The amyloid precursor protein intracellular domain induces sleep disruptions and its nuclear localization fluctuates in circadian pacemaker neurons in Drosophila and mice.

The most prominent symptom of Alzheimer's disease (AD) is cognitive decline; however, sleep and other circadian disruptions are also common in AD patients. Sleep disruptions have been connected with memory problems and therefore the changes in sleep patterns observed in AD patients may also actively contribute to cognitive decline. However, the underlying molecular mechanisms that connect sleep disruptions and AD are unclear. A characteristic feature of AD is the formation of plaques consisting of Amyloid-ß (Aß) peptides generated by cleavage of the Amyloid Precursor Protein (APP). Besides Aß, APP cleavage generates several other fragments, including the APP intracellular domain (AICD) that has been linked to transcriptional regulation and neuronal homeostasis. Here we show that overexpression of the AICD reduces the early evening expression of two core clock genes and disrupts the sleep pattern in flies. Analyzing the subcellular localization of the AICD in pacemaker neurons, we found that the AICD levels in the nucleus are low during daytime but increase at night. While this pattern of nuclear AICD persisted with age, the nighttime levels were higher in aged flies. Increasing the cleavage of the fly APP protein also disrupted AICD nuclear localization. Lastly, we show that the day/nighttime nuclear pattern of the AICD is also detectable in neurons in the suprachiasmatic nucleus of mice and that it also changes with age. Together, these data suggest that AD-associated changes in APP processing and the subsequent changes in AICD levels may cause sleep disruptions in AD.

The role of neuronal excitation and inhibition in the pre-Bötzinger complex on the cough reflex in the cat.

Brainstem respiratory neuronal network significantly contributes to cough motor pattern generation. Neuronal populations in the pre-Bötzinger complex (PreBötC) represent a substantial component for respiratory rhythmogenesis. We studied the role of PreBötC neuronal excitation and inhibition on mechanically induced tracheobronchial cough in 15 spontaneously breathing, pentobarbital anesthetized adult cats (35 mg/kg, iv initially). Neuronal excitation by unilateral microinjection of glutamate analog d,l-homocysteic acid resulted in mild reduction of cough abdominal electromyogram (EMG) amplitudes and very limited temporal changes of cough compared with effects on breathing (very high respiratory rate, high amplitude inspiratory bursts with a short inspiratory phase, and tonic inspiratory motor component). Mean arterial blood pressure temporarily decreased. Blocking glutamate-related neuronal excitation by bilateral microinjections of nonspecific glutamate receptor antagonist kynurenic acid reduced cough inspiratory and expiratory EMG amplitude and shortened most cough temporal characteristics similarly to breathing temporal characteristics. Respiratory rate decreased and blood pressure temporarily increased. Limiting active neuronal inhibition by unilateral and bilateral microinjections of GABAA receptor antagonist gabazine resulted in lower cough number, reduced expiratory cough efforts, and prolongation of cough temporal features and breathing phases (with lower respiratory rate). The PreBötC is important for cough motor pattern generation. Excitatory glutamatergic neurotransmission in the PreBötC is involved in control of cough intensity and patterning. GABAA receptor-related inhibition in the PreBötC strongly affects breathing and coughing phase durations in the same manner, as well as cough expiratory efforts. In conclusion, differences in effects on cough and breathing are consistent with separate control of these behaviors.NEW & NOTEWORTHY This study is the first to explore the role of the inspiratory rhythm and pattern generator, the pre-Bötzinger complex (PreBötC), in cough motor pattern formation. In the PreBötC, excitatory glutamatergic neurotransmission affects cough intensity and patterning but not rhythm, and GABAA receptor-related inhibition affects coughing and breathing phase durations similarly to each other. Our data show that the PreBötC is important for cough motor pattern generation, but cough rhythmogenesis appears to be controlled elsewhere.

Coherence analysis of the calcium activity of putative astrocytic and neuronal cells on the L5 ventral horn and neural output in activated lumbar CPG networks.

