Brainstem Neurons Research Articles

Diverse processing underlying frequency integration in midbrain neurons of barn owls.

Emergent response properties of sensory neurons depend on circuit connectivity and somatodendritic processing. Neurons of the barn owl’s external nucleus of the inferior colliculus (ICx) display emergence of spatial selectivity. These neurons use interaural time difference (ITD) as a cue for the horizontal direction of sound sources. ITD is detected by upstream brainstem neurons with narrow frequency tuning, resulting in spatially ambiguous responses. This spatial ambiguity is resolved by ICx neurons integrating inputs over frequency, a relevant processing in sound localization across species. Previous models have predicted that ICx neurons function as point neurons that linearly integrate inputs across frequency. However, the complex dendritic trees and spines of ICx neurons raises the question of whether this prediction is accurate. Data from in vivo intracellular recordings of ICx neurons were used to address this question. Results revealed diverse frequency integration properties, where some ICx neurons showed responses consistent with the point neuron hypothesis and others with nonlinear dendritic integration. Modeling showed that varied connectivity patterns and forms of dendritic processing may underlie observed ICx neurons’ frequency integration processing. These results corroborate the ability of neurons with complex dendritic trees to implement diverse linear and nonlinear integration of synaptic inputs, of relevance for adaptive coding and learning, and supporting a fundamental mechanism in sound localization.

Stress and central autonomic network.

The central autonomic network (CAN) plays a critical role in the stress response, which is triggered by challenges on the homeostasis (physiological stressors) or unpleasant social or environmental situations. This review focuses on the role of areas of the CAN including the insular and anterior cingulate cortices, extended amygdala, hypothalamus, periaqueductal gray and locus coeruleus in the stress response. These areas are interconnected and affect sympathetic or parasympathetic output via their influence on premotor or preganglionic autonomic neurons in the lower brainstem and spinal cord. The insula integrates multiple inputs to create a sense of the physiological state of the body, whereas the anterior cingulate initiates predictive visceromotor commands. The amygdala and bed nucleus of the stria terminalis provide automatic emotional tagging and trigger automatic survival responses to threat via their outputs to the hypothalamus, periaqueductal gray, and lower brainstem. Several regions of the hypothalamus, including the paraventricular nucleus, dorsomedial nucleus and lateral hypothalamic area participate in different patterns of stress response according to the type of stimulus and projections to premotor and preganglionic autonomic neurons. The periaqueductal gray initiates different patterns of autonomic, pain modulatory, and motor responses, including the "fight or flight" or "playing dead" responses. The locus coeruleus promotes emotional learning in the amygdala associated with states of anxiety. Neurons of the C1 area of the rostral ventrolateral medulla elicit sympathoexcitatory responses to internal stressors such as hypoxia and inflammation. The ventromedial medulla, including the nucleus raphe pallidus, initiates sympathoexcitatory responses to social and other external stressors.

Acute hyperglycemia does not affect central respiratory chemoreflex responsiveness to CO2 in healthy humans

The central respiratory chemoreceptor complex (CCRC) is comprised of brainstem neurons and surrounding interoceptors, which collectively increase ventilation in response to elevated brainstem tissue CO2/[H+] (i.e., central chemoreflex; CCR). The extent that the CCRC detects/responds to other metabolically related chemostimuli is unknown. We aimed to test the effects of acute oral glucose ingestion on CCR reactivity in heathy human participants (n = 38). We instrumented participants with a pneumotachometer (minute ventilation) and a gas sample line connected to a dual gas analyzer (pressure of end-tidal CO2). Following a baseline (BL) period and capillary blood [glucose] (BG) sample, fasted (F) participants underwent a modified hyperoxic rebreathing test to assess CCR reactivity. Participants then consumed a 75 g standard glucose beverage (glucose loaded; GL). Following 30-min, they underwent a second BL, BG sample and hyperoxic rebreathing test. BG and metabolic rate were higher in GL, confirming the metabolic stimulus. However, the ventilatory recruitment threshold and the CCR responses were unchanged between F and GL states.

Semaphorin7A: its role in the control of serotonergic circuits and functional recovery following spinal cord injury.

