Pairing Transcranial Magnetic Stimulation and Loud Sounds Produces Plastic Changes in Motor Output.

Most current methods for neuromodulation target the cortex. Approaches for inducing plasticity in subcortical motor pathways, such as the reticulospinal tract, could help to boost recovery after damage (e.g., stroke). In this study, we paired loud acoustic stimulation (LAS) with transcranial magnetic stimulation (TMS) over the motor cortex in male and female healthy humans. LAS activates the reticular formation; TMS activates descending systems, including corticoreticular fibers. Two hundred paired stimuli were used, with 50 ms interstimulus interval at which LAS suppresses TMS responses. Before and after stimulus pairing, responses in the contralateral biceps muscle to TMS alone were measured. Ten, 20, and 30 min after stimulus pairing ended, TMS responses were enhanced, indicating the induction of LTP. No long-term changes were seen in control experiments which used 200 unpaired TMS or LAS, indicating the importance of associative stimulation. Following paired stimulation, no changes were seen in responses to direct corticospinal stimulation at the level of the medulla, or in the extent of reaction time shortening by a loud sound (StartReact effect), suggesting that plasticity did not occur in corticospinal or reticulospinal synapses. Direct measurements in female monkeys undergoing a similar paired protocol revealed no enhancement of corticospinal volleys after paired stimulation, suggesting no changes occurred in intracortical connections. The most likely substrate for the plastic changes, consistent with all our measurements, is an increase in the efficacy of corticoreticular connections. This new protocol may find utility, as it seems to target different motor circuits compared with other available paradigms.SIGNIFICANCE STATEMENT Induction of plasticity by neurostimulation protocols may be promising to enhance functional recovery after damage such as following stroke, but current protocols mainly target cortical circuits. In this study, we developed a novel paradigm which may generate long-term changes in connections between cortex and brainstem. This could provide an additional tool to modulate and improve recovery.

Properties of the Caudal Pontine Reticular Nucleus Neurons Determine the Acoustic Startle Response in Cntnap2 KO Rats.

BACKGROUND: Rats with a loss-of-function mutation in the contactin-associated protein-like 2 (Cntnap2) gene have been validated as an animal model of autism spectrum disorder (ASD). Similar to many autistic individuals, Cntnap2 knock-out rats (Cntnap2-⁣/-) are hyperreactive to sound as measured through the acoustic startle response. The brainstem region that mediates the acoustic startle response is the caudal pontine reticular nucleus (PnC), specifically giant neurons in the PnC. We previously reported a sex-dependent genotypic effect in the sound-evoked neuronal activity recorded from the PnC, whereby female Cntnap2-⁣/- rats had a dramatic increase in sound-evoked responses compared with wildtype counterparts, but male Cntnap2-⁣/- rats showed only a modest increase in PnC activity that cannot fully explain the largely increased startle in male Cntnap2-⁣/- rats. The present study therefore investigates activation and histological properties of PnC giant neurons in Cntnap2-⁣/- rats and wildtype littermates. METHODS: The acoustic startle response was elicited by presenting rats with 95 dB startle pulses before rats were euthanized. PnC brain sections were stained and analyzed for the total number of PnC giant neurons and the percentage of giant neurons that expressed phosphorylated cAMP response element binding protein (pCREB) in response to startle stimuli. Additionally, in vitro electrophysiology was conducted to assess the resting state activity and intrinsic properties of PnC giant neurons. RESULTS: Wildtype and Cntnap2-⁣/- rats had similar total numbers of PnC giant neurons and similar levels of baseline pCREB expression, as well as similar numbers of giant neurons that were firing at rest. Increased startle magnitudes in Cntnap2-⁣/- rats were associated with increased percentages of pCREB-expressing PnC giant neurons in response to startle stimuli. Male rats had increased pCREB-expressing PnC giant neurons compared with female rats, and the recruited giant neurons in males were also larger in soma size. CONCLUSIONS: Recruitment and size of PnC giant neurons are important factors for regulating the magnitude of the acoustic startle response in Cntnap2-⁣/- rats, particularly in males. These findings allow for a better understanding of increased reactivity to sound in Cntnap2-⁣/- rats and in CNTNAP2-associated disorders such as ASD.

Both Corticospinal and Reticulospinal Tracts Control Force of Contraction.

