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Hypoxia alters cellular metabolism and although the effects of sustained hypoxia (SH) have been extensively studied, less is known about chronic intermittent hypoxia (IH), commonly associated with cardiovascular morbidity and stroke. We hypothesize that impaired glutamate homeostasis after chronic IH may underlie vulnerability to stroke-induced excitotoxicity. P16 organotypic hippocampal slices, cultured for 7 days were exposed for 7 days to IH (alternating 2 min 5% O2-15 min 21% O2), SH (5% O2) or RA (21% O2), then 3 glutamate challenges. The first and last exposures were intended as a metabolic stimulus (200 µM glutamate, 15 min); the second emulated excitotoxicity (10 mM glutamate, 10 min). GFAP, MAP2, and EAAT1, EAAT2 glutamate transporters expression were assessed after exposure to each hypoxic protocol. Additionally, cell viability was determined at baseline and after each glutamate challenge, in presence or absence of ceftriaxone that increases glutamate transporter expression. GFAP and MAP2 decreased after 7 days IH and SH. Long-term IH but not SH decreased EAAT1 and EAAT2. Excitotoxic glutamate challenge decreased cell viability and the following 200 µM exposure further increased cell death, particularly in IH-exposed slices. Ceftriaxone prevented glutamate transporter decrease and improved cell viability after IH and excitotoxicity. We conclude that IH is more detrimental to cell survival and glutamate homeostasis than SH. These findings suggest that impaired regulation of extracellular glutamate levels is implicated in the increased brain susceptibility to excitotoxic insult after long-term IH.

The prevalence of Autism Spectrum Disorder (ASD) has drastically risen over the last two decades and is currently estimated to affect 1 in 36 children in the U.S., according to the center for disease control (CDC). This heterogenous neurodevelopmental disorder is characterized by impaired social interactions, communication deficits, and repetitive behaviors plus restricted interest. Autistic individuals also commonly present with a myriad of comorbidities, such as attention deficit hyperactivity disorder, anxiety, and seizures. To date, a pharmacological intervention for the treatment of core autistic symptoms has not been identified. Cannabidiol (CBD), the major nonpsychoactive constituent of , is suggested to have multiple therapeutic applications, but its effect(s) on idiopathic autism is unknown. We hypothesized that CBD will effectively attenuate the autism-like behaviors and autism-associated comorbid behaviors in BTBR T Itpr3 /J (BTBR) mice, an established mouse model of idiopathic ASD. Male BTBR mice were injected intraperitoneally with either vehicle, 20 mg/kg CBD or 50 mg/kg CBD daily for two weeks beginning at postnatal day 21 ± 3. On the final treatment day, a battery of behavioral assays were used to evaluate the effects of CBD on the BTBR mice, as compared to age-matched, vehicle-treated C57BL/6 J mice. High dose (50 mg/kg) CBD treatment attenuated the elevated repetitive self-grooming behavior and hyperlocomotion in BTBR mice. The social deficits exhibited by the control BTBR mice were rescued by the 20 mg/kg CBD treatment. Our data indicate that different doses for CBD are needed for treating specific ASD-like behaviors. Together, our results suggest that CBD may be an effective drug to ameliorate repetitive/restricted behaviors, social deficits, and autism-associated hyperactivity.

Breathing is a singularly robust behavior, yet this motor pattern is continuously modulated at slow and fast timescales to maintain blood-gas homeostasis, while intercalating orofacial behaviors. This functional multiplexing goes beyond the rhythmogenic function that is typically ascribed to medullary respiration-modulated networks and may explain lack of progress in identifying the mechanism and constituents of the respiratory rhythm generator. By recording optically along the ventral respiratory column in medulla, we found convergent evidence that rhythmogenic function is distributed over a dispersed and heterogeneous network that is synchronized by electrotonic coupling across a neuronal syncytium. First, high-speed recordings revealed that inspiratory onset occurred synchronously along the column and did not emanate from a rhythmogenic core. Second, following synaptic isolation, synchronized stationary rhythmic activity was detected along the column. This activity was attenuated following gap junction blockade and was silenced by tetrodotoxin. The layering of syncytial and synaptic coupling complicates identification of rhythmogenic mechanism, while enabling functional multiplexing.

The normal breathing rhythm in mammals is hypothesized to be generated by neurokinin-1 receptor (NK1R)-expressing neurons in the preBötzinger complex (preBötC), a medullary region proposed to contain the kernel of the circuits generating respiration. If this hypothesis is correct, then complete destruction of preBötC NK1R neurons should severely perturb and perhaps even fatally arrest breathing. Here we show that specific and near complete bilateral (but not unilateral) destruction of preBötC NK1R neurons results in both an ataxic breathing pattern with markedly altered blood gases and pH, and pathological responses to challenges such as hyperoxia, hypoxia and anesthesia. Thus, these ∼600 neurons seem necessary for the generation of normal breathing in rats.

Bath-applied membrane-permeant Ca2+ indicators offer access to network function with single-cell resolution. A barrier to wider and more efficient use of this technique is the difficulty of extracting fluorescence signals from the active constituents of the network under study. Here we present a method for semi-automatic region of interest (ROI) detection that exploits the spatially compact, slowly time-varying character of the somatic signals that these indicators typically produce. First, the image series is differenced to eliminate static and very slowly varying fluorescence values, and then the differenced image series undergoes low-pass filtering in the spatial domain, to eliminate temporally isolated fluctuations in brightness. This processed image series is then thresholded so that pixel regions of fluctuating brightness are set to white, while all other regions are set to black. Binary images are averaged, and then subjected to iterative thresholding to extract ROIs associated with both dim and bright cells. The original image series is then analyzed using the generated ROIs, after which the end-user rejects spurious signals. These methods are applied to respiratory networks in the neonate rat tilted sagittal slab preparation, and to simulations with signal-to-noise ratios ranging between 1.0–0.2. Simulations established that algorithm performance degraded gracefully with increasing noise. Because signal extraction is the necessary first step in the analysis of time-varying Ca2+ signals, semi-automated ROI detection frees the researcher to focus on the next step: selecting traces of interest from the relatively complete set generated using these methods.

Although the mammalian locomotor CPG has been localized to the lumbar spinal cord, the functional-anatomical organization of flexor and extensor interneurons has not been characterized. Here, we tested the hypothesis that flexor and extensor interneuronal networks for walking are physically segregated in the lumbar spinal cord. For this purpose, we performed optical recordings and lesion experiments from a horizontally sectioned lumbar spinal cord isolated from neonate rats. This ventral hemi spinal cord preparation produces well-organized fictive locomotion when superfused with 5-HT/NMDA. The dorsal surface of the preparation was visualized using the Ca(2+) indicator fluo-4 AM, while simultaneously monitoring motor output at ventral roots L2 and L5. Using calcium imaging, we provided a general mapping view of the interneurons that maintained a stable phase relationship with motor output. We showed that the dorsal surface of L1 segment contains a higher density of locomotor rhythmic cells than the other segments. Moreover, L1 segment lesioning induced the most important changes in the locomotor activity in comparison with lesions at the T13 or L2 segments. However, no lesions led to selective disruption of either flexor or extensor output. In addition, this study found no evidence of functional parcellation of locomotor interneurons into flexor and extensor pools at the dorsal-ventral midline of the lumbar spinal cord of the rat.

In vertebrates, respiratory control is ascribed to heterogeneous respiration-modulated neurons along the Ventral Respiratory Column (VRC) in medulla, which includes the preBötzinger Complex (preBötC), the putative respiratory rhythm generator. Here, the functional anatomy of the VRC was characterized via optical recordings in the sagittaly sectioned neonate rat hindbrain, at sampling rates permitting coupling estimation between neuron pairs, so that each neuron was described using unitary, neuron-system, and coupling attributes. Structured coupling relations in local networks, significantly oriented coupling in the peri-inspiratory interval detected in pooled data, and significant correlations between firing rate and expiratory duration in subsets of neurons revealed network regulation at multiple timescales. Spatially averaged neuronal attributes, including coupling vectors, revealed a sharp boundary at the rostral margin of the preBötC, as well as other functional anatomical features congruent with identified structures, including the parafacial respiratory group and the nucleus ambiguus. Cluster analysis of attributes identified two spatially compact, homogenous groups: the first overlapped with the preBötC, and was characterized by strong respiratory modulation and dense bidirectional coupling with itself and other groups, consistent with a central role for the preBötC in respiratory control; the second lay between preBötC and the facial nucleus, and was characterized by weak respiratory modulation and weak coupling with other respiratory neurons, which is congruent with cardiovascular regulatory networks that are found in this region. Other groups identified using cluster analysis suggested that networks along VRC regulated expiratory duration, and the transition to and from inspiration, but these groups were heterogeneous and anatomically dispersed. Thus, by recording local networks in parallel, this study found evidence for respiratory regulation at multiple timescales along the VRC, as well as a role for the preBötC in the integration of functionally disparate respiratory neurons. •High-speed optical recordings of respiratory networks were made in rat hindbrain.•Coupling relations were estimated using the normalized transfer entropy metric.•Deterministic neuronal interactions at multiple time scales were detected.•A functional hub with dense recurrent coupling to the larger network was found.•Structured coupling in networks and macroscopic directed flow were detected.

Opioids are well-known to cause respiratory depression, but despite clinical evidence of dysphagia, the effects of opioids on swallow excitability and motor pattern are unknown. We sought to test the effects of the clinically-relevant opioid buprenorphine on pharyngeal swallow and respiratory drive in male and female rats. We also evaluated utility of serotonin 5-HT1A agonists (8-OH-DPAT and buspirone) to improve swallowing and breathing outcomes following buprenorphine administration. Experiments were performed on 44 freely breathing Sprague Dawley rats anesthetized with sodium pentobarbital. Bipolar fine wire electrodes were inserted into the mylohyoid, thyroarytenoid, posterior cricoarytenoid, thyropharyngeus and diaphragm muscles to measure electromyographic (EMG) activity of swallowing and breathing behaviors. We evaluated the hypotheses that swallow varies by stimulus, opioids depress swallow and breathing, and that 5-HT1A agonists improve these depressions. Our results largely confirmed the hypotheses: 1) Swallow-related muscle activity was larger during swallows elicited by oral water infusion plus esophageal distension than by either stimulus alone. 2) Buprenorphine depressed swallow in both sexes, but most significantly in females. 3) Female animals were more susceptible to buprenorphine-induced respiratory arrest. 4) 8-OH-DPAT rescued breathing following buprenorphine-induced respiratory arrest, and pre-treatment with the partial 5-HT1A agonist buspirone prevented buprenorphine-induced respiratory arrest in female animals. 5) 8-OH-DPAT enhanced swallow-related mylohyoid drive, but did not restore excitability of the swallow pattern generator following total suppression by buprenorphine. Our results highlight sex-specific and behavior-specific effects of buprenorphine and provide pre-clinical evidence of a 5HT1A agonist for the treatment of respiratory depression and dysphagia.Opioids are well-known to cause respiratory depression, but despite clinical evidence of dysphagia, the effects of opioids on swallow excitability and motor pattern are unknown. We sought to test the effects of the clinically-relevant opioid buprenorphine on pharyngeal swallow and respiratory drive in male and female rats. We also evaluated utility of serotonin 5-HT1A agonists (8-OH-DPAT and buspirone) to improve swallowing and breathing outcomes following buprenorphine administration. Experiments were performed on 44 freely breathing Sprague Dawley rats anesthetized with sodium pentobarbital. Bipolar fine wire electrodes were inserted into the mylohyoid, thyroarytenoid, posterior cricoarytenoid, thyropharyngeus and diaphragm muscles to measure electromyographic (EMG) activity of swallowing and breathing behaviors. We evaluated the hypotheses that swallow varies by stimulus, opioids depress swallow and breathing, and that 5-HT1A agonists improve these depressions. Our results largely confirmed the hypotheses: 1) Swallow-related muscle activity was larger during swallows elicited by oral water infusion plus esophageal distension than by either stimulus alone. 2) Buprenorphine depressed swallow in both sexes, but most significantly in females. 3) Female animals were more susceptible to buprenorphine-induced respiratory arrest. 4) 8-OH-DPAT rescued breathing following buprenorphine-induced respiratory arrest, and pre-treatment with the partial 5-HT1A agonist buspirone prevented buprenorphine-induced respiratory arrest in female animals. 5) 8-OH-DPAT enhanced swallow-related mylohyoid drive, but did not restore excitability of the swallow pattern generator following total suppression by buprenorphine. Our results highlight sex-specific and behavior-specific effects of buprenorphine and provide pre-clinical evidence of a 5HT1A agonist for the treatment of respiratory depression and dysphagia.