Astrocytes are thought to play a crucial role in providing structure to the spinal cord and maintaining efficient synaptic function and metabolism because their fine processes envelop the synapses of neurons and form many neuronal networks within the central nervous system (CNS). To investigate whether putative astrocytes and putative neurons distributed on the ventral horn play a role in the modulation of lumbar locomotor central pattern generator (CPG) networks, we used extracellular recording and optical imaging techniques and recorded the neural output from the left L5 ventral root and the calcium activity of putative astrocytes and neurons in the L5 ventral horn at the same time when activating an isolated L1-L5 spinal cord preparation from rats aged 0-2 days. Optical measurements detected cells that showed a fluorescence intensity change under all experimental conditions, namely, (1) 5-HT + NMDA, (2) TTX, and (3) TTX + Low K+. These cells were semiautomatically identified using an in-house MATLAB-based program, as putative astrocytes and neurons according to the cell classification, i.e., increased or decreased fluorescence intensity change (ΔF/F0), and subjective judgment based on their soma size. Coherence and its phase were calculated according to the calcium activity of the putative astrocytes and putative neurons, and neural output was calculated during fictive locomotion with in-house MATLAB-based programs. We found that the number of putative astrocytes activated by applying low K+ tends not to differ from that activated by applying the protease-activated receptor 1 (PAR1) selective agonist TFLLR-NH2 (TFLLR). Moreover, the calcium activity of several putative astrocytes and neurons synchronized with locomotor-like activity at a frequency range below 0.5 Hz and the time lag between peaks of cellular calcium activity and locomotor-like activity ranged from -1000 to + 1000 ms. These findings presumably indicates that these putative astrocytes and neurons in the left L5 ventral horn require -1000 to + 1000 ms to communicate with lumbar CPG networks and maintain efficient synaptic function and metabolism in activated lumbar CPG networks. This finding suggests the possibility that putative astrocytic and neuronal cells in the L5 ventral horn contribute to generating the rhythms and patterns of locomotor-like activity by activated CPG networks in the first to fifth lumbar spinal cord.

The lamprey respiratory network: Some evolutionary aspects.

Breathing is a complex behaviour that involves rhythm generating networks. In this review, we examine the main characteristics of respiratory rhythm generation in vertebrates and, in particular, we describe the main results of our studies on the role of neural mechanisms involved in the neuromodulation of the lamprey respiration. The lamprey respiratory rhythm generator is located in the paratrigeminal respiratory group (pTRG) and shows similarities with the mammalian preBötzinger complex. In fact, within the pTRG a major role is played by glutamate, but also GABA and glycine display important contributions. In addition, neuromodulatory influences are exerted by opioids, substance P, acetylcholine and serotonin. Both structures respond to exogenous ATP with a biphasic response and astrocytes there located strongly contribute to the modulation of the respiratory pattern. The results emphasize that some important characteristics of the respiratory rhythm generating network are, to a great extent, maintained throughout evolution.

Glutamatergic control of a pattern-generating central nucleus in a gymnotiform fish.

