatment trial), and the latency and frequency of aggressive and general exploratory behaviours were recorded. Based on the presence of attack behaviour in the pre-treatment trial, mice were classified as isolation-induced aggressive and non-aggressive. All mice were then treated for 7 days with methamidophos (3.5 mg/kg/day, n = 22, intraperitoneal (i.p.)) or saline (1 mL/kg/day, control group, n = 25, i.p.), and a second trial was performed. Repeated exposure to methamidophos induced attack behaviour in non-aggressive mice. The treatment with methamidophos also decreased plasma butyrylcholinesterase and brain acetylcholinesterase activity. These results suggest that methamidophos has a pro-aggressive effect on socially isolated mice.

Ablation of NK1 receptor bearing neurons in the nucleus of the solitary tract blunts cardiovascular reflexes in awake rats.

The nucleus of the solitary tract (NTS) receives primary afferents involved in cardiovascular regulation. We investigated the role of NK(1)-receptor bearing neurons in the NTS on cardiovascular reflexes in awake rats fitted with chronic venous and arterial cannulae. These neurons were lesioned selectively with saporin conjugated with substance P (SP-SAP, 2 microM, bilateral injections of 20 nL in the subpostremal NTS, or 200 nL in both the subpostremal and the commissural NTS). Before, and 7 and 14 days after injection of SP-SAP, we measured changes in blood pressure and heart rate induced by i.v. injection of phenylephrine and nitroprusside (baroreceptor reflex), cyanide (arterial chemoreceptor reflex), and phenylbiguanide (Bezold-Jarisch reflex). The smaller injections with SP-SAP completely abolished NK1 receptor staining in the subpostremal NTS. The larger injections abolished NK1 receptor immunoreactivity in an area that extended from the commissural NTS to the rostral end of the subpostremal NTS. The lesions seemed to affect only a limited number of neurons, since neutral red stained sections did not show any obvious reduction in cell number. The smaller lesions reduced the gain of baroreflex bradycardia and the hypotension induced by phenylbiguanide. The larger lesions completely abolished the response to phenylbiguanide, blocked the baroreflex bradycardia induced by phenylephrine, severely blunted the baroreflex tachycardia, and blocked the bradycardia and reduced the hypertension induced by cyanide. Thus, these responses depend critically on NK(1)-receptor bearing neurons in the NTS.

Temporal profile and mechanisms of the prompt sympathoexcitation following coronary ligation in Wistar rats.

Our aim was to assess the timing and mechanisms of the sympathoexcitation that occurs immediately after coronary ligation. We recorded thoracic sympathetic (tSNA) and phrenic activities, heart rate (HR) and perfusion pressure in Wistar rats subjected to either ligation of the left anterior descending coronary artery (LAD) or Sham operated in the working heart-brainstem preparation. Thirty minutes after LAD ligation, tSNA had increased (basal: 2.5±0.2 µV, 30 min: 3.5±0.3 µV), being even higher at 60 min (5.2±0.5 µV, P<0.01); while no change was observed in Sham animals. HR increased significantly 45 min after LAD (P<0.01). Sixty minutes after LAD ligation, there was: (i) an augmented peripheral chemoreflex - greater sympathoexcitatory response (50, 45 and 27% of increase to 25, 50 and 75 µL injections of NaCN 0.03%, respectively, when compared to Sham, P<0.01); (ii) an elevated pressor response (32±1 versus 23±1 mmHg in Sham, P<0.01) and a reduced baroreflex sympathetic gain (1.3±0.1 versus Sham 2.0±0.1%.mmHg-1, P<0.01) to phenylephrine injection; (iii) an elevated cardiac sympathetic tone (ΔHR after atenolol: -108±8 versus -82±7 bpm in Sham, P<0.05). In contrast, no changes were observed in cardiac vagal tone and bradycardic response to both baroreflex and chemoreflex between LAD and Sham groups. The immediate sympathoexcitatory response in LAD rats was dependent on an excitatory spinal sympathetic cardiocardiac reflex, whereas at 3 h an angiotensin II type 1 receptor mechanism was essential since Losartan curbed the response by 34% relative to LAD rats administered saline (P<0.05). A spinal reflex appears key to the immediate sympathoexcitatory response after coronary ligation. Therefore, the sympathoexcitatory response seems to be maintained by an angiotensinergic mechanism and concomitant augmentation of sympathoexcitatory reflexes.

