Effect of ambient temperature on sleep breathing phenotype in mice: the role of orexins

The loss of orexinergic neurons, releasing orexins, results in narcolepsy. Orexins participate in the regulation of many physiological functions, and their role as wake-promoting molecules has been widely described. Less is known about the involvement of orexins in body temperature and respiratory regulation. The aim of this study was to investigate whether orexin peptides modulate respiratory regulation as a function of ambient temperature (T°a) during different sleep stages. Respiratory phenotype of male orexin knockout (KO-ORX, n=9) and wild-type (WT, n=8) mice was studied at thermoneutrality (T°a=30°C) or during mild cold exposure (T°a=20°C) inside a whole-body plethysmography chamber. The states of wakefulness (W), non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS) were scored non-invasively, using a previously validated technique. Both in WT and KO-ORX mice T°a strongly and significantly affected ventilatory period and minute ventilation values during NREMS and REMS; moreover, the occurrence rate of sleep apneas in NREMS was significantly reduced at T°a=20°C compared to T°a=30°C. Overall, there were no differences in respiratory regulation during sleep between WT and KO-ORX mice, except for sigh occurrence rate, which was significantly increased at T°a=20°C with respect to T°a =30°C in WT mice, but not in KO-ORX mice. These results do not support a main role for orexin peptides in the temperature-dependent modulation of respiratory regulation during sleep. However, we showed that the occurrence rate of sleep apneas critically depends on T°a, without any significant effect of orexin peptides

Heterozygous CDKL5 Knockout Female Mice Are a Valuable Animal Model for CDKL5 Disorder

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/-) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/- mice. We found that Cdkl5 +/- mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/- mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/- mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder

Sleep and Tibialis Anterior Muscle Activity in Mice With Mild Hypoxia and Iron Deficiency: Implications for the Restless Legs Syndrome

Restless legs syndrome (RLS) is a neurological disorder that entails an urge to move with a circadian pattern during the evening/night. RLS may be accompanied by decreased sleep time and increased occurrence of periodic leg movements during sleep (PLMS), which involve bursts of tibialis anterior (TA) muscle electromyogram (EMG). Mild hypoxia and non-anemic iron deficiency, a highly prevalent nutritional deficiency, are relatively unexplored factors in RLS pathophysiology. We tested whether mice exposed to mild hypoxia, alone or in combination with non-anemic iron deficiency, show decreased sleep time particularly in the light (rest) period and increased occurrence of TA EMG phasic events similar to human PLMS. Female C57BL/6J mice were fed diets with low or normal iron for 6 months from weaning and instrumented with electrodes to record the electroencephalogram and the EMG of both TA muscles. Mice were recorded in a whole-body plethysmograph while breathing a normoxic or mildly hypoxic (15% O2) gas mixture for 48 h. Hypoxia increased minute ventilation during sleep. The low-iron diet decreased liver and serum iron, leaving blood hemoglobin and brainstem iron levels unaffected. Hypoxia, either alone or in combination with non-anemic iron deficiency, decreased non-rapid-eye-movement (non-REM) sleep time, but this occurred irrespective of the light/dark period and was not associated with increased occurrence of TA EMG events during non-REM sleep. These results do not support the hypothesis that mild hypoxia is sufficient to cause signs of RLS, either alone or in combination with non-anemic iron deficiency, pointing to the necessity of further susceptibility factors

Cardiac Functional and Structural Abnormalities in a Mouse Model of CDKL5 Deficiency Disorder

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental disease that mostly affects girls, who are heterozygous for mutations in the X-linked CDKL5 gene. Mutations in the CDKL5 gene lead to a lack of CDKL5 protein expression or function and cause numerous clinical features, including early-onset seizures, marked hypotonia, autistic features, gastrointestinal problems, and severe neurodevelopmental impairment. Mouse models of CDD recapitulate several aspects of CDD symptomology, including cognitive impairments, motor deficits, and autistic-like features, and have been useful to dissect the role of CDKL5 in brain development and function. However, our current knowledge of the function of CDKL5 in other organs/tissues besides the brain is still quite limited, reducing the possibility of broad-spectrum interventions. Here, for the first time, we report the presence of cardiac function/structure alterations in heterozygous Cdkl5 +/- female mice. We found a prolonged QT interval (corrected for the heart rate, QTc) and increased heart rate in Cdkl5 +/- mice. These changes correlate with a marked decrease in parasympathetic activity to the heart and in the expression of the Scn5a and Hcn4 voltage-gated channels. Interestingly, Cdkl5 +/- hearts showed increased fibrosis, altered gap junction organization and connexin-43 expression, mitochondrial dysfunction, and increased ROS production. Together, these findings not only contribute to our understanding of the role of CDKL5 in heart structure/function but also document a novel preclinical phenotype for future therapeutic investigation

