Swallowing-like activity elicited in neonatal rat medullary slice preparation.

Swallowing is induced by a central pattern generator in the nucleus tractus solitarius (NTS). We aimed to create a medullary slice preparation to elucidate the neural architecture of the central pattern generator of swallowing (Sw-CPG) and record its neural activities. Experiments were conducted on 2-day-old Sprague-Dawley rats (n = 46). The brainstem-spinal cord was transected at the pontomedullary and cervicothoracic junctions; the medulla was sliced transversely at thicknesses of 600, 700, or 800 µm. The rostral end of the slice was 100 µm rostral to the vagus nerve. We recorded hypoglossal nerve activity and electrically stimulated the vagus nerve or microinjected bicuculline methiodide (BIC) into the NTS. The 800-µm slices generated both rhythmic respiratory activity and electrically elicited neural activity. The 700-µm slices generated only respiratory activity, while the 600-µm slices did not generate any neural activity. BIC microinjection into the NTS in 800-µm slices resulted in the typical activity that closely resembled the swallowing activity reported in other experiments. This swallowing-like activity consistently lengthened the respiratory interval. Despite complete inhibition of respiratory activity, weak swallowing-like activity was observed under bath application of a non-NMDA receptor antagonist. Contrastingly, bath application of NMDA receptor antagonists resulted in a complete loss of swallowing-like activity and no change in respiratory activity. These results suggest that the 800-µm medullary slice preparation contains both afferent and efferent neural circuits and pattern generators of swallowing activity. Additionally, NMDA receptors may be necessary for generating swallowing activity. This medullary slice preparation can therefore elucidate Sw-CPG neural networks.

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 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.

Neural mechanisms underlying respiratory regulation within the preBötzinger complex of the rabbit.

The preBötzinger complex (preBötC) is a medullary area essential for normal breathing and widely recognized as necessary and sufficient to generate the inspiratory phase of respiration. It has been studied mainly in rodents. Here we report the main results of our studies revealing the characteristics of the rabbit preBötC identified by means of neuronal recordings, D,L-homocysteic acid microinjections and histological controls. A crucial role in the respiratory rhythmogenesis within this neural substrate is played by excitatory amino acids, but also GABA and glycine display important contributions. Increases in respiratory frequency are induced by microinjections of neurokinins, somatostatin as well by serotonin (5-HT) through an action on 5-HT1A and 5-HT3 receptors or the disinhibition of a GABAergic circuit. Respiratory depression is observed in response to microinjections of the µ-opioid receptor agonist DAMGO. Our results show similarities and differences with the rodent preBötC and emphasize the importance of comparative studies on the mechanisms underlying respiratory rhythmogenesis in different animal species.

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.

Neuronal mechanisms underlying opioid-induced respiratory depression: our current understanding.

Opioid-induced respiratory depression (OIRD) represents the primary cause of death associated with therapeutic and recreational opioid use. Within the United States, the rate of death from opioid abuse since the early 1990s has grown disproportionally, prompting the classification as a nationwide "epidemic." Since this time, we have begun to unravel many fundamental cellular and systems-level mechanisms associated with opioid-related death. However, factors such as individual vulnerability, neuromodulatory compensation, and redundancy of opioid effects across central and peripheral nervous systems have created a barrier to a concise, integrative view of OIRD. Within this review, we bring together multiple perspectives in the field of OIRD to create an overarching viewpoint of what we know, and where we view this essential topic of research going forward into the future.

Excitation-inhibition balance regulates the patterning of spinal and cranial inspiratory motor outputs in rats in situ.

Spinal phrenic nerve activity (PNA) drives the diaphragm but cranial hypoglossal nerve activity (HNA) also expresses synchronous activity during inspiration. Here, we investigated the effects of local disinhibition (bilateral microinjections of bicuculline) of the nucleus tractus solitarius (NTS), the pre-Bötzinger complex and Bötzinger complex core circuit (pre-BötC/BötC) and the Kölliker-Fuse nuclei (KFn) on the synchronization of PNA and HNA in arterially-perfused brainstem preparations of rats. To quantitatively analyze the bicuculline effects on a putatively distributed inspiratory central pattern generator (i-CPG), we quantified the phase synchronization properties between PNA and HNA. The analysis revealed that bicuculline-evoked local disinhibition significantly reduced the strength of phase synchronization between PNA and HNA at any target site. However, the emergence of desynchronized HNA following disinhibition was more prevalent after NTS or pre-BötC/BötC microinjections compared to the KFn. We conclude that the primary i-CPG is located in a distributed medullary circuit whereas pontine contributions are restricted to synaptic gating of synchronous HNA and PNA.

