Feedforward inhibition of stress by brainstem neuropeptide Y neurons.

Resistance to stress is a key determinant for mammalian functioning. While many studies have revealed neural circuits and substrates responsible for initiating and mediating stress responses, little is known about how the brain resists to stress and prevents overreactions. Here, we identified a previously uncharacterized neuropeptide Y (NPY) neuronal population in the dorsal raphe nucleus and ventrolateral periaqueductal gray region (DRN/vlPAG) with anxiolytic effects in male mice. NPYDRN/vlPAG neurons are rapidly activated by various stressful stimuli. Inhibiting these neurons exacerbated hypophagic and anxiety responses during stress, while activation significantly ameliorates acute stress-induced hypophagia and anxiety levels and transmits positive valence. Furthermore, NPYDRN/vlPAG neurons exert differential but synergic anxiolytic effects via inhibitory projections to the paraventricular thalamic nucleus (PVT) and the lateral hypothalamic area (LH). Together, our findings reveal a feedforward inhibition neural mechanism underlying stress resistance and suggest NPYDRN/vlPAG neurons as a potential therapeutic target for stress-related disorders.

Binaural interaction in human auditory brainstem and middle-latency responses affected by sound frequency band, lateralization predictability, and attended modality.

The binaural interaction component (BIC) of the auditory evoked potential is the difference between the waveforms of the binaural response and the sum of left and right monaural responses. This investigation examined BICs of the auditory brainstem (ABR) and middle-latency (MLR) responses concerning three objectives: 1) the level of the auditory system at which low-frequency dominance in BIC amplitudes begins when the binaural temporal fine structure is more influential with lower- than higher-frequency content; 2) how BICs vary as a function of frequency and lateralization predictability, as could relate to the improved lateralization of high-frequency sounds; 3) how attention affects BICs. Sixteen right-handed participants were presented with either low-passed (< 1000 Hz) or high-passed (> 2000 Hz) clicks at 30 dB SL with a 38 dB (A) masking noise, at a stimulus onset asynchrony of 180 ms. Further, this repeated-measures design manipulated stimulus presentation (binaural, left monaural, right monaural), lateralization predictability (unpredictable, predictable), and attended modality (either auditory or visual). For the objectives, respectively, the results were: 1) whereas low-frequency dominance in BIC amplitudes began during, and continued after, the Na-BIC, binaural (center) as well as summed monaural (left and right) amplitudes revealed low-frequency dominance only after the Na wave; 2) with a predictable position that was fixed, no BIC exhibited equivalent amplitudes between low- and high-passed clicks; 3) whether clicks were low- or high-passed, selective attention affected the ABR-BIC yet not MLR-BICs. These findings indicate that low-frequency dominance in lateralization begins at the Na latency, being independent of the efferent cortico-collicular pathway's influence.

Brainstem Dbh+ neurons control allergen-induced airway hyperreactivity.

Exaggerated airway constriction triggered by repeated exposure to allergen, also called hyperreactivity, is a hallmark of asthma. Whereas vagal sensory neurons are known to function in allergen-induced hyperreactivity1-3, the identity of downstream nodes remains poorly understood. Here we mapped a full allergen circuit from the lung to the brainstem and back to the lung. Repeated exposure of mice to inhaled allergen activated the nuclei of solitary tract (nTS) neurons in a mast cell-, interleukin-4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA sequencing, followed by RNAscope assay at baseline and allergen challenges, showed that a Dbh+ nTS population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted hyperreactivity whereas chemogenetic activation promoted it. Viral tracing indicated that Dbh+ nTS neurons project to the nucleus ambiguus (NA) and that NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that directly drive airway constriction. Delivery of noradrenaline antagonists to the NA blunted hyperreactivity, suggesting noradrenaline as the transmitter between Dbh+ nTS and NA. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. This knowledge informs how neural modulation could be used to control allergen-induced airway hyperreactivity.

A vagal-brainstem interoceptive circuit for cough-like defensive behaviors in mice.

Coughing is a respiratory behavior that plays a crucial role in protecting the respiratory system. Here we show that the nucleus of the solitary tract (NTS) in mice contains heterogenous neuronal populations that differentially control breathing. Within these subtypes, activation of tachykinin 1 (Tac1)-expressing neurons triggers specific respiratory behaviors that, as revealed by our detailed characterization, are cough-like behaviors. Chemogenetic silencing or genetic ablation of Tac1 neurons inhibits cough-like behaviors induced by tussive challenges. These Tac1 neurons receive synaptic inputs from the bronchopulmonary chemosensory and mechanosensory neurons in the vagal ganglion and coordinate medullary regions to control distinct aspects of cough-like defensive behaviors. We propose that these Tac1 neurons in the NTS are a key component of the airway-vagal-brain neural circuit that controls cough-like defensive behaviors in mice and that they coordinate the downstream modular circuits to elicit the sequential motor pattern of forceful expiratory responses.