The control of contraction strength is a key part of movement control. In primates, both corticospinal and reticulospinal cells provide input to motoneurons. Corticospinal discharge is known to correlate with force, but there are no previous reports of how reticular formation (RF) activity modulates with different contractions. Here we trained two female macaque monkeys (body weight, 5.9-6.9 kg) to pull a handle that could be loaded with 0.5-6 kg weights and recorded from identified pyramidal tract neurons (PTNs) in primary motor cortex and RF cells during task performance. Population-averaged firing rate increased monotonically with higher force for the RF, but showed a complex profile with little net modulation for PTNs. This reflected a more heterogeneous profile of rate modulation across the PTN population, leading to cancellation in the average. Linear discriminant analysis classified the force based on the time course of rate modulation equally well for PTNs and RF cells. Peak firing rate had significant linear correlation with force for 43 of 92 PTNs (46.7%) and 21 of 46 RF cells (43.5%). For almost all RF cells (20 of 21), the correlation coefficient was positive; similar numbers of PTNs (22 vs 21) had positive versus negative coefficients. Considering the timing of force representation, similar fractions (PTNs: 61.2%; RF cells: 55.5%) commenced coding before the onset of muscle activity. We conclude that both corticospinal and reticulospinal tracts contribute to the control of contraction force; the reticulospinal tract seems to specify an overall signal simply related to force, whereas corticospinal cell activity would be better suited for fine-scale adjustments.SIGNIFICANCE STATEMENT For the first time, we compare the coding of force for corticospinal and reticular formation cells in awake behaving monkeys, over a wide range of contraction strengths likely to come close to maximum voluntary contraction. Both cortical and brainstem systems coded similarly well for force, but whereas reticular formation cells carried a simple uniform signal, corticospinal neurons were more heterogeneous. This may reflect a role in the gross specification of a coordinated movement, versus more fine-grained adjustments around individual joints.

Brainstem sources of input to the central mesencephalic reticular formation in the macaque.

Physiological studies indicate that the central mesencephalic reticular formation (cMRF) plays a role in gaze changes, including control of disjunctive saccades. Neuroanatomical studies have demonstrated strong interconnections with the superior colliculus, along with projections to extraocular motor nuclei, the preganglionic nucleus of Edinger-Westphal, the paramedian pontine reticular formation, nucleus raphe interpositus, medullary reticular formation and cervical spinal cord, as might be expected for a structure that is intimately involved in gaze control. However, the sources of input to this midbrain structure have not been described in detail. In the present study, the brainstem cells of origin supplying the cMRF were labeled by retrograde transport of tracer (wheat germ agglutinin conjugated horseradish peroxidase) in macaque monkeys. Within the diencephalon, labeled neurons were noted in the ventromedial nucleus of the hypothalamus, pregeniculate nucleus and habenula. In the midbrain, labeled cells were found in the substantia nigra pars reticulata, medial pretectal nucleus, superior colliculus, tectal longitudinal column, periaqueductal gray, supraoculomotor area, and contralateral cMRF. In the pons they were located in the paralemniscal zone, parabrachial nucleus, locus coeruleus, nucleus prepositus hypoglossi and the paramedian pontine reticular formation. Finally, in the medulla they were observed in the medullary reticular formation. The fact that this list of input sources is very similar to those of the superior colliculus supports the view that the cMRF represents an important gaze control center.

Extensive Cortical Convergence to Primate Reticulospinal Pathways.

Early evolution of the motor cortex included development of connections to brainstem reticulospinal neurons; these projections persist in primates. In this study, we examined the organization of corticoreticular connections in five macaque monkeys (one male) using both intracellular and extracellular recordings from reticular formation neurons, including identified reticulospinal cells. Synaptic responses to stimulation of different parts of primary motor cortex (M1) and supplementary motor area (SMA) bilaterally were assessed. Widespread short latency excitation, compatible with monosynaptic transmission over fast-conducting pathways, was observed, as well as longer latency responses likely reflecting a mixture of slower monosynaptic and oligosynaptic pathways. There was a high degree of convergence: 56% of reticulospinal cells with input from M1 received projections from M1 in both hemispheres; for SMA, the equivalent figure was even higher (70%). Of reticulospinal neurons with input from the cortex, 78% received projections from both M1 and SMA (regardless of hemisphere); 83% of reticulospinal cells with input from M1 received projections from more than one of the tested M1 sites. This convergence at the single cell level allows reticulospinal neurons to integrate information from across the motor areas of the cortex, taking account of the bilateral motor context. Reticulospinal connections are known to strengthen following damage to the corticospinal tract, such as after stroke, partially contributing to functional recovery. Extensive corticoreticular convergence provides redundancy of control, which may allow the cortex to continue to exploit this descending pathway even after damage to one area.SIGNIFICANCE STATEMENT The reticulospinal tract (RST) provides a parallel pathway for motor control in primates, alongside the more sophisticated corticospinal system. We found extensive convergent inputs to primate reticulospinal cells from primary and supplementary motor cortex bilaterally. These redundant connections could maintain transmission of voluntary commands to the spinal cord after damage (e.g., after stroke or spinal cord injury), possibly assisting recovery of function.