The activity of central pattern-generating networks (CPGs) may change under the control exerted by various neurotransmitters and modulators to adapt its behavioral outputs to different environmental demands. Although the mechanisms underlying this control have been well established in invertebrates, most of their synaptic and cellular bases are not yet well understood in vertebrates. Gymnotus omarorum, a pulse-type gymnotiform electric fish, provides a well-suited vertebrate model to investigate these mechanisms. G. omarorum emits rhythmic and stereotyped electric organ discharges (EODs), which function in both perception and communication, under the command of an electromotor CPG. This nucleus is composed of electrotonically coupled intrinsic pacemaker cells, which pace the rhythm, and bulbospinal projecting relay cells that contribute to organize the pattern of the muscle-derived effector activation that produce the EOD. Descending influences target CPG neurons to produce adaptive behavioral electromotor responses to different environmental challenges. We used electrophysiological and pharmacological techniques in brainstem slices of G. omarorum to investigate the underpinnings of the fast transmitter control of its electromotor CPG. We demonstrate that pacemaker, but not relay cells, are endowed with ionotropic and metabotropic glutamate receptor subtypes. We also show that glutamatergic control of the CPG likely involves two types of synapses contacting pacemaker cells, one type containing both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors and the other one only-NMDA receptor. Fast neurotransmitter control of vertebrate CPGs seems to exploit the kinetics of the involved postsynaptic receptors to command different behavioral outputs. The prospect of common neural designs to control CPG activity in vertebrates is discussed.NEW & NOTEWORTHY Underpinnings of neuromodulation of central pattern-generating networks (CPG) have been well characterized in many species. The effects of fast neurotransmitter systems remain, however, poorly understood. This research uses in vitro electrophysiological and pharmacological techniques to show that the neurotransmitter control of a vertebrate CPG in gymnotiform fish involves the convergence of only-NMDA and AMPA-NMDA glutamatergic synapses onto neurons that pace the rhythm. These inputs may organize different behavioral outputs according to their distinct functional properties.

The M-current works in tandem with the persistent sodium current to set the speed of locomotion.

The central pattern generator (CPG) for locomotion is a set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM): a subthreshold noninactivating outward current blocked by 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and enhanced by N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73). Immunostaining and mutant mice highlight an important role of Kv7.2-containing channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities and controls the speed of locomotion. Computational models captured these results and showed how an interplay between IM and INaP endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion.

Neuronal Circuits That Control Rhythmic Pectoral Fin Movements in Zebrafish.

The most basic form of locomotion in limbed vertebrates consists of alternating activities of the flexor and extensor muscles within each limb coupled with left/right limb alternation. Although larval zebrafish are not limbed, their pectoral fin movements exhibit the following fundamental aspects of this basic movement: abductor/adductor alternation (corresponding to flexor/extensor alternation) and left/right fin alternation. Because of the simplicity of their movements and the compact neural organization of their spinal cords, zebrafish can serve as a good model to identify the neuronal networks of the central pattern generator (CPG) that controls rhythmic appendage movements. Here, we set out to investigate neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish, using transgenic fish that specifically express GFP in abductor or adductor motor neurons (MNs) and candidate CPG neurons. First, we showed that spiking activities of abductor and adductor MNs were essentially alternating. Second, both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively, indicating that the MN spiking activities are controlled in a push-pull manner. Further, we obtained the following evidence that dmrt3a-expressing commissural inhibitory neurons are involved in regulating the activities of abductor MNs: (1) strong inhibitory synaptic connections were found from dmrt3a neurons to abductor MNs; and (2) ablation of dmrt3a neurons shifted the spike timing of abductor MNs. Thus, in this simple system of abductor/adductor alternation, the last-order inhibitory inputs originating from the contralaterally located neurons play an important role in controlling the firing timings of MNs.SIGNIFICANCE STATEMENT Pectoral fin movements in larval zebrafish exhibit fundamental aspects of basic rhythmic appendage movement: alternation of the abductor and adductor (corresponding to flexor-extensor alternation) coupled with left-right alternation. We set out to investigate the neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish. We showed that both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively. This indicates that MN activities are controlled in a push-pull manner. We further obtained evidence that dmrt3a-expressing commissural inhibitory neurons exert an inhibitory effect on abductor MNs. The current study marks the first step toward the identification of central pattern generator organization for rhythmic fin movements.

Rapid adaptation to elevated extracellular potassium in the pyloric circuit of the crab, Cancer borealis.