Serotonin is a monoamine neurotransmitter synthetized in various populations of brainstem neurons. In the spinal cord, descending serotonergic projections regulate postural muscle tone, locomotion and rhythm and coordination of movements via the Central Pattern Generator. Following a spinal cord injury, serotonergic projections to the lumbar spinal cord, where the Central Pattern Generators are located, are interrupted resulting in devastating locomotor impairments and changes in the expression and activation of serotonin and its spinal receptors. The molecular cues that control the precise patterning and targeting of serotonergic inputs onto Central Pattern Generator networks in healthy animals or after injury are still unknown. In our recent research work, we have been particularly interested in Semaphorin7A, which belongs to the Semaphorins family involved in guiding growing axons and controlling plasticity of synaptic connections. In this review, we discuss the role of Semaphorin7A signaling as an important molecular actor that instructs the patterning of serotonin inputs to spinal Central Pattern Generator networks. We show that Semaphorin7A controls the wiring of descending serotonin axons in the spinal cord. Our results reveal that mistargetting of serotonin fibers in the spinal cord is compensated in healthy uninjured Semaphorin7A deficient mice so that their gross locomotion proceeds accurately. We also demonstrate that when the system is challenged with a spinal lesion, the pattern of post-injury serotonin expression is significantly altered in Semaphorin7A deficient mice with specific ectopic targeting of serotonin fibers in the lumbar spinal cord. Compensatory mechanisms in place in uninjured Semaphorin7A deficient mice are lost and injured Semaphorin7A deficient mice exhibit a worsening of their post-injury locomotor abilities. Our findings identify Semaphorin7A as a critical determinant of serotonergic circuit formation in healthy or spinal cord injured mice.

Los potenciales evocados auditivos de estado estable a múltiples frecuencias y su valor en la evaluación objetiva de la audición

Uno de los principales objetivos en la realización de un examen electroaudiométrico es la obtención de una evaluación frecuencia específico de los umbrales de audibilidad. En las últimas décadas han sido propuestas múltiples técnicas basadas en el registro de los Potenciales Evocados Auditivos (PEA). Los mismos no son afectados por la sedación ni el sueño y pueden ser detectados a intensidades de estimulación muy cercanas al umbral de audibilidad. Las ventajas antes mencionadas lo han convertido en una útil herramienta para la evaluación objetiva de la audición. No obstante, esta técnica tiene ciertas limitaciones desde el punto de vista electroaudiométrico, debido principalmente a la falta de frecuencia especificada de dicha respuesta.
 Los Potenciales Evocados Auditivos de Estado Estable (PEAee) por estímulos tonales aislados y a frecuencias de estimulación entre 75 y 110 Hz han sido propuestos como una alternativa valida para la realización de una audiometría frecuencia especifica. Este tipo de respuesta representa la descarga sincrónica de las neuronas del tronco cerebral, las cuales siguen la frecuencia de modulación del estimulo que les da origen.
 Múltiples han sido los autores que en los últimos años y usando este tipo de respuesta han obtenido estimaciones confiables del umbral de audibilidad tonal, tanto en niños y adultos sanos como en sujetos hipoacúsicos. Esta técnica tiene múltiples ventajas sobre los PEA transitorios en la determinación de umbrales específicos en frecuencia: 1) Dado la periodicidad de la respuesta esta puede ser representada en el dominio de la frecuencia, minimizando por ende la complejidad de su medición, 2) El estimulo acústico empleado es mas especifico en frecuencia, 3) Debido a las propiedades de rectificación de la coclea la respuesta provocada por un tono modulado en amplitud es representada como un pico espectral a la frecuencia de modulación, 4) Estos picos espectrales pueden ser detectados usando diferentes estadígrafos en el dominio de la frecuencia.
 A pesar de las ventajas anteriormente descritas, la obtención de un audiograma completo mediante el uso de los PEAee provocados por estímulos tonales aislados puede requerir un gran consumo de tiempo. Más recientemente ha sido propuesta una variante optimizada de los PEAee entre 75 y 110 Hz con el empleo simultaneo de múltiples tonos modulados en amplitud. Dado el hecho que cada tono portador es modulado con una frecuencia diferente, múltiples tonos pueden ser entonces sumados, formando un tono complejo compuesto por múltiples tonos modulados en amplitud. Usando entonces como estimulo, una mezcla compuesta por tonos de 500, 1000, 2000 y 4000 Hz podemos activar y evaluar simultáneamente estas cuatro regiones de frecuencia de la cóclea. Por otra parte, estos estímulos complejos pueden ser presentados binauralmente, evaluando ambos oídos simultáneamente.
 Los PEAee provocados por múltiples tonos modulados en amplitud han sido empleados con alentadores resultados en la determinación objetiva de los umbrales de audibilidad en niños y adultos, pacientes hipoacúsicos y en la detección temprana de defectos auditivos. El presente artículo resume algunos conceptos básicos, así como sus aplicaciones clínicas.

