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Background: Chemogenetics is a powerful tool to study the role of specific neuronal populations in physiology and diseases. Of particular interest, in mice, acute and specific activation of parafacial zone (PZ) GABAergic neurons expressing the Designer Receptors Activated by Designer Drugs (DREADD) hM3Dq (PZ GABA-hM3Dq ) enhances slow-wave-sleep (SWS), and this effect lasts for up to 6 h, allowing prolonged and detailed study of SWS. However, the most widely used DREADDs ligand, clozapine N-oxide (CNO), is metabolized into clozapine which has the potential of inducing non-specific effects. In addition, CNO is usually injected intraperitoneally (IP) in mice, limiting the number and frequency of repeated administration., New Methods: The present study is designed to validate the use of alternative DREADDs ligands-deschloroclozapine (DCZ) and compound 21 (C21)-and a new administration route, the voluntary oral administration., Results: We show that IP injections of DCZ and C21 dose-dependently enhance SWS in PZ GABA-hM3Dq mice, similar to CNO. We also show that oral administration of CNO, DCZ and C21 induces the same sleep phenotype as compared with IP injection., Comparison With Existing Methods and Conclusion: Therefore, DCZ and C21 are powerful alternatives to the use of CNO. Moreover, the voluntary oral administration is suitable for repeated dosing of DREADDs ligands., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Many newborns are investigated and empirically treated for suspected early-onset sepsis (EOS). This study aimed to describe neonatologists' self-identified risk thresholds for investigating and treating EOS and assess the consistency of these thresholds with clinical decisions., Study Design: Voluntary online survey, available in two randomized versions, sent to neonatologists from 20 centers of the Brazilian Network on Neonatal Research. The surveys included questions about thresholds for investigating and treating EOS and presented four clinical scenarios with varying calculated risks. In survey version A, only the scenarios were presented, and participants were asked if they would order a blood test or start antibiotics. Survey version B presented the same scenarios and the risk of sepsis. Clinical decisions were compared between survey versions using chi-square tests and agreement between thresholds and clinical decisions were investigated using Kappa coefficients., Results: In total, 293 surveys were completed (145 survey version A and 148 survey version B). The median risk thresholds for blood test and antibiotic treatment were 1:100 and 1:25, respectively. In the high-risk scenario, there was no difference in the proportion choosing antibiotic therapy between the groups. In the moderate-risk scenarios, both tests and antibiotics were chosen more frequently when the calculated risks were included (survey version B). In the low-risk scenario, there was no difference between survey versions. There was poor agreement between the self-described thresholds and clinical decisions., Conclusion: Neonatologists overestimate the risk of EOS and underestimate their risk thresholds. Knowledge of calculated risk may increase laboratory investigation and antibiotic use in infants at moderate risk for EOS., Key Points: · Neonatologists overestimate the risk of EOS.. · There is wide variation in diagnostic/treatment thresholds for EOS.. · Clinical decision on EOS is not consistent with risk thresholds.. · Knowledge of risk may increase investigation and treatment of EOS.., Competing Interests: None declared., (Thieme. All rights reserved.)

Aging and Alzheimer's disease (AD) are both associated with reduced quantity and quality of the deepest stage of sleep, called slow-wave-sleep (SWS). Slow-wave-sleep deficits have been shown to worsen AD symptoms and prevent healthy aging. However, the mechanism remains poorly understood due to the lack of animal models in which SWS can be specifically manipulated. Notably, a mouse model of SWS enhancement has been recently developed in adult mice. As a prelude to studies assessing the impact of SWS enhancement on aging and neurodegeneration, we first asked whether SWS can be enhanced in animal models of aging and AD. The chemogenetic receptor hM3Dq was conditionally expressed in GABAergic neurons of the parafacial zone of aged mice and AD (APP/PS1) mouse model. Sleep-wake phenotypes were analyzed in baseline condition and following clozapine- N -oxide (CNO) and vehicle injections. Both aged and AD mice display deficits in sleep quality, characterized by decreased slow wave activity. Both aged and AD mice show SWS enhancement following CNO injection, characterized by a shorter SWS latency, increased SWS amount and consolidation, and enhanced slow wave activity, compared with vehicle injection. Importantly, the SWS enhancement phenotypes in aged and APP/PS1 model mice are comparable to those seen in adult and littermate wild-type mice, respectively. These mouse models will allow investigation of the role of SWS in aging and AD, using, for the first time, gain-of SWS experiments., (© The Author(s) 2022. Published by Oxford University Press on behalf of Sleep Research Society.)