Elevated potassium concentration ([K+]) is often used to alter excitability in neurons and networks by shifting the potassium equilibrium potential (EK) and, consequently, the resting membrane potential. We studied the effects of increased extracellular [K+] on the well-described pyloric circuit of the crab Cancer borealis. A 2.5-fold increase in extracellular [K+] (2.5×[K+]) depolarized pyloric dilator (PD) neurons and resulted in short-term loss of their normal bursting activity. This period of silence was followed within 5-10 min by the recovery of spiking and/or bursting activity during continued superfusion of 2.5×[K+] saline. In contrast, when PD neurons were pharmacologically isolated from pyloric presynaptic inputs, they exhibited no transient loss of spiking activity in 2.5×[K+], suggesting the presence of an acute inhibitory effect mediated by circuit interactions. Action potential threshold in PD neurons hyperpolarized during an hour-long exposure to 2.5×[K+] concurrent with the recovery of spiking and/or bursting activity. Thus the initial loss of activity appears to be mediated by synaptic interactions within the network, but the secondary adaptation depends on changes in the intrinsic excitability of the pacemaker neurons. The complex sequence of events in the responses of pyloric neurons to elevated [K+] demonstrates that electrophysiological recordings are necessary to determine both the transient and longer term effects of even modest alterations of K+ concentrations on neuronal activity.NEW & NOTEWORTHY Solutions with elevated extracellular potassium are commonly used as a depolarizing stimulus. We studied the effects of high potassium concentration ([K+]) on the pyloric circuit of the crab stomatogastric ganglion. A 2.5-fold increase in extracellular [K+] caused a transient loss of activity that was not due to depolarization block, followed by a rapid increase in excitability and recovery of spiking within minutes. This suggests that changing extracellular potassium can have complex and nonstationary effects on neuronal circuits.

Hormone-mediated modulation of the electromotor CPG in pulse-type weakly electric fish. Commonalities and differences across species.

Like stomatogastric activity in crustaceans, vocalization in teleosts and frogs, and locomotion in mammals, the electric organ discharge (EOD) of weakly electric fish is a rhythmic and stereotyped electromotor pattern. The EOD, which functions in both perception and communication, is controlled by a two-layered central pattern generator (CPG), the electromotor CPG, which modifies its basal output in response to environmental and social challenges. Despite major anatomo-functional commonalities in the electromotor CPG across electric fish species, we show that Gymnotus omarorum and Brachyhypopomus gauderio have evolved divergent neural processes to transiently modify the CPG outputs through descending fast neurotransmitter inputs to generate communication signals. We also present two examples of electric behavioral displays in which it is possible to separately analyze the effects of neuropeptides (mid-term modulation) and gonadal steroid hormones (long-term modulation) upon the CPG. First, the nonbreeding territorial aggression of G. omarorum has been an advantageous model to analyze the status-dependent modulation of the excitability of CPG neuronal components by vasotocin. Second, the seasonal and sexually dimorphic courtship signals of B. gauderio have been useful to understand the effects of sex steroids on the responses to glutamatergic inputs in the CPG. Overall, the electromotor CPG functions in a regime that safeguards the EOD waveform. However, prepacemaker influences and hormonal modulation enable an enormous versatility and allows the EOD to adapt its functional state in a species-, sex-, and social context-specific manners.

Molecular mechanisms of homeostatic plasticity in central pattern generator networks.

Central pattern generator (CPG) networks rely on a balance of intrinsic and network properties to produce reliable, repeatable activity patterns. This balance is maintained by homeostatic plasticity where alterations in neuronal properties dynamically maintain appropriate neural output in the face of changing environmental conditions and perturbations. However, it remains unclear just how these neurons and networks can both monitor their ongoing activity and use this information to elicit homeostatic physiological responses to ensure robustness of output over time. Evidence exists that CPG networks use a mixed strategy of activity-dependent, activity-independent, modulator-dependent, and synaptically regulated homeostatic plasticity to achieve this critical stability. In this review, we focus on some of the current understanding of the molecular pathways and mechanisms responsible for this homeostatic plasticity in the context of central pattern generator function, with a special emphasis on some of the smaller invertebrate networks that have allowed for extensive cellular-level analyses that have brought recent insights to these questions.

Developmental stage-dependent switching in the neuromodulation of vertebrate locomotor central pattern generator networks.

