Locomotor efference copy signaling and gaze control: An evolutionary perspective.

Neural replicas of the spinal motor commands that drive locomotion have become increasingly recognized as an intrinsic neural mechanism for producing gaze-stabilizing eye movements that counteract the perturbing effects of self-generated head/body motion. By pre-empting reactive signaling by motion-detecting vestibular sensors, such locomotor efference copies (ECs) provide estimates of the sensory consequences of behavioral action. Initially demonstrated in amphibian larvae during spontaneous fictive swimming in deafferented in vitro preparations, direct evidence for a contribution of locomotor ECs to gaze stabilization now extends to the ancestral lamprey and to tetrapod adult frogs and mice. Supporting behavioral evidence also exists for other mammals, including humans, therefore further indicating the mechanism's conservation during vertebrate evolution. The relationship between feedforward ECs and vestibular sensory feedback in ocular movement control is variable, ranging from additive to the former supplanting the latter, depending on vestibular sensing ability, and the intensity and regularity of rhythmic locomotor movements.

From tadpole to adult frog locomotion.

The transition from larval to adult locomotion in the anuran, Xenopus laevis, involves a dramatic switch from axial to appendicular swimming including intermediate stages when the tail and hindlimbs co-exist and contribute to propulsion. Hatchling tadpole swimming is generated by an axial central pattern generator (CPG) which matures rapidly during early larval life. During metamorphosis, the developing limbs are controlled by a de novo appendicular CPG driven initially by the axial system before segregating to allow both systems to operate together or independently. Neuromodulation plays important roles throughout, but key modulators switch their effects from early inhibitory influences to facilitating locomotion. Temperature affects the construction and operation of locomotor networks and global changes in environmental temperature place aquatic poikilotherms, like amphibians, at risk. The locomotor control strategy of anurans differs from other amphibian groups such as salamanders, where evolution has acted upon the thyroid hormone pathway to sculpt different developmental outcomes.

Sodium-mediated plateau potentials in an identified decisional neuron contribute to feeding-related motor pattern genesis in Aplysia.

Motivated behaviors such as feeding depend on the functional properties of decision neurons to provide the flexibility required for behavioral adaptation. Here, we analyzed the ionic basis of the endogenous membrane properties of an identified decision neuron (B63) that drive radula biting cycles underlying food-seeking behavior in Aplysia. Each spontaneous bite cycle arises from the irregular triggering of a plateau-like potential and resultant bursting by rhythmic subthreshold oscillations in B63's membrane potential. In isolated buccal ganglion preparations, and after synaptic isolation, the expression of B63's plateau potentials persisted after removal of extracellular calcium, but was completely suppressed in a tetrodotoxin (TTX)- containing bath solution, thereby indicating the contribution of a transmembrane Na+ influx. Potassium outward efflux through tetraethylammonium (TEA)- and calcium-sensitive channels was found to contribute to each plateau's active termination. This intrinsic plateauing capability, in contrast to B63's membrane potential oscillation, was blocked by the calcium-activated non-specific cationic current (ICAN) blocker flufenamic acid (FFA). Conversely, the SERCA blocker cyclopianozic acid (CPA), which abolished the neuron's oscillation, did not prevent the expression of experimentally evoked plateau potentials. These results therefore indicate that the dynamic properties of the decision neuron B63 rely on two distinct mechanisms involving different sub-populations of ionic conductances.

Role of locomotor efference copy in vertebrate gaze stabilization.