Descending pathways from the superior colliculus mediating autonomic and respiratory effects associated with orienting behaviour.

The ability to discriminate competing external stimuli and initiate contextually appropriate behaviours is a key brain function. Neurons in the deep superior colliculus (dSC) integrate multisensory inputs and activate descending projections to premotor pathways responsible for orienting, attention and defence, behaviours which involve adjustments to respiratory and cardiovascular parameters. However, the neural pathways that subserve the physiological components of orienting are poorly understood. We report that orienting responses to optogenetic dSC stimulation are accompanied by short-latency autonomic, respiratory and electroencephalographic effects in awake rats, closely mimicking those evoked by naturalistic alerting stimuli. Physiological responses were not accompanied by detectable aversion or fear, and persisted under urethane anaesthesia, indicating independence from emotional stress. Anterograde and trans-synaptic viral tracing identified a monosynaptic pathway that links the dSC to spinally projecting neurons in the medullary gigantocellular reticular nucleus (GiA), a key hub for the coordination of orienting and locomotor behaviours. In urethane-anaesthetized animals, sympathoexcitatory and cardiovascular, but not respiratory, responses to dSC stimulation were replicated by optogenetic stimulation of the dSC-GiA terminals, suggesting a likely role for this pathway in mediating the autonomic components of dSC-mediated responses. Similarly, extracellular recordings from putative GiA sympathetic premotor neurons confirmed short-latency excitatory inputs from the dSC. This pathway represents a likely substrate for autonomic components of orienting responses that are mediated by dSC neurons and suggests a mechanism through which physiological and motor components of orienting behaviours may be integrated without the involvement of higher centres that mediate affective components of defensive responses. KEY POINTS: Neurons in the deep superior colliculus (dSC) integrate multimodal sensory signals to elicit context-dependent innate behaviours that are accompanied by stereotypical cardiovascular and respiratory activities. The pathways responsible for mediating the physiological components of colliculus-mediated orienting behaviours are unknown. We show that optogenetic dSC stimulation evokes transient orienting, respiratory and autonomic effects in awake rats which persist under urethane anaesthesia. Anterograde tracing from the dSC identified projections to spinally projecting neurons in the medullary gigantocellular reticular nucleus (GiA). Stimulation of this pathway recapitulated autonomic effects evoked by stimulation of dSC neurons. Electrophysiological recordings from putative GiA sympathetic premotor neurons confirmed short latency excitatory input from dSC neurons. This disynaptic dSC-GiA-spinal sympathoexcitatory pathway may underlie autonomic adjustments to salient environmental cues independent of input from higher centres.

Noradrenergic substrates sensing light within brainstem reticular formation as targets for light-induced behavioral and cardiovascular plasticity.

The occurrence of pure light exerts a variety of effects in the human body, which span from behavioral alterations, such as light-driven automatic motor activity, cognition and mood to more archaic vegetative functions, which encompass most organs of the body with remarkable effects on the cardiovascular system. Although empirical evidence clearly indicates occurrence of these widespread effects, the anatomical correlates and long-lasting changes within putatively specific neuronal circuitries remain largely unexplored. A specific role is supposed to take place for catecholamine containing neurons in the core of the brainstem reticular formation, which produces a widespread release of noradrenaline in the forebrain while controlling the vegetative nervous system. An indirect as well as a direct (mono-synaptic) retino-brainstem pathway is hypothesized to rise from a subtype of intrinsically photosensitive retinal ganglion cells (iPRGCs), subtype M1, which do stain for Brn3b, and project to the pre-tectal region (including the olivary pre-tectal nucleus). This pathway provides profuse axon collaterals, which spread to the periacqueductal gray and dorsal raphe nuclei. According to this evidence, a retino-reticular monosynaptic system occurs, which powerfully modulate the noradrenergic hub of reticular nuclei in the lateral column of the brainstem reticular formation. These nuclei, which are evidenced in the present study, provide the anatomical basis to induce behavioral and cardiovascular modulation. The occurrence of a highly interconnected network within these nuclei is responsible for light driven plastic effects, which may alter persistently behavior and vegetative functions as the consequence of long-lasting alterations in the environmental light stimulation of the retina. These changes, which occur within the core of an archaic circuitry such as the noradrenaline-containing neurons of the reticular formation, recapitulate, within the CNS, ancestral effects of light-driven changes, which can be detected already within the retina itself at the level of multipotent photic cells.

Descending Dopaminergic Inputs to Reticulospinal Neurons Promote Locomotor Movements.

