A BDNF loop-domain mimetic acutely reverses spontaneous apneas and respiratory abnormalities during behavioral arousal in a mouse model of Rett syndrome.

Reduced levels of brain-derived neurotrophic factor (BDNF) are thought to contribute to the pathophysiology of Rett syndrome (RTT), a severe neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). In Mecp2 mutant mice, BDNF deficits have been associated with breathing abnormalities, a core feature of RTT, as well as with synaptic hyperexcitability within the brainstem respiratory network. Application of BDNF can reverse hyperexcitability in acute brainstem slices from Mecp2-null mice, suggesting that therapies targeting BDNF or its receptor, TrkB, could be effective at acute reversal of respiratory abnormalities in RTT. Therefore, we examined the ability of LM22A-4, a small-molecule BDNF loop-domain mimetic and TrkB partial agonist, to modulate synaptic excitability within respiratory cell groups in the brainstem nucleus tractus solitarius (nTS) and to acutely reverse abnormalities in breathing at rest and during behavioral arousal in Mecp2 mutants. Patch-clamp recordings in Mecp2-null brainstem slices demonstrated that LM22A-4 decreases excitability at primary afferent synapses in the nTS by reducing the amplitude of evoked excitatory postsynaptic currents and the frequency of spontaneous and miniature excitatory postsynaptic currents. In vivo, acute treatment of Mecp2-null and -heterozygous mutants with LM22A-4 completely eliminated spontaneous apneas in resting animals, without sedation. Moreover, we demonstrate that respiratory dysregulation during behavioral arousal, a feature of human RTT, is also reversed in Mecp2 mutants by acute treatment with LM22A-4. Together, these data support the hypothesis that reduced BDNF signaling and respiratory dysfunction in RTT are linked, and establish the proof-of-concept that treatment with a small-molecule structural mimetic of a BDNF loop domain and a TrkB partial agonist can acutely reverse abnormal breathing at rest and in response to behavioral arousal in symptomatic RTT mice.

Neurotrophic factors in development and regulation of respiratory control.

Neurotrophic factors (NTFs) are a heterogeneous group of extracellular signaling molecules that play critical roles in the development, maintenance, modulation and plasticity of the central and peripheral nervous systems. A subset of these factors, including members of three multigene families-the neurotrophins, neuropoetic cytokines and the glial cell line-derived neurotrophic factor ligands-are particularly important for development and regulation of neurons involved in respiratory control. Here, we review the functional biology of these NTFs and their receptors, as well as their roles in regulating survival, maturation, synaptic strength and plasticity in respiratory control pathways. In addition, we highlight recent progress in identifying the role of abnormal NTF signaling in the molecular pathogenesis of respiratory dysfunction in Rett syndrome and in the development of potential new NTF-targeted therapeutic strategies.

Preclinical research in Rett syndrome: setting the foundation for translational success.

In September of 2011, the National Institute of Neurological Disorders and Stroke (NINDS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the International Rett Syndrome Foundation (IRSF) and the Rett Syndrome Research Trust (RSRT) convened a workshop involving a broad cross-section of basic scientists, clinicians and representatives from the National Institutes of Health (NIH), the US Food and Drug Administration (FDA), the pharmaceutical industry and private foundations to assess the state of the art in animal studies of Rett syndrome (RTT). The aim of the workshop was to identify crucial knowledge gaps and to suggest scientific priorities and best practices for the use of animal models in preclinical evaluation of potential new RTT therapeutics. This review summarizes outcomes from the workshop and extensive follow-up discussions among participants, and includes: (1) a comprehensive summary of the physiological and behavioral phenotypes of RTT mouse models to date, and areas in which further phenotypic analyses are required to enhance the utility of these models for translational studies; (2) discussion of the impact of genetic differences among mouse models, and methodological differences among laboratories, on the expression and analysis, respectively, of phenotypic traits; and (3) definitions of the standards that the community of RTT researchers can implement for rigorous preclinical study design and transparent reporting to ensure that decisions to initiate costly clinical trials are grounded in reliable preclinical data.

Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment.

Excitatory-inhibitory imbalance has been identified within specific brain microcircuits in models of Rett syndrome (RTT) and other autism spectrum disorders (ASDs). However, macrocircuit dysfunction across the RTT brain as a whole has not been defined. To approach this issue, we mapped expression of the activity-dependent, immediate-early gene product Fos in the brains of wild-type (Wt) and methyl-CpG-binding protein 2 (Mecp2)-null (Null) mice, a model of RTT, before and after the appearance of overt symptoms (3 and 6 weeks of age, respectively). At 6 weeks, Null mice exhibit significantly less Fos labeling than Wt in limbic cortices and subcortical structures, including key nodes in the default mode network. In contrast, Null mice exhibit significantly more Fos labeling than Wt in the hindbrain, most notably in cardiorespiratory regions of the nucleus tractus solitarius (nTS). Using nTS as a model, whole-cell recordings demonstrated that increased Fos expression in Nulls at 6 weeks of age is associated with synaptic hyperexcitability, including increased frequency of spontaneous and miniature EPSCs and increased amplitude of evoked EPSCs in Nulls. No such effect of genotype on Fos or synaptic function was seen at 3 weeks. In the mutant forebrain, reduced Fos expression, as well as abnormal sensorimotor function, were reversed by the NMDA receptor antagonist ketamine. In light of recent findings that the default mode network is hypoactive in autism, our data raise the possibility that hypofunction within this meta-circuit is a shared feature of RTT and other ASDs and is reversible.

Expression and function of serotonin 2A and 2B receptors in the mammalian respiratory network.

Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT(2A)Rs, expression and function of 5-HT(2B)Rs within the respiratory network are still unclear. 5-HT(2B)R utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT(2A)Rs and 5-HT(2B)Rs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT(2B)R. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation.

Altered responses of MeCP2-deficient mouse brain stem to severe hypoxia.

Rett syndrome (RTT) patients suffer from respiratory arrhythmias with frequent apneas causing intermittent hypoxia. In a RTT mouse model (methyl-CpG-binding protein 2-deficient mice; Mecp2(-/y)) we recently discovered an enhanced hippocampal susceptibility to hypoxia and hypoxia-induced spreading depression (HSD). In the present study we investigated whether this also applies to infant Mecp2(-/y) brain stem, which could become life-threatening due to failure of cardiorespiratory control. HSD most reliably occurred in the nucleus of the solitary tract (NTS) and the spinal trigeminal nucleus (Sp5). HSD susceptibility of the Mecp2(-/y) NTS and Sp5 was increased on 8 mM K(+)-mediated conditioning. 5-HT(1A) receptor stimulation with 8-hydroxy-2-(di-propylamino)tetralin (8-OH-DPAT) postponed HSD by up to 40%, mediating genotype-independent protection. The deleterious impact of HSD on in vitro respiration became obvious in rhythmically active slices, where HSD propagation into the pre-Bötzinger complex (pre-BötC) immediately arrested the respiratory rhythm. Compared with wild-type, the Mecp2(-/y) pre-BötC was invaded less frequently by HSD, but if so, HSD occurred earlier. On reoxygenation, in vitro rhythms reappeared with increased frequency, which was less pronounced in Mecp2(-/y) slices. 8-OH-DPAT increased respiratory frequency but failed to postpone HSD in the pre-BötC. Repetitive hypoxia facilitated posthypoxic recovery only if HSD occurred. In 57% of Mecp2(-/y) slices, however, HSD spared the pre-BötC. Although this occasionally promoted residual hypoxic respiratory activity ("gasping"), it also prolonged the posthypoxic recovery, and thus the absence of central inspiratory drive, which in vivo would lengthen respiratory arrest. In view of the breathing disorders in RTTs, the increased hypoxia susceptibility of MeCP2-deficient brain stem potentially contributes to life-threatening disturbances of cardiorespiratory control.

Postnatal changes in morphology and dendritic organization of neurones located in the area of the Kölliker-Fuse nucleus of rat.

The Kölliker-Fuse nucleus (KF) is an integral part of the central pattern generator for breathing and shows postnatal development of synaptic functions and cyto-architectural structure. Here, we analyzed the postnatal changes in cell morphology of biocytin-labelled KF neurones. Developmental analyses revealed an increasing size of somas and dendritic length. These changes were accompanied by changes in the orientation of the main dendritic branches from a diffuse orientation in neonates to a predominant medio-lateral orientation in juveniles. These developmental changes may allow for synaptic contacts with multiple ascending fibre tracts required for the processing of multi-modal respiratory inputs in the KF.

Infant brain stem is prone to the generation of spreading depression during severe hypoxia.

Spreading depression (SD) resembles a concerted, massive neuronal/glial depolarization propagating within the gray matter. Being associated with cerebropathology, such as cerebral ischemia or hemorrhage, epileptic seizures, and migraine, it is well studied in cortex and hippocampus. We have now analyzed the susceptibility of rat brain stem to hypoxia-induced spreading depression-like depolarization (HSD), which could critically interfere with cardiorespiratory control. In rat brain stem slices, severe hypoxia (oxygen withdrawal) triggered HSD within minutes. The sudden extracellular DC potential shift of approximately -20 mV showed the typical profile known from other brain regions and was accompanied by an intrinsic optical signal (IOS). Spatiotemporal IOS analysis revealed that in infant brain stem, HSD was preferably ignited within the spinal trigeminal nucleus and then mostly spread out medially, invading the hypoglossal nucleus, the nucleus of the solitary tract (NTS), and the ventral respiratory group (VRG). The neuronal hypoxic depolarizations underlying the generation of HSD were massive, but incomplete. The propagation velocity of HSD and the associated extracellular K(+) rise were also less marked than in other brain regions. In adult brain stem, HSD was mostly confined to the NTS and its occurrence was facilitated by hypotonic solutions, but not by glial poisoning or block of GABAergic and glycinergic synapses. In conclusion, brain stem tissue reliably generates propagating HSD episodes, which may be of interest for basilar-type migraine and brain stem infarcts. The preferred occurrence of HSD in the infant brain stem and its propagation into the VRG may be of importance for neonatal brain stem pathology such as sudden infant death syndrome.

Postnatal emergence of synaptic plasticity associated with dynamic adaptation of the respiratory motor pattern.

The shape of the three-phase respiratory motor pattern (inspiration, postinspiration, late expiration) is controlled by a central pattern generator (CPG) located in the ponto-medullary brainstem. Synaptic interactions between and within specific sub-compartments of the CPG are subject of intensive research. This review addresses the neural control of postinspiratory activity as the essential determinant of inspiratory/expiratory phase duration. The generation of the postinspiratory phase depends on synaptic interaction between neurones of the nucleus tractus solitarii (NTS), which relay afferent inputs from pulmonary stretch receptors, and the pontine Kölliker-Fuse nucleus (KF) as integral parts of the CPG. Both regions undergo significant changes during the first three postnatal weeks in rodents. Developmental changes in glutamatergic synaptic functions and its modulation by brain-derived neurotrophic factor may have implications in synaptic plasticity within the NTS/KF axis. We propose that dependent on these developmental changes, the CPG becomes permissive for short- and long-term plasticity associated with environmental, metabolic and behavioural adaptation of the breathing pattern.

Emergence of brain-derived neurotrophic factor-induced postsynaptic potentiation of NMDA currents during the postnatal maturation of the Kolliker-Fuse nucleus of rat.

The Kölliker-Fuse nucleus (KF) contributes essentially to respiratory pattern formation and adaptation of breathing to afferent information. Systems physiology suggests that these KF functions depend on NMDA receptors (NMDA-R). Recent investigations revealed postnatal changes in the modulation of glutamatergic neurotransmission by brain-derived neurotrophic factor (BDNF) in the KF. Therefore, we investigated postnatal changes in NMDA-R subunit composition and postsynaptic modulation of NMDA-R-mediated currents by BDNF in KF slice preparations derived from three age groups (neonatal: postnatal day (P) 1-5; intermediate: P6-13; juvenile: P14-21). Immunohistochemistry showed a developmental up-regulation of the NR2D subunit. This correlated with a developmental increase in decay time of NMDA currents and a decline of desensitization in response to repetitive exogenous NMDA applications. Thus, developmental up-regulation of the NR2D subunit, which reduces the Mg(2+) block of NMDA-R, causes these specific changes in NMDA current characteristics. This may determine the NMDA-R-dependent function of the mature KF in the control of respiratory phase transition. Subsequent experiments revealed that bath-application of BDNF progressively potentiated these repetitively evoked NMDA currents only in intermediate and juvenile age groups. Pharmacological inhibition of protein kinase C (PKC), as a downstream component of the BDNF-tyrosine kinase B receptor (trkB) signalling, prevented BDNF-induced potentiation of NMDA currents. BDNF-induced potentiation of NMDA currents in later developmental stages might be essential for synaptic plasticity during the adaptation of the breathing pattern in response to peripheral/central commands. The lack of plasticity in neonatal neurones strengthens the hypothesis that the respiratory network becomes permissive for activity-dependent plasticity with ongoing postnatal development.

Developmental changes in the BDNF-induced modulation of inhibitory synaptic transmission in the Kölliker-Fuse nucleus of rat.

The Kölliker-Fuse nucleus (KF), part of the pontine respiratory group, is involved in the control of respiratory phase duration, and receives both excitatory and inhibitory afferent input from various other brain regions. There is evidence for developmental changes in the modulation of excitatory inputs to the KF by the neurotrophin brain-derived neurotrophic factor (BDNF). In the present study we investigated if BDNF exerts developmental effects on inhibitory synaptic transmission in the KF. Recordings of inhibitory postsynaptic currents (IPSCs) in KF neurons in a pontine slice preparation revealed general developmental changes. Recording of spontaneous and evoked IPSCs (sIPSCs, eIPSCS) revealed that neonatally the gamma-aminobutyric acid (GABA)ergic fraction of IPSCs was predominant, while in later developmental stages glycinergic neurotransmission significantly increased. Bath-application of BDNF significantly reduced sIPSC frequency in all developmental stages, while BDNF-mediated modulation on eIPSCs showed developmental differences. The eIPSCs mean amplitude was uniformly and significantly reduced following BDNF application only in neurons from rats younger than postnatal day 10. At later postnatal stages the response pattern became heterogeneous, and both augmentations and reductions of eIPSC amplitudes occurred. All BDNF effects on eIPSCs and sIPSCs were reversed with the tyrosine kinase receptor-B inhibitor K252a. We conclude that developmental changes in inhibitory neurotransmission, including the BDNF-mediated modulation of eIPSCs, relate to the postnatal maturation of the KF. The changes in BDNF-mediated modulation of IPSCs in the KF may have strong implications for developmental changes in synaptic plasticity and the adaptation of the breathing pattern to afferent inputs.

