Unilateral Optogenetic Stimulation of Lhx6 Neurons in the Zona Incerta Increases REM sleep.

To determine how a waking brain falls asleep researchers have monitored and manipulated activity of neurons and glia in various brain regions. While imaging GABA neurons in the zona incerta (ZI) we found a subgroup that anticipates onset of NREM sleep 1. To differentiate the GABA subtype we now image and optogenetically manipulate the ZI neurons containing the transcription factor, Lhx6. In the first study Lhx6-cre mice (n=5; female=4) were given rAAV-DJ-EF1a-DIO-GCaMP6M into the ZI (isofluorane anesthesia), a GRIN lens implanted, and 21d later sleep and fluorescence in individual Lhx6 neurons were recorded for 4h. Calcium fluorescence was detected in 132 neurons. 45.5% of the Lhx6 neurons were REM-max; 30.3% were Wake-max; 11.4% were wake+REM max; 9% were NREM-max; and 3.8% had no change. The NREM-max group of neurons fluoresced 30s ahead of sleep onset. The second study tested the effects of unilateral optogenetic stimulation of the ZI Lhx6 neurons (n=14 mice) (AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato]. Stimulation at 1 and 5Hz (1 minute on- 4 minute off) significantly increased percent REM sleep during the 4h stimulation period (last half of day cycle). The typical experimental approach is to stimulate neurons in both hemispheres, but here we found that low frequency stimulation of ZI Lhx6 neurons in one hemisphere is sufficient to shift states of consciousness. Detailed mapping combined with mechanistic testing is necessary to identify local nodes that can shift the brain between wake-sleep states.

Asma en el adulto: características clínicas, comorbilidades y riesgo para apnea obstructiva de sueño / Asthma in adults: clinical characteristics, comorbidities and risk for obstructivesleep apnea

Introducción:Escasos reportes sobre asma existen en la literatura nacional. El objetivo delestudio es describir ciertas características clínicas, las comorbilidades y el riesgo para apnea obstructiva del sueño (AOS) en este colectivo. Material y métodos:Mediante un estudio observacional tipo caso-control, se incluyeron pacientes portadores de asma de ambos sexos y un grupo control pareado por sexo y edad. Se consignaron datos demográficos, características de la enfermedad, datos relativos al control del asma y espirometría, comorbilidades y riesgo de apnea de sueño medido por el cuestionario STOP-BANG durante una entrevista estructurada. Resultados: Se incluyeron 132 individuos en el grupo caso y 132 en el grupo control. Entre los asmáticos, se consignaron 38,63% de formas no controladas y 9% severas. También se constató mayor frecuencia dehipertensión arterial, obesidad, rinitis alérgicay trastornos de la memoria. Riesgo mayor para AOS, aunque no significativo, se consignó en el grupo de casos. En el subgrupo de asmáticos con obesidad o en adultos mayores, la diferencia fue estadísticamente significativa-Discusión:Aundisponiendo de medicamentos preventivos,la tasa de control de los asmáticos en esta muestra es llamativa y merece estudios sistemáticos. Es extremadamente importante tener en cuenta algunas comorbilidades para el manejo de este grupo de pacientes,incluyendo la estratificación de riesgo para AOS.Palabras clave:asma; comorbilidad; apnea obstructiva del sueño

Introduction.Few reports on asthma exist in the national literature. The objective of our study is to describe certain clinical characteristics, comorbidities, and risk for obstructive sleep apnea (OSA) in this group. Material and methods.Through an observational case-control study, patients with asthma of both sexes and a control group matched by sex and age were included. Demographic data, disease characteristics, data related to asthma control and spirometry, comorbidities, and risk of sleep apnea measured by the STOP-BANG questionnaire were recorded during a structured interview. Results.132 individuals were included in the case group and 132 in the control group. Among the asthmatics, 38.63% of uncontrolled forms and 9% were severe. There was also a higher frequency of arterial hypertension, obesity, allergic rhinitis and memory disorders. Higher risk for OSA, although not significant, was recorded in the case group. In the subgroup of asthmatics with obesity or in older adults, the difference was relevant to the statistical analysis. Discussion.Even with preventive medications available, the control rate of asthmatics in this sample is striking and deserves systematic studies. It is extremely important to take into account some comorbidities for the management ofthis group of patients, including risk stratification for OSA.Key Words:asthma; comorbidity;obstructive sleep apnea

Activity of GABA neurons in the zona incerta and ventral lateral periaqueductal grey is biased towards sleep.