Vertebrate locomotion presents a major challenge for maintaining visual acuity due to head movements resulting from the intimate biomechanical coupling with the propulsive musculoskeletal system. Retinal image stabilization has been traditionally ascribed to the transformation of motion-related sensory feedback into counteracting ocular motor commands. However, extensive exploration of spontaneously active semi-intact and isolated brain/spinal cord preparations of the amphibian Xenopus laevis, have revealed that efference copies (ECs) of the spinal motor program that generates axial- or limb-based propulsion directly drive compensatory eye movements. During fictive locomotion in larvae, ascending ECs from rostral spinal central pattern generating (CPG) circuitry are relayed through a defined ascending pathway to the mid- and hindbrain ocular motor nuclei to produce conjugate eye rotations during tail-based undulatory swimming in the intact animal. In post-metamorphic adult frogs, this spinal rhythmic command switches to a bilaterally-synchronous burst pattern that is appropriate for generating convergent eye movements required for maintaining image stability during limb kick-based rectilinear forward propulsion. The transition between these two fundamentally different coupling patterns is underpinned by the emergence of altered trajectories in spino-ocular motor coupling pathways that occur gradually during metamorphosis, providing a goal-specific, morpho-functional plasticity that ensures retinal image stability irrespective of locomotor mode. Although the functional impact of predictive ECs produced by the locomotory CPG matches the spatio-temporal specificity of reactive sensory-motor responses, rather than contributing additively to image stabilization, horizontal vestibulo-ocular reflexes (VORs) are selectively suppressed during intense locomotor CPG activity. This is achieved at least in part by an EC-mediated attenuation of mechano-electrical encoding at the vestibular sensory periphery. Thus, locomotor ECs and their potential suppressive impact on vestibular sensory-motor processing, both of which have now been reported in other vertebrates including humans, appear to play an important role in the maintenance of stable vision during active body displacements.

Microglia shape the embryonic development of mammalian respiratory networks.

Microglia, brain-resident macrophages, play key roles during prenatal development in defining neural circuitry function, including ensuring proper synaptic wiring and maintaining homeostasis. Mammalian breathing rhythmogenesis arises from interacting brainstem neural networks that are assembled during embryonic development, but the specific role of microglia in this process remains unknown. Here, we investigated the anatomical and functional consequences of respiratory circuit formation in the absence of microglia. We first established the normal distribution of microglia within the wild-type (WT, Spi1+/+ (Pu.1 WT)) mouse (Mus musculus) brainstem at embryonic ages when the respiratory networks are known to emerge (embryonic day (E) 14.5 for the parafacial respiratory group (epF) and E16.5 for the preBötzinger complex (preBötC)). In transgenic mice depleted of microglia (Spi1-/- (Pu.1 KO) mutant), we performed anatomical staining, calcium imaging, and electrophysiological recordings of neuronal activities in vitro to assess the status of these circuits at their respective times of functional emergence. Spontaneous respiratory-related activity recorded from reduced in vitro preparations showed an abnormally slow rhythm frequency expressed by the epF at E14.5, the preBötC at E16.5, and in the phrenic motor nerves from E16.5 onwards. These deficits were associated with a reduced number of active epF neurons, defects in commissural projections that couple the bilateral preBötC half-centers, and an accompanying decrease in their functional coordination. These abnormalities probably contribute to eventual neonatal death, since plethysmography revealed that E18.5 Spi1-/- embryos are unable to sustain breathing activity ex utero. Our results thus point to a crucial contribution of microglia in the proper establishment of the central respiratory command during embryonic development.

Perinatal development of central vestibular neurons in mice.

Central circuitry of the vestibular nuclei integrates sensory inputs in the adaptive control of motor behaviors such as posture, locomotion, and gaze stabilization. Thus far, such circuits have been mostly examined at mature stages, whereas their emergence and early development have remained poorly described. Here, we focused on the perinatal period of murine development, from embryonic day E14.5 to post-natal day P5, to investigate the ontogeny of two functionally distinct vestibular neuronal groups, neurons projecting to the spinal cord via the lateral vestibulospinal tract (LVST) and commissural neurons of the medial vestibular nucleus that cross the midline to the contralateral nucleus. Using transgenic mice and retrograde labeling, we found that network-constitutive GABAergic and glycinergic neurons are already established in the two vestibular groups at embryonic stages. Although incapable of repetitive firing at E14.5, neurons of both groups can generate spike trains from E15.5 onward and diverge into previously established A or B subtypes according to the absence (A) or presence (B) of a two-stage spike after hyperpolarization. Investigation of several voltage-dependent membrane properties indicated that solely LVST neurons undergo significant maturational changes in their electrophysiological characteristics during perinatal development. The proportions of A vs B subtypes also evolve in both groups, with type A neurons remaining predominant at all stages, and type B commissural neurons appearing only post-natally. Together, our results indicate that vestibular neurons acquire their distinct morpho-functional identities after E14.5 and that the early maturation of membrane properties does not emerge uniformly in the different functional subpopulations of vestibulo-motor pathways.