Evaluation of G protein bias and β-arrestin 2 signaling in opioid-induced respiratory depression

Respiratory depression is a potentially fatal side effect of opioid analgesics and a major limitation to their use. G protein-biased opioid agonists have been proposed as "safer" analgesics with less respiratory depression. These agonists are biased to activate G proteins rather than β-arrestin signaling. Respiratory depression has been shown to correlate with both G protein bias and intrinsic efficacy, and recent work has refuted the role of β-arrestin signaling in opioid-induced respiratory depression. In addition, there is substantial evidence that G proteins do, in fact, mediate respiratory depression by actions in respiratory-controlling brainstem neurons. Based on these studies, we provide the perspective that protection from respiratory depression displayed by newly developed G protein-biased agonists is due to factors other than G protein versus arrestin bias.

MicroRNA expression in animal models of amyotrophic lateral sclerosis and potential therapeutic approaches.

A review of recent animal models of amyotrophic lateral sclerosis showed a large number of miRNAs had altered levels of expression in the brain and spinal cord, motor neurons of spinal cord and brainstem, and hypoglossal, facial, and red motor nuclei and were mostly upregulated. Among the miRNAs found to be upregulated in two of the studies were miR-21, miR-155, miR-125b, miR-146a, miR-124, miR-9, and miR-19b, while those downregulated in two of the studies included miR-146a, miR-29, miR-9, and miR-125b. A change of direction in miRNA expression occurred in some tissues when compared (e.g., miR-29b-3p in cerebellum and spinal cord of wobbler mice at 40 days), or at different disease stages (e.g., miR-200a in spinal cord of SOD1(G93A) mice at 95 days vs. 108 and 112 days). In the animal models, suppression of miR-129-5p resulted in increased lifespan, improved muscle strength, reduced neuromuscular junction degeneration, and tended to improve motor neuron survival in the SOD1(G93A) mouse model. Suppression of miR-155 was also associated with increased lifespan, while lowering of miR-29a tended to improve lifespan in males and increase muscle strength in SOD1(G93A) mice. Overexpression of members of miR-17~92 cluster improved motor neuron survival in SOD1(G93A) mice. Treatment with an artificial miRNA designed to target hSOD1 increased lifespan and improved muscle strength in SOD1(G93A) animals. Further studies with animal models of amyotrophic lateral sclerosis are warranted to validate these findings and identify specific miRNAs whose suppression or directed against hSOD1 results in increased lifespan, improved muscle strength, reduced neuromuscular junction degeneration, and improved motor neuron survival in SOD1(G93A) animals.

CRISPR/Cas9 mediated somatic gene therapy for insertional mutations: the vibrator mouse model.

Somatic gene therapy remains technically challenging, especially in the central nervous system (CNS). Efficiency of gene delivery, efficacy in recipient cells, and proportion of cells required for overall benefit are the key points needed to be considered in any therapeutic approach. Recent efforts have demonstrated the efficacy of RNA-guided nucleases such as CRISPR/Cas9 in correcting point mutations or removing dominant mutations. Here we used viral delivered Cas9 plasmid and two guide RNAs to remove a recessive insertional mutation, vibrator (vb), in the mouse brain. The vb mice expressed ∼20% of normal levels of phosphatidylinositol transfer protein, α (PITPα) RNA and protein due to an endogenous retrovirus inserted in intron 4, resulting in early-onset tremor, degeneration of brainstem and spinal cord neurons, and juvenile death. The in situ CRISPR/Cas9 viral treatment effectively delayed neurodegeneration, attenuated tremor, and bypassed juvenile death. Our studies demonstrate the potential of CRISPR/Cas9-mediated gene therapy for insertional mutations in the postnatal brain.

