Synchronization of inspiratory burst onset along the ventral respiratory column in the neonate mouse is mediated by electrotonic coupling.

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.

Evidence of intermediate reticular formation involvement in swallow pattern generation, recorded optically in the neonate rat sagittally sectioned hindbrain.

Swallow is a primitive behavior regulated by medullary networks, responsible for movement of food/liquid from the oral cavity to the esophagus. To investigate how functionally heterogeneous networks along the medullary intermediate reticular formation (IRt) and ventral respiratory column (VRC) control swallow, we electrically stimulated the nucleus tractus solitarius to induce fictive swallow between inspiratory bursts, with concurrent optical recordings using a synthetic Ca2+ indicator in the neonatal sagittally sectioned rat hindbrain (SSRH) preparation. Simultaneous recordings from hypoglossal nerve rootlet (XIIn) and ventral cervical spinal root C1-C2 enabled identification of the system-level correlates of 1) swallow (identified as activation of the XIIn but not the cervical root) and 2) Breuer-Hering expiratory reflex (BHE; lengthened expiration in response to stimuli during expiration). Optical recording revealed reconfiguration of respiration-modulated networks in the ventrolateral medulla during swallow and the BHE reflex. Recordings identified novel spatially compact networks in the IRt near the facial nucleus (VIIn) that were active during fictive swallow, suggesting that the swallow network is not restricted to the caudal medulla. These findings also establish the utility of using this in vitro preparation to investigate how functionally heterogeneous medullary networks interact and reconfigure to enable a repertoire of orofacial behaviors.NEW & NOTEWORTHY For the first time, medullary networks that control breathing and swallow are recorded optically. Episodic swallows are induced via electrical stimulation along the dorsal medulla, in and near the NTS, during spontaneously occurring fictive respiration. These findings establish that networks regulating both orofacial behaviors and breathing are accessible for optical recording at the surface of the sagittally sectioned rodent hindbrain preparation.

Diabetic cardiomyopathy: role of the E3 ubiquitin ligase.

Diabetic cardiomyopathy (DCM) is the leading cause of mortality in diabetes. As the number of cases of diabetes continues to rise, it is urgent to develop new strategies to protect against DCM, which is characterized by cardiac hypertrophy, increased apoptosis, fibrosis, and altered insulin metabolism. The E3 ubiquitin ligases (E3s), one component of the ubiquitin-proteasome system, play vital roles in all of the features of DCM listed above. They also modulate the activity of several transcription factors involved in the pathogenesis of DCM. In addition, the E3s degrade both insulin receptor and insulin receptor substrates and also regulate insulin gene transcription, leading to insulin resistance and insulin deficiency. Therefore, the E3s may be a driving force for DCM. This review summarizes currently available studies to analyze the roles of the E3s in DCM, enriches our knowledge of how DCM develops, and provides a novel strategy to protect heart from diabetes.

Fenofibrate increases cardiac autophagy via FGF21/SIRT1 and prevents fibrosis and inflammation in the hearts of Type 1 diabetic mice.

Fenofibrate (FF), as a peroxisome-proliferator-activated receptor α (PPARα) agonist, has been used clinically for decades to lower lipid levels. In the present study, we examined whether FF can be repurposed to prevent the pathogenesi of the heart in Type 1 diabetes and to describe the underlying mechanism of its action. Streptozotocin (STZ)-induced diabetic mice and their age-matched control mice were treated with vehicle or FF by gavage every other day for 3 or 6 months. FF prevented diabetes-induced cardiac dysfunction (e.g. decreased ejection fraction and hypertrophy), inflammation and remodelling. FF also increased cardiac expression of fibroblast growth factor 21 (FGF21) and sirtuin 1 (Sirt1) in non-diabetic and diabetic conditions. Deletion of FGF21 gene (FGF21-KO) worsened diabetes-induced pathogenic effects in the heart. FF treatment prevented heart deterioration in the wild-type diabetic mice, but could not do so in the FGF21-KO diabetic mice although the systemic lipid profile was lowered in both wild-type and FGF21-KO diabetic mice. Mechanistically, FF treatment prevented diabetes-impaired autophagy, reflected by increased microtubule-associated protein 1A/1B-light chain 3, in the wild-type diabetic mice but not in the FGF21-KO diabetic mice. Studies with H9C2 cells in vitro demonstrated that exposure to high glucose (HG) significantly increased inflammatory response, oxidative stress and pro-fibrotic response and also significantly inhibited autophagy. These effects of HG were prevented by FF treatment. Inhibition of either autophagy by 3-methyladenine (3MA) or Sirt1 by sirtinol (SI) abolished FF's prevention of HG-induced effects. These results suggested that FF could prevent Type 1 diabetes-induced pathological and functional abnormalities of the heart by increasing FGF21 that may up-regulate Sirt1-mediated autophagy.