Fentanyl-induced hyperalgesia and analgesic tolerance in male rats: common underlying mechanisms and prevention by a polyamine deficient diet.

Opioids are a mainstay of pain management but can induce unwanted effects, including analgesic tolerance and paradoxical hyperalgesia, either of which leads to increased pain. Clinically, however, the relationship between these two phenomena remains elusive. By evaluating changes in mechanical nociceptive threshold in male rats, we found that in contrast to a purely analgesic control response to a single subcutaneous administration of fentanyl (25 µg/kg), in rats subjected to inflammatory pain 2 weeks previously (Day0), the same test dose (D13) induced a bi-phasic response: initial decreased analgesia (tolerance) followed by hyperalgesia lasting several hours. Both the tolerance and hyperalgesia were further enhanced in rats that had additionally received fentanyl on D0. The dose-response profiles (5 fg to 50 µg/kg) of pain- and opioid-experienced rats were very different from pain/drug-naive rats. At ultra-low fentanyl doses (<5 ng/kg and <500 ng/kg for naïve control and pain/drug-experienced rats, respectively), solely hyperalgesia was observed in all cases. At higher doses, which now produced analgesia alone in naive rats, reduced analgesia (tolerance) coupled with hyperalgesia occurred in pain/fentanyl-experienced rats, with both phases increasing with dose. Transcriptomic and pharmacological data revealed that an overactivation of the spinal N-methyl-D-aspartate receptor-inducible NO synthase cascade plays a critical role in both acute tolerance and hyperalgesia, and together with the finding that the magnitudes of analgesia and associated hyperalgesia are negatively correlated, is indicative of closely related phenomena. Finally, a polyamine deficient diet prevented inducible NO synthase transcript upregulation, restored fentanyl's analgesic efficacy and suppressed the emergence of hyperalgesia.

Synergistic interaction between sensory inputs and propriospinal signalling underlying quadrupedal locomotion.

KEY POINTS: Stimulation of hindlimb afferent fibres can both stabilize and increase the activity of fore- and hindlimb motoneurons during fictive locomotion. The increase in motoneuron activity is at least partially due to the production of doublets of action potentials in a subpopulation of motoneurons. These results were obtained using an in vitro brainstem/spinal cord preparation of neonatal rat. ABSTRACT: Quadrupedal locomotion relies on a dynamic coordination between central pattern generators (CPGs) located in the cervical and lumbar spinal cord, and controlling the fore- and hindlimbs, respectively. It is assumed that this CPG interaction is achieved through separate closed-loop processes involving propriospinal and sensory pathways. However, the functional consequences of a concomitant involvement of these different influences on the degree of coordination between the fore- and hindlimb CPGs is still largely unknown. Using an in vitro brainstem/spinal cord preparation of neonatal rat, we found that rhythmic, bilaterally alternating stimulation of hindlimb sensory input pathways elicited coordinated hindlimb and forelimb CPG activity. During pharmacologically induced fictive locomotion, lumbar dorsal root (DR) stimulation entrained and stabilized an ongoing cervico-lumbar locomotor-like rhythm and increased the amplitude of both lumbar and cervical ventral root bursting. The increase in cervical burst amplitudes was correlated with the occurrence of doublet action potential firing in a subpopulation of motoneurons, enabling the latter to transition between low and high frequency discharge according to the intensity of DR stimulation. Moreover, our data revealed that propriospinal and sensory pathways act synergistically to strengthen cervico-lumbar interactions. Indeed, split-bath experiments showed that fully coordinated cervico-lumbar fictive locomotion was induced by combining pharmacological stimulation of either the lumbar or cervical CPGs with lumbar DR stimulation. This study thus highlights the powerful interactions between sensory and propriospinal pathways which serve to ensure the coupling of the fore- and hindlimb CPGs for effective quadrupedal locomotion.

