Epidural electrical stimulation of the cervical spinal cord opposes opioid-induced respiratory depression.

Opioid overdose suppresses brainstem respiratory circuits, causes apnoea and may result in death. Epidural electrical stimulation (EES) at the cervical spinal cord facilitated motor activity in rodents and humans, and we hypothesized that EES of the cervical spinal cord could antagonize opioid-induced respiratory depression in humans. Eighteen patients requiring surgical access to the dorsal surface of the spinal cord between C2 and C7 received EES or sham stimulation for up to 90 s at 5 or 30 Hz during complete (OFF-State) or partial suppression (ON-State) of respiration induced by remifentanil. During the ON-State, 30 Hz EES at C4 and 5 Hz EES at C3/4 increased tidal volume and decreased the end-tidal carbon dioxide level compared to pre-stimulation control levels. EES of 5 Hz at C5 and C7 increased respiratory frequency compared to pre-stimulation control levels. In the OFF-State, 30 Hz cervical EES at C3/4 terminated apnoea and induced rhythmic breathing. In cadaveric tissue obtained from a brain bank, more neurons expressed both the neurokinin 1 receptor (NK1R) and somatostatin (SST) in the cervical spinal levels responsive to EES (C3/4, C6 and C7) compared to a region non-responsive to EES (C2). Thus, the capacity of cervical EES to oppose opioid depression of respiration may be mediated by NK1R+/SST+ neurons in the dorsal cervical spinal cord. This study provides proof of principle that cervical EES may provide a novel therapeutic approach to augment respiratory activity when the neural function of the central respiratory circuits is compromised by opioids or other pathological conditions. KEY POINTS: Epidural electrical stimulation (EES) using an implanted spinal cord stimulator (SCS) is an FDA-approved method to manage chronic pain. We tested the hypothesis that cervical EES facilitates respiration during administration of opioids in 18 human subjects who were treated with low-dose remifentanil that suppressed respiration (ON-State) or high-dose remifentanil that completely inhibited breathing (OFF-State) during the course of cervical surgery. Dorsal cervical EES of the spinal cord augmented the respiratory tidal volume or increased the respiratory frequency, and the response to EES varied as a function of the stimulation frequency (5 or 30 Hz) and the cervical level stimulated (C2-C7). Short, continuous cervical EES restored a cyclic breathing pattern (eupnoea) in the OFF-State, suggesting that cervical EES reversed the opioid-induced respiratory depression. These findings add to our understanding of respiratory pattern modulation and suggest a novel mechanism to oppose the respiratory depression caused by opioids.

Dendritic and Axonal L-Type Calcium Channels Cooperate to Enhance Motoneuron Firing Output during Drosophila Larval Locomotion.

Behaviorally adequate neuronal firing patterns are critically dependent on the specific types of ion channel expressed and on their subcellular localization. This study combines in situ electrophysiology with genetic and pharmacological intervention in larval Drosophila melanogaster of both sexes to address localization and function of L-type like calcium channels in motoneurons. We demonstrate that Dmca1D (Cav1 homolog) L-type like calcium channels localize to both the somatodendritic and the axonal compartment of larval crawling motoneurons. In situ patch-clamp recordings in genetic mosaics reveal that Dmca1D channels increase burst duration and maximum intraburst firing frequencies during crawling-like motor patterns in semi-intact animals. Genetic and acute pharmacological manipulations suggest that prolonged burst durations are caused by dendritically localized Dmca1D channels, which activate upon cholinergic synaptic input and amplify EPSPs, thus indicating a conserved function of dendritic L-type channels from Drosophila to vertebrates. By contrast, maximum intraburst firing rates require axonal calcium influx through Dmca1D channels, likely to enhance sodium channel de-inactivation via a fast afterhyperpolarization through BK channel activation. Therefore, in unmyelinated Drosophila motoneurons different functions of axonal and dendritic L-type like calcium channels likely operate synergistically to maximize firing output during locomotion.SIGNIFICANCE STATEMENT Nervous system function depends on the specific excitabilities of different types of neurons. Excitability is largely shaped by different combinations of voltage-dependent ion channels. Despite a high degree of conservation, the huge diversity of ion channel types and their differential localization pose challenges in assigning distinct functions to specific channels across species. We find a conserved role, from fruit flies to mammals, for L-type calcium channels in augmenting motoneuron excitability. As in spinal cord, dendritic L-type channels amplify excitatory synaptic input. In contrast to spinal motoneurons, axonal L-type channels enhance firing rates in unmyelinated Drosophila motoraxons. Therefore, enhancing motoneuron excitability by L-type channels seems an old strategy, but localization and interactions with other channels are tuned to species-specific requirements.

