In-vitro Recordings of Neural Magnetic Activity From the Auditory Brainstem Using Color Centers in Diamond: A Simulation Study

Magnetometry based on nitrogen-vacancy (NV) centers in diamond is a novel technique capable of measuring magnetic fields with high sensitivity and high spatial resolution. With the further advancements of these sensors, they may open up novel approaches for the 2D imaging of neural signals in vitro. In the present study, we investigate the feasibility of NV-based imaging by numerically simulating the magnetic signal from the auditory pathway of a rodent brainstem slice (ventral cochlear nucleus, VCN, to the medial trapezoid body, MNTB) as stimulated by both electric and optic stimulation. The resulting signal from these two stimulation methods are evaluated and compared. A realistic pathway model was created based on published data of the neural morphologies and channel dynamics of the globular bushy cells in the VCN and their axonal projections to the principal cells in the MNTB. The pathway dynamics in response to optic and electric stimulation and the emitted magnetic fields were estimated using the cable equation. For simulating the optic stimulation, the light distribution in brain tissue was numerically estimated and used to model the optogenetic neural excitation based on a four state channelrhodopsin-2 (ChR2) model. The corresponding heating was also estimated, using the bio-heat equation and was found to be low (<2°C) even at excessively strong optic signals. A peak magnetic field strength of ∼0.5 and ∼0.1 nT was calculated from the auditory brainstem pathway after electrical and optical stimulation, respectively. By increasing the stimulating light intensity four-fold (far exceeding commonly used intensities) the peak magnetic signal strength only increased to 0.2 nT. Thus, while optogenetic stimulation would be favorable to avoid artefacts in the recordings, electric stimulation achieves higher peak fields. The present simulation study predicts that high-resolution magnetic imaging of the action potentials traveling along the auditory brainstem pathway will only be possible for next generation NV sensors. However, the existing sensors already have sufficient sensitivity to support the magnetic sensing of cumulated neural signals sampled from larger parts of the pathway, which might be a promising intermediate step toward further maturing this novel technology.

TRPM3 expression and control of glutamate release from primary vagal afferent neurons.

Vagal afferent fibers contact neurons in the nucleus of the solitary tract (NTS) and release glutamate via three distinct release pathways: synchronous, asynchronous, and spontaneous. The presence of TRPV1 in vagal afferents is predictive of activity-dependent asynchronous glutamate release along with temperature-sensitive spontaneous vesicle fusion. However, pharmacological blockade or genetic deletion of TRPV1 does not eliminate the asynchronous profile and only attenuates the temperature-dependent spontaneous release at high temperatures (>40˚C), indicating additional temperature-sensitive calcium conductance(s) contributing to these release pathways. The transient receptor potential cation channel melastatin subtype 3 (TRPM3) is a calcium-selective channel which functions as a thermosensor (30-37˚C) in somatic primary afferent neurons. We predict TRPM3 is expressed in vagal afferent neurons and contributes to asynchronous and spontaneous glutamate release pathways. We investigated these hypotheses via measurements on cultured nodose neurons and in brainstem slice preparations containing vagal afferent to NTS synaptic contacts. We found histological and genetic evidence that TRPM3 is highly expressed in vagal afferent neurons. The TRPM3-selective agonist, pregnenolone sulfate, rapidly and reversibly activated the majority (~70%) of nodose neurons; most of which also contained TRPV1. We confirmed the role of TRPM3 with pharmacological blockade and genetic deletion. In the brain, TRPM3 signaling strongly controlled both basal and temperature-driven spontaneous glutamate release. Surprisingly, genetic deletion of TRPM3 did not alter synchronous nor asynchronous glutamate release. These results provide convergent evidence that vagal afferents express functional TRPM3 that serves as an additional temperature-sensitive calcium conductance involved in controlling spontaneous glutamate release onto neurons in the NTS.

Mechanism of central hypopnoea induced by organic phosphorus poisoning

Whether central apnoea or hypopnoea can be induced by organophosphorus poisoning remains unknown to date. By using the acute brainstem slice method and multi-electrode array system, we established a paraoxon (a typical acetylcholinesterase inhibitor) poisoning model to investigate the time-dependent changes in respiratory burst amplitudes of the pre-Bötzinger complex (respiratory rhythm generator). We then determined whether pralidoxime or atropine, which are antidotes of paraoxon, could counteract the effects of paraoxon. Herein, we showed that paraoxon significantly decreased the respiratory burst amplitude of the pre-Bötzinger complex (p < 0.05). Moreover, pralidoxime and atropine could suppress the decrease in amplitude by paraoxon (p < 0.05). Paraoxon directly impaired the pre-Bötzinger complex, and the findings implied that this impairment caused central apnoea or hypopnoea. Pralidoxime and atropine could therapeutically attenuate the impairment. This study is the first to prove the usefulness of the multi-electrode array method for electrophysiological and toxicological studies in the mammalian brainstem.

Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices

Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.

Astrocytes modulate baroreceptor reflex sensitivity at the level of the nucleus of the solitary tract

Astrocytes play an important role in cardiovascular reflex integration at the level of the nucleus tractus solitarii (NTS). Existing reports from brainstem slice preparations suggest that astrocytes here respond to input from the solitary tract by increasing intracellular calcium. However, the physiological significance of this neuron-astrocyte signaling in vivo remains unknown. Here, we report that stimulation of the vagus nerve in an anesthetized rat induced rapid [Ca2+]i increases in astrocytes transduced to express calcium sensor GCaMP6. The receptors involved were determined using brainstem-derived astroglial cell cultures were loaded with [Ca2+] indicator Fura-2. 5-HT (10 µM) caused robust increases in [Ca2+]i, and pharmacological interrogation revealed the expression of functional 5-HT2A receptors. This observation was confirmed in vivo: intravenous administration of ketanserin decreased the magnitude of [Ca2+]i responses, induced by vagal afferent simulation, by ∼50%. However, the response was completely blocked by topical application of the AMPA receptor antagonist CNQX alone. To investigate the role of astrocyte-neuron communication, the vesicular release in the NTS astrocytes was blocked by virally driven expression of a dominant-negative SNARE protein in vivo. This increased baroreflex sensitivity in awake animals, which was also observed in anesthetized animals after topical application of the P2Y1 receptor antagonist MDS-2500 to the NTS. We hypothesize that NTS astrocytes respond to incoming afferent release of glutamate and this response is modulated by 5-HT originating from vagal afferents or other sources. ATP is then released, which acts on inhibitory interneurons via P2Y1 receptors and thus modulates the expression of cardiovascular reflexes.Significance statementCardiorespiratory nuclei in the brainstem integrate cardiovascular sensory information to optimise tissue perfusion and blood gas concentrations. We describe experimental evidence that NTS astrocytes participate in setting the baroreflex sensitivity by release of ATP acting on P2Y1 receptors on inhibitory interneurons. Activation of astrocytes is partly under control of 5-HT co-released with glutamate from vagal afferents, which allows modulation of autonomic response to high frequency/duration of afferent stimulation by monitoring extra-synaptic 5-HT acting on glial 5-HT2A receptors. This could represent a signaling pathway that is activated under pathological conditions and is responsible for baroreflex impairment in conditions that result in astrogliosis, for example from systemic inflammatory response or chronic hypoxia/hypercapnia.

Role of synaptic inhibition in the coupling of the respiratory rhythms that underlie eupnea and sigh behaviors

ABSTRACTThe preBötzinger Complex (preBötC) gives rise to two types of breathing behavior: eupnea and sighing. Here, we examine the neural mechanisms that couple their underlying rhythms by recording from the preBötC in neonatal mouse brainstem slice preparations. It has been proposed that chloride-mediated synaptic inhibition couples inspiratory (eupnea-related) bursts and sigh bursts, but we find no evidence to support that notion. First, we characterize a fluctuating temporal relationship between sigh bursts and their preceding inspiratory bursts; their coupling is far weaker than previously described. Surprisingly, selective blockade of inhibitory synapses strengthened (rather than weakened) that phasic inspiratory-sigh burst relationship. Furthermore, pharmacological disinhibition did not alter the duration of the prolonged interval that follows a sigh burst prior to resumption of the inspiratory rhythm. These results demonstrate that coupling between inspiratory and sigh rhythms does not depend on synaptic inhibition.SIGNIFICANCE STATEMENTBreathing consists of eupnea and sigh breaths, which differ in their magnitude and frequency. Both breath types emerge from a brainstem microcircuit that coordinates their timing. Here, we advance understanding of these rhythms by assessing the nature and strength of their coordination, and by showing that synaptic inhibition does not enforce their temporal coupling in contrast to conventional understanding. This study provides insights into the basic neural mechanisms that link oscillations of different amplitude and frequency in a core oscillator.