Meso-diencephalic dopaminergic neurons are known to modulate locomotor behaviors through their ascending projections to the basal ganglia, which in turn project to the mesencephalic locomotor region, known to control locomotion in vertebrates. In addition to their ascending projections, dopaminergic neurons were found to increase locomotor movements through direct descending projections to the mesencephalic locomotor region and spinal cord. Intriguingly, fibers expressing tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, were also observed around reticulospinal neurons of lampreys. We now examined the origin and the role of this innervation. Using immunofluorescence and tracing experiments, we found that fibers positive for dopamine innervate reticulospinal neurons in the four reticular nuclei of lampreys. We identified the dopaminergic source using tracer injections in reticular nuclei, which retrogradely labeled dopaminergic neurons in a caudal diencephalic nucleus (posterior tuberculum [PT]). Using voltammetry in brain preparations isolated in vitro, we found that PT stimulation evoked dopamine release in all four reticular nuclei, but not in the spinal cord. In semi-intact preparations where the brain is accessible and the body moves, PT stimulation evoked swimming, and injection of a D1 receptor antagonist within the middle rhombencephalic reticular nucleus was sufficient to decrease reticulospinal activity and PT-evoked swimming. Our study reveals that dopaminergic neurons have access to command neurons that integrate sensory and descending inputs to activate spinal locomotor neurons. As such, our findings strengthen the idea that dopamine can modulate locomotor behavior both via ascending projections to the basal ganglia and through descending projections to brainstem motor circuits.SIGNIFICANCE STATEMENT Meso-diencephalic dopaminergic neurons play a key role in modulating locomotion by releasing dopamine in the basal ganglia, spinal networks, and the mesencephalic locomotor region, a brainstem region that controls locomotion in a graded fashion. Here, we report in lampreys that dopaminergic neurons release dopamine in the four reticular nuclei where reticulospinal neurons are located. Reticulospinal neurons integrate sensory and descending suprareticular inputs to control spinal interneurons and motoneurons. By directly modulating the activity of reticulospinal neurons, meso-diencephalic dopaminergic neurons control the very last instructions sent by the brain to spinal locomotor circuits. Our study reports on a new direct descending dopaminergic projection to reticulospinal neurons that modulates locomotor behavior.

EphA4 Is Required for Neural Circuits Controlling Skilled Reaching.

Skilled forelimb movements are initiated by feedforward motor commands conveyed by supraspinal motor pathways. The accuracy of reaching and grasping relies on internal feedback pathways that update ongoing motor commands. In mice lacking the axon guidance molecule EphA4, axonal misrouting of the corticospinal tract and spinal interneurons is manifested, leading to a hopping gait in hindlimbs. Moreover, mice with a conditional forebrain deletion of EphA4, display forelimb hopping in adaptive locomotion and exploratory reaching movements. However, it remains unclear how loss of EphA4 signaling disrupts function of forelimb motor circuit and skilled reaching and grasping movements. Here we investigated how neural circuits controlling skilled reaching were affected by the loss of EphA4. Both male and female C57BL/6 wild-type, heterozygous EphA4+/-, and homozygous EphA4-/- mice were used in behavioral and in vivo electrophysiological investigations. We found that EphA4 knock-out (-/-) mice displayed impaired goal-directed reaching movements. In vivo intracellular recordings from forelimb motor neurons demonstrated increased corticoreticulospinal excitation, decreased direct reticulospinal excitation, and reduced direct propriospinal excitation in EphA4 knock-out mice. Cerebellar surface recordings showed a functional perturbation of the lateral reticular nucleus-cerebellum internal feedback pathway in EphA4 knock-out mice. Together, our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts the function of both feedforward and feedback motor pathways, resulting in deficits in skilled reaching.SIGNIFICANCE STATEMENT The central advances of this study are the demonstration that null mutation in the axon guidance molecule EphA4 gene impairs the ability of mice to perform skilled reaching, and identification of how these behavioral deficits correlates with discrete neurophysiological changes in central motor pathways involved in the control of reaching. Our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts both feedforward and feedback motor pathways, resulting in deficits in skilled reaching. This analysis of motor circuit function may help to understand the pathophysiological mechanisms underlying movement disorders in humans.

Evidence of intermediate reticular formation involvement in swallow pattern generation, recorded optically in the neonate rat sagittally sectioned hindbrain.