Organelle calcium-derived voltage oscillations in pacemaker neurons drive the motor program for food-seeking behavior in Aplysia.

The expression of motivated behaviors depends on both external and internally arising neural stimuli, yet the intrinsic releasing mechanisms for such variably occurring behaviors remain elusive. In isolated nervous system preparations of Aplysia, we have found that irregularly expressed cycles of motor output underlying food-seeking behavior arise from regular membrane potential oscillations of varying magnitude in an identified pair of interneurons (B63) in the bilateral buccal ganglia. This rhythmic signal, which is specific to the B63 cells, is generated by organelle-derived intracellular calcium fluxes that activate voltage-independent plasma membrane channels. The resulting voltage oscillation spreads throughout a subset of gap junction-coupled buccal network neurons and by triggering plateau potential-mediated bursts in B63, can initiate motor output driving food-seeking action. Thus, an atypical neuronal pacemaker mechanism, based on rhythmic intracellular calcium store release and intercellular propagation, can act as an autonomous intrinsic releaser for the occurrence of a motivated behavior.

Role of the K+-Cl- Cotransporter KCC2a Isoform in Mammalian Respiration at Birth.

In central respiratory circuitry, synaptic excitation is responsible for synchronizing neuronal activity in the different respiratory rhythm phases, whereas chloride-mediated inhibition is important for shaping the respiratory pattern itself. The potassium chloride cotransporter KCC2, which serves to maintain low intraneuronal Cl- concentration and thus render chloride-mediated synaptic signaling inhibitory, exists in two isoforms, KCC2a and KCC2b. KCC2 is essential for functional breathing motor control at birth, but the specific contribution of the KCC2a isoform remains unknown. Here, to address this issue, we investigated the respiratory phenotype of mice deficient for KCC2a. In vivo plethysmographic recordings revealed that KCC2a-deficient pups at P0 transiently express an abnormally low breathing rate and a high occurrence of apneas. Immunostainings confirmed that KCC2a is normally expressed in the brainstem neuronal groups involved in breathing (pre-Bötzinger complex, parafacial respiratory group, hypoglossus nucleus) and is absent in these regions in the KCC2a-/- mutant. However, in variously reduced in vitro medullary preparations, spontaneous rhythmic respiratory activity is similar to that expressed in wild-type preparations, as is hypoglossal motor output, and no respiratory pauses are detected, suggesting that the rhythm-generating networks are not intrinsically affected in mutants at P0. In contrast, inhibitory neuromodulatory influences exerted by the pons on respiratory rhythmogenesis are stronger in the mutant, thereby explaining the breathing anomalies observed in vivo. Thus, our results indicate that the KCC2a isoform is important for establishing proper breathing behavior at the time of birth, but by acting at sites that are extrinsic to the central respiratory networks themselves.

Functional limb muscle innervation prior to cholinergic transmitter specification during early metamorphosis in Xenopus.

In vertebrates, functional motoneurons are defined as differentiated neurons that are connected to a central premotor network and activate peripheral muscle using acetylcholine. Generally, motoneurons and muscles develop simultaneously during embryogenesis. However, during Xenopus metamorphosis, developing limb motoneurons must reach their target muscles through the already established larval cholinergic axial neuromuscular system. Here, we demonstrate that at metamorphosis onset, spinal neurons retrogradely labeled from the emerging hindlimbs initially express neither choline acetyltransferase nor vesicular acetylcholine transporter. Nevertheless, they are positive for the motoneuronal transcription factor Islet1/2 and exhibit intrinsic and axial locomotor-driven electrophysiological activity. Moreover, the early appendicular motoneurons activate developing limb muscles via nicotinic antagonist-resistant, glutamate antagonist-sensitive, neuromuscular synapses. Coincidently, the hindlimb muscles transiently express glutamate, but not nicotinic receptors. Subsequently, both pre- and postsynaptic neuromuscular partners switch definitively to typical cholinergic transmitter signaling. Thus, our results demonstrate a novel context-dependent re-specification of neurotransmitter phenotype during neuromuscular system development.