Expression of a Secretable, Cell-Penetrating CDKL5 Protein Enhances the Efficacy of Gene Therapy for CDKL5 Deficiency Disorder

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases

Validation of ‘Somnivore’, a Machine Learning Algorithm for Automated Scoring and Analysis of Polysomnography Data

Manual scoring of polysomnography data is labor-intensive and time-consuming, and most existing software does not account for subjective differences and user variability. Therefore, we evaluated a supervised machine learning algorithm, SomnivoreTM, for automated wake–sleep stage classification. We designed an algorithm that extracts features from various input channels, following a brief session of manual scoring, and provides automated wake-sleep stage classification for each recording. For algorithm validation, polysomnography data was obtained from independent laboratories, and include normal, cognitively-impaired, and alcohol-treated human subjects (total n = 52), narcoleptic mice and drug-treated rats (total n = 56), and pigeons (n = 5). Training and testing sets for validation were previously scored manually by 1–2 trained sleep technologists from each laboratory. F-measure was used to assess precision and sensitivity for statistical analysis of classifier output and human scorer agreement. The algorithm gave high concordance with manual visual scoring across all human data (wake 0.91 ± 0.01; N1 0.57 ± 0.01; N2 0.81 ± 0.01; N3 0.86 ± 0.01; REM 0.87 ± 0.01), which was comparable to manual inter-scorer agreement on all stages. Similarly, high concordance was observed across all rodent (wake 0.95 ± 0.01; NREM 0.94 ± 0.01; REM 0.91 ± 0.01) and pigeon (wake 0.96 ± 0.006; NREM 0.97 ± 0.01; REM 0.86 ± 0.02) data. Effects of classifier learning from single signal inputs, simple stage reclassification, automated removal of transition epochs, and training set size were also examined. In summary, we have developed a polysomnography analysis program for automated sleep-stage classification of data from diverse species. Somnivore enables flexible, accurate, and high-throughput analysis of experimental and clinical sleep studies

Sleep-related changes in blood pressure in hypocretin-deficient narcoleptic mice

Objectives. Blood pressure (BP) physiologically has higher and lower values during the active and rest period, respectively. Subjects failing to show the appropriate BP decrease (10-20%) on passing form diurnal activity to nocturnal rest and sleep have increased risk of target organ damage at the cardiac, vascular and cerebrovascular levels. Hypocretin (HCRT) releasing neurons, mainly located in the lateral hypothalamus, project widely to the central nervous system. Thus HCRT neurons are involved in several autonomic functions, including BP regulation. HCRT neurons also play a key role in wake-sleep cycle regulation, the lack of which becomes evident in HCRT-deficient narcoleptic patients. I investigated whether chronic lack of HCRT signaling alters BP during sleep in mouse models of narcolepsy. Methods. The main study was performed on HCRT-ataxin3 transgenic mice (TG) with selective post-natal ablation of HCRT neurons, HCRT gene knockout mice (KO) with preserved HCRT neurons, and Wild-Type control mice (WT) with identical genetic background. Experiments where replicated on TG and WT mice with hybrid genetic background (hTG and hWT, respectively). Mice were implanted with a telemetric pressure transducer (TA11PA-C10, DSI) and electrodes for discriminating wakefulness (W), rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Signals were recorded for 3 days. Mean BP values were computed in each wake-sleep state and analyzed by ANOVA and t-test with significance at p<0.05. Results. The decrease in BP between either NREMS or REMS and W was significantly blunted in TG and KO with respect to WT as well as in hTG with respect to hWT. Conclusions. Independently from the genetic background, chronic HCRT deficiency leads to a decreased BP difference between W and sleep potentially adverse in narcoleptic subjects. These data suggest that HCRT play an important role in the sleep-dependent cardiovascular control

The arterial blood supply of the pancreas: a review. V. The dorsal pancreatic artery. An anatomic and a radiologic study

The present article is the fifth part of a comprehensive review on the arterial blood supply of the pancreas and deals with the dorsal pancreatic artery. The aim of this review is to summarise the anatomic studies, starting from Haller's reports, and to supply, as far as possible with original material, angiographic evidence for the classic anatomic notions. For this purpose, the overall research was carried out by studying 1015 selective angiographies (celiac trunk and its branches, superior mesenteric artery) taken from the angiographic archives of the Institutes of Radiology of Siena, Rome (Catholic University), and Perugia. Angiographically, the authors could demonstrate the dorsal pancreatic artery, present in most instances, as arising from the splenic artery, common hepatic artery, superior mesenteric artery or celiac trunk and accessory right hepatic artery as coming from the superior mesenteric artery. Variations in the course and length of the dorsal pancreatic artery were demonstrated as well as some collateral branches. The authors underline the discordant opinions still existing regarding the incidence of the different ways the dorsal pancreatic artery arises, and discuss its uncertain embryologic development and surgical relevance