Interaction between nesfatin-1 and oxytocin in the modulation of the swallowing reflex.

Nesfatin-1, an 82-amino acid peptide encoded by the secreted precursor nucleobinin-2 (NUCB2), exerts potent anorexigenic action independently of leptin signaling. This propensity has propelled this peptide and its analogues as potential anti-obesity drug candidates. However, a more extensive comprehension of its biological actions is needed prior to envisaging its potential use in the treatment of metabolic diseases. Swallowing is an essential motor component of ingestive behavior, which induces the propulsion of the alimentary bolus from the mouth to the esophagus. The dorsal swallowing group (DSG) which constitutes a part of the central pattern generator of swallowing (SwCPG) is located within the solitary tract nucleus (STN), a region reported to contain nesfatin-1/NUCB2 expressing neurons. In this context, we investigate here the possible effects of nesfatin-1 on swallowing discharge. Nesfatin-1 dose-dependently inhibited swallowing reflex and activated neurons located in the DSG region. In addition, we provide evidences that strongly suggest that this nesfatin-1 inhibitory effect involved an oxytocinergic relay. Indeed, oxytocin (OT) injection at the brainstem level inhibited swallowing reflex and OT receptor antagonist prevented nesfatin-1 inhibitory action. Altogether, these data constitute the first demonstration that nesfatin-1 modulates swallowing reflex by acting at the brainstem level via an oxytocinergic relay.

Doxapram stimulates respiratory activity through distinct activation of neurons in the nucleus hypoglossus and the pre-Bötzinger complex.

Doxapram is a respiratory stimulant used for decades as a treatment option in apnea of prematurity refractory to methylxanthine treatment. Its mode of action, however, is still poorly understood. We investigated direct effects of doxapram on the pre-Bötzinger complex (PreBötC) and on a downstream motor output system, the hypoglossal nucleus (XII), in the transverse brainstem slice preparation. While doxapram has only a modest stimulatory effect on frequency of activity generated within the PreBötC, a much more robust increase in the amplitude of population activity in the subsequent motor output generated in the XII was observed. In whole cell patch-clamp recordings of PreBötC and XII neurons, we confirmed significantly increased firing of evoked action potentials in XII neurons in the presence of doxapram, while PreBötC neurons showed no significant alteration in firing properties. Interestingly, the amplitude of activity in the motor output was not increased in the presence of doxapram compared with control conditions during hypoxia. We conclude that part of the stimulatory effects of doxapram is caused by direct input on brainstem centers with differential effects on the rhythm generating kernel (PreBötC) and the downstream motor output (XII). NEW & NOTEWORTHY The clinically used respiratory stimulant doxapram has distinct effects on the rhythm generating kernel (pre-Bötzinger complex) and motor output centers (nucleus hypoglossus). These effects are obliterated during hypoxia and are mediated by distinct changes in the intrinsic properties of neurons of the nucleus hypoglossus and synaptic transmission received by pre-Bötzinger complex neurons.

Graded Transmission without Action Potentials Sustains Rhythmic Activity in Some But Not All Modulators That Activate the Same Current.

Neurons in the central pattern-generating circuits in the crustacean stomatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide Ia (CabTRP1a), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (IMI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRP1a and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRP1a in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP1a in the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na+ current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na+ channels, and one that can persist in their absence.SIGNIFICANCE STATEMENT The pyloric rhythm of the crab stomatogastric ganglion depends both on spike-mediated and graded synaptic transmission. We activate the pyloric rhythm with a wide variety of different neuromodulators, all of which converge on the same voltage-dependent inward current. Interestingly, when action potentials and spike-mediated transmission are blocked using TTX, we find that the muscarinic agonist oxotremorine and the neuropeptide CabTRP1a sustain rhythmic alternations and appropriate phases of activity in the absence of action potentials. In contrast, TTX blocks rhythmic activity in the presence of other modulators. This demonstrates fundamental differences in the burst-generation mechanisms in different modulators that would not be suspected on the basis of their cellular actions at the level of the targeted current.

