MYTX-011: A pH-Dependent Anti-c-MET Antibody-Drug Conjugate Designed for Enhanced Payload Delivery to c-MET-Expressing Tumor Cells.

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a c-mesenchymal-epithelial transition (MET)-targeting ADC (MYTX-011) can overcome the requirement for high c-MET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower c-MET levels. MYTX-011 drove fourfold higher net internalization than a non-pH-engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least threefold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high c-MET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other monomethyl auristatin E-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of patients with NSCLC with c-MET expression than other c-MET-targeting ADCs. A first-in-human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

MYTX-011: a pH-dependent anti-cMET antibody-drug conjugate designed for enhanced payload delivery to cMET expressing tumor cells.

Advances in linker payload technology and target selection have been at the forefront of recent improvements in antibody-drug conjugate (ADC) design, leading to several approvals over the last decade. In contrast, the potential of novel ADC technologies to enhance payload delivery to tumors is relatively underexplored. We demonstrate that incorporation of pH-dependent binding in the antibody component of a cMET targeting ADC (MYTX-011) can overcome the requirement for high cMET expression on tumors, an innovation that has the potential to benefit a broader population of patients with lower cMET levels. MYTX-011 drove four-fold higher net internalization than a non-pH engineered parent ADC in non-small cell lung cancer (NSCLC) cells and showed increased cytotoxicity against a panel of cell lines from various solid tumors. A single dose of MYTX-011 showed at least three-fold higher efficacy than a benchmark ADC in mouse xenograft models of NSCLC ranging from low to high cMET expression. Moreover, MYTX-011 showed improved pharmacokinetics over parent and benchmark ADCs. In a repeat dose toxicology study, MYTX-011 exhibited a toxicity profile similar to other MMAE-based ADCs. These results highlight the potential of MYTX-011 for treating a broader range of NSCLC patients with cMET expression than other cMET targeting ADCs. A first in human study is ongoing to determine the safety, tolerability, and preliminary efficacy of MYTX-011 in patients with NSCLC (NCT05652868).

Painful to Discuss: The Intersection of Chronic Pain, Mental Health, and Analgesic Use among People with HIV.

Objective: This retrospective chart review study aims to identify patients in an HIV clinical setting in an area of high HIV prevalence in Atlanta, Georgia, USA who have chronic pain, analgesic prescriptions, and/or mental health diagnoses. Design: People living with HIV (PLWH) are at higher risk for experiencing trauma, mental health conditions, and chronic pain than their HIV-negative counterparts. This study was designed to evaluate the intersection of these factors within an urban HIV clinic. Methods: Retrospective chart review study. Results: Of the adult patients enrolled at an HIV clinic in Atlanta, Georgia USA between 2011-2022 (n=15,970), 93.7% were prescribed analgesics, 40.5% had documented pain diagnoses, and 23.5% had documented mental health diagnoses. Additionally, 14.3% of all enrolled patients had all three factors concurrently. Conclusions: The complexity of HIV, chronic pain, mental health challenges, and analgesic use demand a patient-centered, collaborative approach including a multidisciplinary care team. Seeing persistent pain among PLWH with a trauma-informed approach to care within the lens of co-occurring mental health diagnoses will allow us to better understand, treat, and sustain patients in life-saving HIV care.

Limits to the accurate and generalizable use of soundscapes to monitor biodiversity.

Although eco-acoustic monitoring has the potential to deliver biodiversity insight on vast scales, existing analytical approaches behave unpredictably across studies. We collated 8,023 audio recordings with paired manual avifaunal point counts to investigate whether soundscapes could be used to monitor biodiversity across diverse ecosystems. We found that neither univariate indices nor machine learning models were predictive of species richness across datasets but soundscape change was consistently indicative of community change. Our findings indicate that there are no common features of biodiverse soundscapes and that soundscape monitoring should be used cautiously and in conjunction with more reliable in-person ecological surveys.

pyResearchInsights-An open-source Python package for scientific text analysis.

With an increasing number of scientific articles published each year, there is a need to synthesize and obtain insights across ever-growing volumes of literature. Here, we present pyResearchInsights, a novel open-source automated content analysis package that can be used to analyze scientific abstracts within a natural language processing framework.The package collects abstracts from scientific repositories, identifies topics of research discussed in these abstracts, and presents interactive concept maps to visualize these research topics. To showcase the utilities of this package, we present two examples, specific to the field of ecology and conservation biology.First, we demonstrate the end-to-end functionality of the package by presenting topics of research discussed in 1,131 abstracts pertaining to birds of the Tropical Andes. Our results suggest that a large proportion of avian research in this biodiversity hotspot pertains to species distributions, climate change, and plant ecology.Second, we retrieved and analyzed 22,561 abstracts across eight journals in the field of conservation biology to identify twelve global topics of conservation research. Our analysis shows that conservation policy and landscape ecology are focal topics of research. We further examined how these conservation-associated research topics varied across five biodiversity hotspots.Lastly, we compared the utilities of this package with existing tools that carry out automated content analysis, and we show that our open-source package has wider functionality and provides end-to-end utilities that seldom exist across other tools.