Sulforaphane prevents the development of cardiomyopathy in type 2 diabetic mice probably by reversing oxidative stress-induced inhibition of LKB1/AMPK pathway.

Type 2 diabetes mellitus (T2DM)-induced cardiomyopathy is associated with cardiac oxidative stress, inflammation, and remodeling. Sulforaphane (SFN), an isothiocyanate naturally presenting in widely consumed vegetables, particularly broccoli, plays an important role in cardiac protection from diabetes. We investigated the effect of SFN on T2DM-induced cardiac lipid accumulation and subsequent cardiomyopathy. Male C57BL/6J mice were fed a high-fat diet for 3months to induce insulin resistance, followed by a treatment with 100mg/kg body-weight streptozotocin to induce hyperglycemia; we referred to it as the T2DM mouse model. Other age-matched mice were fed a normal diet as control. T2DM and control mice were treated with or without 4-month SFN at 0.5mg/kg daily five days a week. At the study's end, cardiac function was assessed. SFN treatment significantly attenuated cardiac remodeling and dysfunction induced by T2DM. SFN treatment also significantly inhibited cardiac lipid accumulation, measured by Oil Red O staining, and improved cardiac inflammation oxidative stress and fibrosis, shown by down-regulating diabetes-induced PAI-1, TNF-α, CTGF, TGF-ß, 3-NT, and 4-HNE expression. Elevated 4-HNE resulted in the increase of 4-HNE-LKB1 adducts that should inhibit LKB1 and subsequent AMPK activity. SFN upregulated the expression of Nrf2 and its downstream genes, NQO1 and HO-1, decreased 4-HNE-LKB1 adducts and then reversed diabetes-induced inhibition of LKB1/AMPK and its downstream targets, including sirtuin 1, PGC-1α, phosphorylated acetyl-CoA carboxylase, carnitine palmitoyl transferase-1, ULK1, and light chain-3 II. These results suggest that SFN treatment to T2DM mice may attenuate the cardiac oxidative stress-induced inhibition of LKB1/AMPK signaling pathway, thereby preventing T2DM-induced lipotoxicity and cardiomyopathy.

Zinc rescue of Akt2 gene deletion-linked murine cardiac dysfunction and pathological changes is metallothionein-dependent.

We have demonstrated that zinc supplementation provides cardiac protection from diabetes in mice, but its underlying mechanism remains unclear. Since zinc mimics the function of insulin, it may provide benefit to the heart via stimulating Akt-mediated glucose metabolism. Akt2 plays an important role in cardiac glucose metabolism and mice with Akt2 gene deletion (Akt2-KO) exhibit a type 2 diabetes phenotype; therefore, we assumed that no cardiac protection by zinc supplementation from diabetes would be observed in Akt2-KO mice. Surprisingly, despite Akt2 gene deletion, zinc supplementation provided protection against cardiac dysfunction and other pathological changes in Akt2-KO mice, which were accompanied by significant decreases in Akt and GSK-3ß phosphorylation. Correspondingly, glycogen synthase phosphorylation and hexokinase II and PGC-1α expression, all involved in the regulation of glucose metabolism, were significantly altered in diabetic hearts, along with a significantly increased expression of Akt negative regulators: PTEN, PTP1B, and TRB3. All these molecular, pathological, and functional changes were significantly prevented by 3-month zinc supplementation. Furthermore, the stimulation of Akt-mediated glucose metabolic kinases or enzymes by zinc treatment was metallothionein-dependent since it could not be observed in metallothionein-knockout mice. These results suggest that zinc preserves cardiac function and structure in Akt2-KO mice presumably due to its insulin mimetic effect on cardiac glucose-metabolism. The cardioprotective effects of zinc are metallothionein-dependent. This is very important since zinc supplementation may be required for patients with Akt2 gene deficiency or insulin resistance.

The preBötzinger complex as a hub for network activity along the ventral respiratory column in the neonate 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.

Serotonin therapies for opioid-induced disordered swallow and respiratory depression.