Background and Objectives: The Brazilian Neonatal Resuscitation Program releases guidelines based on local interpretation of international consensus on science and treatment recommendations. We aimed to analyze whether guidelines for preterm newborns were applied to practice in the 20 Brazilian Network on Neonatal Research centers of this middle-income country., Methods: Prospectively collected data from 2014 to 2020 were analyzed for 8514 infants born at 230/7 to 316/7 weeks' gestation. The frequency of procedures was evaluated by gestational age (GA) category, including use of a thermal care bundle, positive pressure ventilation (PPV), PPV with a T-piece resuscitator, maximum fraction of inspired oxygen (Fio2) concentration during PPV, tracheal intubation, chest compressions and medications, and use of continuous positive airway pressure in the delivery room. Logistic regression, adjusted by center and year, was used to estimate the probability of receiving recommended treatment., Results: For 3644 infants 23 to 27 weeks' GA and 4870 infants 28 to 31 weeks' GA, respectively, the probability of receiving care consistent with guidelines per year increased, including thermal care (odds ratio [OR], 1.52 [95% confidence interval (CI) 1.44-1.61] and 1.45 [1.38-1.52]) and PPV with a T-piece (OR, 1.45 [95% CI 1.37-1.55] and 1.41 [1.32-1.51]). The probability of receiving PPV with Fio2 1.00 decreased equally in both GA groups (OR, 0.89; 95% CI, 0.86-0.93)., Conclusions: Between 2014 and 2020, the resuscitation guidelines for newborns <32 weeks' GA on thermal care, PPV with a T-piece resuscitator, and decreased use of Fio2 1.00 were translated into clinical practice., (Copyright © 2022 by the American Academy of Pediatrics.)

Parafacial zone (PZ) GABAergic neurons play a major role in slow-wave-sleep (SWS), also called non-rapid eye movement (NREM) sleep. The PZ also contains glutamatergic neurons expressing the vesicular transporter for glutamate, isoform 2 (Vglut2). We hypothesized that PZ Vglut2-expressing (PZ Vglut2 ) neurons are also involved in sleep control, playing a synergistic role with PZ GABAergic neurons. To test this hypothesis, we specifically activated PZ Vglut2 neurons using the excitatory chemogenetic receptor hM3Dq. Anatomical inspection of the injection sites revealed hM3Dq transfection in PZ, parabrachial nucleus (PB), sublaterodorsal nucleus (SLD) or various combinations of these three brain areas. Consistent with the known wake- and REM sleep-promoting role of PB and SLD, respectively, chemogenetic activation of PB Vglut2 or SLD Vglut2 resulted in wake or REM sleep enhancement. Chemogenetic activation of PZ Vglut2 neurons did not affect sleep-wake phenotype during the mouse active period but increased wakefulness and REM sleep, similar to PB Vglut2 and SLD Vglut2 activation, during the rest period. To definitively confirm the role of PZ Vglut2 neurons, we used a specific marker for PZ Vglut2 neurons, Phox2B. Chemogenetic activation of PZ Phox2B neurons did not affect sleep-wake phenotype, indicating that PZ glutamatergic neurons are not sufficient to affect sleep-wake cycle. These results indicate that PZ glutamatergic neurons are not involved in sleep-wake control.

The preoptic area (POA) is necessary for sleep, but the fundamental POA circuits have remained elusive. Previous studies showed that galanin (GAL)- and GABA-producing neurons in the ventrolateral preoptic nucleus (VLPO) express cFos after periods of increased sleep and innervate key wake-promoting regions. Although lesions in this region can produce insomnia, high frequency photostimulation of the POA GAL neurons was shown to paradoxically cause waking, not sleep. Here we report that photostimulation of VLPO GAL neurons in mice promotes sleep with low frequency stimulation (1-4 Hz), but causes conduction block and waking at frequencies above 8 Hz. Further, optogenetic inhibition reduces sleep. Chemogenetic activation of VLPO GAL neurons confirms the increase in sleep, and also reduces body temperature. In addition, chemogenetic activation of VLPO GAL neurons induces short-latency sleep in an animal model of insomnia. Collectively, these findings establish a causal role of VLPO GAL neurons in both sleep induction and heat loss.