Dedicated C-fiber vagal sensory afferent pathways to the paraventricular nucleus of the hypothalamus

The nucleus of the solitary tract (NTS) receives viscerosensory information from the vagus nerve to regulate diverse homeostatic reflex functions. The NTS projects to a wide network of other brain regions, including the paraventricular nucleus of the hypothalamus (PVN). Here we examined the synaptic characteristics of primary afferent pathways to PVN-projecting NTS neurons in rat brainstem slices.Expression of the Transient Receptor Potential Vanilloid receptor (TRPV1+ ) distinguishes C-fiber afferents within the solitary tract (ST) from A-fibers (TRPV1-). We used resiniferatoxin (RTX), a TRPV1 agonist, to differentiate the two. The variability in the latency (jitter) of evoked excitatory postsynaptic currents (ST-EPSCs) distinguished monosynaptic from polysynaptic ST-EPSCs. Rhodamine injected into PVN was retrogradely transported to identify PVN-projecting NTS neurons within brainstem slices. Graded shocks to the ST elicited all-or-none EPSCs in rhodamine-positive NTS neurons with latencies that had either low jitter (<200 µs – monosynaptic), high jitter (>200 µs - polysynaptic inputs) or both. RTX blocked ST-evoked TRPV1 + EPSCs whether mono- or polysynaptic. Most PVN-projecting NTS neurons (17/21 neurons) had at least one input polysynaptically connected to the ST. Compared to unlabeled NTS neurons, PVN-projecting NTS neurons were more likely to receive indirect inputs and be higher order. Surprisingly, sEPSC rates for PVN-projecting neurons were double that of unlabeled NTS neurons. The ST synaptic responses for PVN-projecting NTS neurons were either all TRPV1+ or all TRPV1-, including neurons that received both direct and indirect inputs. Overall, PVN-projecting NTS neurons received direct and indirect vagal afferent information with strict segregation regarding TRPV1 expression.

Tonotopic distribution and inferior colliculus projection pattern of inhibitory and excitatory cell types in the lateral superior olive of Mongolian gerbils.

Sound localization critically relies on brainstem neurons that compare information from the two ears. The conventional role of the lateral superior olive (LSO) is extraction of intensity differences; however, it is increasingly clear that relative timing, especially of transients, is also an important function. Cellular diversity within the LSO that is not well understood may underlie its multiple roles. There are glycinergic inhibitory and glutamatergic excitatory principal neurons in the LSO, however, there is some disagreement regarding their relative distribution and projection pattern. Here we employ in situ hybridization to definitively identify transmitter types combined with retrograde labeling of projections to the inferior colliculus (IC) to address these questions. Excitatory LSO neurons were more numerous (76%) than inhibitory ones. A smaller proportion of inhibitory neurons were IC-projecting (45% vs. 64% for excitatory) suggesting that inhibitory LSO neurons may have more projections to other regions such the lateral lemniscus or more distributed IC projections. Inhibitory LSO neurons almost exclusively projected ipsilaterally making up a sizeable proportion (41%) of the transmitter type-labeled ipsilateral IC projection from LSO and exhibited a moderate low frequency bias (10% difference H-L). Two thirds of excitatory neurons projected contralaterally and had a slight high frequency bias (4%). One third of excitatory LSO neurons projected ipsilaterally to the IC and these cells were strongly biased toward the low frequency limb of the LSO (37%). This projection appears to be species specific in animals with good low frequency hearing suggesting that it may be a specialization for such ability.

