Jan-Marino Ramirez

Breathing is a fundamental motor rhythm necessary to sustain life. The rhythm and pattern of breathing arises from the coordination of a rostrocaudally extended ventral respiratory column (VRC) in the medulla. Although recurrent excitation is sufficient for rhythmic bursting in reduced in vitro preparations, comparatively less is understood about how inhibition and excitation interact with feedback circuits to generate the breathing rhythm in vivo. Here, we expand on the classical “inspiratory-off switch” model that inhibitory signaling from lung stretch afferent pathways plays a critical role in generating the breathing rhythm in vivo. We perform high-density extracellular recordings along the length of the VRC during optogenetic and physiological manipulations and apply computational analyses to characterize how these perturbations govern low-dimensional neural dynamics. We show that stimulation of GABAergic afferents of the nucleus of the solitary tract advance or delay breathing in a manner similar to, but quantitatively distinct from, inhibition local to the VRC. We use generative dynamical models to show that inhibitory activity controls respiratory activity through transient stabilization of an attractive region of the low-dimensional manifold corresponding to expiration. Together, these results propose an “inspiratory-off attractor” model that underlies network level control of breathing in vivo.

The preBötzinger Complex is among the few neural circuits where selective elimination of defined neuronal subpopulation is sufficient to destabilize a core autonomic function and can fatally impair breathing. Within this circuitry, neurons expressing the neurokinin-1 receptor ( Tacr1/NK1R ) and somatostatin ( Sst ) are critical subpopulations; Tacr1 ⁺ neurons respond to the neuropeptide substance P and are necessary for maintaining inspiratory rhythms, and ablation of Sst ⁺ neurons results in apneas. To further dissect and analyze the specific roles for Tacr1 ⁺ and Sst ⁺ cell types, we conducted a comprehensive transcriptomic analysis using single-nucleus RNA sequencing of enriched preBötC from neonatal C57BL/6J mice. Because respiratory rhythmogenesis is an inherently electrophysiological process, we focused on the ion channel transcriptomes of Tacr1 + and Sst+ populations to resolve the molecular underpinnings of their distinct contributions to breathing. A balanced Random Forest classifier distinguished Tacr1⁺ from Sst⁺ neurons with higher accuracy, indicating a distinct and relatively homogeneous ion channel identity in Tacr1⁺ neurons. Differential expression analyses identified coordinated upregulation of Trpc5 , Kcnc2 and Cacna2d2 genes in Tacr1⁺ neurons. Tacr1⁺ neurons further exhibited elevated expression of the NALCN channelosome subunits and selective enrichment genes of Htr2c and Adra1a neuromodulatory receptor genes. Together, these findings define a molecularly distinct ion channel composition of Tacr1⁺ neurons and imply convergent substance P linked mechanisms: TRPC5-mediated I _CAN and NALCN-mediated sodium leak conductance supporting rhythmic inspiratory activity, providing a molecular framework for targeted interrogation of respiratory circuit function.

Astrocytes play vital roles in regulating brain states across organisms. Specifically, they regulate breathing behaviors and associated brain states, including facilitating transitions between breathing phases by sensing changes in O₂ and CO₂ levels, regulating the sleep-wake cycle, and impacting arousal and wakefulness. Here, we test the hypothesis that astrocytes in the ventral respiratory column (VRC) are important for arousal and sigh generation in alert mice. Our results reveal that a subset of Aldh1l1 cells in the VRC are activated prior to sigh generation and are recruited by hypoxia. Chemogenetic or optogenetic activation of Aldh1l1 astrocytes in the VRC increased the probability of evoking arousals with sighs. We also demonstrated that activating Aldh1l1 astrocytes increased calcium transients in catecholaminergic neurons in the VRC immediately before arousal with sighs. We conclude that medullary astrocytes can modulate sigh generation and arousal transitions, and are important for the ventilatory and arousal response to hypoxia.

Rett syndrome is an x-linked genetic neurological disorder primarily caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. This progressive neurodevelopmental condition hinders patients’ ability to breathe and eat normally. It remains unclear how Mecp2 deficiency leads to the high prevalence of dysphagia and aspiration pneumonia observed in individuals with Rett syndrome. This study aims to determine the effects of Mecp2 deficiency on swallow-related neuromuscular mechanisms that contribute to dysphagia in Rett syndrome. Swallow-related submental complex duration and amplitude were significantly decreased in both Mecp2 ^−/y and Mecp2 ^+/− mice compared to wild-type, likely due to reduced motor unit activation. In Mecp2- deficient mice, cholinergic immunoreactivity in the hypoglossal, facial, and trigeminal motor nuclei was decreased in postsymptomatic, but not presymptomatic mice. We also observed a significant increase in the transition time from inspiration to swallow, swallow to the subsequent inspiration, and impaired post swallow respiratory rhythm resumption in Mecp2 ^−/y , but not Mecp2 ^+/− mice. The combination of decreased ChAT ⁺ cells in brainstem motor nuclei and reduced submental muscle complex activity suggest impaired swallow-related hyolaryngeal elevation and laryngeal vestibular closure. These results provide insight into a neuromuscular mechanism underlying dysphagia in Rett syndrome and support the use of Mecp2- deficient mice as a viable preclinical model for further investigation of swallow and upper airway dysfunction in Rett syndrome.