Swallow is a primitive behavior regulated by medullary networks, responsible for movement of food/liquid from the oral cavity to the esophagus. To investigate how functionally heterogeneous networks along the medullary intermediate reticular formation (IRt) and ventral respiratory column (VRC) control swallow, we electrically stimulated the nucleus tractus solitarius to induce fictive swallow between inspiratory bursts, with concurrent optical recordings using a synthetic Ca2+ indicator in the neonatal sagittally sectioned rat hindbrain (SSRH) preparation. Simultaneous recordings from hypoglossal nerve rootlet (XIIn) and ventral cervical spinal root C1-C2 enabled identification of the system-level correlates of 1) swallow (identified as activation of the XIIn but not the cervical root) and 2) Breuer-Hering expiratory reflex (BHE; lengthened expiration in response to stimuli during expiration). Optical recording revealed reconfiguration of respiration-modulated networks in the ventrolateral medulla during swallow and the BHE reflex. Recordings identified novel spatially compact networks in the IRt near the facial nucleus (VIIn) that were active during fictive swallow, suggesting that the swallow network is not restricted to the caudal medulla. These findings also establish the utility of using this in vitro preparation to investigate how functionally heterogeneous medullary networks interact and reconfigure to enable a repertoire of orofacial behaviors.NEW & NOTEWORTHY For the first time, medullary networks that control breathing and swallow are recorded optically. Episodic swallows are induced via electrical stimulation along the dorsal medulla, in and near the NTS, during spontaneously occurring fictive respiration. These findings establish that networks regulating both orofacial behaviors and breathing are accessible for optical recording at the surface of the sagittally sectioned rodent hindbrain preparation.

Neurons in the Intermediate Reticular Nucleus Coordinate Postinspiratory Activity, Swallowing, and Respiratory-Sympathetic Coupling in the Rat.

Breathing results from sequential recruitment of muscles in the expiratory, inspiratory, and postinspiratory (post-I) phases of the respiratory cycle. Here we investigate whether neurons in the medullary intermediate reticular nucleus (IRt) are components of a central pattern generator (CPG) that generates post-I activity in laryngeal adductors and vasomotor sympathetic nerves and interacts with other members of the central respiratory network to terminate inspiration. We first identified the region of the (male) rat IRt that contains the highest density of lightly cholinergic neurons, many of which are glutamatergic, which aligns well with the putative postinspiratory complex in the mouse (Anderson et al., 2016). Acute bilateral inhibition of this region reduced the amplitudes of post-I vagal and sympathetic nerve activities. However, although associated with reduced expiratory duration and increased respiratory frequency, IRt inhibition did not affect inspiratory duration or abolish the recruitment of post-I activity during acute hypoxemia as predicted. Rather than representing an independent CPG for post-I activity, we hypothesized that IRt neurons may instead function as a relay that distributes post-I activity generated elsewhere, and wondered whether they could be a site of integration for para-respiratory CPGs that drive the same outputs. Consistent with this idea, IRt inhibition blocked rhythmic motor and autonomic components of fictive swallow but not swallow-related apnea. Our data support a role for IRt neurons in the transmission of post-I and swallowing activity to motor and sympathetic outputs, but suggest that other mechanisms also contribute to the generation of post-I activity.SIGNIFICANCE STATEMENT Interactions between multiple coupled oscillators underlie a three-part respiratory cycle composed from inspiratory, postinspiratory (post-I), and late-expiratory phases. Central post-I activity terminates inspiration and activates laryngeal motoneurons. We investigate whether neurons in the intermediate reticular nucleus (IRt) form the central pattern generator (CPG) responsible for post-I activity. We confirm that IRt activity contributes to post-I motor and autonomic outputs, and find that IRt neurons are necessary for activation of the same outputs during swallow, but that they are not required for termination of inspiration or recruitment of post-I activity during hypoxemia. We conclude that this population may not represent a distinct CPG, but instead may function as a premotor relay that integrates activity generated by diverse respiratory and nonrespiratory CPGs.

Classification of Neurons in the Primate Reticular Formation and Changes after Recovery from Pyramidal Tract Lesion.

The reticular formation is important in primate motor control, both in health and during recovery after brain damage. Little is known about the different neurons present in the reticular nuclei. Here we recorded extracellular spikes from the reticular formation in five healthy female awake behaving monkeys (193 cells), and in two female monkeys 1 year after recovery from a unilateral pyramidal tract lesion (125 cells). Analysis of spike shape and four measures derived from the interspike interval distribution identified four clusters of neurons in control animals. Cluster 1 cells had a slow firing rate. Cluster 2 cells had narrow spikes and irregular firing, which often included high-frequency bursts. Cluster 3 cells were highly rhythmic and fast firing. Cluster 4 cells showed negative spikes. A separate population of 42 cells was antidromically identified as reticulospinal neurons in five anesthetized female monkeys. The distribution of spike width in these cells closely overlaid the distribution for cluster 2, leading us tentatively to suggest that cluster 2 included neurons with reticulospinal projections. In animals after corticospinal lesion, cells could be identified in all four clusters. The firing rate of cells in clusters 1 and 2 was increased in lesioned animals relative to control animals (by 52% and 60%, respectively); cells in cluster 2 were also more regular and more bursting in the lesioned animals. We suggest that changes in both membrane properties and local circuits within the reticular formation occur following lesioning, potentially increasing reticulospinal output to help compensate for lost corticospinal descending drive.SIGNIFICANCE STATEMENT This work is the first to subclassify neurons in the reticular formation, providing insights into the local circuitry of this important but little understood structure. The approach developed can be applied to any extracellular recording from this region, allowing future studies to place their data within our current framework of four neural types. Changes in reticular neurons may be important to subserve functional recovery after damage in human patients, such as after stroke or spinal cord injury.