Modulation of respiratory output by cervical epidural stimulation in the anesthetized mouse.

Respiration is produced and controlled by well-characterized brain stem nuclei, but the contributions of spinal circuits to respiratory control and modulation remain under investigation. Many respiratory studies are conducted in in vitro preparations (e.g., brain stem slice) obtained from neonatal rodents. While informative, these studies do not fully recapitulate the complex afferent and efferent neural circuits that are likely to be involved in eupnea (i.e., quiet breathing). To begin to investigate spinal contributions to respiration, we electrically stimulated the cervical spinal cord during unassisted respiration in anesthetized, intact mice. Specifically, we used epidermal electrical stimulation at 20 Hz and varied current intensity to map changes in respiration. Stimulating at 1.5 mA at cervical level 3 (C3) consistently caused a significant increase in respiratory frequency compared with prestimulation baseline and when compared with sham stimulations. The increase in respiratory frequency persisted for several minutes after epidural stimulation ceased. There was no change in tidal volume, and the estimated minute ventilation was increased as a consequence of the increase in respiratory frequency. Sigh frequency also increased during epidural stimulation at C3. Neither the increase in respiratory frequency nor the increase in sighing were observed after stimulation at other dorsal cervical levels. These findings suggest that the spinal circuits involved in the modulation of eupnea and sighing may be preferentially activated by specific endogenous inputs. Moreover, the cervical spinal cord may play a role in respiratory modulation that affects both eupneic respiration and sigh production in intact, adult mice.

Optogenetic perturbation of preBötzinger complex inhibitory neurons modulates respiratory pattern.

Inhibitory neurons make up a substantial fraction of the neurons in the preBötzinger complex (preBötC), a site that is critical for mammalian eupneic breathing. We investigated the role of glycinergic preBötC neurons in respiratory rhythmogenesis in mice using optogenetically targeted excitation and inhibition. Channelrhodopsin-2 (ChR2) or Archaerhodopsin (Arch) were expressed in glycinergic preBötC neurons of glycine transporter 2 (Glyt2, also known as Slc6a5)-Cre mice. In ChR2-transfected mice, brief inspiratory-phase bilateral photostimulation targeting the preBötC prematurely terminated inspiration, whereas expiratory-phase photostimulation delayed the onset of the next inspiration. Prolonged photostimulation produced apneas lasting as long as the light pulse. Inspiratory-phase photoinhibition in Arch-transfected mice during inspiration increased tidal volume without altering inspiratory duration, whereas expiratory-phase photoinhibition shortened the latency until the next inspiration. During persistent apneas, prolonged photoinhibition restored rhythmic breathing. We conclude that glycinergic preBötC neurons modulate inspiratory pattern and are important for reflex apneas, but that the rhythm can persist after substantial dampening of their activity.

Distinct inspiratory rhythm and pattern generating mechanisms in the preBötzinger complex.

In the mammalian respiratory central pattern generator, the preBötzinger complex (preBötC) produces rhythmic bursts that drive inspiratory motor output. Cellular mechanisms initiated by each burst are hypothesized to be necessary to determine the timing of the subsequent burst, playing a critical role in rhythmogenesis. To explore mechanisms relating inspiratory burst generation to rhythmogenesis, we compared preBötC and hypoglossal (XII) nerve motor activity in medullary slices from neonatal mice in conditions where periods between successive inspiratory XII bursts were highly variable and distributed multimodally. This pattern resulted from rhythmic preBötC neural population activity that consisted of bursts, concurrent with XII bursts, intermingled with significantly smaller "burstlets". Burstlets occurred at regular intervals during significantly longer XII interburst intervals, at times when a XII burst was expected. When a preBötC burst occurred, its high amplitude inspiratory component (I-burst) was preceded by a preinspiratory component that closely resembled the rising phase of burstlets. Cadmium (8 µM) eliminated preBötC and XII bursts, but rhythmic preBötC burstlets persisted. Burstlets and preinspiratory activity were observed in ~90% of preBötC neurons that were active during I-bursts. When preBötC excitability was raised significantly, burstlets could leak through to motor output in medullary slices and in vivo in adult anesthetized rats. Thus, rhythmic bursting, a fundamental mode of nervous system activity and an essential element of breathing, can be deconstructed into a rhythmogenic process producing low amplitude burstlets and preinspiratory activity that determine timing, and a pattern-generating process producing suprathreshold I-bursts essential for motor output.