What makes the human brain special? Human neurons, glia cells, and cortical circuits have been shown to be significantly different from those of other species, including mammals. This has led to a massive effort by the neuroscience community to directly study these differences in a multimodal approach. The studies conducted include single-cell and network recordings of human tissue samples, single-cell transcriptomics, and morphological analysis of the distinct cells to better understand the underlying differences from the cellular to the systems level. Furthermore, to overcome the translational gap from animal studies to patient care, the development of disease modeling in human tissue samples is of utmost interest. Here, we review and highlight research that focuses on the specialization of the human brain from molecular expression, cellular properties, to the challenges and promises of clinical translation.

Summary: Breathing is a fundamental motor rhythm necessary to sustain life. The rhythm and pattern of breathing arises from the coordination of a bilaterally symmetric, rostro-caudally extended column of heterogeneous neural populations in the medulla called the Ventral Respiratory Column (VRC). By recording from the extent of the VRC using Neuropixels during optogenetic and physiological manipulations, and projecting the population activity into a dynamical latent space, we find that GABAergic lung-stretch feedback circuits promote rhythmic population activity by creating a temporary stable fixed point in the latent state that terminates diaphragmatic contraction. Stimulation of GABAergic circuits either intrinsic to the VRC (VRC ^GABA ) or via afferent pathways from GABAergic neurons of the Nucleus of the Solitary Tract (NTS ^GABA ) advance or delay breathing when activated with specific respiratory phase through temporary stabilization of this fixed point. Lastly, we show that activation of glutamatergic signaling opposes the effects of inhibitory signaling by destabilizing this expiratory fixed point to promote rapid inspiration. Together, we decompose the functional impact of different respiratory control subpopulations to reveal an integrated network level mechanism for respiratory control in vivo .

Opioid-induced respiratory depression (OIRD) suppresses breathing and destabilizes ventilation. Using optogenetic and chemogenetic tools, we demonstrated that stimulating retrotrapezoid nucleus (RTN) Phox2b⁺/ Nmb⁺ neurons enhances breathing, even after fentanyl administration, whereas their inhibition exacerbates hypoventilation. These findings reveal that RTN neurons retain their ability to drive ventilation during OIRD, highlighting their potential as a therapeutic target to restore respiratory function in opioid overdose cases.

Breathing regulation depends on a highly intricate and precise network within the brainstem, requiring the identification of all neuronal elements in the brainstem respiratory circuits and a comprehensive understanding of their organization into distinct functional compartments. These compartments play a pivotal role by providing essential input to three main targets: cranial motoneurons that regulate airway control, spinal motoneurons that activate the inspiratory and expiratory muscles, and higher brain structures that influence breathing behavior and integrate it with other physiological and behavioral processes. This review offers a comprehensive examination of the phenotypes, connections, and functional roles of the major compartments within the brainstem and forebrain respiratory circuits. In addition, it summarizes the diverse neurotransmitters used by neurons in these regions, highlighting their contributions to the coordination and modulation of respiratory activity.

Astrocytes play vital roles in regulating brain states across organisms. Specifically, they serve several roles in regulating breathing behaviors and associated brain states, including facilitating transitions between phases of breathing by sensing small changes in O₂ and CO₂ levels, regulating the sleep-wake cycle, and impacting arousal and wakefulness. Here, we tested the hypothesis that astrocytes in the ventral respiratory column (VRC) are important for arousal and sigh generation in alert mice (Aldh1l1^Cre). Using calcium imaging we show that some Aldh1l1 cells are phase-locked with sigh generation and are activated in the VRC by hypoxia. Optogenetic activation (AAV-CAG-ChR2-EYFP) of astrocytes in the VRC increased the probability of evoking sighs and arousal while awake and during non-rapid eye movement (NREM) sleep. Depletion of astrocytes in the VRC by an AAV-CAG-Caspase3 virus (ablation of 77%) does not impact the probability of sigh generation in any sleep-wake state under control conditions. However following the depletion of astrocytes, arousal and sigh generation is significantly delayed in response to hypoxia (65.3 ± 5.5 vs. control: 21.7 ± 1.9 seconds). We conclude that medullary astrocytes play a critical role in the generation of arousal and sighs particularly in response to hypoxia.