Determining levels of cryptic diversity within the endemic frog genera, Indirana and Walkerana, of the Western Ghats, India.

A large number of species in the tropics are awaiting discovery, many due to their cryptic morphology ie. lack of discernable morphological difference. We explored the presence of cryptic lineages within the frog genera, Indirana and Walkerana, which are endemic to the Western Ghats of Peninsular India. By reconstructing a phylogeny using 5 genes and robust geographic sampling, we delimited 19 lineages along a population-species continuum, using multiple criteria including haplotype clusters, genetic distance, morphological distinctness, and geographical separation. Of these 19 lineages, 14 belonged to the genus Indirana and 5 to the genus Walkerana. Divergence dating analyses revealed that the clade comprising Indirana and Walkerana began diversifying around 71 mya and the most recent common ancestor of Indirana and Walkerana split around 43 mya. We tested for the presence of cryptic lineages by examining the relationship between genetic and morphological divergence among related pairs within a pool of 15 lineages. The pairs showed strong morphological conservatism across varying levels of genetic divergence. Our results highlight the prevalence of morphologically cryptic lineages in these ancient endemic clades of the Western Ghats. This emphasizes the significance of other axes, such as geography, in species delimitation. With increasing threats to amphibian habitats, it is imperative that cryptic lineages are identified so that appropriate conservation measures can be implemented.

A deeply divergent lineage of Walkerana (Anura: Ranixalidae) from the Western Ghats of Peninsular India.

The frog family Ranixalidae is endemic to the Western Ghats of Peninsular India and contains two genera, Indirana and Walkerana. The three known species of Walkerana are restricted to different hill ranges south of the Palghat Gap, an ancient valley in the Western Ghats. In this study, we report the discovery of a deeply divergent lineage of Walkerana from the high elevations of the Elivalmalai hill range. This finding extends the geographic range of the Walkerana clade to the north of the Palghat Gap. The new species Walkerana muduga sp. nov. is genetically and morphologically divergent, and geographically isolated from its sister lineages. We also recovered a potential new lineage in the adjoining hill ranges suggesting the presence of additional new species in this genus north of the Palghat Gap.

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

Cognitive Deficits Are Attenuated in Neuroglobin Overexpressing Mice Exposed to a Model of Obstructive Sleep Apnea.

Background: Obstructive sleep apnea (OSA) is a highly prevalent disease manifesting as intermittent hypoxia during sleep (IH) and is increasingly recognized as being independently associated with neurobehavioral deficits. These deficits may be due to increased apoptosis in the hippocampus and cerebral cortex, as well as increased oxidative stress and inflammation. It has been reported that neuroglobin (Ngb) is upregulated in response to hypoxia-ischemia insults and exhibits a protective role in ischemia-reperfusion brain injury. We hypothesized that transgenic overexpression of Ngb would attenuate spatial learning deficits in a murine model of OSA. Methods:Wild-type mice and Ngb overexpressing male mice (Ngb-TG) were randomly assigned to either IH or room air (RA) exposures. The effects of IH during the light period on performance in a water maze spatial task were assessed, as well as anxiety and depressive-like behaviors using elevated plus maze (EPM) and forced swim tests. Cortical tissues from all the mice were extracted for biochemical studies for lipid peroxidation. Results:Ngb TG mice exhibited increased Ngb immunoreactivity in brain tissues and IH did not elicit significant changes in Ngb expression in either Ngb-TG mice or WT mice. On a standard place training task in the water maze, Ngb-TG mice displayed preserved spatial learning, and were protected from the reduced spatial learning performances observed in WT mice exposed to IH. Furthermore, anxiety and depression levels were enhanced in WT mice exposed to IH as compared to RA controls, while alterations emerged in Ngb-TG mice exposed to IH. Furthermore, WT mice, but not Ngb-TG mice had significantly elevated levels of malondialdehyde in cortical lysates following IH exposures. Conclusions:In a murine model of OSA, oxidative stress responses and neurocognitive and behavioral impairments induced by IH during sleep are attenuated by the neuroprotective effects of Ngb.

Growth hormone releasing hormone (GHRH) signaling modulates intermittent hypoxia-induced oxidative stress and cognitive deficits in mouse.