The breath shape controls intonation of mouse vocalizations.

Intonation in speech is the control of vocal pitch to layer expressive meaning to communication, like increasing pitch to indicate a question. Also, stereotyped patterns of pitch are used to create distinct sounds with different denotations, like in tonal languages and, perhaps, the 10 sounds in the murine lexicon. A basic tone is created by exhalation through a constricted laryngeal voice box, and it is thought that more complex utterances are produced solely by dynamic changes in laryngeal tension. But perhaps, the shifting pitch also results from altering the swiftness of exhalation. Consistent with the latter model, we describe that intonation in most vocalization types follows deviations in exhalation that appear to be generated by the re-activation of the cardinal breathing muscle for inspiration. We also show that the brainstem vocalization central pattern generator, the iRO, can create this breath pattern. Consequently, ectopic activation of the iRO not only induces phonation, but also the pitch patterns that compose most of the vocalizations in the murine lexicon. These results reveal a novel brainstem mechanism for intonation.

Real-time closed-loop brainstem stimulation modality for enhancing temporal blood pressure reduction.

BACKGROUND: Traditional pharmacological interventions are well tolerated in the management of elevated blood pressure (BP) for individuals with resistant hypertension. Although neuromodulation has been investigated as an alternative solution, its open-loop (OL) modality cannot follow the patient's physiological state. In fact, neuromodulation for controlling highly fluctuating BP necessitates a closed-loop (CL) stimulation modality based on biomarkers to monitor the patient's continuously varying physiological state. OBJECTIVE: By leveraging its intuitive linkage with BP responses in ongoing efforts aimed at developing a CL system to enhance temporal BP reduction effect, this study proposes a CL neuromodulation modality that controls nucleus tractus solitarius (NTS) activity to effectively reduce BP, thus reflecting continuously varying physiological states. METHOD: While performing neurostimulation targeting the NTS in the rat model, the arterial BP response and neural activity of the NTS were simultaneously measured. To evaluate the temporal BP response effect of CL neurostimulation, OL (constant parameter; 20 Hz, 200 µA) and CL (Initial parameter; 11 Hz, 112 µA) stimulation protocols were performed with stimulation 180 s and rest 600 s, respectively, and examined NTS activity and BP response to the protocols. RESULTS: In-vivo experiments for OL versus CL protocol for direct NTS stimulation in rats demonstrated an enhancement in temporal BP reduction via the CL modulation of NTS activity. CONCLUSION: This study proposes a CL stimulation modality that enhances the effectiveness of BP control using a feedback control algorithm based on neural signals, thereby suggesting a new approach to antihypertensive neuromodulation.

Brainstem neuronal responses to transcutaneous auricular and cervical vagus nerve stimulation in rats.

Transcutaneous auricular vagus nerve stimulation (taVNS) targets subcutaneous axons in the auricular branch of the vagus nerve at the outer ear. Its non-invasive nature makes it a potential treatment for various disorders. taVNS induces neuromodulatory effects within the nucleus of the solitary tract (NTS), and due to its widespread connectivity, the NTS acts as a gateway to elicit neuromodulation in both higher-order brain regions and other brainstem nuclei (e.g. spinal trigeminal nucleus; Sp5). Our objective was to examine stimulation parameters on single-neuron electrophysiological responses in α-chloralose-anaesthetized Sprague-Dawley rats within NTS and Sp5. taVNS was also compared to traditional cervical VNS (cVNS) on single neuronal activation. Specifically, electrophysiological extracellular recordings were evaluated for a range of frequency and intensity parameters (20-250 Hz, 0.5-1.0 mA). Neurons were classified as positive, negative or non-responders based on increased activity, decreased activity or no response during stimulation, respectively. Frequency-dependent analysis showed that 20 and 100 Hz generated the highest proportion of positive responders in NTS and Sp5 with 1.0 mA intensities eliciting the greatest magnitude of response. Comparisons between taVNS and cVNS revealed similar parameter-specific activation for caudal NTS neuronal populations; however, individual neurons showed different activation profiles. The latter suggests that cVNS and taVNS send afferent input to NTS via different neuronal pathways. This study demonstrates differential parameter-specific taVNS responses and begins an investigation of the mechanisms responsible for taVNS modulation. Understanding the neuronal pathways responsible for eliciting neuromodulatory effects will enable more tailored taVNS treatments in various clinical disorders. KEY POINTS: Transcutaneous auricular vagus nerve stimulation (taVNS) offers a non-invasive alternative to invasive cervical vagus nerve stimulation (cVNS) by activating vagal afferents in the ear to induce neuromodulation. Our study evaluated taVNS effects on neuronal firing patterns in the nucleus of the solitary tract (NTS) and spinal trigeminal nucleus (Sp5) and found that 20 and 100 Hz notably increased neuronal activity during stimulation in both nuclei. Increasing taVNS intensity not only increased the number of neurons responding in Sp5 but also increased the magnitude of response, suggesting a heightened sensitivity to taVNS compared to NTS. Comparisons between cVNS and taVNS revealed similar overall activation but different responses on individual neurons, indicating distinct neural pathways. These results show parameter-specific and nuclei-specific responses to taVNS and confirm that taVNS can elicit responses comparable to cVNS at the neuronal level, but it does so through different neuronal pathways.