A New Perspective on Predictive Motor Signaling.

Adaptive behavior relies on complex neural processing in multiple interacting networks of both motor and sensory systems. One such interaction employs intrinsic neuronal signals, so-called 'corollary discharge' or 'efference copy', that may be used to predict the sensory consequences of a specific behavioral action, thereby enabling self-generated (reafferent) sensory information and extrinsic (exafferent) sensory inflow to be dissociated. Here, by using well-established examples, we seek to identify the distinguishing features of corollary discharge and efference copy within the framework of predictive motor-to-sensory system coordination. We then extend the more general concept of predictive signaling by showing how neural replicas of a particular motor command not only inform sensory pathways in order to gate reafferent stimulation, but can also be used to directly coordinate distinct and otherwise independent behaviors to the original motor task. Moreover, this motor-to-motor pairing may additionally extend to a gating of sensory input to either or both of the coupled systems. The employment of predictive internal signaling in such motor systems coupling and remote sensory input control thus adds to our understanding of how an organism's central nervous system is able to coordinate the activity of multiple and generally disparate motor and sensory circuits in the production of effective behavior.

The embryonic development of hindbrain respiratory networks is unaffected by mutation of the planar polarity protein Scribble.

The central command for breathing arises mainly from two interconnected rhythmogenic hindbrain networks, the parafacial respiratory group (pFRG or epF at embryonic stages) and the preBötzinger complex (preBötC), which are comprised of a limited number of neurons located in confined regions of the ventral medulla. In rodents, both networks become active toward the end of gestation but little is known about the signaling pathways involved in their anatomical and functional establishment during embryogenesis. During embryonic development, epF and preBötC neurons migrate from their territories of origin to their final positions in ventral brainstem areas. Planar Cell Polarity (PCP) signaling, including the molecule Scrib, is known to control the developmental migration of several hindbrain neuronal groups. Accordingly, a homozygous mutation of Scrib leads to severe disruption of hindbrain anatomy and function. Here, we aimed to determine whether Scrib is also involved in the prenatal development of the hindbrain nuclei controlling breathing. We combined immunostaining, calcium imaging and electrophysiological recordings of neuronal activity in isolated in vitro preparations. In the Scrib mutant, despite severe neural tube defects, epF and preBötC neurons settled at their expected hindbrain positions. Furthermore, both networks remained capable of generating rhythmically organized, respiratory-related activities and exhibited normal sensitivity to pharmacological agents known to modify respiratory circuit function. Thus Scrib is not required for the proper migration of epF and preBötC neurons during mouse embryogenesis. Our findings thus further illustrate the robustness and specificity of the developmental processes involved in the establishment of hindbrain respiratory circuits.

Development of pacemaker properties and rhythmogenic mechanisms in the mouse embryonic respiratory network.

Breathing is a vital rhythmic behavior generated by hindbrain neuronal circuitry, including the preBötzinger complex network (preBötC) that controls inspiration. The emergence of preBötC network activity during prenatal development has been described, but little is known regarding inspiratory neurons expressing pacemaker properties at embryonic stages. Here, we combined calcium imaging and electrophysiological recordings in mouse embryo brainstem slices together with computational modeling to reveal the existence of heterogeneous pacemaker oscillatory properties relying on distinct combinations of burst-generating INaP and ICAN conductances. The respective proportion of the different inspiratory pacemaker subtypes changes during prenatal development. Concomitantly, network rhythmogenesis switches from a purely INaP/ICAN-dependent mechanism at E16.5 to a combined pacemaker/network-driven process at E18.5. Our results provide the first description of pacemaker bursting properties in embryonic preBötC neurons and indicate that network rhythmogenesis undergoes important changes during prenatal development through alterations in both circuit properties and the biophysical characteristics of pacemaker neurons.