STUDY OBJECTIVES: As in various brain regions the activity of gamma-aminobutyric acid (GABA) neurons is largely unknown, we measured in vivo changes in calcium fluorescence in GABA neurons in the zona incerta (ZI) and the ventral lateral periaqueductal grey (vlPAG), two areas that have been implicated in regulating sleep. METHODS: vGAT-Cre mice were implanted with sleep electrodes, microinjected with rAAV-DIO-GCaMP6 into the ZI (n = 6) or vlPAG (n = 5) (isoflurane anesthesia) and a GRIN (Gradient-Index) lens inserted atop the injection site. Twenty-one days later, fluorescence in individual vGAT neurons was recorded over multiple REM cycles. Regions of interest corresponding to individual vGAT somata were automatically extracted with PCA-ICA analysis. RESULTS: In the ZI, 372 neurons were identified. Previously, we had recorded the activity of 310 vGAT neurons in the ZI and we combined the published dataset with the new dataset to create a comprehensive dataset of ZI vGAT neurons (total neurons = 682; mice = 11). In the vlPAG, 169 neurons (mice = 5) were identified. In both regions, most neurons were maximally active in REM sleep (R-Max; ZI = 51.0%, vlPAG = 60.9%). The second most abundant group was W-Max (ZI = 23.9%, vlPAG = 25.4%). In the ZI, but not in vlPAG, there were neurons that were NREMS-Max (11.7%). vlPAG had REMS-Off neurons (8.3%). In both areas, there were two minor classes: wake/REMS-Max and state indifferent. In the ZI, the NREMS-Max neurons fluoresced 30 s ahead of sleep onset. CONCLUSIONS: These descriptive data show that the activity of GABA neurons is biased in favor of sleep in two brain regions implicated in sleep.

Mapping Network Activity in Sleep.

It was in the influenza pandemic of 1918 that von Economo identified specific brain regions regulating sleep and wake. Since then researchers have used a variety of tools to determine how the brain shifts between states of consciousness. In every enterprise new tools have validated existing data, corrected errors and made new discoveries to advance science. The brain is a challenge but new tools can disentangle the brain network. We summarize the newest tool, a miniature microscope, that provides unprecedented view of activity of glia and neurons in freely behaving mice. With this tool we have observed that the activity of a majority of GABA and MCH neurons in the lateral hypothalamus is heavily biased toward sleep. We suggest that miniscope data identifies activity at the cellular level in normal versus diseased brains, and also in response to specific hypnotics. Shifts in activity in small networks across the brain will help identify point of criticality that switches the brain from wake to sleep.

Activity of a subset of vesicular GABA-transporter neurons in the ventral zona incerta anticipates sleep onset.

STUDY OBJECTIVES: Sleep and wake are opposing behavioral states controlled by the activity of specific neurons that need to be located and mapped. To better understand how a waking brain falls asleep it is necessary to identify activity of individual phenotype-specific neurons, especially neurons that anticipate sleep onset. In freely behaving mice, we used microendoscopy to monitor calcium (Ca2+) fluorescence in individual hypothalamic neurons expressing the vesicular GABA transporter (vGAT), a validated marker of GABA neurons. METHODS: vGAT-Cre mice (male = 3; female = 2) transfected with rAAV-FLEX-GCaMP6M in the lateral hypothalamus were imaged 30 days later during multiple episodes of waking (W), non-rapid-eye movement sleep (NREMS) or REMS (REMS). RESULTS: 372 vGAT neurons were recorded in the zona incerta. 23.9% of the vGAT neurons showed maximal fluorescence during wake (classified as wake-max), 4% were NREM-max, 56.2% REM-max, 5.9% wake/REM max, while 9.9% were state-indifferent. In the NREM-max group, Ca2+ fluorescence began to increase before onset of NREM sleep, remained high throughout NREM sleep, and declined in REM sleep. CONCLUSIONS: We found that 60.2% of the vGAT GABA neurons in the zona incerta had activity that was biased towards sleep (NREM and REMS). A subset of vGAT neurons (NREM-max) became active in advance of sleep onset and may induce sleep by inhibiting the activity of the arousal neurons. Abnormal activation of the NREM-max neurons may drive sleep attacks and hypersomnia.

Activity dynamics of amygdala GABAergic neurons during cataplexy of narcolepsy.

Recent studies showed activation of the GABAergic neurons in the central nucleus of the amygdala (CeA) triggered cataplexy of sleep disorder narcolepsy. However, there is still no direct evidence on CeA GABAergic neurons' real-time dynamic during cataplexy. We used a deep brain calcium imaging tool to image the intrinsic calcium transient as a marker of neuronal activity changes in the narcoleptic VGAT-Cre mice by expressing the calcium sensor GCaMP6 into genetically defined CeA GABAergic neurons. Two distinct GABAergic neuronal groups involved in cataplexy were identified: spontaneous cataplexy-ON and predator odor-induced cataplexy-ON neurons. Majority in the latter group were inactive during regular sleep/wake cycles but were specifically activated by predator odor and continued their intense activities into succeeding cataplexy bouts. Furthermore, we found that CeA GABAergic neurons became highly synchronized during predator odor-induced cataplexy. We suggest that the abnormal activation and synchronization of CeA GABAergic neurons may trigger emotion-induced cataplexy.

Amygdala GABA Neurons Project To vlPAG And mPFC.

The amygdala regulates multiple behaviors and emotions by projecting to multiple brain regions. However, the topographical distribution of amygdala neurons projecting to specific brain areas is still unclear. In the present study, we focus on determining whether single amygdala neurons project to the brain stem ventrolateral periaqueductal grey (vlPAG) and to the medial prefrontal cortex (mPFC). The mPFC neurons are involved in detecting emotional content while the vlPAG neurons are involved in regulating muscle tone. In VGAT-Cre mice a cre-inducible retrograde AAV tracer expressing tdTomato was microinjected into the ventrolateral periaqueductal grey matter (vlPAG), while a second retrograde AAV tracer with generic expression of GFP was delivered into the medial prefrontal cortex (mPFC). The results identified a subgroup of bifurcating GABAergic neurons in the central nucleus (CeA) and basolateral amygdala (BLA) that projects to vlPAG and mPFC. Based on these projections we suggest that amygdala GABA neurons may be involved in triggering emotionally-induced cataplexy in the sleep disorder, narcolepsy.

Dynamic Network Activation of Hypothalamic MCH Neurons in REM Sleep and Exploratory Behavior.

Most brain neurons are active in waking, but hypothalamic neurons that synthesize the neuropeptide melanin-concentrating hormone (MCH) are claimed to be active only during sleep, particularly rapid eye movement (REM) sleep. Here we use deep-brain imaging to identify changes in fluorescence of the genetically encoded calcium (Ca2+) indicator GCaMP6 in individual hypothalamic neurons that contain MCH. An in vitro electrophysiology study determined a strong relationship between depolarization and Ca2+ fluorescence in MCH neurons. In 10 freely behaving MCH-cre mice (male and female), the highest fluorescence occurred in all recorded neurons (n = 106) in REM sleep relative to quiet waking or non-REM sleep. Unexpectedly, 70% of the MCH neurons had strong fluorescence activity when the mice explored novel objects. Spatial and temporal mapping of the change in fluorescence between pairs of MCH neurons revealed dynamic activation of MCH neurons during REM sleep and activation of a subset of the same neurons during exploratory behavior. Functional network activity maps will facilitate comparisons of not only single-neuron activity, but also network responses in different conditions and disease.SIGNIFICANCE STATEMENT Functional activity maps identify brain circuits responding to specific behaviors, including rapid eye movement sleep (REM sleep), a sleep phase when the brain is as active as in waking. To provide the first activity map of individual neurons during REM sleep, we use deep-brain calcium imaging in unrestrained mice to map the activity of hypothalamic melanin-concentrating hormone (MCH) neurons. MCH neurons were found to be synchronously active during REM sleep, and also during the exploration of novel objects. Spatial mapping revealed dynamic network activation during REM sleep and activation of a subset of the neurons during exploratory behavior. Functional activity maps at the cellular level in specific behaviors, including sleep, are needed to establish a brain connectome.