Effects of linalool on respiratory neuron activity in the brainstem-spinal cord preparation from newborn rats.

Linalool and linalyl acetate are major components of lavender essential oil. These substances possess many biological activities, such as anti-inflammatory activity, analgesic and anxiolytic effects, and anticonvulsant properties, and they also induce modulation of neuronal activity in the autonomic nervous system. However, there are no reports of the direct effects of linalool on respiratory activity. In the present study, we analyzed the effects of linalool and linalyl acetate on central respiratory activity in the brainstem-spinal cord preparation isolated from newborn rats. Linalool dose-dependently decreased the rate of respiratory activity. This effect was reversed by bicuculline, suggesting that linalool enhanced inhibitory synaptic connections via GABAA receptors. In addition, linalool reduced the coefficient of variation of inspiratory burst intervals and thus could work to stabilize the respiratory rhythm. Linalyl acetate did not cause inhibitory effects as observed in linalool treatment. Linalool depressed burst activity of pre-inspiratory neurons in the medullary respiratory networks and increased the amplitude of inspiratory inhibitory postsynaptic potentials of pre-inspiratory neurons. We concluded that linalool caused inhibitory effects on respiratory rhythm generation mainly through activation of presynaptic GABAA receptors of pre-inspiratory neurons.

A brainstem maestro conducting the somatic and autonomic motor symphony.

Somatic and sympathetic tones fluctuate together seamlessly across daily behaviors. In this issue of Cell, Zhang et al. describe populations of spinal projecting neurons in the rostral ventromedial medulla (rVMM) that harmonize somatic motor function and sympathetic activation. The coordinated regulation plays a vital role in supporting behaviors associated with various arousal states.

Chloride Modulates Central pH Sensitivity and Plasticity of Brainstem Breathing-Related Biorhythms in Zebra Finch Embryos.

All terrestrial vertebrate life must transition from aquatic gas exchange in the embryonic environment to aerial or pulmonary respiration at birth. In addition to being able to breathe air, neonates must possess functional sensory feedback systems for maintaining acid-base balance. Respiratory neurons in the brainstem act as pH sensors that can adjust breathing to regulate systemic pH. The central pH sensitivity of breathing-related motor output develops over the embryonic period in the zebra finch (Taeniopygia guttata). Due to the key role of chloride ions in electrochemical stability and developmental plasticity, we tested chloride's role in the development of central pH sensitivity. We blocked gamma-aminobutyric acid-A receptors and cation-chloride cotransport that subtly modulated the low-pH effects on early breathing biorhythms. Further, chloride-free artificial cerebrospinal fluid altered the pattern and timing of breathing biorhythms and blocked the stimulating effect of acidosis in E12-14 brainstems. Early and middle stage embryos exhibited rebound plasticity in brainstem motor outputs during low-pH treatment, which was eliminated by chloride-free solution. Results show that chloride modulates low-pH sensitivity and rebound plasticity in the zebra finch embryonic brainstem, but work is needed to determine the cellular and circuit mechanisms that control functional chloride balance during acid-base disturbances.

Simultaneous cortical, subcortical, and brainstem mapping of sensory activation.