Orexin (also known as hypocretin) neurons are considered a key component of the ascending arousal system. They are active during wakefulness, at which time they drive and maintain arousal, and are silent during sleep. Their activity is controlled by long-range inputs from many sources, as well as by more short-range inputs, including from presumptive GABAergic neurons in the lateral hypothalamus/perifornical region (LH/PF). To characterize local GABAergic input to orexin neurons, we used channelrhodopsin-2-assisted circuit mapping in brain slices. We expressed channelrhodopsin-2 in GABAergic neurons (Vgat + ) in the LH/PF and recorded from genetically identified surrounding orexin neurons (LH/PF Vgat → Orx). We performed all experiments in mice of either sex. Photostimulation of LH/PF GABAergic neurons inhibited the firing of orexin neurons through the release of GABA, evoking GABA A -mediated IPSCs in orexin neurons. These photo-evoked IPSCs were maintained in the presence of TTX, indicating direct connectivity. Carbachol inhibited LH/PF Vgat → Orx input through muscarinic receptors. By contrast, application of orexin was without effect on LH/PF Vgat → Orx input, whereas dynorphin, another peptide produced by orexin neurons, inhibited LH/PF Vgat → Orx input through κ-opioid receptors. Our results demonstrate that orexin neurons are under inhibitory control by local GABAergic neurons and that this input is depressed by cholinergic signaling, unaffected by orexin and inhibited by dynorphin. We propose that local release of dynorphin may, via collaterals, provides a positive feedback to orexin neurons and that, during wakefulness, orexin neurons may be disinhibited by acetylcholine and by their own release of dynorphin. SIGNIFICANCE STATEMENT The lateral hypothalamus contains important wake-promoting cell populations, including orexin-producing neurons. Intermingled with the orexin neurons, there are other cell populations that selectively discharge during nonrapid eye movement or rapid eye movement sleep. Some of these sleep-active neurons release GABA and are thought to inhibit wake-active neurons during rapid eye movement and nonrapid eye movement sleep. However, this hypothesis had not been tested. Here we show that orexin neurons are inhibited by a local GABAergic input. We propose that this local GABAergic input inhibits orexin neurons during sleep but that, during wakefulness, this input is depressed, possibly through cholinergically mediated disinhibition and/or by release of dynorphin from orexin neurons themselves., (Copyright © 2018 the authors 0270-6474/18/381588-12$15.00/0.)