Brainstem Steering of Locomotor Activity in the Newborn Rat.

Control of locomotion relies on motor loops conveying modulatory signals between brainstem and spinal motor circuits. We investigated the steering control of the brainstem reticular formation over the spinal locomotor networks using isolated brainstem-spinal cord preparations of male and female neonatal rats. First, we performed patch-clamp recordings of identified reticulospinal cells during episodes of fictive locomotion. This revealed that a spinal ascending phasic modulation of reticulospinal cell activity is already present at birth. Half of the cells exhibited tonic firing during locomotion, while the other half emitted phasic discharges of action potentials phase locked to ongoing activity. We next showed that mimicking the phasic activity of reticulospinal neurons by applying patterned electrical stimulation bilaterally at the ventral caudal medulla level triggered fictive locomotion efficiently. Moreover, the brainstem stimuli-induced locomotor rhythm was entrained in a one-to-one coupling over a range of cycle periods (2-6 s). Additionally, we induced turning like motor outputs by either increasing or decreasing the relative duration of the stimulation trains on one side of the brainstem compared to the other. The ability of the patterned descending command to control the locomotor output depended on the functional integrity of ventral reticulospinal pathways and the involvement of local spinal central pattern generator circuitry. Altogether, this study provides a mechanism by which brainstem reticulospinal neurons relay steering and speed commands to the spinal locomotor networks.SIGNIFICANCE STATEMENT Locomotor function allows the survival of most animal species while sustaining the expression of fundamental behaviors. Locomotor activities adapt from moment to moment to behavioral and environmental changes. We show that the brainstem can control the spinal locomotor network outputs through phasic descending commands that alternate bilaterally. Manipulating the periodicity and/or the relative durations of the left and right descending commands at the brainstem level is efficient to set the locomotor speed and sustain directional changes.

Tenuous Inhibitory GABAergic Signaling in the Reticular Thalamus.

Maintenance of a low intracellular Cl- concentration ([Cl-]i) is critical for enabling inhibitory neuronal responses to GABAA receptor-mediated signaling. Cl- transporters, including KCC2, and extracellular impermeant anions ([A]o) of the extracellular matrix are both proposed to be important regulators of [Cl-]i Neurons of the reticular thalamic (RT) nucleus express reduced levels of KCC2, indicating that GABAergic signaling may produce excitation in RT neurons. However, by performing perforated patch recordings and calcium imaging experiments in rats (male and female), we find that [Cl-]i remains relatively low in RT neurons. Although we identify a small contribution of [A]o to a low [Cl-]i in RT neurons, our results also demonstrate that reduced levels of KCC2 remain sufficient to maintain low levels of Cl- Reduced KCC2 levels, however, restrict the capacity of RT neurons to rapidly extrude Cl- following periods of elevated GABAergic signaling. In a computational model of a local RT network featuring slow Cl- extrusion kinetics, similar to those we found experimentally, model RT neurons are predisposed to an activity-dependent switch from GABA-mediated inhibition to excitation. By decreasing the activity threshold required to produce excitatory GABAergic signaling, weaker stimuli are able to propagate activity within the model RT nucleus. Our results indicate the importance of even diminished levels of KCC2 in maintaining inhibitory signaling within the RT nucleus and suggest how this important activity choke point may be easily overcome in disorders such as epilepsy.SIGNIFICANCE STATEMENT Precise regulation of intracellular Cl- levels ([Cl-]i) preserves appropriate, often inhibitory, GABAergic signaling within the brain. However, there is disagreement over the relative contribution of various mechanisms that maintain low [Cl-]i We found that the Cl- transporter KCC2 is an important Cl- extruder in the reticular thalamic (RT) nucleus, despite this nucleus having remarkably low KCC2 immunoreactivity relative to other regions of the adult brain. We also identified a smaller contribution of fixed, impermeant anions ([A]o) to lowering [Cl-]i in RT neurons. Inhibitory signaling among RT neurons is important for preventing excessive activation of RT neurons, which can be responsible for generating seizures. Our work suggests that KCC2 critically restricts the spread of activity within the RT nucleus.

Crossed activation of thoracic trunk motoneurons by medullary reticulospinal neurons.