Attenuation of Sensory Transmission Through the Rat Trigeminal Ganglion by GABA Receptor Activation

While the trigeminal ganglion is often considered a passive conduit of sensory transmission, neurons and satellite glial cells (SGCs) within it can release neurotransmitters and express neuroreceptors. Some trigeminal ganglion neurons contain the neurotransmitter γ-aminobutyric acid (GABA) and express GABA receptors. There is behavioral evidence that increased GABA levels in the trigeminal ganglion decreases nociception, while a loss of GABA receptors results in hyperalgesia, although the neural mechanisms for this remain to be investigated. In this study, the expression of GABA receptors by trigeminal ganglion neurons that innervate rat labial skin and masseter muscle was compared using immunohistochemistry. The effect of intraganglionic administration of GABA receptor agonists was investigated by single unit recording of trigeminal brainstem and ganglion neuron responses to stimulation of the labial skin and/or masseter muscle in anesthetized rats. The mean frequency of expression of GABAA and GABAB receptors by masseter and labial skin ganglion neurons was 62.5% and 92.7%, and 55.4% and 20.3%, respectively. The expression of both GABA receptors was significantly greater in skin ganglion neurons. Masticatory muscle evoked brainstem trigeminal neuron responses were significantly attenuated by intraganglionic injection of muscimol (GABAA) but not baclofen (GABAB). The mechanical sensitivity of slow and fast conducting masticatory muscle afferent fibers was decreased and increased, respectively, by intraganglionic injection of both muscimol and baclofen. Activation of GABAA receptors may exert a gating effect on sensory transmission through the trigeminal ganglion by decreasing putative nociceptive input and enhancing innocuous sensory input.

Chronic, Episodic Nicotine Alters Hypoglossal Motor Neuron Function at a Critical Developmental Time Point in Neonatal Rats.

Developmental nicotine exposure (DNE), alters brainstem neurons that control breathing, including hypoglossal motor neurons (XIIMNs), which innervate the tongue. Here, we tested the hypothesis that chronic, episodic DNE (eDNE), which mimics nicotine replacement therapies such as e-cigarettes or nicotine gum, alters the function of nicotinic acetylcholine receptors (nAChRs), XIIMN intrinsic properties, and tongue muscle function in vivo similar to what we have observed with a chronic, sustained exposure model. We delivered nicotine to pregnant Sprague Dawley rats through drinking water and studied pups of either sex in two age groups: postnatal day (P)1–P5 and P10–P12, which encompasses a critical period in brain development. At P1–P5, eDNE was associated with delayed recovery of nAChRs from desensitization; however, there were no changes in the magnitude of desensitization, XIIMN intrinsic properties, or tongue muscle function in vivo. By P10–P12, eDNE XIIMNs had lower peak firing frequencies in response to depolarizing current injection, larger delayed rectifier potassium currents, and continued to exhibit delayed nAChR recovery. Moreover, this age group exhibited a blunted and delayed tongue muscle response to nasal occlusion in vivo, indicating that changes to XIIMN intrinsic properties is an important mechanism behind this effect, as it is not produced by altered nAChR function alone. Together, these results show that eDNE alters XIIMNs and tongue muscle function during a critical period in brain development and that the specific effects of chronic nicotine exposure may be pattern dependent.

The diverse effects of brain glucagon‐like peptide 1 receptors on ingestive behaviour

Glucagon-like peptide 1 (GLP-1) is well known as a gut hormone and also acts as a neuropeptide, produced in a discrete population of caudal brainstem neurons that project widely throughout the brain. GLP-1 receptors are expressed in many brain areas of relevance to energy balance, and stimulation of these receptors at many of these sites potently suppresses food intake. This review surveys the current evidence for effects mediated by GLP-1 receptors on feeding behaviour at a wide array of brain sites and discusses behavioural and neurophysiological mechanisms for the effects identified thus far. Taken together, it is clear that GLP-1 receptor activity in the brain can influence feeding by diverse means, including mediation of gastrointestinal satiation and/or satiety signalling, suppression of motivation for food reward, induction of nausea and mediation of restraint stress-induced hypophagia, but many questions about the organization of this system remain.