The orexin (hypocretin) neurons play an essential role in promoting arousal, and loss of the orexin neurons results in narcolepsy, a condition characterized by chronic sleepiness and cataplexy. The orexin neurons excite wake-promoting neurons in the basal forebrain (BF), and a reciprocal projection from the BF back to the orexin neurons may help promote arousal and motivation. The BF contains at least three different cell types (cholinergic, glutamatergic, and γ-aminobutyric acid (GABA)ergic neurons) across its different regions (medial septum, diagonal band, magnocellular preoptic area, and substantia innominata). Given the neurochemical and anatomical heterogeneity of the BF, we mapped the pattern of BF projections to the orexin neurons across multiple BF regions and neuronal types. We performed conditional anterograde tracing using mice that express Cre recombinase only in neurons producing acetylcholine, glutamate, or GABA. We found that the orexin neurons are heavily apposed by axon terminals of glutamatergic and GABAergic neurons of the substantia innominata (SI) and magnocellular preoptic area, but there was no innervation by the cholinergic neurons. Channelrhodopsin-assisted circuit mapping (CRACM) demonstrated that glutamatergic SI neurons frequently form functional synapses with the orexin neurons, but, surprisingly, functional synapses from SI GABAergic neurons were rare. Considering their strong reciprocal connections, BF and orexin neurons likely work in concert to promote arousal, motivation, and other behaviors. J. Comp. Neurol. 525:1668-1684, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The pedunculopontine tegmental (PPT) nucleus has long been implicated in the regulation of cortical activity and behavioral states, including rapid eye-movement (REM) sleep. For example, electrical stimulation of the PPT region during sleep leads to rapid awakening, whereas lesions of the PPT in cats reduce REM sleep. Though these effects have been linked with the activity of cholinergic PPT neurons, the PPT also includes intermingled glutamatergic and GABAergic cell populations, and the precise roles of cholinergic, glutamatergic, and GABAergic PPT cell groups in regulating cortical activity and behavioral state remain unknown. Using a chemogenetic approach in three Cre-driver mouse lines, we found that selective activation of glutamatergic PPT neurons induced prolonged cortical activation and behavioral wakefulness, whereas inhibition reduced wakefulness and increased non-REM (NREM) sleep. Activation of cholinergic PPT neurons suppressed lower-frequency electroencephalogram rhythms during NREM sleep. Last, activation of GABAergic PPT neurons slightly reduced REM sleep. These findings reveal that glutamatergic, cholinergic, and GABAergic PPT neurons differentially influence cortical activity and sleep/wake states., Significance Statement: More than 40 million Americans suffer from chronic sleep disruption, and the development of effective treatments requires a more detailed understanding of the neuronal mechanisms controlling sleep and arousal. The pedunculopontine tegmental (PPT) nucleus has long been considered a key site for regulating wakefulness and REM sleep. This is mainly because of the cholinergic neurons contained in the PPT nucleus. However, the PPT nucleus also contains glutamatergic and GABAergic neurons that likely contribute to the regulation of cortical activity and sleep-wake states. The chemogenetic experiments in the present study reveal that cholinergic, glutamatergic, and GABAergic PPT neurons each have distinct effects on sleep/wake behavior, improving our understanding of how the PPT nucleus regulates cortical activity and behavioral states., (Copyright © 2017 the authors 0270-6474/17/371352-15$15.00/0.)

Currently available evidence indicates that neurons containing melanin-concentrating hormone (MCH) in the lateral hypothalamus are critical modulators of sleep-wakefulness, but their precise role in this function is not clear. Studies employing optogenetic stimulation of MCH neurons have yielded inconsistent results, presumably due to differences in the optogenetic stimulation protocols, which do not approximate normal patterns of cell firing. In order to resolve this discrepancy, we (1) selectively activated the MCH neurons using a chemogenetic approach (Cre-dependent hM3Dq expression) and (2) selectively destroyed MCH neurons using a genetically targeted diphtheria toxin deletion method, and studied the changes in sleep-wake in mice. Our results indicate that selective activation of MCH neurons causes specific increases in rapid eye movement (REM) sleep without altering wake or non-REM (NREM) sleep. On the other hand, selective deletions of MCH neurons altered the diurnal rhythm of wake and REM sleep without altering their total amounts. These results indicate that activation of MCH neurons primarily drives REM sleep and their presence may be necessary for normal expression of diurnal variation of REM sleep and wake., Competing Interests: The authors declare no competing financial interests., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Key Points: The basal forebrain is an important component of the ascending arousal system and may be a key site through which the orexin neurons promote arousal. It has long been known that orexin-A and -B excite basal forebrain cholinergic neurons, but orexin-producing neurons also make the inhibitory peptide dynorphin. Using whole-cell recordings in brain slices, we found that dynorphin-A directly inhibits basal forebrain cholinergic neurons via κ-opioid receptors, and decreases afferent excitatory synaptic input to these neurons. While the effects of dynorphin-A and orexin-A desensitize over multiple applications, co-application of dynorphin-A and orexin-A produces a sustained response that reverses depending on the membrane potential of basal forebrain cholinergic neurons. At -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails., Abstract: The basal forebrain (BF) is an essential component of the ascending arousal systems and may be a key site through which the orexin (also known as hypocretin) neurons drive arousal and promote the maintenance of normal wakefulness. All orexin neurons also make dynorphin, and nearly all brain regions innervated by the orexin neurons express kappa opiate receptors, the main receptor for dynorphin. This is remarkable because orexin excites target neurons including BF neurons, but dynorphin has inhibitory effects. We identified the sources of dynorphin input to the magnocellular preoptic nucleus and substantia innominata (MCPO/SI) in mice and determined the effects of dynorphin-A on MCPO/SI cholinergic neurons using patch-clamp recordings in brain slices. We found that the orexin neurons are the main source of dynorphin input to the MCPO/SI region, and dynorphin-A inhibits MCPO/SI cholinergic neurons through κ-opioid receptors by (1) activation of a G protein-coupled inwardly rectifying potassium current, (2) inhibition of a voltage-gated Ca(2+) current and (3) presynaptic depression of the glutamatergic input to these neurons. The responses both to dynorphin-A and to orexin-A desensitize, but co-application of dynorphin-A and orexin-A produces a sustained response. In addition, the polarity of the response to the co-application depends on the membrane potential of BF neurons; at -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails. This suggests that depending on their state of activation, BF cholinergic neurons can be excited or inhibited by signals from the orexin neurons., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Wakefulness, along with fast cortical rhythms and associated cognition, depend on the basal forebrain (BF). BF cholinergic cell loss in dementia and the sedative effect of anti-cholinergic drugs have long implicated these neurons as important for cognition and wakefulness. The BF also contains intermingled inhibitory GABAergic and excitatory glutamatergic cell groups whose exact neurobiological roles are unclear. Here we show that genetically targeted chemogenetic activation of BF cholinergic or glutamatergic neurons in behaving mice produced significant effects on state consolidation and/or the electroencephalogram but had no effect on total wake. Similar activation of BF GABAergic neurons produced sustained wakefulness and high-frequency cortical rhythms, whereas chemogenetic inhibition increased sleep. Our findings reveal a major contribution of BF GABAergic neurons to wakefulness and the fast cortical rhythms associated with cognition. These findings may be clinically applicable to manipulations aimed at increasing forebrain activation in dementia and the minimally conscious state.