Nonpainful tactile sensory stimuli are processed in the cortex, subcortex, and brainstem. Recent functional magnetic resonance imaging studies have highlighted the value of whole-brain, systems-level investigation for examining sensory processing. However, whole-brain functional magnetic resonance imaging studies are uncommon, in part due to challenges with signal to noise when studying the brainstem. Furthermore, differentiation of small sensory brainstem structures such as the cuneate and gracile nuclei necessitates high-resolution imaging. To address this gap in systems-level sensory investigation, we employed a whole-brain, multi-echo functional magnetic resonance imaging acquisition at 3T with multi-echo independent component analysis denoising and brainstem-specific modeling to enable detection of activation across the entire sensory system. In healthy participants, we examined patterns of activity in response to nonpainful brushing of the right hand, left hand, and right foot (n = 10 per location), and found the expected lateralization, with distinct cortical and subcortical responses for upper and lower limb stimulation. At the brainstem level, we differentiated the adjacent cuneate and gracile nuclei, corresponding to hand and foot stimulation respectively. Our findings demonstrate that simultaneous cortical, subcortical, and brainstem mapping at 3T could be a key tool to understand the sensory system in both healthy individuals and clinical cohorts with sensory deficits.

Noradrenergic current responses of neurons in rat oculomotor neural integrators.

The prepositus hypoglossi nucleus (PHN) and the interstitial nucleus of Cajal (INC) are involved in the control of horizontal and vertical gaze, respectively. A previous study showed that PHN neurons exhibit depolarized or hyperpolarized responses to noradrenaline (NA). However, the adrenoceptor types that participate in NA-induced responses and the effects of NA on INC neurons have not yet been investigated. Furthermore, the relationship between NA-induced responses and neuron types defined by neurotransmitter phenotypes has not been determined. In this study, we investigated NA-induced current responses in PHN and INC neurons and the relationships between these responses and neuron types using whole cell recordings in wild-type and transgenic rat brainstem slices. Local application of NA to the cell soma induced slow inward (SI) and slow outward (SO) currents that were mainly mediated by α1 and α2 adrenoceptors, respectively. These current responses were observed in both PHN and INC neurons, although the proportion of INC neurons that responded to NA was low. Analyses of the distributions of the current responses revealed that in the PHN, all fluorescently identified inhibitory neurons exhibited SI currents, whereas glutamatergic and cholinergic neurons exhibited both SI and SO currents. In the INC, glutamatergic and inhibitory neurons preferentially exhibited SI and SO currents, respectively. When the PHN and INC neurons were characterized by their firing pattern, we found that the proportions of the currents depended on their firing pattern. These results suggest that various modes of noradrenergic modulation in horizontal and vertical neural integrators are dependent on neuron type.NEW & NOTEWORTHY Noradrenergic modulation of oculomotor neural integrators involved in gaze control has not been elucidated. Here, we report that noradrenaline (NA)-induced slow inward (SI) and outward (SO) currents are mediated mainly by α1 and α2 adrenoceptors in neurons that participate in horizontal and vertical gaze control. The NA-induced current responses differed depending on the neurotransmitter phenotype and firing pattern. These results suggest various modes of noradrenergic modulation in horizontal and vertical integrator neurons.

Neurexins control the strength and precise timing of glycinergic inhibition in the auditory brainstem.

Neurexins play diverse functions as presynaptic organizers in various glutamatergic and GABAergic synapses. However, it remains unknown whether and how neurexins are involved in shaping functional properties of the glycinergic synapses, which mediate prominent inhibition in the brainstem and spinal cord. To address these issues, we examined the role of neurexins in a model glycinergic synapse between the principal neuron in the medial nucleus of the trapezoid body (MNTB) and the principal neuron in the lateral superior olive (LSO) in the auditory brainstem. Combining RNAscope with stereotactic injection of AAV-Cre in the MNTB of neurexin1/2/3 conditional triple knockout mice, we showed that MNTB neurons highly express all isoforms of neurexins although their expression levels vary remarkably. Selective ablation of all neurexins in MNTB neurons not only reduced the amplitude but also altered the kinetics of the glycinergic synaptic transmission at LSO neurons. The synaptic dysfunctions primarily resulted from an impaired Ca2+ sensitivity of release and a loosened coupling between voltage-gated Ca2+ channels and synaptic vesicles. Together, our current findings demonstrate that neurexins are essential in controlling the strength and temporal precision of the glycinergic synapse, which therefore corroborates the role of neurexins as key presynaptic organizers in all major types of fast chemical synapses.

Multimodal MRI reveals brainstem connections that sustain wakefulness in human consciousness.