Distribution of nitric oxide in the rock cavy (Kerodon rupestris) brain II: The brainstem

In a recent paper, we described the distribution of Nitric oxide (NO) in the diencephalon of the rock cavy (Kerodon rupestris). This present paper follows this work, showing the distribution of NO synthesizing neurons in the rock cavy's brainstem. For this, we used immunohistochemistry against the neuronal form of nitric oxide synthase (NOS) and NADPH diaphorase histochemistry. In contrast to the diencephalon in the rock cavy, where the NOS neurons were seen to be limited to some nuclei in the thalamus and hypothalamus, the distribution of NOS in the brainstem is widespread. Neurons immunoreactive to NOS (NOS-ir) were seen as rostral as the precommissural nuclei and as caudal as the caudal and gelatinous parts of the spinal trigeminal nucleus. Places such as the raphe nuclei, trigeminal complex, superior and inferior colliculus, oculomotor complex, periaqueductal grey matter, solitary tract nucleus, laterodorsal tegmental nucleus, pedunculopontine tegmental, and other nuclei of the reticular formation are among the locations with the most NOS-ir neurons. This distribution is similar, but with some differences, to those described for other rodents, indicating that NO also has an important role in rock cavy's physiology.

α-Synuclein Deposition in Sympathetic Nerve Fibers in Genetic Forms of Parkinson's Disease.

Cytoplasmic inclusions of α-synuclein (α-syn) in brainstem neurons are characteristic of idiopathic Parkinson's disease (PD). PD also entails α-syn buildup in sympathetic nerves. Among genetic forms of PD, the relative extents of sympathetic intraneuronal accumulation of α-syn have not been reported. This cross-sectional observational study compared magnitudes of intraneuronal deposition of α-syn in common and rare genetic forms of PD. α-Syn deposition was quantified by the α-syn-tyrosine hydroxylase colocalization index in C2 cervical skin biopsies from 65 subjects. These included 30 subjects with pathogenic mutations in SNCA (n=3), PRKN [biallelic (n=7) and monoallelic (n=3)], LRRK2 (n=7), GBA (n=7), or PARK7/DJ1 [biallelic (n=1) and monoallelic (n=2)]. Twenty-five of the mutation carriers had PD and five did not. Data were also analyzed from 19 patients with idiopathic PD and 16 control participants. α-Syn deposition varied as a function of genotype (F=16.7, P< 0.0001). It was above the control range in 100% of subjects with SNCA mutations, 100% with LRRK2 mutations, 95% with idiopathic PD, 83% with GBA mutations, and 0% with biallelic PRKN mutations. α-Syn deposition in the biallelic PRKN group was significantly higher than in the control group. In addition, patients with biallelic PRKN mutations had higher α-syn deposition than their unaffected siblings. Individuals with SNCA, DJ-1, LRRK2, or GBA mutations have substantial intraneuronal α-syn deposition in sympathetic noradrenergic nerves in skin biopsies, whereas those with biallelic PRKN mutations do not. Biallelic PRKN patients may have mildly increased α-syn deposition compared with control subjects. © 2021 International Parkinson and Movement Disorder Society.

Parafacial neurons in the human brainstem express specific markers for neurons of the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Neuronal Loss in the Nucleus Tractus Solitarius and Associated Disturbances of the Hypoxic Ventilatory Response in Rat Offspring Following in‐utero Morphine Exposure

The incidence of opioid use during pregnancy, including prescriptions, has become a growing public health concern and has been linked to neonatal abstinence syndrome (NAS), poor neurodevelopmental outcome and increased risk of sudden infant death syndrome (SIDS). Abnormal cardiorespiratory regulation in infants born following in-utero opioid exposure includes reduced vagal tone, delayed arousal, and altered ventilatory responses to hypoxia and hypercapnia. Impairments in ventilatory defense responses and abnormal development of brainstem cardiorespiratory control regions may contribute to infant vulnerability and dampen responses to exogenous stressors leading to SIDS. Although infants with NAS may be treated with morphine, not all infants require pharmacologic treatment. To mimic the latter group of infants, we used a rat model of in utero morphine exposure to investigate the long-term effects it has on respiratory control and brainstem neuronal development of the offspring. Using a rat model of in utero morphine exposure to characterize long-term (10 days) effects on the offspring including: 1) the acute hypoxic (HVR) and hypercapnic ventilatory response (HCVR) as indices of respiratory control (dys)function; and 2) brainstem neuron counts as an indicator of neural development. Pregnant dams received twice daily (morning and afternoon) subcutaneous injections of various doses of morphine (0.5, 1, and 5mg/kg) or saline (control group) during the final week of pregnancy to mimic 3rd trimester maternal opioid use. Following birth, nursing dams and rat pups were left untreated. On postnatal day 10, whole body plethysmography was used to assess the HVR and HCVR in the pups. Brains were removed and prepared for immunohistochemical assessment of neuron cell counts in key brainstem cardiorespiratory control regions. In-utero morphine exposure at any dose (0.5, 1, 5mg/kg) caused a long-term attenuation of the HVR (Fig. 1A), but not the HCVR when compared to pups from in-utero saline treated rats. At the highest dose (5mg/kg), neuron cell numbers were reduced by approximately 50% compared to saline rats (Fig. 1B-C). These data demonstrate a long-term effect of in-utero morphine exposure on respiratory control, which may in part be explained by disrupted brainstem neural development. These cardiorespiratory anomalies could offer insight into the pathophysiology of the increased risk of SIDS seen in infants born from mothers with opioid use.