Nicotine-mediated neuroprotection of rat spinal networks against excitotoxicity.

Activation of neuronal nicotinic acetylcholine receptors (nAChRs) by nicotine is reported to protect brain neurons from glutamate excitotoxicity. We inquired whether a similar phenomenon can occur in the rat isolated spinal cord (or spinal slice culture) challenged by a transient (1 hr) application of kainate (a powerful glutamate receptor agonist) to induce excitotoxicity mimicking spinal injury in vitro. We recorded spinal reflexes and fictive locomotion generated by the locomotor central pattern generator before and 24 hr after applying kainate. We also monitored network activity with Ca2+ imaging and counted neurons and glia with immunohistochemical methods. In control conditions, nicotine (1 µM; 4 hr) depressed reflexes and fictive locomotion with slow recovery and no apparent neurotoxicity at 24 hr although synchronous Ca2+ transients appeared in slice cultures. Kainate nearly halved neuron numbers (while sparing glia), decreased reflexes and Ca2+ transients, and suppressed fictive locomotion. When nicotine was applied (4 hr) after washout of kainate, fictive locomotor cycles appeared 24 hr later though with low periodicity, and significant protection of neurons, including motoneurons, was observed. Nicotine applied together with kainate and maintained for further 4 hr yielded better neuroprotection, improved fictive locomotion expression and reversed the depression of Ca2+ transients. nAChR antagonists did not intensify kainate neurotoxicity and inhibited the neuroprotective effects of nicotine. These data suggest that nicotine was efficacious to limit histological and functional excitotoxic damage probably because it activated and then desensitized nAChRs on excitatory and inhibitory network neurons to prevent triggering intracellular cell death pathways.

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.

Rapid recovery and altered neurochemical dependence of locomotor central pattern generation following lumbar neonatal spinal cord injury.

KEY POINTS: Spinal compression injury targeted to the neonatal upper lumbar spinal cord, the region of highest hindlimb locomotor rhythmogenicity, leads to an initial paralysis of the hindlimbs. Behavioural recovery is evident within a few days and approaches normal function within about 3 weeks. Fictive locomotion in the isolated injured spinal cord cannot be elicited by a neurochemical cocktail containing NMDA, dopamine and serotonin 1 day post-injury, but can 3 days post-injury as readily as in the uninjured spinal cord. Low frequency coordinated rhythmic activity can be elicited in the isolated uninjured spinal cord by NMDA + dopamine (without serotonin), but not in the isolated injured spinal cord. In both the injured and uninjured spinal cord, eliciting bona fide fictive locomotion requires the additional presence of serotonin. ABSTRACT: Following incomplete compression injury in the thoracic spinal cord of neonatal mice 1 day after birth (P1), we previously reported that virtually normal hindlimb locomotor function is recovered within about 3 weeks despite substantial permanent thoracic tissue loss. Here, we asked whether similar recovery occurs following lumbar injury that impacts more directly on the locomotor central pattern generator (CPG). As in thoracic injuries, lumbar injuries caused about 90% neuronal loss at the injury site and increased serotonergic innervation below the injury. Motor recovery was slower after lumbar than thoracic injury, but virtually normal function was attained by P25 in both cases. Locomotor CPG status was tested by eliciting fictive locomotion in isolated spinal cords using a widely used neurochemical cocktail (NMDA, dopamine, serotonin). No fictive locomotion could be elicited 1 day post-injury, but could within 3 days post-injury as readily as in age-matched uninjured control spinal cords. Burst patterning and coordination were largely similar in injured and control spinal cords but there were differences. Notably, in both groups there were two main locomotor frequencies, but injured spinal cords exhibited a shift towards the higher frequency. Injury also altered the neurochemical dependence of locomotor CPG output, such that injured spinal cords, unlike control spinal cords, were incapable of generating low frequency rhythmic coordinated activity in the presence of NMDA and dopamine alone. Thus, the neonatal spinal cord also exhibits remarkable functional recovery after lumbar injuries, but the neurochemical sensitivity of locomotor circuitry is modified in the process.

Modulation of Rhythmic Activity in Mammalian Spinal Networks Is Dependent on Excitability State.