Activation of contralateral muscles by supraspinal neurons, or crossed activation, is critical for bilateral coordination. Studies in mammals have focused on the neural circuits that mediate cross activation of limb muscles, but the neural circuits involved in crossed activation of trunk muscles are still poorly understood. In this study, we characterized functional connections between reticulospinal (RS) neurons in the medial and lateral regions of the medullary reticular formation (medMRF and latMRF) and contralateral trunk motoneurons (MNs) in the thoracic cord (T7 and T10 segments). To do this, we combined electrical microstimulation of the medMRF and latMRF and calcium imaging from single cells in an ex vivo brain stem-spinal cord preparation of neonatal mice. Our findings substantiate two spatially distinct RS pathways to contralateral trunk MNs. Both pathways originate in the latMRF and are midline crossing, one at the level of the spinal cord via excitatory descending commissural interneurons (reticulo-commissural pathway) and the other at the level of the brain stem (crossed RS pathway). Activation of these RS pathways may enable different patterns of bilateral trunk coordination. Possible implications for recovery of trunk function after stroke or spinal cord injury are discussed.NEW & NOTEWORTHY We identify two spatially distinct reticulospinal pathways for crossed activation of trunk motoneurons. Both pathways cross the midline, one at the level of the brain stem and the other at the level of the spinal cord via excitatory commissural interneurons. Jointly, these pathways provide new opportunities for repair interventions aimed at recovering trunk functions after stroke or spinal cord injury.

Multimodal stimuli modulate rapid visual responses during reaching.

The reticulospinal tract plays an important role in primate upper limb function, but methods for assessing its activity are limited. One promising approach is to measure rapid visual responses (RVRs) in arm muscle activity during a visually cued reaching task; these may arise from a tecto-reticulospinal pathway. We investigated whether changes in reticulospinal excitability can be assessed noninvasively using RVRs, by pairing the visual stimuli of the reaching task with electrical stimulation of the median nerve, galvanic vestibular stimulation, or loud sounds, all of which are known to activate the reticular formation. Surface electromyogram (EMG) recordings were made from the right deltoid of healthy human subjects as they performed fast reaching movements toward visual targets. Stimuli were delivered up to 200 ms before target appearance, and RVR was quantified as the EMG amplitude in a window 75-125 ms after visual target onset. Median nerve, vestibular, and auditory stimuli all consistently facilitated the RVRs, as well as reducing the latency of responses. We propose that this facilitation reflects modulation of tecto-reticulospinal excitability, which is consistent with the idea that the amplitude of RVRs can be used to assess changes in brain stem excitability noninvasively in humans.NEW & NOTEWORTHY Short-latency responses in arm muscles evoked during a visually driven reaching task have previously been proposed to be tecto-reticulospinal in origin. We demonstrate that these responses can be facilitated by pairing the appearance of a visual target with stimuli that activate the reticular formation: median nerve, vestibular, and auditory stimuli. We propose that this reflects noninvasive measurement and modulation of reticulospinal excitability.

Central mesencephalic reticular formation control of the near response: lens accommodation circuits.

To view a nearby target, the three components of the near response are brought into play: 1) the eyes are converged through contraction of the medial rectus muscles to direct both foveae at the target, 2) the ciliary muscle contracts to allow the lens to thicken, increasing its refractive power to focus the near target on the retina, and 3) the pupil constricts to increase depth of field. In this study, we utilized retrograde transsynaptic transport of the N2c strain of rabies virus injected into the ciliary body of one eye of macaque monkeys to identify premotor neurons that control lens accommodation. We previously used this approach to label a premotor population located in the supraoculomotor area. In the present report, we describe a set of neurons located bilaterally in the central mesencephalic reticular formation that are labeled in the same time frame as the supraoculomotor area population, indicating their premotor character. The labeled premotor neurons are mostly multipolar cells, with long, very sparsely branched dendrites. They form a band that stretches across the core of the midbrain reticular formation. This population appears to be continuous with the premotor near-response neurons located in the supraoculomotor area at the level of the caudal central subdivision of the oculomotor nucleus. The central mesencephalic reticular formation has previously been associated with horizontal saccadic eye movements, so these premotor cells might be involved in controlling lens accommodation during disjunctive saccades. Alternatively, they may represent a population that controls vergence velocity. NEW & NOTEWORTHY This report uses transsynaptic transport of rabies virus to provide new evidence that the central mesencephalic reticular formation (cMRF) contains premotor neurons controlling lens accommodation. When combined with other recent reports that the cMRF also contains premotor neurons supplying medial rectus motoneurons, these results indicate that this portion of the reticular formation plays an important role in directing the near response and disjunctive saccades when viewers look between targets located at different distances.