Neuromodulation plays important and stage-dependent roles in regulating locomotor central pattern (CPG) outputs during vertebrate motor system development. Dopamine, serotonin and nitric oxide are three neuromodulators that potently influence CPG outputs in the development of Xenopus frog tadpole locomotion. However, their roles switch from predominantly inhibitory early in development to mainly excitatory at later stages. In this review, we compare the stage-dependent switching in neuromodulation in Xenopus with other vertebrate systems, notably the mouse and the zebrafish, and highlight features that appear to be phylogenetically conserved.

Neuromodulation of central pattern generators and its role in the functional recovery of central pattern generator activity.

Neuromodulators play an important role in how the nervous system organizes activity that results in behavior. Disruption of the normal patterns of neuromodulatory release or production is known to be related to the onset of severe pathologies such as Parkinson's disease, Rett syndrome, Alzheimer's disease, and affective disorders. Some of these pathologies involve neuronal structures that are called central pattern generators (CPGs), which are involved in the production of rhythmic activities throughout the nervous system. Here I discuss the interplay between CPGs and neuromodulatory activity, with particular emphasis on the potential role of neuromodulators in the recovery of disrupted neuronal activity. I refer to invertebrate and vertebrate model systems and some of the lessons we have learned from research on these systems and propose a few avenues for future research. I make one suggestion that may guide future research in the field: neuromodulators restrict the parameter landscape in which CPG components operate, and the removal of neuromodulators may enable a perturbed CPG in finding a new set of parameter values that can allow it to regain normal function.

The Expression of Galanin in the Parafacial Respiratory Group and its Effects on Respiration in Neonatal Rats.

The inhibitory peptide galanin is expressed within the retrotrapezoidal nucleus (RTN) - a key central chemoreceptor site that also contains the active expiratory oscillator. It was previously reported that microinjection of galanin into pre-Bötzinger complex - containing the inspiratory oscillator - exerts inhibitory effects on inspiratory motor output and respiratory rhythm. In neonatal rats, the present study aimed to investigate: (1) expression of galanin within the parafacial respiratory group (pFRG), which overlaps anatomically and functionally with the adult RTN, and; (2) effects of galanin on respiratory rhythm using the in vitro brainstem-spinal cord preparation. We showed that 14 ±â€¯2% of Phox2b-immunoreactive (ir) neurons in the parafacial region were also galanin-ir. Galanin peptide expression was confirmed within 3/9 CO2-sensitive, Phox2b-ir Pre-Inspiratory neurons (Pre-I) recorded in parafacial region. Bath application of galanin (0.1-0.2 µM): (1) decreased the duration of membrane depolarization in both Pre-I and inspiratory pFRG neurons, and; (2) decreased the number of C4 bursts that were associated with each burst in Pre-I neurons within the pFRG. In preparations showing episodic breathing at baseline, the respiratory patterning reverted to the 'normal' pattern of single, uniformly rhythmic C4 bursts (n = 10). In preparations with normal respiratory patterning at baseline, slowing of C4 rhythm (n = 7) resulted although rhythmic bursting in recorded Pre-I neurons remained unperturbed (n = 6). This study therefore demonstrates that galanin is expressed within the pFRG of neonatal rats, including neurons that are intrinsically chemosensitive. Overall the peptide has an inhibitory effect on inspiratory motor output, as previously shown in adults.

Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits.

