Intravenous chloral hydrate anesthesia provides appropriate analgesia for surgical interventions in male Sprague-Dawley rats.

BACKGROUND: The use of chloral hydrate as a sole maintenance anesthetic agent in rodent research has been controversial due to statements made in reference literature conflicting with results of primary research studies regarding its analgesic efficacy, and because of its associated tissue damage when administered intraperitoneally. OBJECTIVE: Our aim was to assess the analgesic efficacy of chloral hydrate using an intravenous (i.v.) route of administration, in order to prevent the local tissue irritation or ileus that has been previously reported using intraperitoneal (i.p.) routes. METHODS: We measured tail withdrawal latencies to a nociceptive thermal stimulus (infrared beam) in Sprague-Dawley rats-first when awake (unanesthetized), and then subsequently during i.v. chloral hydrate anesthesia. During anesthesia we also measured ongoing heart and respiration rates. RESULTS: Withdrawal latencies during chloral hydrate anesthesia were significantly higher, and often maximal, indicating a robust analgesic effect. Importantly, both respiration and heart rate remained unchanged following exposure to the nociceptive stimulus, and were comparable to values observed under other anesthetics and during natural sleep. CONCLUSIONS: Together with previous studies, these results demonstrate that i.v. chloral hydrate provides excellent anesthetic depth and analgesic efficacy for surgical manipulations in rats.

Tonic excitation of nucleus reuniens decreases prefrontal-hippocampal coordination during slow-wave states.

The nucleus reuniens of the thalamus (RE) is an important node between the medial prefrontal cortex (mPFC) and the hippocampus (HPC). Previously, we have shown that its mode of activity and its influence in mPFC-HPC communication is dependent upon brain state. During slow-wave states, RE units are closely and rhythmically coupled to the ongoing mPFC-slow oscillation (SO), while during activated (theta) states, RE neurons fire in an arrhythmic and tonically active manner. Inactivating the RE selectively impoverishes coordination of the SO between mPFC and HPC and interestingly, both mPFC and RE stimulation during the SO cause larger responses in the HPC than during theta. It is unclear if the activity patterns within the RE across states may play a role in both phenomena. Here, we optogenetically excited RE neurons in a tonic fashion to assess the impact on mPFC-HPC coupling. This stimulation decreased the influence of mPFC stimulation in the HPC during SO states, in a manner similar to what is observed across state changes into theta. Importantly, this type of stimulation had no effect on evoked responses during theta. Perhaps more interestingly, tonic optogenetic excitation of the RE also decreased mPFC-HPC SO coherence. Thus, it may not be the integrity of the RE per se that is responsible for efficient communication between mPFC and HPC, but rather the particular state in which RE neurons find themselves. Our results have direct implications for how distant brain regions can communicate most effectively, an issue that is ultimately important for activity-dependent processes occurring during slow-wave sleep-dependent memory consolidation.

A Comparison of Brain-State Dynamics across Common Anesthetic Agents in Male Sprague-Dawley Rats.

Anesthesia is a powerful tool in neuroscientific research, especially in sleep research where it has the experimental advantage of allowing surgical interventions that are ethically problematic in natural sleep. Yet, while it is well documented that different anesthetic agents produce a variety of brain states, and consequently have differential effects on a multitude of neurophysiological factors, these outcomes vary based on dosages, the animal species used, and the pharmacological mechanisms specific to each anesthetic agent. Thus, our aim was to conduct a controlled comparison of spontaneous electrophysiological dynamics at a surgical plane of anesthesia under six common research anesthetics using a ubiquitous animal model, the Sprague-Dawley rat. From this direct comparison, we also evaluated which anesthetic agents may serve as pharmacological proxies for the electrophysiological features and dynamics of unconscious states such as sleep and coma. We found that at a surgical plane, pentobarbital, isoflurane and propofol all produced a continuous pattern of burst-suppression activity, which is a neurophysiological state characteristically observed during coma. In contrast, ketamine-xylazine produced synchronized, slow-oscillatory activity, similar to that observed during slow-wave sleep. Notably, both urethane and chloral hydrate produced the spontaneous, cyclical alternations between forebrain activation (REM-like) and deactivation (non-REM-like) that are similar to those observed during natural sleep. Thus, choice of anesthesia, in conjunction with continuous brain state monitoring, are critical considerations in order to avoid brain-state confounds when conducting neurophysiological experiments.

Long-term stability of physiological signals within fluctuations of brain state under urethane anesthesia.

Urethane, an acute laboratory anesthetic, produces distinct neurophysiological and physiological effects creating an effective model of the dynamics of natural sleep. As a model of both sleep-like neurophysiological activity and the downstream peripheral function urethane is used to model a variety of physiological and pathophysiological processes. As urethane is typically administered as a single-bolus dose, it is unclear the stability of peripheral physiological functions both within and between brain-states under urethane anesthesia. In this present study, we recorded respiration rate and heart rate concurrently with local field potentials from the neocortex and hippocampus to determine the stability of peripheral physiological functions within and between brain-states under urethane anesthesia. Our data shows electroencephalographic characteristics and breathing rate are remarkable stable over long-term recordings within minor reductions in heart rate on the same time scale. Our findings indicate that the use of urethane to model peripheral physiological functions associated with changing brain states are stable during long duration experiments.

Membrane Resonance in Pyramidal and GABAergic Neurons of the Mouse Perirhinal Cortex.

The perirhinal cortex (PRC) is a polymodal associative region of the temporal lobe that works as a gateway between cortical areas and hippocampus. In recent years, an increasing interest arose in the role played by the PRC in learning and memory processes, such as object recognition memory, in contrast with certain forms of hippocampus-dependent spatial and episodic memory. The integrative properties of the PRC should provide all necessary resources to select and enhance the information to be propagated to and from the hippocampus. Among these properties, we explore in this paper the ability of the PRC neurons to amplify the output voltage to current input at selected frequencies, known as membrane resonance. Within cerebral circuits the resonance of a neuron operates as a filter toward inputs signals at certain frequencies to coordinate network activity in the brain by affecting the rate of neuronal firing and the precision of spike timing. Furthermore, the ability of the PRC neurons to resonate could have a fundamental role in generating subthreshold oscillations and in the selection of cortical inputs directed to the hippocampus. Here, performing whole-cell patch-clamp recordings from perirhinal pyramidal neurons and GABAergic interneurons of GAD67-GFP+ mice, we found, for the first time, that the majority of PRC neurons are resonant at their resting potential, with a resonance frequency of 0.5-1.5 Hz at 23°C and of 1.5-2.8 Hz at 36°C. In the presence of ZD7288 (blocker of HCN channels) resonance was abolished in both pyramidal neurons and interneurons, suggesting that Ih current is critically involved in resonance generation. Otherwise, application of TTx (voltage-dependent Na+ channel blocker) attenuates the resonance in pyramidal neurons but not in interneurons, suggesting that only in pyramidal neurons the persistent sodium current has an amplifying effect. These experimental results have also been confirmed by a computational model. From a functional point of view, the resonance in the PRC would affect the reverberating activity between neocortex and hippocampus, especially during slow wave sleep, and could be involved in the redistribution and strengthening of memory representation in cortical regions.

Prefrontal-Hippocampal Pathways Through the Nucleus Reuniens Are Functionally Biased by Brain State.

Circuit-level communication between disparate brain regions is fundamental for the complexities of the central nervous system operation. Co-ordinated bouts of rhythmic activity between the prefrontal cortex (PFC) and hippocampus (HPC), in particular, are important for mnemonic processes. This is true during awake behavior, as well as during offline states like sleep. We have recently shown that the anatomically interposed thalamic nucleus reuniens (RE) has a role in coordinating slow-wave activity between the PFC and HPC. Here, we took advantage of spontaneous brain state changes occurring during urethane anesthesia in order to assess if PFC-HPC communication was modified during activated (theta) vs. deactivated (slow oscillation: SO) states. These forebrain states are highly similar to those expressed during rapid eye movement (REM) and non-REM stages of natural sleep, respectively. Evoked potentials and excitatory current sinks in the HPC were consistently larger during SO states, regardless of whether PFC or RE afferents were stimulated. Interestingly, PFC stimulation during theta appeared to preferentially use a cortico-cortical pathway, presumably involving the entorhinal cortex as opposed to the more direct RE to HPC conduit. Optogenetic and chemogenetic manipulations of the RE suggested that this state-dependent biasing was mediated by responding in the RE itself. Finally, the phase of both ongoing rhythms also appeared to be an important factor in modulating HPC responses, with maximal field excitatory postsynaptic potentials (EPSPs) occurring during the negative-going phase of both rhythms. Thus, forebrain state plays an important role in how communication takes place across the PFC and HPC, with the RE as a determining factor in how this is shaped. Furthermore, ongoing sleep-like rhythms influence the coordination and perhaps potentiate excitatory processing in this extended episodic memory circuit. Our results have direct implications for activity-dependent processes relevant to sleep-dependent memory consolidation.

In vivo assessment of mechanisms underlying the neurovascular basis of postictal amnesia.

Long-lasting confusion and memory difficulties during the postictal state remain a major unmet problem in epilepsy that lacks pathophysiological explanation and treatment. We previously identified that long-lasting periods of severe postictal hypoperfusion/hypoxia, not seizures per se, are associated with memory impairment after temporal lobe seizures. While this observation suggests a key pathophysiological role for insufficient energy delivery, it is unclear how the networks that underlie episodic memory respond to vascular constraints that ultimately give rise to amnesia. Here, we focused on cellular/network level analyses in the CA1 of hippocampus in vivo to determine if neural activity, network oscillations, synaptic transmission, and/or synaptic plasticity are impaired following kindled seizures. Importantly, the induction of severe postictal hypoperfusion/hypoxia was prevented in animals treated by a COX-2 inhibitor, which experimentally separated seizures from their vascular consequences. We observed complete activation of CA1 pyramidal neurons during brief seizures, followed by a short period of reduced activity and flattening of the local field potential that resolved within minutes. During the postictal state, constituting tens of minutes to hours, we observed no changes in neural activity, network oscillations, and synaptic transmission. However, long-term potentiation of the temporoammonic pathway to CA1 was impaired in the postictal period, but only when severe local hypoxia occurred. Lastly, we tested the ability of rats to perform object-context discrimination, which has been proposed to require temporoammonic input to differentiate between sensory experience and the stored representation of the expected object-context pairing. Deficits in this task following seizures were reversed by COX-2 inhibition, which prevented severe postictal hypoxia. These results support a key role for hypoperfusion/hypoxia in postictal memory impairments and identify that many aspects of hippocampal network function are resilient during severe hypoxia except for long-term synaptic plasticity.

Prenatal fruit juice exposure enhances memory consolidation in male post-weanling Sprague-Dawley rats.

OBJECTIVES: Nutritional intake during gestation is known to impact health outcomes for progeny. Correlational evidence in humans suggests that increased fruit consumption of pregnant mothers enhances infant cognitive development. Moreover, wild-type Drosophila supplemented with a combination of orange and tomato juice showed robust enhancements in performance on an associative olfactory memory task. The current study aimed to experimentally test the effects of prenatal fruit juice exposure in a non-human, mammalian model of learning and memory. METHODS: Across three separate birth cohorts, pregnant rats were given access to diluted tomato and orange juice (N = 2 per cohort), with control rats (N = 2 per cohort) receiving only water, in addition to standard rodent chow, throughout the duration of gestation, ending at parturition. Following weaning, male offspring were tested for learning and memory in a spatial version of the circular water maze and an auditory-cued fear-conditioning task. RESULTS: All pregnant rats increased fluid and food intake over the gestational period. Fruit juice-fed pregnant rats had increased fluid intake compared to control pregnant rats. When testing progeny, there were no effects of prenatal fruit juice on spatial learning, while it appeared to impair learning in fear conditioning relative to controls. However, we measured significant enhancements in both spatial memory and conditioned fear memory in the prenatal fruit-juice group compared to controls. Measures of vigilance, in response to the conditioned cue, were increased in prenatal fruit rats compared to controls, suggesting less generalized, and more adaptive, anxiety behaviours. DISCUSSION: Our results corroborate the human and Drosophila findings of prenatal fruit effects on behaviour, specifically that prenatal fruit juice exposure may be beneficial for early-life memory consolidation in rats.

The collagenase model of intracerebral hemorrhage in awake, freely moving animals: The effects of isoflurane.

Intracerebral hemorrhage (ICH) is a devastating stroke often modelled in rats. Isoflurane anesthetic, commonly used in preclinical research, affects general physiology (e.g., blood pressure) and electrophysiology (e.g., burst suppression) in many ways. These physiological changes may detract from the clinical relevance of the model. Here, we revised the standard collagenase model to produce an ICH in rats without anesthetic. Guide cannulas were implanted stereotaxically under anesthetic. After 3 days of recovery, collagenase was infused through an internal cannula into the striatum of animals randomly assigned to the non-anesthetized or isoflurane group. We assessed whether isoflurane affected hematoma volume, core temperature, movement activity, pain, blood pressure, and seizure activity. With a small ICH, there was a hematoma volume increased from 8.6 (±3.3, 95% confidence interval) µL in anesthetized rats to 13.2 (±3.1) µL in non-anesthetized rats (P = 0.008), but with a larger ICH, hematoma volumes were similar. Isoflurane decreased temperature by 1.3 °C (±0.16 °C, P < 0.001) for 2 h and caused a 35.1 (±1.7) mmHg group difference in blood pressure (P < 0.007) for 12 m. Blood glucose increased twofold after isoflurane procedures (P < 0.001). Pain, as assessed with the rat grimace scale, did not differ between groups. Seizure incidence rate (62.5%) in non-anesthetized ICH rats was similar to historic amounts (61.3%). In conclusion, isoflurane appears to have some significant and injury size-dependent effects on the collagenase model. Thus, when anesthetic effects are a known concern, the use of the standardized cannula infusion approach is scientifically and ethically acceptable.

Differential effects of L- and D-lactate on memory encoding and consolidation: Potential role of HCAR1 signaling.

The process of memory consolidation is energy-demanding and brain energy deficits result in memory impairments. Indeed, L-lactate, a preferred neuronal energy substrate, enhances the formation of memory, while blockade of the neuronal uptake of L-lactate by either pharmacological means or using its enantiomer D-lactate, impairs memory. Beyond metabolism, both enantiomers of lactate also have signaling properties through the hydroxycarboxylic acid receptor 1 (HCAR1). Thus far, paradigms testing for an effect of lactate on memory modulation have ignored HCAR1 signaling while also mainly performing manipulations before learning and using intracranial administration techniques. Using an inhibitory avoidance (IA) memory protocol, the present study examined the effects of systemic administration of both L- and D-lactate as well as the specific HCAR1 agonist 3,5-dihydroxybenzoic acid (3,5-DHBA) across pre- and post-training periods. We found that post-training subcutaneous injections of either 3,5-DHBA or D-lactate significantly enhanced memory compared to saline controls, whereas L-lactate had no effect, suggesting that HCAR1 signaling in the absence of lactate metabolism supports memory consolidation processes. When administered 15 minutes prior to training, D-lactate and 3,5-DHBA impaired memory compared to saline controls. In contrast, L-lactate treated rats showed memory enhancements as compared to D-lactate-treated rats. Taken together, these results suggest different roles for lactate at different memory stages. It is likely that a metabolic role is at play during learning while HCAR1 signaling may play a greater role during consolidation.

The Reuniens Nucleus of the Thalamus Has an Essential Role in Coordinating Slow-Wave Activity between Neocortex and Hippocampus.

Sleep is a period of profound neural synchrony throughout the brain, a phenomenon involved in various physiological functions. The coordination between neocortex and hippocampus, in particular, appears to be critical for episodic memory, and, indeed, enhanced synchrony in this circuit is a hallmark of slow-wave sleep. However, it is unclear how this coordination is mediated. To this end, we examined the role of the thalamic nucleus reuniens (RE), a midline body with reciprocal connections to both prefrontal and hippocampal cortices. Using a combination of electrophysiological, optogenetic, and chemogenetic techniques in the urethane-anesthetized rat (a model of forebrain sleep activity), we directly assessed the role of the RE in mediating slow oscillatory synchrony. Using unit recording techniques, we confirmed that RE neurons showed slow rhythmic activity patterns during deactivated forebrain states that were coupled to ongoing slow oscillations. Optogenetic activation of RE neurons or their projection fibers in the cingulum bundle caused an evoked potential in hippocampus that was maximal at the level of stratum lacunosum-moleculare of CA1. A similar but longer-latency response could be evoked by stimulation of the medial prefrontal cortex that was then abolished by chemogenetic inhibition of the RE. Inactivation of the RE also severely reduced the coherence of the slow oscillation across cortical and hippocampal sites, suggesting that its activity is necessary to couple slow-wave activity across these regions. These results indicate an essential role of the RE in coordinating neocortico-hippocampal slow oscillatory activity, which may be fundamental for slow-wave sleep-related episodic memory consolidation.

Postnatal development of persistent inward currents in rat XII motoneurons and their modulation by serotonin, muscarine and noradrenaline.

KEY POINTS: Persistent inward currents (PICs) in spinal motoneurons are long-lasting, voltage-dependent currents that increase excitability; they are dramatically potentiated by serotonin, muscarine, and noradrenaline (norepinephrine). Loss of these modulators (and the PIC) during sleep is hypothesized as a major contributor to REM sleep atonia. Reduced excitability of XII motoneurons that drive airway muscles and maintain airway patency is causally implicated in obstructive sleep apnoea (OSA), but whether XII motoneurons possess a modulator-sensitive PIC that could be a factor in the reduced airway tone of sleep is unknown. Whole-cell recordings from rat XII motoneurons in brain slices indicate that PIC amplitude increases ∼50% between 1 and 23 days of age, when potentiation of the PIC by 5HT2 , muscarinic, or α1 noradrenergic agonists peaks at <50%, manyfold lower than the potentiation observed in spinal motoneurons. α1 noradrenergic receptor activation produced changes in XII motoneuron firing behaviour consistent with PIC involvement, but indicators of strong PIC activation were never observed; in vivo experiments are needed to determine the role of the modulator-sensitive PIC in sleep-dependent reductions in airway tone. ABSTRACT: Hypoglossal (XII) motoneurons play a key role in maintaining airway patency; reductions in their excitability during sleep through inhibition and disfacilitation, i.e. loss of excitatory modulation, is implicated in obstructive sleep apnoea. In spinal motoneurons, 5HT2 , muscarinic and α1 noradrenergic modulatory systems potentiate persistent inward currents (PICs) severalfold, dramatically increasing excitability. If the PICs in XII and spinal motoneurons are equally sensitive to modulation, loss of the PIC secondary to reduced modulatory tone during sleep could contribute to airway atonia. Modulatory systems also change developmentally. We therefore characterized developmental changes in magnitude of the XII motoneuron PIC and its sensitivity to modulation by comparing, in neonatal (P1-4) and juvenile (P14-23) rat brainstem slices, the PIC elicited by slow voltage ramps in the absence and presence of agonists for 5HT2 , muscarinic, and α1 noradrenergic receptors. XII motoneuron PIC amplitude increased developmentally (from -195 ± 12 to -304 ± 19 pA). In neonatal XII motoneurons, the PIC was only potentiated by α1 receptor activation (5 ± 4%). In contrast, all modulators potentiated the juvenile XII motoneurons PIC (5HT2 , 5 ± 5%; muscarine, 22 ± 11%; α1 , 18 ± 5%). These data suggest that the influence of the PIC and its modulation on XII motoneuron excitability will increase with postnatal development. Notably, the modulator-induced potentiation of the PIC in XII motoneurons was dramatically smaller than the 2- to 6-fold potentiation reported for spinal motoneurons. In vivo measurements are required to determine if the modulator-sensitive, XII motoneuron PIC is an important factor in sleep-state dependent reductions in airway tone.

Electrophysiological correlates of hyperoxia during resting-state EEG in awake human subjects.

Recreational use of concentrated oxygen has increased. Claims have been made that hyperoxic breathing can help reduce fatigue, increase alertness, and improve attentional capacities; however, few systematic studies of these potential benefits exist. Here, we examined the effects of short-term (15 min) hyperoxia on resting states in awake human subjects by measuring spontaneous EEG activity between normoxic and hyperoxic situations, using a within-subject design for both eyes-opened and eyes-closed conditions. We also measured respiration rate, heart rate, and blood oxygen saturation levels to correlate basic physiological changes due to the hyperoxic challenge with any brain activity changes. Our results show that breathing short-term 100% oxygen led to increased blood oxygen saturation levels, decreased heart rate, and a slight, but nonsignificant, decrease in breathing rate. Changes of brain activity were apparent, including decreases in low-alpha (7-10 Hz), high-alpha (10-14 Hz), beta (14-30 Hz), and gamma (30-50 Hz) frequency ranges during eyes-opened hyperoxic conditions. During eyes-closed hyperoxia, increases in the delta (0.5-3.5 Hz) and theta (3.5-7 Hz) frequency range were apparent together with decreases in the beta range. Hyperoxia appeared to accentuate the decrease of low alpha and gamma ranges across the eyes-opened and eyes-closed conditions, suggesting that it modulated brain state itself. As decreased alpha during eyes-opened conditions has been associated with increased attentional processing and selective attention, and increased delta and theta during eyes-closed condition are typically associated with the initiation of sleep, our results suggest a state-specific and perhaps opposing influence of short-term hyperoxia.

Hyperoxia enhances slow-wave forebrain states in urethane-anesthetized and naturally sleeping rats.

Oxygen (O2) is a crucial element for physiological functioning in mammals. In particular, brain function is critically dependent on a minimum amount of circulating blood levels of O2 and both immediate and lasting neural dysfunction can result following anoxic or hypoxic episodes. Although the effects of deficiencies in O2 levels on the brain have been reasonably well studied, less is known about the influence of elevated levels of O2 (hyperoxia) in inspired gas under atmospheric pressure. This is of importance due to its typical use in surgical anesthesia, in the treatment of stroke and traumatic brain injury, and even in its recreational or alternative therapeutic use. Using local field potential (EEG) recordings in spontaneously breathing urethane-anesthetized and naturally sleeping rats, we characterized the influence of different levels of O2 in inspired gases on brain states. While rats were under urethane anesthesia, administration of 100% O2 elicited a significant and reversible increase in time spent in the deactivated (i.e., slow-wave) state, with concomitant decreases in both heartbeat and respiration rates. Increasing the concentration of carbon dioxide (to 5%) in inspired gas produced the opposite result on EEG states, mainly a decrease in the time spent in the deactivated state. Consistent with this, decreasing concentrations of O2 (to 15%) in inspired gases decreased time spent in the deactivated state. Further confirmation of the hyperoxic effect was found in naturally sleeping animals where it similarly increased time spent in slow-wave (nonrapid eye movement) states. Thus alterations of O2 in inspired air appear to directly affect forebrain EEG states, which has implications for brain function, as well as for the regulation of brain states and levels of forebrain arousal during sleep in both normal and pathological conditions. NEW & NOTEWORTHY We show that alterations of oxygen concentration in inspired air biases forebrain EEG state. Hyperoxia increases the prevalence of slow-wave states. Hypoxia and hypercapnia appear to do the opposite. This suggests that oxidative metabolism is an important stimulant for brain state.

New waves: Rhythmic electrical field stimulation systematically alters spontaneous slow dynamics across mouse neocortex.

The signature rhythm of slow-wave forebrain activity is the large amplitude, slow oscillation (SO: ∼1 Hz) made up of alternating synchronous periods of activity and silence at the single cell and network levels. On each wave, the SO originates at a unique location and propagates across the neocortex. Attempts to manipulate SO activity using electrical fields have been shown to entrain cortical networks and enhance memory performance. However, neural activity during this manipulation has remained elusive due to methodological issues in typical electrical recordings. Here we took advantage of voltage-sensitive dye (VSD) imaging in a bilateral cortical preparation of urethane-anesthetized mice to track SO cortical activity and its modulation by sinusoidal electrical field stimulation applied to frontal regions. We show that under spontaneous conditions, the SO propagates in two main opposing directional patterns along an anterior lateral - posterior medial axis, displaying a rich variety of possible trajectories on any given wave. Under rhythmic field stimulation, new propagation patterns emerge, which are not observed under spontaneous conditions, reflecting stimulus-entrained activity with distributed and varied anterior initiation zones and a consistent termination zone in the posterior somatosensory cortex. Furthermore, stimulus-induced activity patterns tend to repeat cycle after cycle, showing higher stereotypy than during spontaneous activity. Our results show that slow electrical field stimulation robustly entrains and alters ongoing slow cortical dynamics during sleep-like states, suggesting a mechanism for targeting specific cortical representations to manipulate memory processes.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism.

KEY POINTS: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. ABSTRACT: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

Cognitive Enhancement in Infants Associated with Increased Maternal Fruit Intake During Pregnancy: Results from a Birth Cohort Study with Validation in an Animal Model.

In-utero nutrition is an under-studied aspect of cognitive development. Fruit has been an important dietary constituent for early hominins and humans. Among 808 eligible CHILD-Edmonton sub-cohort subjects, 688 (85%) had 1-year cognitive outcome data. We found that each maternal daily serving of fruit (sum of fruit plus 100% fruit juice) consumed during pregnancy was associated with a 2.38 point increase in 1-year cognitive development (95% CI 0.39, 4.37; p<0.05). Consistent with this, we found 30% higher learning Performance index (PI) scores in Drosophila offspring from parents who consumed 30% fruit juice supplementation prenatally (PI: 85.7; SE 1.8; p<0.05) compared to the offspring of standard diet parents (PI: 65.0 SE 3.4). Using the Drosophila model, we also show that the cyclic adenylate monophosphate (cAMP) pathway may be a major regulator of this effect, as prenatal fruit associated cognitive enhancement was blocked in Drosophila rutabaga mutants with reduced Ca(2+)-Calmodulin-dependent adenylyl cyclase. Moreover, gestation is a critical time for this effect as postnatal fruit intake did not enhance cognitive performance in either humans or Drosophila. Our study supports increased fruit consumption during pregnancy with significant increases in infant cognitive performance. Validation in Drosophila helps control for potential participant bias or unmeasured confounders.

ZIP It: Neural Silencing Is an Additional Effect of the PKM-Zeta Inhibitor Zeta-Inhibitory Peptide.

UNLABELLED: Protein kinase M ζ (PKMζ), an atypical isoform of protein kinase C, has been suggested to be necessary and sufficient for the maintenance of long-term potentiation (LTP) and long-term memory (LTM). This evidence is heavily based on the use of ζ inhibitory peptide (ZIP), a supposed specific inhibitor of PKMζ that interferes with both LTP and LTM. Problematically, both LTP and LTM are unaffected in both constitutive and conditional PKMζ knock-out mice, yet both are still impaired by ZIP application, suggesting a nonspecific mechanism of action. Because translational interference can disrupt neural activity, we assessed network activity after a unilateral intrahippocampal infusion of ZIP in anesthetized rats. ZIP profoundly reduced spontaneous hippocampal local field potentials, comparable in magnitude to infusions of lidocaine, but with a slower onset and longer duration. Our results highlight a serious confound in interpreting the behavioral effects of ZIP. We suggest that future molecular approaches in neuroscience consider the intervening level of cellular and systems neurophysiology before claiming influences on behavior. SIGNIFICANCE STATEMENT: Long-term memory in the brain is thought to arise from a sustained molecular process that can maintain changes in synaptic plasticity. A so-called candidate for the title of "the memory molecule" is protein kinase M ζ (PKMζ), mainly because its inhibition by ζ inhibitory peptide (ZIP) interferes with previously established synaptic plasticity and memory. We show that brain applications of ZIP that can impair memory actually profoundly suppress spontaneous brain activity directly or can cause abnormal seizure activity. We suggest that normal brain activity occurring after learning may be a more primary element of memory permanence.