Activation of Orexin B receptors in the pontine Kölliker-Fuse nucleus modulates pre-inspiratory hypoglossal motor activity in rat.

Orexins (splice variants A and B) are hypothalamic neuropeptides that have essential functions in control of arousal and nutrition. Lack of Orexins is strongly associated with narcolepsy and sleep disordered breathing. However, the role of Orexins and particularly that of Orexin-B (OXB), in respiratory centres controlling upper-airway patency are less defined. In the present study we performed microinjections of OXB into the pontine Kölliker-Fuse nucleus (KF) of the dorsolateral pons, since this nucleus is particularly involved in the pre-motor control of upper airway muscles. The OXB mediated effects on heart, phrenic (PNA) and hypoglossal (XII-A) nerve activities were analysed in an in situ perfused brainstem preparation. Injection of OXB into the KF evoked significant augmentation of the respiratory frequency. Importantly, OXB provoked particularly prolonged pre-inspiratory discharge of the XII nerve, while no cardiovascular response was observed after KF microinjections. In summary, OXB in the KF exerts an excitatory effect on XII pre-motoneurones. Since pre-inspiratory activity of the XII is important for the decrease in upper airway resistance during inspiration, we conclude that OXB release in the KF has strong implications in the state-dependent control of upper airway patency under physiological and pathophysiological conditions.

Developmental changes in brain-derived neurotrophic factor-mediated modulations of synaptic activities in the pontine Kölliker-Fuse nucleus of the rat.

The Kölliker-Fuse nucleus (KF), part of the respiratory network, is involved in the modulation of respiratory phase durations in response to peripheral and central afferent inputs. The KF is immature at birth. Developmental changes in its physiological and anatomical properties have yet to be investigated. Since brain-derived neurotrophic factor (BDNF) is of major importance for the maturation of neuronal networks, we investigated its effects on developmental changes in the KF on different postnatal days (neonatal, P1-5; intermediate, P6-13; juvenile, P14-21) by analysing single neurones in the in vitro slice preparation and network activities in the perfused brainstem preparation in situ. The BDNF had only weak effects on the frequency of mixed excitatory and inhibitory spontaneous postsynaptic currents (sPSCs) in neonatal slice preparations. Postnatally, in the intermediate and juvenile age groups, a significant augmentation of the sPSC frequency was observed in the presence of 100 pm BDNF (+23.5+/-12.6 and +76.7+/-28.4%, respectively). Subsequent analyses of BDNF effects on evoked excitatory postsynaptic currents (eEPSCs) revealed significant enhancement of eEPSC amplitude of +20.8+/-7.0% only in juvenile stages (intermediates, -13.2+/-4.8%). On the network level, significant modulation of phrenic nerve activity following BDNF microinjection into the KF was also observed only in juveniles. The data suggest that KF neurones are subject to BDNF-mediated fast synaptic modulation after completion of postnatal maturation. After maturation, BDNF contributes to modulation of fast excitatory neurotransmission in respiratory-related KF neurones. This may be important for network plasticity associated with the processing of afferent information.

Development of adaptive behaviour of the respiratory network: implications for the pontine Kolliker-Fuse nucleus.

Breathing is constantly modulated by afferent sensory inputs in order to adapt to changes in behaviour and environment. The pontine respiratory group, in particular the Kolliker-Fuse nucleus, might be a key structure for adaptive behaviours of the respiratory network. Here, we review the anatomical connectivity of the Kolliker-Fuse nucleus with primary sensory structures and with the medullary respiratory centres and focus on the importance of pontine and medullary postinspiratory neurones in the mediation of respiratory reflexes. Furthermore, we will summarise recent findings from our group regarding ontogenetic changes of respiratory reflexes (e.g., the diving response) and provide evidence that immaturity of the Kolliker-Fuse nucleus might account in neonates for a lack of plasticity in sensory evoked modulations of respiratory activity. We propose that a subpopulation of neurones within the Kolliker-Fuse nucleus represent command neurones for sensory processing which are capable of initiating adaptive behaviour in the respiratory network. Recent data from our laboratory suggest that these command neurones undergo substantial postnatal maturation.