Network synchronization and synchrony propagation: emergent elements of inspiration

SUMMARYThe preBötzinger Complex (preBötC) – the kernel of breathing rhythmogenesis in mammals – is a non-canonical central pattern generator with undetermined mechanisms. We assessed preBötC network dynamics under respiratory rhythmic and nonrhythmic conditions in vitro. In each cycle under rhythmic conditions, an inspiratory burst emerges as (presumptive) preBötC rhythmogenic neurons transition from aperiodic uncorrelated population spike activity to become increasingly synchronized during preinspiration, triggering bursts; burst activity subsides and the cycle repeats. In a brainstem slice in nonrhythmic conditions, antagonizing GABAA receptors can initiate this periodic synchronization and consequent rhythm coincident with inducing a higher conductance state in nonrhythmogenic preBötC output neurons. Furthermore, when input synchrony onto these neurons was weak, preBötC activity failed to propagate to motor nerves. Our analyses uncover a dynamic reorganization of preBötC network activity – underpinning intricate cyclic neuronal interactions leading to network synchronization and its efficient propagation – correlated with and, we postulate, essential to, rhythmicity.

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

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

Adrenergic agonist induces rhythmic firing in quiescent cardiac preganglionic neurons in nucleus ambiguous via activation of intrinsic membrane excitability

Cholinergic vagal nerves projecting from neurons in the brainstem nucleus ambiguus (NAm) play a predominant role in cardiac parasympathetic pacemaking control. Central adrenergic signaling modulates the tone of this vagal output; however the exact excitability mechanisms are not fully understood. We investigated responses of NAm neurons to adrenergic agonists using in vitro mouse brainstem slices. Preganglionic NAm neurons were identified by Chat-tdtomato fluorescence in young adult transgenic mice and their cardiac projection confirmed by retrograde dye tracing. Juxtacellular recordings detected sparse or absent spontaneous action potentials (AP) in NAm neurons. However bath application of epinephrine or norepinephrine strongly and reversibly activated most NAm neurons regardless of their basal firing rate. Epinephrine was more potent than norepinephrine, and this activation largely depends on α1-adrenoceptors. Interestingly, adrenergic activation of NAm neurons does not require an ionotropic synaptic mechanism, since postsynaptic excitatory or inhibitory receptor blockade did not occlude the excitatory effect, and bath-applied adrenergic agonists did not alter excitatory or inhibitory synaptic transmission. Instead, adrenergic agonists significantly elevated intrinsic membrane excitability to facilitate generation of recurrent action potentials. T-type calcium current (ICaT) and hyperpolarization-activated current (Ih) are involved in this excitation pattern, while not required for spontaneous AP induction by epinephrine. In contrast, pharmacological blockade of persistent sodium current (INaP) significantly inhibited the adrenergic effects. Our results demonstrate that central adrenergic signaling enhances the intrinsic excitability of NAm neurons, and persistent sodium current is required for this effect. This central balancing mechanism may counteract excessive peripheral cardiac excitation during increased sympathetic tone.New & NoteworthyCardiac preganglionic cholinergic neurons in the Nucleus ambiguus (NAm) are responsible for slowing cardiac pacemaking. This study identified that adrenergic agonists can induce rhythmic action potentials in otherwise quiescent cholinergic NAm preganglionic neurons in brainstem slice preparation. The modulatory influence of adrenaline on central parasympathetic outflow may contribute to both physiological and deleterious cardiovascular regulation.

Mechanisms Underlying Adaptation of Respiratory Network Activity to Modulatory Stimuli in the Mouse Embryo

Breathing is a rhythmic behavior that requires organized contractions of respiratory effector muscles. This behavior must adapt to constantly changing conditions in order to ensure homeostasis, proper body oxygenation, and CO2/pH regulation. Respiratory rhythmogenesis is controlled by neural networks located in the brainstem. One area considered to be essential for generating the inspiratory phase of the respiratory rhythm is the preBötzinger complex (preBötC). Rhythmogenesis emerges from this network through the interplay between the activation of intrinsic cellular properties (pacemaker properties) and intercellular synaptic connections. Respiratory activity continuously changes under the impact of numerous modulatory substances depending on organismal needs and environmental conditions. The preBötC network has been shown to become active during the last third of gestation. But only little is known regarding the modulation of inspiratory rhythmicity at embryonic stages and even less on a possible role of pacemaker neurons in this functional flexibility during the prenatal period. By combining electrophysiology and calcium imaging performed on embryonic brainstem slice preparations, we provide evidence showing that embryonic inspiratory pacemaker neurons are already intrinsically sensitive to neuromodulation and external conditions (i.e., temperature) affecting respiratory network activity, suggesting a potential role of pacemaker neurons in mediating rhythm adaptation to modulatory stimuli in the embryo.