Ascending vestibular pathways to parietal areas MIP and LIPv and efference copy inputs from the medial reticular formation: Functional frameworks for body representations updating and online movement guidance.

The posterior parietal cortex (PPC) serves as a sensorimotor interface by integrating multisensory signals with motor related information for generating and updating body representations and movement plans. Using retrograde transneuronal transfer of rabies virus combined with a conventional tracer, we identified direct and polysynaptic pathways to two PPC areas, the rostral medial intraparietal area (MIP) and the ventral part of the lateral intraparietal area (LIPv) in macaque monkeys. We found that rostral MIP and LIPv receive ascending vestibular pathways, and putative efference copy inputs disynaptically from the medullary medial reticular formation (MRF) where reticulospinal pathways to neck and arm motoneurons originate. LIPv receives minor disynaptic vestibular inputs, and substantial projections from the head movement-related rostral MRF, consistent with head gain modulation of LIPv activity and a role in planning gaze shifts. Rostral MIP is the target of prominent disynaptic pathways from reaching- and head movement-related MRF domains, and major ascending vestibular pathways trisynaptically from both labyrinths, explaining prominent vestibular responses and discrimination between active and passive movements demonstrated in rostral MIP and in the neighboring ventral intraparietal area, which are heavily interconnected. The findings that rostral MIP (belonging to the 'parietal reach region'), receives vestibular inputs as directly as classical vestibular areas, via a parallel channel, and efference copy signals pathways from MRF reticulospinal domains that belong to reach and head movement networks have important implications for the understanding of the role of the PPC in updating body representations and internal models for online guidance of movement.

The Ascending Reticular Activating System.

Discovery of the ascending reticular activating system (ARAS) can be attributed to work done in research neuroscientist Horace Magoun's laboratory. Before this finding, most scientists would focus on the diencephalon (and anterior midbrain) but not more caudally. Stimulation of the medial bulbar reticular formation in the pontine and midbrain tegmentum resulted disappearance of synchronized discharge and low-voltage fast activity. The effects were mediated by a thalamic projection system. This finding was a dramatic departure from the early philosophers' ascription of the awake soul to the ventricles (Galen), lumbosacral cord (Plato), pineal gland (Descartes), and even from more modern nineteenth- and twentieth-century hypotheses that the corpus striatum or periaqueductal gray matter housed the "seat of awareness." Magoun and his collaborators closed in on its true location in the cephalic brainstem-clinicians and neuropathologists would soon follow.

Carotid chemoreceptors tune breathing via multipath routing: reticular chain and loop operations supported by parallel spike train correlations.

We tested the hypothesis that carotid chemoreceptors tune breathing through parallel circuit paths that target distinct elements of an inspiratory neuron chain in the ventral respiratory column (VRC). Microelectrode arrays were used to monitor neuronal spike trains simultaneously in the VRC, peri-nucleus tractus solitarius (p-NTS)-medial medulla, the dorsal parafacial region of the lateral tegmental field (FTL-pF), and medullary raphe nuclei together with phrenic nerve activity during selective stimulation of carotid chemoreceptors or transient hypoxia in 19 decerebrate, neuromuscularly blocked, and artificially ventilated cats. Of 994 neurons tested, 56% had a significant change in firing rate. A total of 33,422 cell pairs were evaluated for signs of functional interaction; 63% of chemoresponsive neurons were elements of at least one pair with correlational signatures indicative of paucisynaptic relationships. We detected evidence for postinspiratory neuron inhibition of rostral VRC I-Driver (pre-Bötzinger) neurons, an interaction predicted to modulate breathing frequency, and for reciprocal excitation between chemoresponsive p-NTS neurons and more downstream VRC inspiratory neurons for control of breathing depth. Chemoresponsive pericolumnar tonic expiratory neurons, proposed to amplify inspiratory drive by disinhibition, were correlationally linked to afferent and efferent "chains" of chemoresponsive neurons extending to all monitored regions. The chains included coordinated clusters of chemoresponsive FTL-pF neurons with functional links to widespread medullary sites involved in the control of breathing. The results support long-standing concepts on brain stem network architecture and a circuit model for peripheral chemoreceptor modulation of breathing with multiple circuit loops and chains tuned by tegmental field neurons with quasi-periodic discharge patterns. NEW & NOTEWORTHY We tested the long-standing hypothesis that carotid chemoreceptors tune the frequency and depth of breathing through parallel circuit operations targeting the ventral respiratory column. Responses to stimulation of the chemoreceptors and identified functional connectivity support differential tuning of inspiratory neuron burst duration and firing rate and a model of brain stem network architecture incorporating tonic expiratory "hub" neurons regulated by convergent neuronal chains and loops through rostral lateral tegmental field neurons with quasi-periodic discharge patterns.