Adaptive plasticity of spino-extraocular motor coupling during locomotion in metamorphosing Xenopus laevis.

During swimming in the amphibian ITALIC! Xenopus laevis, efference copies of rhythmic locomotor commands produced by the spinal central pattern generator (CPG) can drive extraocular motor output appropriate for producing image-stabilizing eye movements to offset the disruptive effects of self-motion. During metamorphosis, ITALIC! X. laevisremodels its locomotor strategy from larval tail-based undulatory movements to bilaterally synchronous hindlimb kicking in the adult. This change in propulsive mode results in head/body motion with entirely different dynamics, necessitating a concomitant switch in compensatory ocular movements from conjugate left-right rotations to non-conjugate convergence during the linear forward acceleration produced during each kick cycle. Here, using semi-intact or isolated brainstem/spinal cord preparations at intermediate metamorphic stages, we monitored bilateral eye motion along with extraocular, spinal axial and limb motor nerve activity during episodes of spontaneous fictive swimming. Our results show a progressive transition in spinal efference copy control of extraocular motor output that remains adapted to offsetting visual disturbances during the combinatorial expression of bimodal propulsion when functional larval and adult locomotor systems co-exist within the same animal. In stages at metamorphic climax, spino-extraocular motor coupling, which previously derived from axial locomotor circuitry alone, can originate from both axial and ITALIC! de novohindlimb CPGs, although the latter's influence becomes progressively more dominant and eventually exclusive as metamorphosis terminates with tail resorption. Thus, adaptive interactions between locomotor and extraocular motor circuitry allows CPG-driven efference copy signaling to continuously match the changing spatio-temporal requirements for visual image stabilization throughout the transitional period when one propulsive mechanism emerges and replaces another.

A behaviorally related developmental switch in nitrergic modulation of locomotor rhythmogenesis in larval Xenopus tadpoles.

Locomotor control requires functional flexibility to support an animal's full behavioral repertoire. This flexibility is partly endowed by neuromodulators, allowing neural networks to generate a range of motor output configurations. In hatchling Xenopus tadpoles, before the onset of free-swimming behavior, the gaseous modulator nitric oxide (NO) inhibits locomotor output, shortening swim episodes and decreasing swim cycle frequency. While populations of nitrergic neurons are already present in the tadpole's brain stem at hatching, neurons positive for the NO-synthetic enzyme, NO synthase, subsequently appear in the spinal cord, suggesting additional as yet unidentified roles for NO during larval development. Here, we first describe the expression of locomotor behavior during the animal's change from an early sessile to a later free-swimming lifestyle and then compare the effects of NO throughout tadpole development. We identify a discrete switch in nitrergic modulation from net inhibition to overall excitation, coincident with the transition to free-swimming locomotion. Additionally, we show in isolated brain stem-spinal cord preparations of older larvae that NO's excitatory effects are manifested as an increase in the probability of spontaneous swim episode occurrence, as found previously for the neurotransmitter dopamine, but that these effects are mediated within the brain stem. Moreover, while the effects of NO and dopamine are similar, the two modulators act in parallel rather than NO operating serially by modulating dopaminergic signaling. Finally, NO's activation of neurons in the brain stem also leads to the release of NO in the spinal cord that subsequently contributes to NO's facilitation of swimming.

Spinal corollary discharge modulates motion sensing during vertebrate locomotion.

During active movements, neural replicas of the underlying motor commands may assist in adapting motion-detecting sensory systems to an animal's own behaviour. The transmission of such motor efference copies to the mechanosensory periphery offers a potential predictive substrate for diminishing sensory responsiveness to self-motion during vertebrate locomotion. Here, using semi-isolated in vitro preparations of larval Xenopus, we demonstrate that shared efferent neural pathways to hair cells of vestibular endorgans and lateral line neuromasts express cyclic impulse bursts during swimming that are directly driven by spinal locomotor circuitry. Despite common efferent innervation and discharge patterns, afferent signal encoding at the two mechanosensory peripheries is influenced differentially by efference copy signals, reflecting the different organization of body/water motion-detecting processes in the vestibular and lateral line systems. The resultant overall gain reduction in sensory signal encoding in both cases, which likely prevents overstimulation, constitutes an adjustment to increased stimulus magnitudes during locomotion.