Neuromodulators play an important role in activating rhythmically active motor networks; however, what remains unclear are the network interactions whereby neuromodulators recruit spinal motor networks to produce rhythmic activity. Evidence from invertebrate systems has demonstrated that the effect of neuromodulators depends on the pre-existing state of the network. We explored how network excitation state affects the ability of dopamine to evoke rhythmic locomotor activity in the neonatal mouse isolated spinal cord. We found that dopamine can evoke unique patterns of motor activity that are dependent on the excitability state of motor networks. Different patterns of motor activity ranging from tonic, nonrhythmic activity to multirhythmic, nonlocomotor activity to locomotor activity were produced by altering global motor network excitability through manipulations of the extracellular potassium and bath NMDA concentration. A similar effect was observed when network excitation was manipulated during an unstable multirhythm evoked by a low concentration (15 µm) of 5-HT, suggesting that our results are not neuromodulator specific. Our data show in vertebrate systems that modulation is a two-way street and that modulatory actions are largely influenced by the network state. The level of network excitation can account for variability between preparations and is an additional factor to be considered when circuit elements are removed from the network.

Rhythm generation, coordination, and initiation in the vocal pathways of male African clawed frogs.

Central pattern generators (CPGs) in the brain stem are considered to underlie vocalizations in many vertebrate species, but the detailed mechanisms underlying how motor rhythms are generated, coordinated, and initiated remain unclear. We addressed these issues using isolated brain preparations of Xenopus laevis from which fictive vocalizations can be elicited. Advertisement calls of male X. laevis that consist of fast and slow trills are generated by vocal CPGs contained in the brain stem. Brain stem central vocal pathways consist of a premotor nucleus [dorsal tegmental area of medulla (DTAM)] and a laryngeal motor nucleus [a homologue of nucleus ambiguus (n.IX-X)] with extensive reciprocal connections between the nuclei. In addition, DTAM receives descending inputs from the extended amygdala. We found that unilateral transection of the projections between DTAM and n.IX-X eliminated premotor fictive fast trill patterns but did not affect fictive slow trills, suggesting that the fast and slow trill CPGs are distinct; the slow trill CPG is contained in n.IX-X, and the fast trill CPG spans DTAM and n.IX-X. Midline transections that eliminated the anterior, posterior, or both commissures caused no change in the temporal structure of fictive calls, but bilateral synchrony was lost, indicating that the vocal CPGs are contained in the lateral halves of the brain stem and that the commissures synchronize the two oscillators. Furthermore, the elimination of the inputs from extended amygdala to DTAM, in addition to the anterior commissure, resulted in autonomous initiation of fictive fast but not slow trills by each hemibrain stem, indicating that the extended amygdala provides a bilateral signal to initiate fast trills. NEW & NOTEWORTHY: Central pattern generators (CPGs) are considered to underlie vocalizations in many vertebrate species, but the detailed mechanisms underlying their functions remain unclear. We addressed this question using an isolated brain preparation of African clawed frogs. We discovered that two vocal phases are mediated by anatomically distinct CPGs, that there are a pair of CPGs contained in the left and right half of the brain stem, and that mechanisms underlying initiation of the two vocal phases are distinct.

Repetition priming of motor activity mediated by a central pattern generator: the importance of extrinsic vs. intrinsic program initiators.

Repetition priming is characterized by increased performance as a behavior is repeated. Although this phenomenon is ubiquitous, mediating mechanisms are poorly understood. We address this issue in a model system, the feeding network of Aplysia This network generates both ingestive and egestive motor programs. Previous data suggest a chemical coding model: ingestive and egestive inputs to the feeding central pattern generator (CPG) release different modulators, which act via different second messengers to prime motor activity in different ways. The ingestive input to the CPG (neuron CBI-2) releases the peptides feeding circuit activating peptide and cerebral peptide 2, which produce an ingestive pattern of activity. The egestive input to the CPG (the esophageal nerve) releases the peptide small cardioactive peptide. This model is based on research that focused on a single aspect of motor control (radula opening). Here we ask whether repetition priming is observed if activity is triggered with a neuron within the core CPG itself and demonstrate that it is not. Moreover, previous studies demonstrated that effects of modulatory neurotransmitters that induce repetition priming persist. This suggests that it should be possible to "prime" motor programs triggered from within the CPG by first stimulating extrinsic modulatory inputs. We demonstrate that programs triggered after ingestive input activation are ingestive and programs triggered after egestive input activation are egestive. We ask where this priming occurs and demonstrate modifications within the CPG itself. This arrangement is likely to have important consequences for "task" switching, i.e., the cessation of one type of motor activity and the initiation of another.