Cardiovascular responses to substance P in the nucleus tractus solitarii: microinjection study in conscious rats.

The cardiovascular effects of substance P (SP) microinjections in the nucleus tractus solitarii (NTS) were evaluated in conscious rats. We chose this model because it is an effective way to access some of the cardiovascular effects of neurotransmitters in the NTS without the inconvenience of blunting pathways with anesthetic agents or removing forebrain projections by decerebration. The cardiovascular responses to SP injections were also evaluated after chronic nodose ganglionectomy. We found that, in conscious rats, SP microinjections into the NTS induced hypertension and tachycardia. Unilateral and bilateral SP injections into the NTS caused a slow increase in blood pressure and heart rate that peaked 1.5-5 min after injection and lasted for 20-30 min. Nodose ganglionectomy increased the duration of the pressor and tachycardic effects of SP and enhanced the pressor response. These data show that SP in the NTS is involved in pressor pathways. The supersensitivity to SP seen after nodose ganglionectomy suggests that vagal afferent projections are involved in those pressor pathways activated by SP in the NTS.

Participação da substância P e dos receptores NK1 na modulação da pressão arterial e dos reflexos cardiovasculares nos núcleos do tracto solitário de ratos: estudo em modelos experimentais livres de anestesia / Participation of substance P and NK1 receptors on the modulation of arterial pressure and cardiovascular reflexes in the nucleus of the solitary tract o rats: study in experimental models free of anaesthetics

A substância P (SP) tem sido caracterizada como um possível modulador envolvido controle cardiovascular nos núcleos do tracto solitário (NTS). Há evidências de que a SP modula o barorreflexo e os quimiorreflexos carotídeo a cardiopulmonar. Entretanto imprescindibilidade e a relevância da SP para a transmissão e modulação, respectivamente, desses reflexos geraram controvérsia. Uma fonte de controvérsia foi a observação de que os efeitos deste neuropeptídeo no NTS são influenciados por agentes anestésicos. Neste estudo avaliamos, em ratos não anestesiados, a participação de neurônios que expressam receptores NK1 no NTS para a transmissão do barorreflexo e dos quimiorreflexos carotídeo e cardiopulmonar (Bezold-Jarisch). Para tanto, utilizamos a SP conjugada a uma to inativadora do ribossomo, a saporina, para lesar estes neurônios seletivamente Também avaliamos o efeito do bloqueio farmacológicos de receptores GABAA sobre as respostas cardiovasculares induzidas pela microinjeção de SP no NTS de ratos não anestesiados (ABDALA et al., 2003). Em uma preparação in situ de ratos descerebrados e artificialmente perfundidos, que nos permitiu a utilização de protocolos mais invasivos, estudamos a importância da SP para a modulação do barorreflexo durante situações de hipóxia e participação no controle da atividade simpática em ratos espontaneamente hipertensos (SHR). Observamos que a lesão de neurônios que expressam receptores NK1 no NTS atenuou significantemente o barorreflexo e o quimiorreflexo cardiopulmonar, afetando de maneira menos intensa o quimiorreflexo carotídeo. Isso é sugestivo de que a SP modula população significativa de neurônios do NTS envolvidos na mediação destes reflexos. Mostramos que as respostas pressora e taquicárdica induzidas pela microinjeção de SP no NTS de ratos não anestesiados dependem da liberação endógena de GABA, e que a SP passa a induzir hipotensão após o bloqueio farmacológico de receptores GABAA. Isto confirma a participação da SP na modulação de vias funcionalmente antagônicas no NTS. Observamos que a injeção de SP no NTS comissural é capaz de reduzir a atividade simpática e provocar vasodilatação em SHR jovens por um longo período de tempo, mas não em ratos normotensos…(au)