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 tested the effects of the clinically relevant opioid buprenorphine on pharyngeal swallow and respiratory drive in male and female rats. We also evaluated the utility of 5-HT1A agonists (8-OH-DPAT and buspirone) to improve swallowing and breathing 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. We evaluated the hypotheses that swallowing varies by stimulus, opioids depress swallowing and breathing, and that 5-HT1A agonists improve these depressions. Our results largely confirmed the following hypotheses: 1) swallow-related EMG activity was larger during swallows elicited by esophageal distension plus oral water infusion than by either stimulus alone. 2) Buprenorphine depressed swallow in both sexes, but females were more susceptible to total swallow suppression. 3) Female animals were also more vulnerable to opioid-induced respiratory depression. 4) 8-OH-DPAT rescued breathing following buprenorphine-induced respiratory arrest, and pretreatment with the partial 5-HT1A agonist buspirone prevented buprenorphine-induced respiratory arrest in female animals. 5) 8-OH-DPAT enhanced mylohyoid and thyropharyngeus EMG amplitude during swallow 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 preclinical evidence of a 5HT1A agonist for the treatment of respiratory depression and dysphagia.NEW & NOTEWORTHY This is the first study, to our knowledge, to evaluate sex-specific effects of opioid administration on pharyngeal swallow. We expand on a small but growing number of studies that report a lower threshold for opioid-induced respiratory depression in females compared with males, and we are the first to produce this effect with the partial µ-opioid-receptor agonist buprenorphine. This is the first demonstration, to our knowledge, that activation of 5-HT1A receptors can improve swallow and breathing outcomes following systemic buprenorphine administration.

Cannabidiol is a behavioral modulator in BTBR mouse model of idiopathic autism.

Introduction: 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 Cannabis sativa, 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+Itpr3tf/J (BTBR) mice, an established mouse model of idiopathic ASD. Methods: 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. Results: 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. Discussion: 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.

Serotonin therapies for opioid-induced dysphagia and respiratory depression: sex differences in a rat electromyography model.

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.

Perinatal methadone exposure attenuates myelination and induces oligodendrocyte apoptosis in neonatal rat brain.

Methadone (MTD) is a commonly prescribed treatment for opioid use disorder in pregnancy, despite limited information on the effects of passive exposure on fetal brain development. Animal studies suggest a link between perinatal MTD exposure and impaired white matter development. In this study, we characterized the effect of perinatal MTD exposure through the evaluation of oligodendrocyte development and glial cell activation in the neonatal rat brain. Six pregnant Sprague Dawley rat dams were randomized to MTD (0.2 mL/L) or untreated drinking water from embryonic day 7. Pups were terminated at postnatal day 7 and tissue sections were harvested from six randomly selected pups (one male and one female per litter) of each experimental group for immunohistochemistry in areas of corpus callosum (CC), lateral CC, external capsule (EC), and cerebellar white matter. In the MTD-exposed rat pups, myelination was significantly decreased in the CC, lateral CC, EC, and arbor vitae compared with the controls. The increased density and percentage of oligodendrocyte precursor cells (OPCs) were observed in the CC and cerebellar white matter. The highly active proliferation of OPCs as well as decreased density and percentage of differentiated oligodendrocytes were found in the cerebellum but no differences in the cerebrum. Apoptotic activities of both differentiated oligodendrocytes and myelinating oligodendrocytes were significantly increased in all regions of the cerebrum and cerebellum after MTD exposure. There was no quantitative difference in astrocyte, however, cell density and/or morphologic difference consistent with activation were observed in microglia throughout MTD-exposed CC and cerebellum. Taken together, perinatal MTD exposure reveals global attenuation of myelination, accelerated apoptosis of both differentiated and myelinating oligodendrocytes, and microglia activation, supporting an association between antenatal MTD exposure and impaired myelination in the developing brain.