Objective: To analyze the effects of treatment approach on the outcomes of newborns (birth weight [BW] < 1,000 g) with patent ductus arteriosus (PDA), from the Brazilian Neonatal Research Network (BNRN) on: death, bronchopulmonary dysplasia (BPD), severe intraventricular hemorrhage (IVH III/IV), retinopathy of prematurity requiring surgical (ROPsur), necrotizing enterocolitis requiring surgery (NECsur), and death/BPD., Methods: This was a multicentric, cohort study, retrospective data collection, including newborns (BW < 1000 g) with gestational age (GA) < 33 weeks and echocardiographic diagnosis of PDA, from 16 neonatal units of the BNRN from January 1, 2010 to Dec 31, 2011. Newborns who died or were transferred until the third day of life, and those with presence of congenital malformation or infection were excluded. Groups: G1 - conservative approach (without treatment), G2 - pharmacologic (indomethacin or ibuprofen), G3 - surgical ligation (independent of previous treatment). Factors analyzed: antenatal corticosteroid, cesarean section, BW, GA, 5 min. Apgar score < 4, male gender, Score for Neonatal Acute Physiology Perinatal Extension (SNAPPE II), respiratory distress syndrome (RDS), late sepsis (LS), mechanical ventilation (MV), surfactant (< 2 h of life), and time of MV., Outcomes: death, O2 dependence at 36 weeks (BPD36wks), IVH III/IV, ROPsur, NECsur, and death/BPD36wks., Statistics: Student's t-test, chi-squared test, or Fisher's exact test; Odds ratio (95% CI); logistic binary regression and backward stepwise multiple regression. Software: MedCalc (Medical Calculator) software, version 12.1.4.0. p-values < 0.05 were considered statistically significant., Results: 1,097 newborns were selected and 494 newborns were included: G1 - 187 (37.8%), G2 - 205 (41.5%), and G3 - 102 (20.6%). The highest mortality was observed in G1 (51.3%) and the lowest in G3 (14.7%). The highest frequencies of BPD36wks (70.6%) and ROPsur were observed in G3 (23.5%). The lowest occurrence of death/BPD36wks occurred in G2 (58.0%). Pharmacological (OR 0.29; 95% CI: 0.14-0.62) and conservative (OR 0.34; 95% CI: 0.14-0.79) treatments were protective for the outcome death/BPD36wks., Conclusion: The conservative approach of PDA was associated to high mortality, the surgical approach to the occurrence of BPD36wks and ROPsur, and the pharmacological treatment was protective for the outcome death/BPD36wks., (Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.)