Consciousness is composed of arousal (i.e., wakefulness) and awareness. Substantial progress has been made in mapping the cortical networks that underlie awareness in the human brain, but knowledge about the subcortical networks that sustain arousal in humans is incomplete. Here, we aimed to map the connectivity of a proposed subcortical arousal network that sustains wakefulness in the human brain, analogous to the cortical default mode network (DMN) that has been shown to contribute to awareness. We integrated data from ex vivo diffusion magnetic resonance imaging (MRI) of three human brains, obtained at autopsy from neurologically normal individuals, with immunohistochemical staining of subcortical brain sections. We identified nodes of the proposed default ascending arousal network (dAAN) in the brainstem, hypothalamus, thalamus, and basal forebrain. Deterministic and probabilistic tractography analyses of the ex vivo diffusion MRI data revealed projection, association, and commissural pathways linking dAAN nodes with one another and with DMN nodes. Complementary analyses of in vivo 7-tesla resting-state functional MRI data from the Human Connectome Project identified the dopaminergic ventral tegmental area in the midbrain as a widely connected hub node at the nexus of the subcortical arousal and cortical awareness networks. Our network-based autopsy methods and connectivity data provide a putative neuroanatomic architecture for the integration of arousal and awareness in human consciousness.

Cell-type-specific hypothalamic pathways to brainstem drive context-dependent strategies in response to stressors.

Adaptive behavioral responses to stressors are critical for survival. However, which brain areas orchestrate switching the appropriate stress responses to distinct contexts is an open question. This study aimed to identify the cell-type-specific brain circuitry governing the selection of distinct behavioral strategies in response to stressors. Through novel mouse behavior paradigms, we observed distinct stressor-evoked behaviors in two psycho-spatially distinct contexts characterized by stressors inside or outside the safe zone. The identification of brain regions activated in both conditions revealed the involvement of the dorsomedial hypothalamus (DMH). Further investigation using optogenetics, chemogenetics, and photometry revealed that glutamatergic projections from the DMH to periaqueductal gray (PAG) mediated responses to inside stressors, while GABAergic projections, particularly from tachykinin1-expressing neurons, played a crucial role in coping with outside stressors. These findings elucidate the role of cell-type-specific circuitry from the DMH to the PAG in shaping behavioral strategies in response to stressors. These findings have the potential to advance our understanding of fundamental neurobiological processes and inform the development of novel approaches for managing context-dependent and anxiety-associated pathological conditions such as agoraphobia and claustrophobia.

Mapping Early Brain-Body Interactions: Associations of Fetal Heart Rate Variation with Newborn Brainstem, Hypothalamic, and Dorsal Anterior Cingulate Cortex Functional Connectivity.

The autonomic nervous system (ANS) regulates the body's physiology, including cardiovascular function. As the ANS develops during the second to third trimester, fetal heart rate variability (HRV) increases while fetal heart rate (HR) decreases. In this way, fetal HR and HRV provide an index of fetal ANS development and future neurobehavioral regulation. Fetal HR and HRV have been associated with child language ability and psychomotor development behavior in toddlerhood. However, their associations with postbirth autonomic brain systems, such as the brainstem, hypothalamus, and dorsal anterior cingulate cortex (dACC), have yet to be investigated even though brain pathways involved in autonomic regulation are well established in older individuals. We assessed whether fetal HR and HRV were associated with the brainstem, hypothalamic, and dACC functional connectivity in newborns. Data were obtained from 60 pregnant individuals (ages 14-42) at 24-27 and 34-37 weeks of gestation using a fetal actocardiograph to generate fetal HR and HRV. During natural sleep, their infants (38 males and 22 females) underwent a fMRI scan between 40 and 46 weeks of postmenstrual age. Our findings relate fetal heart indices to brainstem, hypothalamic, and dACC connectivity and reveal connections with widespread brain regions that may support behavioral and emotional regulation. We demonstrated the basic physiologic association between fetal HR indices and lower- and higher-order brain regions involved in regulatory processes. This work provides the foundation for future behavioral or physiological regulation research in fetuses and infants.

A conserved brainstem region for instinctive behaviour control: The vertebrate periaqueductal gray.

Instinctive behaviours have evolved across animal phyla and ensure the survival of both the individual and species. They include behaviours that achieve defence, feeding, aggression, sexual reproduction, or parental care. Within the vertebrate subphylum, the brain circuits that support instinctive behaviour output are evolutionarily conserved, being present in the oldest group of living vertebrates, the lamprey. Here, I will provide an evolutionary and comparative perspective on the function of a conserved brainstem region central to the initiation and execution of virtually all instinctive behaviours-the periaqueductal gray. In particular, I will focus on recent advances on the neural mechanisms in the periaqueductal gray that underlie the production of different instinctive behaviours within and across species.