VGAT and VGLUT2 expression in MCH and orexin neurons in double transgenic reporter mice.

The neuropeptides orexin and melanin-concentrating hormone (MCH), as well as the neurotransmitters GABA (γ-Aminobutyric acid) and glutamate are chief modulators of the sleep-wake states in the posterior hypothalamus. To investigate co-expression of vesicular GABA transporter (VGAT, a marker of GABA neurons) and the vesicular glutamate transporter-2 (VGLUT2, a marker of glutamate neurons) in orexin and MCH neurons, we generated two transgenic mouse lines. One line selectively expressed the reporter gene EYFP in VGAT+ neurons, whereas the other line expressed reporter gene tdTomato in VGLUT2+ neurons. Co-localization between these genetic reporters and orexin or MCH immunofluorescent tags was determined using 3D computer reconstructions of Z stacks that were acquired using a multiphoton laser confocal microscope. Our results demonstrated that MCH neurons expressed neither VGAT nor VGLUT2, suggesting MCH neurons are a separate cluster of cells from VGAT+ GABAergic neurons and VGLUT2+ glutamatergic neurons. Moreover, most orexin neurons expressed VGLUT2, indicating these neurons are glutamatergic. Our data suggested that in the posterior hypothalamus there are four major distinct groups of neurons: VGAT+, orexin+/VGLUT2+, orexin-/VGLUT2+, and MCH neurons. This study facilitated our understanding of the role of these neurotransmitters and neuropeptides in relation to sleep/wake regulation.

Fighting obesity: Non-pharmacological interventions.

The abnormal or excessive fat accumulation that impairs health is one of the criteria that fulfills obesity. According to epidemiological data, obesity has become a worldwide public health problem that in turn would trigger additional pathologies such as cardiorespiratory dysfunctions, cancer, gastrointestinal disturbances, depression, sleep disorders, just to mention a few. Then, the search for a therapeutical intervention aimed to prevent and manage obesity has been the focus of study during the last years. As one can assume, the increased prevalence of obesity has translated to search of efficient pharmaceuticals designed to manage this health issue. However, to further complicate the scenario, scientific literature has described that obesity is the result of interaction between multiple events. Therefore, pharmacological approaches have faced a serious challenge for develop the adequate treatment. Here, we argue that a wide range of non-pharmacological/invasive techniques can be used to manage obesity, such as diets, cognitive behavioral interventions, exercise and transcranial direct current stimulation. Combining these techniques may allow improving quality of life of obese patients.

Rewiring brain circuits to block cataplexy in murine models of narcolepsy.

Narcolepsy was first identified almost 130 years ago, but it was only 15 years ago that it was identified as a neurodegenerative disease linked to a loss of orexin neurons in the brain. It is unclear what causes the orexin neurons to die, but our strategy has been to place the gene for orexin into surrogate neurons in the validated mouse models of narcolepsy, and test whether it can block narcolepsy symptoms, such as cataplexy. In both the orexin knockout and the orexin-ataxin-3 mouse models of narcolepsy we have found that cataplexy can be blocked if the surrogate neurons are part of the circuit responsible for cataplexy. We have also determined that the orexin gene can be inserted into surrogate neurons in the amygdala to block emotion-induced cataplexy. Through the use of optogenetics we anticipate that it will be possible to preemptively block cataplexy.

Optogenetic activation of melanin-concentrating hormone neurons increases non-rapid eye movement and rapid eye movement sleep during the night in rats.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake-active period for nocturnal rodents [R. R. Konadhode et al. (2013) J. Neurosci., 33, 10257-10263]. Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin-2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV-MCHp-ChR2-EYFP into the hypothalamus of Long-Evans rats. An in vitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long-Evans rats, rAAV-MCH-ChR2, or the control vector, rAAV-MCH-EYFP, were delivered into the hypothalamus. Three weeks later, baseline sleep was recorded for 48 h without optogenetic stimulation (0 Hz). Subsequently, at the start of the lights-off cycle, the MCH neurons were stimulated at 5, 10, or 30 Hz (1 mW at tip; 1 min on - 4 min off) for 24 h. Sleep was recorded during the 24-h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle, only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep-active MCH neurons contribute to drive sleep in mammals.