Synergy temporal sequences and topography in the spinal cord: evidence for a traveling wave in frog locomotion.

Locomotion is produced by a central pattern generator. Its spinal cord organization is generally considered to be distributed, with more rhythmogenic rostral lumbar segments. While this produces a rostrocaudally traveling wave in undulating species, this is not thought to occur in limbed vertebrates, with the exception of the interneuronal traveling wave demonstrated in fictive cat scratching (Cuellar et al. J Neurosci 29:798-810, 2009). Here, we reexamine this hypothesis in the frog, using the seven muscle synergies A to G previously identified with intraspinal NMDA (Saltiel et al. J Neurophysiol 85:605-619, 2001). We find that locomotion consists of a sequence of synergy activations (A-B-G-A-F-E-G). The same sequence is observed when focal NMDA iontophoresis in the spinal cord elicits a caudal extension-lateral force-flexion cycle (flexion onset without the C synergy). Examining the early NMDA-evoked motor output at 110 sites reveals a rostrocaudal topographic organization of synergy encoding by the lumbar cord. Each synergy is preferentially activated from distinct regions, which may be multiple, and partially overlap between different synergies. Comparing the sequence of synergy activation in locomotion with their spinal cord topography suggests that the locomotor output is achieved by a rostrocaudally traveling wave of activation in the swing-stance cycle. A two-layer circuitry model, based on this topography and a traveling wave reproduces this output and explores its possible modifications under different afferent inputs. Our results and simulations suggest that a rostrocaudally traveling wave of excitation takes advantage of the topography of interneuronal regions encoding synergies, to activate them in the proper sequence for locomotion.

The role of the Kölliker-Fuse nuclei in the determination of abdominal motor output in a perfused brainstem preparation of juvenile rat.

The abdominal muscles are largely quiescent during normal breathing but may exhibit tonic activity or subtle respiratory modulation. The origin of baseline abdominal motor nerve activity (AbNA) if present remains uncharacterised. The contribution of the Kölliker-Fuse nucleus (KF) in the dorsolateral pons in the patterning and amplitude of AbNA was investigated using in situ perfused brainstem preparations of juvenile rats (n=12). Two types of AbNA were observed: Type I - expiratory-modulated (n=7), and Type II - weakly inspiratory/post-inspiratory-modulated (n=5). Despite this, all preparations exhibited the same bi-phasic late expiratory/postinspiratory bursts upon elicitation of the peripheral chemoreflex. Interestingly, the type of AbNA exhibited correlated with postinspiratory duration. Targeted microinjections of GABA-A receptor agonist isoguvacine (10mM; 70nl) into KF however did not significantly modify pattern or amplitude of baseline AbNA in either Type besides the selective abolition of the postinspiratory phase and, consequently, postinspiratory modulation in AbNAwhen present. In sum, the KF is not a major contributorin setting baseline abdominal motor output.

Microglia modulate respiratory rhythm generation and autoresuscitation.

Inflammation has been linked to the induction of apneas and Sudden Infant Death Syndrome, whereas proinflammatory mediators inhibit breathing when applied peripherally or directly into the CNS. Considering that peripheral inflammation can activate microglia in the CNS and that this cell type can directly release all proinflammatory mediators that modulate breathing, it is likely that microglia can modulate breathing generation. It might do so also in hypoxia, since microglia are sensitive to hypoxia, and peripheral proinflammatory conditions affect gasping generation and autoresuscitation. Here, we tested whether microglial activation or inhibition affected respiratory rhythm generation. By measuring breathing as well as the activity of the respiratory rhythm generator (the preBötzinger complex), we found that several microglial activators or inhibitors, applied intracisternally in vivo or in the recording bath in vitro, affect the generation of the respiratory rhythms both in normoxia and hypoxia. Furthermore, microglial activation with lipopolysaccharide affected the ability of the animals to autoresuscitate after hypoxic conditions, an effect that is blocked when lipopolysaccharide is co-applied with the microglial inhibitor minocycline. Moreover, we found that the modulation of respiratory rhythm generation induced in vitro by microglial inhibitors was reproduced by microglial depletion. In conclusion, our data show that microglia can modulate respiratory rhythm generation and autoresuscitation.