The Current View on the Paradox of Pain in Autism Spectrum Disorders.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, which affects 1 in 44 children and may cause severe disabilities. Besides socio-communicational difficulties and repetitive behaviors, ASD also presents as atypical sensorimotor function and pain reactivity. While chronic pain is a frequent co-morbidity in autism, pain management in this population is often insufficient because of difficulties in pain evaluation, worsening their prognosis and perhaps driving higher mortality rates. Previous observations have tended to oversimplify the experience of pain in autism as being insensitive to painful stimuli. Various findings in the past 15 years have challenged and complicated this dogma. However, a relatively small number of studies investigates the physiological correlates of pain reactivity in ASD. We explore the possibility that atypical pain perception in people with ASD is mediated by alterations in pain perception, transmission, expression and modulation, and through interactions between these processes. These complex interactions may account for the great variability and sometimes contradictory findings from the studies. A growing body of evidence is challenging the idea of alterations in pain processing in ASD due to a single factor, and calls for an integrative view. We propose a model of the pain cycle that includes the interplay between the molecular and neurophysiological pathways of pain processing and it conscious appraisal that may interfere with pain reactivity and coping in autism. The role of social factors in pain-induced response is also discussed. Pain assessment in clinical care is mostly based on subjective rather than objective measures. This review clarifies the strong need for a consistent methodology, and describes innovative tools to cope with the heterogeneity of pain expression in ASD, enabling individualized assessment. Multiple measures, including self-reporting, informant reporting, clinician-assessed, and purely physiological metrics may provide more consistent results. An integrative view on the regulation of the pain cycle offers a more robust framework to characterize the experience of pain in autism.

Functional anatomical evidence for respiratory rhythmogenic function of endogenous bursters in rat medulla.

Endogenous burster neurons (EBs) have been found at the level of the facial nucleus (VIIn), and 500 mum caudally, within the pre-Bötzinger complex (preBötC). They have been proposed as either causal to or playing no role in respiratory rhythmogenesis. Little is known about their broader distribution in ventrolateral medulla. Here, a Ca(2+) indicator was used to record respiratory network activity in ventrolateral medulla, and, following synaptic blockade, to identify EBs active at perfusate K(+) concentrations ([K(+)](o)) of 3, 6, and 9 mm. Recordings were made along the respiratory column, extending 300 mum rostrally, and 1100 mum caudally from the caudal pole of VIIn (VIIc), in the in vitro tilted sagittal slab preparation, isolated from neonate male and female Sprague Dawley rats. Activity under matching [K(+)](o) in the intact respiratory network was subsequently investigated. Respiratory neurons (n = 401) formed statistically significant clusters at the VIIc, within the preBötC, and 100 mum caudal to the preBötC. EBs (n = 693) formed statistically significant clusters that overlapped with respiratory clusters at the VIIc and preBötC. EB activity increased significantly as [K(+)](o) was increased, as did neurons that remained coupled following synaptic blockade. The overlap between respiratory and EB clusters in regions of ventrolateral medulla identified as rhythmogenic supports the hypothesis that EBs are constituents of rhythmogenic networks. In addition, the observation of truncated inspiratory bursts and ectopic bursting in respiratory neurons when [K(+)](o) was elevated in the intact network is consistent with a causal role for EBs in respiratory rhythmogenesis.

Evidence for the emergence of an opioid-resistant respiratory rhythm following fentanyl overdose.

Breathing resumes within one to two minutes following fentanyl overdose induced apnea in spontaneously breathing rats. As this regular rhythm is produced at a time wherein fentanyl concentrations and receptor occupancy are likely to be extremely high, the mechanisms initiating and sustaining such a respiratory activity remain unclear. Forty-four un-anesthetized adult rats were studied in an open-flow plethysmograph. Regardless of the dose of fentanyl that was used, i.e. 50 µg.kg-1 (n = 8), 100 µg.kg-1 (n = 8) or 300 µg.kg-1 (n = 7), all rats developed an immediate central apnea followed by a depressed regular rhythm that was produced 118, 97 and 81 s (median) later, respectively. Only one rat did not recover. This inspiratory and regular activity consisted of a low frequency and tidal volume pattern with a significant reduction in V̇E/V̇CO2 ratio, which persisted for at least 30 min and that was not different between 100 or 300 µg.kg-1. The time at which this respiratory rhythm emerged, following the highest dose of fentanyl, was not affected by 100 % O2 or 8% CO2/15 % O2. The absolute level of ventilation was however higher in hypercapnic and moderately hypoxic conditions than in hyperoxia. When a second injection of the highest dose of fentanyl (300 µg.kg-1) was performed at 10 min, ventilation was not significantly affected and no apnea was produced in major contrast to the first injection. When a similar injection was performed 30 min after the first injection, in a separate group of rats, an apnea and breathing depression was produced in 30 % of the animals, while in the other rats, ventilation was unaffected. We conclude that the depressed regular respiratory activity emerging during and following fentanyl overdose is uniquely resistant to fentanyl.

Semi-automated region of interest generation for the analysis of optically recorded neuronal activity.

Bath-applied membrane-permeant Ca(2+) 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 Ca(2+) 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.

Diverse changes in myelin protein expression in rat brain after perinatal methadone exposure.