Neostigmine Microinjected into Prefrontal Cortex of C57BL/6J (B6) Mice Stimulates Breathing

The prefrontal cortex modulates breathing (Neuroscience 123: 17, 2004; J Physiol 591: 6069, 2013) via neurochemical mechanisms that remain to be fully characterized. This study is testing the hypothesis that microinjection of neostigmine bromide, an acetylcholinesterase inhibitor, into the prefrontal cortex of freely behaving mice alters breathing. All procedures involving mice were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Adult male mice (n = 8) were anesthetized and implanted with a 26-gauge guide cannula aimed at the medial prefrontal cortex. One week after recovering from surgery, mice were microinjected (50 nL) with saline (vehicle control) and two concentrations of neostigmine (0.005 and 0.016 nmol/50 nL; 1.5 and 4.8 ng/50 nL). Each mouse received saline and neostigmine in a repeated measures within-subjects design. Respiratory rate (RR) expressed as breaths/min was quantified using whole-body plethysmography. Repeated measures one-way ANOVA showed neostigmine significantly (F(1.9,13.9) = 6.3, p = 0.0116) altered RR. Relative to saline, RR was increased 11.9% by 0.005 nmol and 18.2% by 0.016 nmol neostigmine. Dunnett's post hoc comparisons test revealed that 0.016 nmol significantly (p < 0.05) increased RRwhen compared to saline. Cohen's d = 1.34 for saline vs. 0.016 nmol neostigmine. Histological analyses of microinjection sites confirmed drug delivery into the prelimbic region of the prefrontal cortex. Mean ± S.D. coordinates of the injection sites were 1.8 ± 0.4 mm anterior, 0.3 ± 0.1 mm lateral, and 2.6 ± 0.6 mm ventral relative to bregma (Paxinos and Franklin, 2008). The mechanism by which cholinergic transmission in the prefrontal cortex stimulates breathing remains to be identified. We speculate that the neostigmine-induced increase in RR evoked from the prefrontal cortex may be modulated by projections from the prefrontal cortex to arousal promoting regions of the nucleus pontis oralis (PnO) (J. Neurosci. 29: 871, 2009). Neuroanatomical studies show that the PnO shares reciprocal connections with respiratory neurons in the ponto-medullary brainstem (Lung Cell. Mol. Physiol. 2: L251, 1995). These anatomical data are consistent with functional studies showing that respiratory rate in B6 mice significantly increased in tandem with increases in prefrontal cortex ACh release (Neuroscience 123: 17, 2004). Considered together, the present results support the interpretation that cholinergic neurotransmission in the prefrontal cortex stimulates the drive to breathe. Ongoing studies are further characterizing the site-dependent and concentration-dependent stimulation of breathing by cholinergic transmission in the prefrontal cortex.