Transient receptor potential channel, TRPM4, the putative molecular substrate for Ca2+-activated nonselective cation current (ICAN), is hypothesized to generate bursting activity of pre-Bötzinger complex (pre-BötC) inspiratory neurons and critically contribute to respiratory rhythmogenesis. Another TRP channel, TRPC3, which mediates Na+/Ca2+ fluxes, may be involved in regulating Ca2+-related signaling, including affecting TRPM4/ICAN in respiratory pre-BötC neurons. However, TRPM4 and TRPC3 expression in pre-BötC inspiratory neurons and functional roles of these channels remain to be determined. By single-cell multiplex RT-PCR, we show mRNA expression for these channels in pre-BötC inspiratory neurons in rhythmically active medullary in vitro slices from neonatal rats and mice. Functional contributions were analyzed with pharmacological inhibitors of TRPM4 or TRPC3 in vitro as well as in mature rodent arterially perfused in situ brainstem-spinal cord preparations. Perturbations of respiratory circuit activity were also compared with those by a blocker of ICAN. Pharmacologically attenuating endogenous activation of TRPM4, TRPC3, or ICANin vitro similarly reduced the amplitude of inspiratory motoneuronal activity without significant perturbations of inspiratory frequency or variability of the rhythm. Amplitude perturbations were correlated with reduced inspiratory glutamatergic pre-BötC neuronal activity, monitored by multicellular dynamic calcium imaging in vitro. In more intact circuits in situ, the reduction of pre-BötC and motoneuronal inspiratory activity amplitude was accompanied by reduced post-inspiratory motoneuronal activity, without disruption of rhythm generation. We conclude that endogenously activated TRPM4, which likely mediates ICAN, and TRPC3 channels in pre-BötC inspiratory neurons play fundamental roles in respiratory pattern formation but are not critically involved in respiratory rhythm generation.

Three members of a peptide family are differentially distributed and elicit differential state-dependent responses in a pattern generator-effector system.

C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides. NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.

ATP and astrocytes play a prominent role in the control of the respiratory pattern generator in the lamprey.

KEY POINTS: The paratrigeminal respiratory group (pTRG) is responsible for the respiratory pattern generation in the lamprey. The role of ATP and astrocytes, known to control respiratory activity in mammals, was investigated in the lamprey respiratory network. ATP microinjected into the pTRG induces a biphasic response consisting of marked increases in respiratory frequency mediated by P2X receptors followed by a decrease in the respiratory motor output due to the ATP metabolite adenosine. We provide evidence that astrocytes are involved in the genesis of the normal respiratory pattern, ATP-induced responses and acidification-induced increases of the respiratory activity. The function of astrocytes in rhythmic networks appears to be phylogenetically conserved. ABSTRACT: The role of ATP and astrocytes in respiratory rhythm modulation has been recently investigated in neonatal rodents. However, no information on the role of ATP and astrocytes within the respiratory network of the lamprey is available, particularly within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator. To address these issues, the present study was carried out on isolated brainstems of the adult lamprey. Bath application of ATP caused marked increases in respiratory frequency followed by decreases in the respiratory motor output, mediated by the ATP metabolite adenosine at the level of the pTRG. Bath applications and microinjections of agonists and antagonists of purinergic receptors showed that ATP increased respiratory activity through an action on pTRG P2X receptors. To disclose the respiratory role of astrocytes, we used bath application of the gliotoxin aminoadipic acid, which dramatically depressed the respiratory motor output that, however, promptly recovered following glutamine application. Furthermore, the excitatory responses to ATP-γ-S (a non-hydrolysable ATP analogue), but not to substance P, microinjected into the pTRG, were abolished. Finally, we also demonstrated that acidification-induced increases in respiratory activity were ATP-independent, but mediated by the astrocytes' glutamate-glutamine cycle. The results show for the first time that ATP and especially astrocytes strongly contribute to the modulation of the lamprey respiratory pattern. Their role in the modulation or maintenance of rhythmic neuronal activities appears to be phylogenetically conserved.

Modulatory and plastic effects of kinins on spinal cord networks.