Orexin gene transfer into the amygdala suppresses both spontaneous and emotion-induced cataplexy in orexin-knockout mice.

Narcolepsy is a chronic sleep disorder linked to the loss of orexin-producing neurons in the hypothalamus. Cataplexy, a sudden loss of muscle tone during waking, is an important distinguishing symptom of narcolepsy and it is often triggered by strong emotions. The neural circuit underlying cataplexy attacks is not known, but is likely to involve the amygdala, a region implicated in regulating emotions. In mice models of narcolepsy, transfer of the orexin gene into surrogate neurons has been successful in ameliorating narcoleptic symptoms. However, it is not known whether this method also blocks cataplexy triggered by strong emotions. To examine this possibility, the gene encoding mouse prepro-orexin was transferred into amygdala neurons of orexin-knockout (KO) mice (rAAV-orexin; n = 8). Orexin-KO mice that did not receive gene transfer (no-rAAV; n = 7) or received only the reporter gene (rAAV-GFP; n = 7) served as controls. Three weeks later, the animal's sleep and behaviour were recorded at night (no-odour control night), followed by another recording at night in the presence of predator odour (odour night). Orexin-KO mice given the orexin gene transfer into surrogate amygdala neurons had significantly less spontaneous bouts of cataplexy, and predator odour did not induce cataplexy compared with control mice. Moreover, the mice with orexin gene transfer were awake more during the odour night. These results demonstrate that orexin gene transfer into amygdala neurons can suppress both spontaneous and emotion-induced cataplexy attacks in narcoleptic mice. It suggests that manipulating amygdala pathways is a potential strategy for treating cataplexy in narcolepsy.

Endocannabinoid modulation of cortical up-states and NREM sleep.

Up-/down-state transitions are a form of network activity observed when sensory input into the cortex is diminished such as during non-REM sleep. Up-states emerge from coordinated signaling between glutamatergic and GABAergic synapses and are modulated by systems that affect the balance between inhibition and excitation. We hypothesized that the endocannabinoid (EC) system, a neuromodulatory system intrinsic to the cortical microcircuitry, is an important regulator of up-states and sleep. To test this hypothesis, up-states were recorded from layer V/VI pyramidal neurons in organotypic cultures of wild-type or CB1R knockout (KO) mouse prefrontal cortex. Activation of the cannabinoid 1 receptor (CB1) with exogenous agonists or by blocking metabolism of endocannabinoids, anandamide or 2-arachidonoyl glycerol, increased up-state amplitude and facilitated action potential discharge during up-states. The CB1 agonist also produced a layer II/III-selective reduction in synaptic GABAergic signaling that may underlie its effects on up-state amplitude and spiking. Application of CB1 antagonists revealed that an endogenous EC tone regulates up-state duration. Paradoxically, the duration of up-states in CB1 KO cultures was increased suggesting that chronic absence of EC signaling alters cortical activity. Consistent with increased cortical excitability, CB1 KO mice exhibited increased wakefulness as a result of reduced NREM sleep and NREM bout duration. Under baseline conditions, NREM delta (0.5-4 Hz) power was not different in CB1 KO mice, but during recovery from forced sleep deprivation, KO mice had reduced NREM delta power and increased sleep fragmentation. Overall, these findings demonstrate that the EC system actively regulates cortical up-states and important features of NREM sleep such as its duration and low frequency cortical oscillations.

Optogenetic stimulation of MCH neurons increases sleep.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

Effects of orexin gene transfer in the dorsolateral pons in orexin knockout mice.