Vestibular Brainstem Dysmorphology in an Animal Model of Autism Spectrum Disorder

Introduction - Prenatal exposure to the antiepileptic drug valproic acid (VPA) is associated with an elevated risk of a diagnosis of autism spectrum disorder (ASD) in humans. Accordingly, in utero exposure to VPA is a clinically relevant and validated animal model of ASD. Previous work from our lab on VPA-exposed animals revealed significant changes in the auditory brainstem and cerebellum. Specifically, we have shown fewer neurons in the auditory brainstem, reduced expression of calcium binding proteins, reduced connectivity and hyperactivation in response to pure tone stimuli. In the cerebellum, we have found smaller Purkinje cells, reduced expression of calbindin (CB) and significant gait ataxia. Based on these findings, we rationalized that brainstem vestibular pathways might similarly be affected in VPA-exposed animals. We therefore hypothesized that in utero VPA exposure will result in fewer neurons in the vestibular nuclei, reduced CB+ immunolabeling, postural instability and poor gait performance after vestibular challenge. Methods - After timed mating, pregnant rats were fed peanut butter meals from embryonic days 7 through 12. Dams in the VPA groups were fed peanut butter with 800mg/kg VPA on E10 and 12. Pups were delivered without interference and were handled by experimenters from postnatal day (P) 15-20. Starting on P21, control and VPA-exposed animals underwent a battery of motor tests and vestibular challenges from P21-28. All morphological studies were done using brain tissue from P28 animals. Results - Compared to control animals, we found significantly fewer neurons in the spinal, lateral and superior vestibular nuclei and larger neuronal cell bodies in the spinal and lateral vestibular nuclei in VPA-exposed animals. In the lateral nucleus we also found significantly more stellate and fusiform neurons but fewer round/oval neurons. Preliminary examination of the number of CB+ terminals in the vestibular nuclei suggests that afferent inputs to these neurons may have reduced CB expression. Behavioral tests suggest that VPA-exposed animals are more severely impacted by vestibular challenge than control animals. Conclusion and Significance - Our results are consistent with both structural and functional changes in animals exposed to VPA in utero. Together, our research provides evidence for significant brainstem dysfunction and we propose these alterations extend to human cases of ASD. This work will clarify the brainstem centers impacted in ASD and may lead to the development of simple, non-invasive tests of vestibular function to screen for ASD.

Autopsy indicates brainstem and spinal neuropathology in late‐onset Pompe Disease

Pompe disease (PD) is a neuromuscular disorder caused by a mutation in the gene encoding acid alpha-glucosidase (GAA), the enzyme responsible for lysosomal breakdown of glycogen. The rate of PD progression corresponds to the amount of residual GAA enzyme activity. Infantile-onset Pompe disease is caused a by a complete lack of GAA activity, with patients typically progressing to cardiorespiratory failure within the first year of life. Late-onset PD patients retain some GAA activity and present symptoms later in life, with fatality mainly associated with respiratory failure. This case study presents a histological analysis of the brainstem and spinal cord from a male PD patient who was diagnosed with late-onset PD at age 35. The patient was wheelchair dependent by age 38, displayed progressive respiratory insufficiency with requirement for nocturnal ventilation at age 40, required 24-hour non-invasive ventilation by age 43, and passed away from respiratory failure at age 54. Autopsy was performed approximately 8 hours after death with informed consent. The brain and spinal cord were preserved in a 20% formalin solution and embedded in paraffin for subsequent histological assessment. Tissue sections were cut at 4 µm and stained with periodic acid-Schiff (PAS) to assess glycogen content, cresyl violet to assess neuronal morphology, and Iba-1 to characterize microglia as a marker of neuroinflammation. Positive PAS confirmed glycogen accumulation in neurons throughout the medulla and spinal cord. However, striking differences in neuronal morphology were present across the neuraxis. Hypoglossal motoneurons in the medulla (n=46, soma size: 421 ± 159 µm2) and putative motoneurons in the ventral cervical horn (n=50, soma size: 487 ± 189 µm2) had an atrophied, elongated appearance. In contrast, lumbar (n=63, soma size: 1363 ± 677 µm2) and sacral motoneurons (n=31, soma size: 1411 ± 633 µm2) had the classic ballooned morphology seen in early-onset PD. The Iba-1 staining showed an accumulation of microglia in the white matter of the cervical cord. In contrast, Iba-1 staining was localized to the gray matter in the lumbo-sacral spinal cord. These histological data confirm glycogen accumulation and pathology in brainstem and spinal neurons in late-onset PD. Further, the markedly atrophied appearance of hypoglossal and mid-cervical motoneurons is consistent with neurodegeneration and the concept that effective PD therapy will need to target the central nervous system, in addition to skeletal and cardiac muscle.