Histaminergic neurons in the tuberomammillary nucleus (TMN) are an important component of the ascending arousal system and may form part of a "flip-flop switch" hypothesized to regulate sleep and wakefulness. Anatomical studies have shown that the wake-active TMN and sleep-active ventrolateral preoptic nucleus (VLPO) are reciprocally connected, suggesting that each region can inhibit its counterpart when active. In this study, we determined how histamine affects the two branches of this circuit. We selectively expressed channelrhodopsin-2 (ChR2) in TMN neurons and used patch-clamp recordings in mouse brain slices to examine the effects of photo-evoked histamine release in the ventrolateral TMN and VLPO. Photostimulation decreased inhibitory GABAergic inputs to the ventrolateral TMN neurons but produced a membrane hyperpolarization and increased inhibitory synaptic input to the VLPO neurons. We found that in VLPO the response to histamine was indirect, most likely via a GABAergic interneuron. Our experiments demonstrate that release of histamine from TMN neurons can disinhibit the TMN and suppresses the activity of sleep-active VLPO neurons to promote TMN neuronal firing. This further supports the sleep-wake "flip-flop switch" hypothesis and a role for histamine in stabilizing the switch to favor wake states.

Avoidable infant mortality was compared in the cohorts of live births in 2000/20001 and 2007/2008 in Londrina in the state of Paraná. Data on live births and infant deaths were matched in a single database and the underlying causes of death were classified according to the Brazilian List of Avoidable Causes of Mortality of the Brazilian Unified Health System. The proportional variations of death rates were calculated and the technique of correspondence analysis was used. The proportions of avoidable deaths were 71.6% in 2000/2001 and 65.5% in 2007/2008. Avoidable mortality rate due to "care of the woman during pregnancy" was the most frequent in the two biennials with an increase of 16.7%. There was a decline in avoidable mortality rates due to other causes. In 2000/2001, correspondence analysis revealed a profile of deaths characterized mainly by causes avoidable by "care of the woman during labor" and "health promotion/care" while, in 2007/2008, by causes avoidable by "care of the woman during pregnancy," particularly among low birth weight newborns and "not clearly avoidable," among those with adequate weight. Despite a reduction in some avoidable causes of deaths, improvements in prenatal care are required.

Adenosine has been proposed as an endogenous homeostatic sleep factor that accumulates during waking and inhibits wake-active neurons to promote sleep. It has been specifically hypothesized that adenosine decreases wakefulness and promotes sleep recovery by directly inhibiting wake-active neurons of the basal forebrain (BF), particularly BF cholinergic neurons. We previously showed that adenosine directly inhibits BF cholinergic neurons. Here, we investigated 1) how adenosine modulates glutamatergic input to BF cholinergic neurons and 2) how adenosine uptake and adenosine metabolism are involved in regulating extracellular levels of adenosine. Our experiments were conducted using whole cell patch-clamp recordings in mouse brain slices. We found that in BF cholinergic neurons, adenosine reduced the amplitude of AMPA-mediated evoked glutamatergic excitatory postsynaptic currents (EPSCs) and decreased the frequency of spontaneous and miniature EPSCs through presynaptic A(1) receptors. Thus we have demonstrated that in addition to directly inhibiting BF cholinergic neurons, adenosine depresses excitatory inputs to these neurons. It is therefore possible that both direct and indirect inhibition may synergistically contribute to the sleep-promoting effects of adenosine in the BF. We also found that blocking the influx of adenosine through the equilibrative nucleoside transporters or inhibiting adenosine kinase and adenosine deaminase increased endogenous adenosine inhibitory tone, suggesting a possible mechanism through which adenosine extracellular levels in the basal forebrain are regulated.