Neuromodulation in developing motor microcircuits.

Neuromodulation confers operational flexibility on motor network output and resulting behaviour. Furthermore, neuromodulators play crucial long-term roles in the assembly and maturational shaping of the same networks as they develop. Although previous studies have identified such modulator-dependent contributions to microcircuit ontogeny, some of the underlying mechanisms are only now being elucidated. Deciphering the role of neuromodulatory systems in motor network development has potentially important implications for post-lesional regenerative strategies in adults.

Differential roles of nonsynaptic and synaptic plasticity in operant reward learning-induced compulsive behavior.

BACKGROUND: Rewarding stimuli in associative learning can transform the irregularly and infrequently generated motor patterns underlying motivated behaviors into output for accelerated and stereotyped repetitive action. This transition to compulsive behavioral expression is associated with modified synaptic and membrane properties of central neurons, but establishing the causal relationships between cellular plasticity and motor adaptation has remained a challenge. RESULTS: We found previously that changes in the intrinsic excitability and electrical synapses of identified neurons in Aplysia's central pattern-generating network for feeding are correlated with a switch to compulsive-like motor output expression induced by in vivo operant conditioning. Here, we used specific computer-simulated ionic currents in vitro to selectively replicate or suppress the membrane and synaptic plasticity resulting from this learning. In naive in vitro preparations, such experimental manipulation of neuronal membrane properties alone increased the frequency but not the regularity of feeding motor output found in preparations from operantly trained animals. On the other hand, changes in synaptic strength alone switched the regularity but not the frequency of feeding output from naive to trained states. However, simultaneously imposed changes in both membrane and synaptic properties reproduced both major aspects of the motor plasticity. Conversely, in preparations from trained animals, experimental suppression of the membrane and synaptic plasticity abolished the increase in frequency and regularity of the learned motor output expression. CONCLUSIONS: These data establish direct causality for the contributions of distinct synaptic and nonsynaptic adaptive processes to complementary facets of a compulsive behavior resulting from operant reward learning.

Emergence of sigh rhythmogenesis in the embryonic mouse.

In mammals, eupnoeic breathing is periodically interrupted by spontaneous augmented breaths (sighs) that include a larger-amplitude inspiratory effort, typically followed by a post-sigh apnoea. Previous in vitro studies in newborn rodents have demonstrated that the respiratory oscillator of the pre-Bötzinger complex (preBötC) can generate the distinct inspiratory motor patterns for both eupnoea- and sigh-related behaviour. During mouse embryonic development, the preBötC begins to generate eupnoeic rhythmicity at embryonic day (E) 15.5, but the network's ability to also generate sigh-like activity remains unexplored at prenatal stages. Using transverse brainstem slice preparations we monitored the neuronal population activity of the preBötC at different embryonic ages. Spontaneous sigh-like rhythmicity was found to emerge progressively, being expressed in 0/32 slices at E15.5, 7/30 at E16.5, 9/22 at E17.5 and 23/26 at E18.5. Calcium imaging showed that the preBötC cell population that participates in eupnoeic-like discharge was also active during fictive sighs. However, patch-clamp recordings revealed the existence of an additional small subset of neurons that fired exclusively during sigh activity. Changes in glycinergic inhibitory synaptic signalling, either by pharmacological blockade, functional perturbation or natural maturation of the chloride co-transporters KCC2 or NKCC1 selectively, and in an age-dependent manner, altered the bi-phasic nature of sigh bursts and their coordination with eupnoeic bursting, leading to the generation of an atypical monophasic sigh-related event. Together our results demonstrate that the developmental emergence of a sigh-generating capability occurs after the onset of eupnoeic rhythmogenesis and requires the proper maturation of chloride-mediated glycinergic synaptic transmission.