STUDY OBJECTIVES: Narcolepsy is a sleep disorder characterized by loss of orexin neurons. Previously, our group demonstrated that transfer of the orexin gene into surrogate neurons in the lateral hypothalamus and the zona incerta significantly reduced cataplexy bouts in the orexin-ataxin-3 mice model of narcolepsy. The current study determined the effects of orexin gene transfer into the dorsolateral pontine neurons in the orexin knockout (KO) mice model of narcolepsy. The dorsolateral pons was chosen because it plays a critical role in regulating muscle tone and thus it is conceivable to be involved in cataplexy as well. Cataplexy is the pathognomonic symptom in narcolepsy. DESIGN: Independent groups of orexin KO mice were given bilateral microinjections (0.75 µL each side) of either recombinant adenoassociated virus-orexin (rAAV-orexin; n = 7), or rAAV-green fluorescent protein (rAAV-GFP; n = 7) into the dorsolateral pons. A group of orexin KO mice that did not receive rAAV (n = 7) and a group of wild-type mice (C57BL/J6; n = 5) were used as controls. Three weeks after rAAV-mediated gene transfer narcolepsy symptoms were examined using sleep and behavioral recordings. Number, location of the orexin-immunoreactive neurons, and relative density of orexin immunoreactive fibers were determined. MEASUREMENTS AND RESULTS: Orexin gene transfer into the dorsolateral pons significantly decreased cataplexy and modestly improved wake maintenance compared to the orexin KO mice that did not receive rAAV. In contrast, GFP gene transfer worsened narcoleptic symptoms compared to the no-rAAV orexin KO group. CONCLUSION: Orexin gene transfer into the dorsolateral pontine neurons can control cataplexy attacks and modestly improve wake maintenance.

Cardiac and skeletal myopathy in Fabry disease: a clinicopathologic correlative study.

Skeletal muscle disturbances are commonly reported in patients with Fabry disease. Whether they derive from cardiac dysfunction or direct muscle involvement is still unclear. Clinical, noninvasive, and invasive cardiac and muscle studies, including an endomyocardial and muscle biopsy, were obtained in 12 patients (mean age, 42.1 ± 12.6 years; range, 24-58 years) with Fabry disease. In the youngest patients (group A, 4 men aged <35 years), results of cardiac and skeletal noninvasive studies were normal, except for reduced velocities in tissue Doppler imaging. Histologic examination indicated that muscle myocytes were unaffected, whereas muscle vessels showed the presence of mild glycosphingolipid accumulation in endothelial and smooth muscle cells. In the heart, cardiomyocytes and endothelial and smooth muscle cells of intramural cardiac vessels were involved by the disease. The oldest patients (group B, 6 men and 2 women aged >35 years) showed ultrasound muscle disarray and electromyography signs of myopathy, increased left ventricular mass, and normal cardiac function. Histologic examination showed that muscle myocytes contained mild glycosphingolipid accumulation compared with severe engulfment of cardiomyocytes. Moreover, similar infiltration of myocardial and muscle intramural vessels, causing lumen narrowing and fibrofatty tissue replacement, was observed. Direct muscle involvement occurs in patients with Fabry disease. It is milder and delayed compared with that in the heart. The difference in organ function and the need of residual α-galactosidase A activity are the likely causes.

A community demand-driven approach toward sustainable water and sanitation infrastructure development.

In September 2001, Cooperative Assistance and Relief Everywhere, Peru Country Office (CARE Peru), obtained funding from the United States Agency for International Development (USAID) to implement community-supported, condominial water and sanitation interventions in Manuel Cardozo Dávila, a settlement in Iquitos, Peru. With technical support from the Centers for Disease Control and Prevention (CDC), CARE Peru's Urban Environmental Health Models (Modelos Urbanos de Salud Ambiental [MUSA]) project built on previous work from implementing the Protocol for Assessing Community Excellence in Environmental Health in this same community. The project led to the municipal water supply distribution system being extended 1.3 kilometers into the Southern zone of Iquitos, where it connected to the condominial water system. Altogether, 1030 households were connected to the water supply system after the installation of a condominial water and sewerage system in Cardozo. Diarrheal disease decreased by 37% for children less than 5 years of age from 2003 to 2004. This paper illustrates the strategy used by CARE Peru in conjunction with the Cardozo community to assure that the local demand for improved water and sanitation was met.

Orexin gene transfer into zona incerta neurons suppresses muscle paralysis in narcoleptic mice.

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep-wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.