KEY POINTS: Inflammatory kinins are released following spinal cord injury or neurotrauma. The effects of these kinins on ongoing locomotor activity of central pattern generator networks are unknown. In the present study, kinins were shown to have short- and long-term effects on motor networks. The short-term effects included direct depolarization of interneurons and motoneurons in the ventral horn accompanied by modulation of transient receptor potential vanilloid 1-sensitive nociceptors in the dorsal horn. Over the long-term, we observed a bradykinin-mediated effect on promoting plasticity in the spinal cord. In a model of spinal cord injury, we observed an increase in microglia numbers in both the dorsal and ventral horn and, in a microglia cell culture model, we observed bradykinin-induced expression of glial-derived neurotrophic factor. ABSTRACT: The expression and function of inflammatory mediators in the developing spinal cord remain poorly characterized. We discovered novel, short and long-term roles for the inflammatory nonapeptide bradykinin (BK) and its receptor bradykinin receptor B2 (B2R) in the neuromodulation of developing sensorimotor networks following a spinal cord injury (SCI), suggesting that BK participates in an excitotoxic cascade. Functional expression of B2R was confirmed by a transient disruptive action of BK on fictive locomotion generated by a combination of NMDA, 5-HT and dopamine. The role of BK in the dorsal horn nociceptive afferents was tested using spinal cord attached to one-hind-limb (HL) preparations. In the HL preparations, BK at a subthreshold concentration induced transient disruption of fictive locomotion only in the presence of: (1) noxious heat applied to the hind paw and (2) the heat sensing ion channel transient receptor potential vanilloid 1 (TRPV1), known to be restricted to nociceptors in the superficial dorsal horn. BK directly depolarized motoneurons and ascending interneurons in the ventrolateral funiculus. We found a key mechanism for BK in promoting long-term plasticity within the spinal cord. Using a model of neonatal SCI and a microglial cell culture model, we examined the role of BK in inducing activation of microglia and expression of glial-derived neurotrophic factor (GDNF). In the neonatal SCI model, we observed an increase in microglia numbers and increased GDNF expression restricted to microglia. In the microglia cell culture model, we observed a BK-induced increased expression of GDNF via B2R, suggesting a novel mechanism for BK spinal-mediated plasticity.

Ion Homeostasis in Rhythmogenesis: The Interplay Between Neurons and Astroglia.

Proper function of all excitable cells depends on ion homeostasis. Nowhere is this more critical than in the brain where the extracellular concentration of some ions determines neurons' firing pattern and ability to encode information. Several neuronal functions depend on the ability of neurons to change their firing pattern to a rhythmic bursting pattern, whereas, in some circuits, rhythmic firing is, on the contrary, associated to pathologies like epilepsy or Parkinson's disease. In this review, we focus on the four main ions known to fluctuate during rhythmic firing: calcium, potassium, sodium, and chloride. We discuss the synergistic interactions between these elements to promote an oscillatory activity. We also review evidence supporting an important role for astrocytes in the homeostasis of each of these ions and describe mechanisms by which astrocytes may regulate neuronal firing by altering their extracellular concentrations. A particular emphasis is put on the mechanisms underlying rhythmogenesis in the circuit forming the central pattern generator (CPG) for mastication and other CPG systems. Finally, we discuss how an impairment in the ability of glial cells to maintain such homeostasis may result in pathologies like epilepsy and Parkinson's disease.

Transient BK outward current enhances motoneurone firing rates during Drosophila larval locomotion.