Emergence of population bursts from simultaneous activation of small subsets of preBötzinger complex inspiratory neurons.

During rhythmic movements, central pattern generators (CPGs) trigger bursts of motor activity with precise timing. However, the number of neurons that must be activated within CPGs to generate motor output is unknown. In the mammalian breathing rhythm, a fundamentally important motor behavior, the preBötzinger Complex (preBötC) produces synchronous population-wide bursts of activity to control inspiratory movements. We probed mechanisms underlying inspiratory burst generation in the preBötC using holographic photolysis of caged glutamate in medullary slices from neonatal mice. With stimulation parameters determined to confine photoactivation to targeted neurons, simultaneous excitation of 4-9 targeted neurons could initiate ectopic, endogenous-like bursts with delays averaging 255 ms, placing a critical and novel boundary condition on the microcircuit underlying respiratory rhythmogenesis.

Role of intrinsic properties in Drosophila motoneuron recruitment during fictive crawling.

Motoneurons in most organisms conserve a division into low-threshold and high-threshold types that are responsible for generating powerful and precise movements. Drosophila 1b and 1s motoneurons may be analogous to low-threshold and high-threshold neurons, respectively, based on data obtained at the neuromuscular junction, although there is little information available on intrinsic properties or recruitment during behavior. Therefore in situ whole cell patch-clamp recordings were used to compare parameters of 1b and 1s motoneurons in Drosophila larvae. We find that resting membrane potential, voltage threshold, and delay-to-spike distinguish 1b from 1s motoneurons. The longer delay-to-spike in 1s motoneurons is a result of the shal-encoded A-type K(+) current. Functional differences between 1b and 1s motoneurons are behaviorally relevant because a higher threshold and longer delay-to-spike are observed in MNISN-1s in pairwise whole cell recordings of synaptically evoked activity during bouts of fictive locomotion.

Normal dendrite growth in Drosophila motor neurons requires the AP-1 transcription factor.

During learning and memory formation, information flow through networks is regulated significantly through structural alterations in neurons. Dendrites, sites of signal integration, are key targets of activity-mediated modifications. Although local mechanisms of dendritic growth ensure synapse-specific changes, global mechanisms linking neural activity to nuclear gene expression may have profound influences on neural function. Fos, being an immediate-early gene, is ideally suited to be an initial transducer of neural activity, but a precise role for the AP-1 transcription factor in dendrite growth remains to be elucidated. Here we measure changes in the dendritic fields of identified Drosophila motor neurons in vivo and in primary culture to investigate the role of the immediate-early transcription factor AP-1 in regulating endogenous and activity-induced dendrite growth. Our data indicate that (a) increased neural excitability or depolarization stimulates dendrite growth, (b) AP-1 (a Fos, Jun hetero-dimer) is required for normal motor neuron dendritic growth during development and in response to activity induction, and (c) neuronal Fos protein levels are rapidly but transiently induced in motor neurons following neural activity. Taken together, these results show that AP-1 mediated transcription is important for dendrite growth, and that neural activity influences global dendritic growth through a gene-expression dependent mechanism gated by AP-1.

Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ.

Voltage-dependent Ca2+ channels contribute to neurotransmitter release, integration of synaptic information, and gene regulation within neurons. Thus understanding where diverse Ca2+ channels are expressed is an important step toward understanding neuronal function within a network. Drosophila provides a useful model for exploring the function of voltage-dependent Ca2+ channels in an intact system, but Ca2+ currents within the central processes of Drosophila neurons in situ have not been well described. The aim of this study was to characterize voltage-dependent Ca2+ currents in situ from identified larval motoneurons. Whole cell recordings from the somata of identified motoneurons revealed a significant influence of extracellular Ca2+ on spike shape and firing rate. Using whole cell voltage clamp, along with blockers of Na+ and K+ channels, a Ca2+-dependent inward current was isolated. The Drosophila genome contains three genes with homology to vertebrate voltage-dependent Ca2+ channels: Dmca1A, Dmca1D, and Dmalpha1G. We used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+ current recorded from two identified motoneurons. Our results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties.