GABA system as the cause and effect in early development.

GABA is the primary inhibitory neurotransmitter in the adult brain and through its actions on GABAARs, it protects against excitotoxicity and seizure activity, ensures temporal fidelity of neurotransmission, and regulates concerted rhythmic activity of neuronal populations. In the developing brain, the development of GABAergic neurons precedes that of glutamatergic neurons and the GABA system serves as a guide and framework for the development of other brain systems. Despite this early start, the maturation of the GABA system also continues well into the early postnatal period. In this review, we organize evidence around two scenarios based on the essential and protracted nature of GABA system development: 1) disruptions in the development of the GABA system can lead to large scale disruptions in other developmental processes (i.e., GABA as the cause), 2) protracted maturation of this system makes it vulnerable to the effects of developmental insults (i.e., GABA as the effect). While ample evidence supports the importance of GABA/GABAAR system in both scenarios, large gaps in existing knowledge prevent strong mechanistic conclusions.

Rhythmic firing of neurons in the medulla of conscious freely behaving rats: rhythmic coupling with baroreceptor input.

Recent investigations emphasized the importance of neural control of cardiovascular adjustments in complex behaviors, including stress, exercise, arousal, sleep-wake states, and different tasks. Baroreceptor feedback is an essential component of this system acting on different time scales from maintaining stable levels of cardiovascular parameters on the long-term to rapid alterations according to behavior. The baroreceptor input is essentially rhythmic, reflecting periodic fluctuations in arterial blood pressure. Cardiac rhythm is a prominent feature of the autonomic control system, present on different levels, including neuron activity in central circuits. The mechanism of rhythmic entrainment of neuron firing by the baroreceptor input was studied in great detail under anesthesia, but recordings of sympathetic-related neuron firing in freely moving animals remain extremely scarce. In this study, we recorded multiple single neuron activity in the reticular formation of the medulla in freely moving rats during natural behavior. Neurons firing in synchrony with the cardiac rhythm were detected in each experiment (n = 4). In agreement with prior observations in anesthetized cats, we found that neurons in this area exhibited high neuron-to-neuron variability and temporal flexibility in their coupling to cardiac rhythm in freely moving rats, as well. This included firing in bursts at multiples of cardiac cycles, but not directly coupled to the heartbeat, supporting the concept of baroreceptor input entraining intrinsic neural oscillations rather than imposing a rhythm of solely external origin on these networks. It may also point to a mechanism of maintaining the basic characteristics of sympathetic neuron activity, i.e., burst discharge and cardiac-related rhythmicity, on the background of behavior-related adjustments in their firing rate.

Validation of PiezoSleep Scoring Against EEG/EMG Sleep Scoring in Rats.

Introduction: Current methods of sleep research in rodents involve invasive surgical procedures of EEG and EMG electrodes implantation. Recently, a new method of measuring sleep, PiezoSleep, has been validated against implanted electrodes in mice and rats. PiezoSleep uses a piezoelectric film transducer to detect the rodent's movements and respiration and employs an algorithm to automatically score sleep. Here, we validate PiezoSleep scoring versus EEG/EMG implanted electrodes sleep scoring in rats. Methods: Adult male Brown Norway and Wistar Kyoto rats were implanted with bilateral stainless-steel screws into the skull for EEG recording and bilateral wire electrodes into the nuchal muscles for EMG assessment. In Brown Norway rats, the EEG/EMG electrode leads were soldered to a miniature connector plug and fixed to the skull. In Wistar Kyoto rats, the EEG/EMG leads were tunneled subcutaneously to a telemetry transmitter implanted in the flank. Rats were allowed to recover from surgery for one week. Brown Norway rats were placed in PiezoSleep cages, and had their headsets connected to cable for recording EEG/EMG signals, which were then manually scored by a human scorer in 10-sec epochs. Wistar Kyoto rats were placed in PiezoSleep cages, and EEG/EMG signals were recorded using a telemetry system (DSI). Sleep was scored automatically in 4-sec epochs using NeuroScore software. PiezoSleep software recorded and scored sleep in the rats. Results: Rats implanted with corded EEG/EMG headsets had 85.6% concurrence of sleep-wake scoring with PiezoSleep. Rats implanted with EEG/EMG telemetry had 80.8% concurrence sleep-wake scoring with PiezoSleep. Sensitivity and specificity rates were similar between the EEG/EMG recording systems. Total sleep time and hourly sleep times did not differ in all three systems. However, automatic sleep detection by NeuroScore classified more sleep during the light period compared to the PiezoSleep. Conclusion: We showed that PiezoSleep system can be a reliable alternative to both automatic and visual EEG/EMG- based sleep-wake scoring in rat.

Reciprocal Interactions between Medial Septum and Hippocampus in Theta Generation: Granger Causality Decomposition of Mixed Spike-Field Recordings.

The medial septum (MS) plays an essential role in rhythmogenesis in the hippocampus (HIPP); theta-rhythmic bursts of MS neurons are believed to drive theta oscillations in rats' HIPP. The MS theta pacemaker hypothesis has solid foundation but the MS-hippocampal interactions during different behavioral states are poorly understood. The MS and the HIPP have reciprocal connections and it is not clear in particular what role, if any, the strong HIPP to MS projection plays in theta generation. To study the functional interactions between MS and HIPP during different behavioral states, this study investigated the relationship between MS single-unit activity and HIPP field potential oscillations during theta states of active waking and REM sleep and non-theta states of slow wave sleep (SWS) and quiet waking (QW), i.e., sleep-wake states that comprise the full behavioral repertoire of undisturbed, freely moving rats. We used non-parametric Granger causality (GC) to decompose the MS-HIPP synchrony into its directional components, MS→HIPP and HIPP→MS, and to examine the causal interactions between them within the theta frequency band. We found a significant unidirectional MS→HIPP influence in non-theta states which switches to bidirectional theta drive during theta states with MS→HIPP and HIPP→MS GC being of equal magnitude. In non-theta states, unidirectional MS→HIPP influence was accompanied by significant MS-HIPP coherence, but no signs of theta oscillations in the HIPP. In theta states of active waking and REM sleep, sharp theta coherence and strong theta power in both structures was associated with a rise in HIPP→MS to the level of the MS→HIPP drive. Thus, striking differences between waking and REM sleep theta states and non-theta states of SWS and QW were primarily observed in activation of theta influence carried by the descending HIPP→MS pathway associated with more regular rhythmic bursts in the MS and sharper MS→HIPP GC spectra without a significant increase in MS→HIPP GC magnitude. The results of this study suggest an essential role of descending HIPP to MS projections in theta generation.

Acute exacerbation of sleep apnea by hyperoxia impairs cognitive flexibility in Brown-Norway rats.

STUDY OBJECTIVES: To determine whether learning deficits occur during acute exacerbation of spontaneous sleep related breathing disorder (SRBD) in rats with high (Brown Norway; BN) and low (Zucker Lean; ZL) apnea propensity. DESIGN: Spatial acquisition (3 days) and reversal learning (3 days) in the Morris water maze (MWM) with polysomnography (12:00-08:00): (1) with acute SRBD exacerbation (by 20-h hyperoxia immediately preceding reversal learning) or (2) without SRBD exacerbation (room air throughout). SETTING: Randomized, placebo-controlled, repeated-measures design. PARTICIPANTS: 14 BN rats; 16 ZL rats. INTERVENTIONS: 20-h hyperoxia. MEASUREMENTS AND RESULTS: Apneas were detected as cessation of respiration ≥ 2 sec. Swim latency in MWM, apnea indices (AI; apneas/hour of sleep) and percentages of recording time for nonrapid eye movement (NREM), rapid eye movement (REM), and total sleep were assessed. Baseline AI in BN rats was more than double that of ZL rats (22.46 ± 2.27 versus 10.7 ± 0.9, P = 0.005). Hyperoxia increased AI in both BN (34.3 ± 7.4 versus 22.46 ± 2.27) and ZL rats (15.4 ± 2.7 versus 10.7 ± 0.9) without changes in sleep stage percentages. Control (room air) BN and ZL rats exhibited equivalent acquisition and reversal learning. Acute exacerbation of AI by hyperoxia produced a reversal learning performance deficit in BN but not ZL rats. In addition, the percentage of REM sleep and REM apnea index in BN rats during hyperoxia negatively correlated with reversal learning performance. CONCLUSIONS: Acute exacerbation of sleep related breathing disorder by hyperoxia impairs reversal learning in a rat strain with high apnea propensity, but not a strain with a low apnea propensity. This suggests a non-linear threshold effect may contribute to the relationships between sleep apnea and cognitive dysfunctions, but strain-specific differences also may be important.

Brown Norway and Zucker Lean rats demonstrate circadian variation in ventilation and sleep apnea.

STUDY OBJECTIVES: Circadian rhythms influence many biological systems, but there is limited information about circadian and diurnal variation in sleep related breathing disorder. We examined circadian and diurnal patterns in sleep apnea and ventilatory patterns in two rat strains, one with high sleep apnea propensity (Brown Norway [BN]) and the other with low sleep apnea propensity (Zucker Lean [ZL]). DESIGN/SETTING: Chronically instrumented rats were randomized to breathe room air (control) or 100% oxygen (hyperoxia), and we performed 20-h polysomnography beginning at Zeitgeber time 4 (ZT 4; ZT 0 = lights on, ZT12 = lights off). We examined the effect of strain and inspired gas (twoway analysis of variance) and analyzed circadian and diurnal variability. MEASUREMENTS AND RESULTS: Strain and inspired gas-dependent differences in apnea index (AI; apneas/h) were particularly prominent during the light phase. AI in BN rats (control, 16.9 ± 0.9; hyperoxia, 34.0 ± 5.8) was greater than in ZL rats (control, 8.5 ± 1.0; hyperoxia, 15.4 ± 1.1, [strain effect, P < 0.001; gas effect, P = 0.001]). Hyperoxia reduced respiratory frequency in both strains, and all respiratory pattern variables demonstrated circadian variability. BN rats exposed to hyperoxia demonstrated the largest circadian fluctuation in AI (amplitude = 17.9 ± 3.7 apneas/h [strain effect, P = 0.01; gas effect, P < 0.001; interaction, P = 0.02]; acrophase = 13.9 ± 0.7 h; r (2) = 0.8 ± 1.4). CONCLUSIONS: Inherited, environmental, and circadian factors all are important elements of underlying sleep related breathing disorder. Our method to examine sleep related breathing disorder phenotypes in rats may have implications for understanding vulnerability for sleep related breathing disorder in humans.

Impact of the vagal feedback on cardiorespiratory coupling in anesthetized rats.

Cardiorespiratory coupling can be significantly influenced by both pontine and vagal modulation of medullary motor and premotor areas. We investigated influences of the pontine intertrigeminal region (ITR) and peripheral vagal pathways on the coupling between systolic blood pressure (SBP) and respiration in 9 anesthetized rats. Glutamate injection into the ITR perturbed both respiration and SBP and decreased SBP-respiratory coherence (0.95±0.01 vs 0.89±0.02; (p=0.01). Intravenous infusion of serotonin (5-HT) produced apnea and hypertension and also decreased SBP-respiratory coherence (0.95±0.01 vs 0.72±0.06; p=0.04). Bilateral vagotomy eliminated the cardiorespiratory coherence perturbations induced by central (glutamate injection into the ITR: 0.89±0.03 vs 0.86±0.03; p=0.63) and peripheral (5-HT infusion: 0.89±0.03 vs 0.88±0.02; p=0.98) pharmacologic manipulations. Glutamate stimulation of the ITR postvagotomy increased the relative spectral power density of SBP in the respiratory frequency range (0.25±0.08 vs 0.55±0.06; p=0.01). The data suggest that SBP-respiratory coupling is largely mediated within the central nervous system, with vagal systems acting in a way that disrupts coherence during transient cardiorespiratory disturbances. Although decreased cardiorespiratory coherence may increase cardiac work during perturbations, this may be physiologically advantageous in restoring homeostatic equilibrium of respiration and blood pressure.

Functional topography of respiratory, cardiovascular and pontine-wave responses to glutamate microstimulation of the pedunculopontine tegmentum of the rat.

Functionally distinct areas were mapped within the pedunculopontine tegmentum (PPT) of 42 ketamine/xylazine anesthetized rats using local stimulation by glutamate microinjection (10 mM, 5-12 nl). Functional responses were classified as: (1) apnea; (2) tachypnea; (3) hypertension (HTN); (4) sinus tachycardia; (5) genioglossus electromyogram activation or (6) pontine-waves (p-waves) activation.We found that short latency apneas were predominantly elicited by stimulation in the lateral portion of the PPT, in close proximity to cholinergic neurons. Tachypneic responses were elicited from ventral regions of the PPT and HTN predominated in the ventral portion of the antero-medial PPT. We observed sinus tachycardia after stimulation of the most ventral part of the medial PPT at the boundary with nucleus reticularis pontis oralis, whereas p-waves were registered predominantly following stimulation in the dorso-caudal portion of the PPT. Genioglossus EMG activation was evoked from the medial PPT. Our results support the existence of the functionally distinct areas within the PPT affecting respiration, cardiovascular function, EEG and genioglossus EMG.

Cardiorespiratory effects of intertrigeminal area stimulation in vagotomized rats.

It has been recently shown that the pontine intertrigeminal region (ITR) plays an important role in respiratory regulation, including vagally mediated apneic reflexes. Neurons of the ITR have connections with the nucleus tractus solitarius and projections to the ventrolateral medulla. However, the functional targets of these projections are not fully defined. Stimulation of ITR neurons produced respiratory effects, but cardiovascular responses have not been explored. We investigated impact of bilateral vagotomy on respiratory and cardiovascular responses to glutamate microinjections within the ITR in ketamine/xylazine anesthetized rats. Cardiorespiratory indices, including breath duration (TT), tidal volume (VT), mean cardiac intervals (RR), systolic blood pressure (SBP), pulse pressure (PP) and their coefficients of variation (CVTT, CVVT, CVSBP, CVPP, respectively) were analyzed in 30 s segments before and after injection of glutamate (10 mM, 30 L) into the ITR. This assessment was carried out both before and after bilateral vagotomy. Glutamate injection evoked apnea and increased CVTT, but these responses were not altered by bilateral vagotomy. In contrast, removing vagal pathways significantly increased volume variability (CVVT), making tidal volume more vulnerable to perturbation from the ITR. Vagotomy prolonged the increase of mean systolic blood pressure observed after glutamate injection and unmasked a delayed but sustained elevation of PP and CVPP after ITR stimulation. The present findings indicate a broad involvement of the ITR in autonomic regulation, including at least cardiovascular and respiratory effects.

Conditioned lick behavior and evoked responses using whisker twitches in head restrained rats.

To examine whisker barrel evoked response potentials in chronically implanted rats during behavioral learning with very fast response times, rats must be calm while immobilized with their head restrained. We quantified their behaviors during training with an ethogram and measured each individual animals' progress over the training period. Once calm under restraint, rats were conditioned to differentiate between a reward and control whisker twitch, then provide a lick response when presented with the correct stimulus, rewarded by a drop of water. Rats produced the correct licking response (after reward whisker twitch), and learned not to lick after a control whisker was twitched. By implementing a high-density 64-channel electrocorticogram (ECoG) electrode array, we mapped the barrel field of the somatosensory cortex with high spatial and temporal resolution during conditioned lick behaviors. In agreement with previous reports, we observe a larger evoked response after training, probably related to mechanisms of cortical plasticity.

The effect of atropine administered in the medial septum or hippocampus on high- and low-frequency theta rhythms in the hippocampus of urethane anesthetized rats.

Cholinergic mechanisms are critical for the generation of hippocampal theta rhythm. Cholinergic innervation of the hippocampus originates from the medial septum (MS) and cholinergic receptors are expressed in both the MS and hippocampus. In this study, we compared the effects of the muscarinic receptor antagonist atropine in the MS and the hippocampus on theta generation. Hippocampal theta rhythm was elicited by electrical stimulation of the pontine reticular formation using series of stimuli with varying intensities. Atropine was administered either systemically (50 mg/kg i.p.) or locally in the MS (microdialysis; 25 and 75 mM for 30 or 90 min) or in the hippocampus on one side (microinjection; 20 or 40 ug). The relative power at the peak theta frequency was calculated and averaged over episodes of low-intensity and high-intensity stimulations. We found that atropine drastically reduced theta rhythmic synchronization when injected in either location. After MS administration of atropine, however, high-frequency theta elicited by high-intensity stimuli was more resistant (58% and 67% decrease after 25 mM and 75 mM atropine, respectively) than slow theta elicited by low-intensity stimuli (86% and 91% decrease). There was no significant difference between the powers of the two oscillations after hippocampal injections (70-75% decrease). We conclude that the theta suppressing effect of atropine involves both hippocampal and septal mechanisms and that low-frequency theta as compared with fast theta rhythm is more sensitive to muscarinic acetylcholine receptor antagonism in the MS but not in the hippocampus.

Local functional state differences between rat cortical columns.

Surface evoked potentials (SEPs) during auditory clicks and whisker twitches are usually larger during quiet sleep (QS) over waking and REM sleep. However, SEP amplitudes from single trials fluctuate periodically between high and low values regardless of sleep-wake cycle. To test the hypothesis that state-independent fluctuations represent local functional sleep-like states of individual cortical columns, we examined single trial SEP amplitudes from multiple cortical locations across sleep-wake cycles. Bilateral stimuli produced SEP amplitude fluctuations in each hemisphere that usually covaried (r = 0.4), but with frequent hemispheric differences. Two neighboring whiskers, twitched simultaneously on the same side, produced highly correlated SEPs in neighboring cortical columns (r = 0.9) with frequent divergences. We found 50% more disparity during QS over waking, indicating that the differences did not result from recording noise or stimulus inconsistency. Local SEP fluctuations also followed local differences in the delta wave signal during QS (r = 0.4), suggesting that similar mechanisms may modulate the SEP. The duration of the localized sleep-like (high SEP amplitude) state was dependent on the duration of prior wake-like (low SEP amplitude) state (r = 0.5), suggesting a use dependence of prior functional state period. Since SEP indicators fluctuated independently from whole animal sleep state, and were frequently different between hemispheres and nearby cortical columns, these data support the theory that sleep-like functional states may be localized to brain regions at least as small as cortical columns.