KEY POINTS: We combine in situ electrophysiology with genetic manipulation in Drosophila larvae aiming to investigate the role of fast calcium-activated potassium currents for motoneurone firing patterns during locomotion. We first demonstrate that slowpoke channels underlie fast calcium-activated potassium currents in these motoneurones. By conducting recordings in semi-intact animals that produce crawling-like movements, we show that slowpoke channels are required specifically in motoneurones for maximum firing rates during locomotion. Such enhancement of maximum firing rates occurs because slowpoke channels prevent depolarization block by limiting the amplitude of motoneurone depolarization in response to synaptic drive. In addition, slowpoke channels mediate a fast afterhyperpolarization that ensures the efficient recovery of sodium channels from inactivation during high frequency firing. The results of the present study provide new insights into the mechanisms by which outward conductances facilitate neuronal excitability and also provide direct confirmation of the functional relevance of precisely regulated slowpoke channel properties in motor control. ABSTRACT: A large number of voltage-gated ion channels, their interactions with accessory subunits, and their post-transcriptional modifications generate an immense functional diversity of neurones. Therefore, a key challenge is to understand the genetic basis and precise function of specific ionic conductances for neuronal firing properties in the context of behaviour. The present study identifies slowpoke (slo) as exclusively mediating fast activating, fast inactivating BK current (ICF ) in larval Drosophila crawling motoneurones. Combining in vivo patch clamp recordings during larval crawling with pharmacology and targeted genetic manipulations reveals that ICF acts specifically in motoneurones to sculpt their firing patterns in response to a given input from the central pattern generating (CPG) networks. First, ICF curtails motoneurone postsynaptic depolarizations during rhythmical CPG drive. Second, ICF is activated during the rising phase of the action potential and mediates a fast afterhyperpolarization. Consequently, ICF is required for maximal intraburst firing rates during locomotion, probably by allowing recovery from inactivation of fast sodium channels and decreased potassium channel activation. This contrasts the common view that outward conductances oppose excitability but is in accordance with reports on transient BK and Kv3 channel function in multiple types of vertebrate neurones. Therefore, our finding that ICF enhances firing rates specifically during bursting patterns relevant to behaviour is probably of relevance to all brains.

State-dependent control of breathing by the retrotrapezoid nucleus.

KEY POINTS: This study explores the state dependence of the hypercapnic ventilatory reflex (HCVR). We simulated an instantaneous increase or decrease of central chemoreceptor activity by activating or inhibiting the retrotrapezoid nucleus (RTN) by optogenetics in conscious rats. During quiet wake or non-REM sleep, hypercapnia increased both breathing frequency (fR ) and tidal volume (VT ) whereas, in REM sleep, hypercapnia increased VT exclusively. Optogenetic inhibition of RTN reduced VT in all sleep-wake states, but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Phasic RTN stimulation produced active expiration and reduced early expiratory airflow (i.e. increased upper airway resistance) only during wake. We conclude that the HCVR is highly state-dependent. The HCVR is reduced during REM sleep because fR is no longer under chemoreceptor control and thus could explain why central sleep apnoea is less frequent in REM sleep. ABSTRACT: Breathing has different characteristics during quiet wake, non-REM or REM sleep, including variable dependence on PCO2. We investigated whether the retrotrapezoid nucleus (RTN), a proton-sensitive structure that mediates a large portion of the hypercapnic ventilatory reflex, regulates breathing differently during sleep vs. wake. Electroencephalogram, neck electromyogram, blood pressure, respiratory frequency (fR ) and tidal volume (VT ) were recorded in 28 conscious adult male Sprague-Dawley rats. Optogenetic stimulation of RTN with channelrhodopsin-2, or inhibition with archaerhodopsin, simulated an instantaneous increase or decrease of central chemoreceptor activity. Both opsins were delivered with PRSX8-promoter-containing lentiviral vectors. RTN and catecholaminergic neurons were transduced. During quiet wake or non-REM sleep, hypercapnia (3 or 6% FI,CO2 ) increased both fR and VT whereas, in REM sleep, hypercapnia increased VT exclusively. RTN inhibition always reduced VT but reduced fR only during quiet wake and non-REM sleep. RTN stimulation always increased VT but raised fR only in quiet wake and non-REM sleep. Blood pressure was unaffected by either stimulation or inhibition. Except in REM sleep, phasic RTN stimulation entrained and shortened the breathing cycle by selectively shortening the post-inspiratory phase. Phasic stimulation also produced active expiration and reduced early expiratory airflow but only during wake. VT is always regulated by RTN and CO2 but fR is regulated by CO2 and RTN only when the brainstem pattern generator is in autorhythmic mode (anaesthesia, non-REM sleep, quiet wake). The reduced contribution of RTN to breathing during REM sleep could explain why certain central apnoeas are less frequent during this sleep stage.