The national incidence of neonatal abstinence syndrome has dramatically increased over the last decade due to an increase in antenatal opioid exposure. Recent human and animal studies suggest that antenatal opioid exposure impacts the developing brain. The purpose of this study is to evaluate the effects of perinatal methadone exposure on myelination in multiple regions in the developing rat brain. Pregnant Sprague-Dawley rats were randomly assigned into three experimental groups and subsequently exposed to drinking water alone or drinking water containing methadone from 7 days post coitum through day 7 or through day 19 after delivery. Two male neonatal rats were randomly selected from each litter and terminated at day 19. The cerebral cortex, hippocampus, cerebellum, and brainstem were dissected and analyzed for three myelin specific proteins - CNP, PLP, and MBP - by Western blot analysis. All pups with exposure to methadone demonstrated decreased expression of CNP, PLP, and MBP in the cerebral cortex and hippocampus. In the cerebellum, PLP expression was down­regulated without apparent alteration of CNP and MBP expression. PLP and MBP expression, but not CNP expression, were significantly inhibited in the brainstem. Compared to the pups with postnatal methadone exposure via maternal milk through day 7, partial recovery of CNP and PLP expression only occurred in the cerebral cortices of the pups exposed through day 19. The findings show that antenatal opioid exposure in rat pups is associated with regionally­specific alterations in brain myelination that diversely affects myelin proteins.

Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation.

Current consensus holds that a single medullary network generates respiratory rhythm in mammals. Pre-Bötzinger Complex inspiratory (I) neurons, isolated in transverse slices, and preinspiratory (pre-I) neurons, found only in more intact en bloc preparations and in vivo, are each proposed as necessary for rhythm generation. Opioids slow I, but not pre-I, neuronal burst periods. In slices, opioids gradually lengthened respiratory periods, whereas in more intact preparations, periods jumped nondeterministically to integer multiples of the control period (quantal slowing). These findings suggest that opioid-induced quantal slowing results from transmission failure of rhythmic drive from pre-I neurons to preBötC I networks, depressed below threshold for spontaneous rhythmic activity. Thus, both I (in the slice), and pre-I neurons are sufficient for respiratory rhythmogenesis.

A vibrating microtome attachment for cutting brain slice preparations at reproducible compound angles relative to the midline.

Slice preparations isolate functional networks, permitting single unit recording under visual control, and the use of fluorescent indicators. Circuits of interest often lie at a tilt in both the rostrocaudal and ventrodorsal axis, thus exposing circuits of interest at the cut surface of a slice would require a device for tilting a preparation along two orthogonal axes relative to the blade. Such a device, designed to be used in conjunction with a vibrating microtome, permitting the isolation of slice preparations at reproducible angles, is described here. Because the two orthogonal axes of tilt can be independently and continuously adjusted, it is possible to use this device to successively refine tilt parameters from preparation to preparation for optimal exposure of circuits of interest, facilitating the development of new slice preparations. Its use in cutting a thick medullary slab preparation, isolated from the neonate rat, which exposes respiratory networks at the cut surface is described.

Belt-and-suspenders as a biological design principle.

Recent studies have shown both the pFRG and the preBötC are sufficient to generate respiratory rhythm, and are hypothesized to do so via distinct mechanisms (Onimaru and Homma 2003; Mellen, Janczewski, Bocchiaro and Feldman 2003). The coexistence of mechanistically distinct, functionally matching networks (defined as degeneracy, Edelman and Gally 2001) is a ubiquitous feature of motor networks in both invertebrates (Selverston and Miller 1980) and vertebrates (DiDomenico, Nissanov and Eaton 1988). In almost all cases, a consensus exists about which subsystem is the "primary" rhythm generator, yet consistently, the effect of modulators on the isolated primary rhythm generator is qualitatively different than their effect on the more intact network (Ayali and Harris-Warrick 1999) and, in the intact animal, all rhythmogenic networks are active during motor pattern generation. Thus, at best, ascribing primacy to a particular network has weak support (since the other networks can produce qualitatively similar patterns; Prinz, Bucher and Marder 2004) and little explanatory power (since effects of modulatory inputs on the isolated "primary" rhythm generator do not persist in more intact networks). The ubiquity of degenerate networks for motor pattern generation suggests that a more useful question is why such an organization exists. We propose that degeneracy is ubiquitous because it reduces the phenotype's sensitivity to genetic mutation and environmental perturbation, and broadens the adaptiveness of motor patterns.