Intermittent hypoxia (IH) during sleep, such as occurs in obstructive sleep apnea (OSA), leads to degenerative changes in the hippocampus, and is associated with spatial learning deficits in adult mice. In both patients and murine models of OSA, the disease is associated with suppression of growth hormone (GH) secretion, which is actively involved in the growth, development, and function of the central nervous system (CNS). Recent work showed that exogenous GH therapy attenuated neurocognitive deficits elicited by IH during sleep in rats. Here, we show that administration of the Growth Hormone Releasing Hormone (GHRH) agonist JI-34 attenuates IH-induced neurocognitive deficits, anxiety, and depression in mice along with reduction in oxidative stress markers such as MDA and 8-hydroxydeoxyguanosine, and increases in hypoxia inducible factor-1α DNA binding and up-regulation of insulin growth factor-1 and erythropoietin expression. In contrast, treatment with a GHRH antagonist (MIA-602) during intermittent hypoxia did not affect any of the IH-induced deleterious effects in mice. Thus, exogenous GHRH administered as the formulation of a GHRH agonist may provide a viable therapeutic intervention to protect IH-vulnerable brain regions from OSA-associated neurocognitive dysfunction. Sleep apnea, characterized by chronic intermittent hypoxia (IH), is associated with substantial cognitive and behavioral deficits. Here, we show that administration of a GHRH agonist (JI-34) reduces oxidative stress, increases both HIF-1α nuclear binding and downstream expression of IGF1 and erythropoietin (EPO) in hippocampus and cortex, and markedly attenuates water maze performance deficits in mice exposed to intermittent hypoxia during sleep.

TNF-α and temporal changes in sleep architecture in mice exposed to sleep fragmentation.

TNF-α plays critical roles in host-defense, sleep-wake regulation, and the pathogenesis of various disorders. Increases in the concentration of circulating TNF-α after either sleep deprivation or sleep fragmentation (SF) appear to underlie excessive daytime sleepiness in patients with sleep apnea (OSA). Following baseline recordings, mice were subjected to 15 days of SF (daily for 12 h/day from 07.00 h to 19.00 h), and sleep parameters were recorded on days1, 7 and 15. Sleep architecture and sleep propensity were assessed in both C57BL/6J and in TNF-α double receptor KO mice (TNFR KO). To further confirm the role of TNF-α, we also assessed the effect of treatment with a TNF- α neutralizing antibody in C57BL/6J mice. SF was not associated with major changes in global sleep architecture in C57BL/6J and TNFR KO mice. TNFR KO mice showed higher baseline SWS delta power. Further, following 15 days of SF, mice injected with TNF-α neutralizing antibody and TNFR KO mice showed increased EEG SWS activity. However, SWS latency, indicative of increased propensity to sleep, was only decreased in C57BL/6J, and was unaffected in TNFR KO mice as well as in C57BL/6J mice exposed to SF but treated with TNF-α neutralizing antibody. Taken together, our findings show that the excessive sleepiness incurred by recurrent arousals during sleep may be due to activation of TNF-alpha-dependent inflammatory pathways, despite the presence of preserved sleep duration and global sleep architecture.

Disrupted sleep without sleep curtailment induces sleepiness and cognitive dysfunction via the tumor necrosis factor-α pathway.

BACKGROUND: Sleepiness and cognitive dysfunction are recognized as prominent consequences of sleep deprivation. Experimentally induced short-term sleep fragmentation, even in the absence of any reductions in total sleep duration, will lead to the emergence of excessive daytime sleepiness and cognitive impairments in humans. Tumor necrosis factor (TNF)-α has important regulatory effects on sleep, and seems to play a role in the occurrence of excessive daytime sleepiness in children who have disrupted sleep as a result of obstructive sleep apnea, a condition associated with prominent sleep fragmentation. The aim of this study was to examine role of the TNF-α pathway after long-term sleep fragmentation in mice. METHODS: The effect of chronic sleep fragmentation during the sleep-predominant period on sleep architecture, sleep latency, cognitive function, behavior, and inflammatory markers was assessed in C57BL/6 J and in mice lacking the TNF-α receptor (double knockout mice). In addition, we also assessed the above parameters in C57BL/6 J mice after injection of a TNF-α neutralizing antibody. RESULTS: Mice subjected to chronic sleep fragmentation had preserved sleep duration, sleep state distribution, and cumulative delta frequency power, but also exhibited excessive sleepiness, altered cognitive abilities and mood correlates, reduced cyclic AMP response element-binding protein phosphorylation and transcriptional activity, and increased phosphodiesterase-4 expression, in the absence of AMP kinase-α phosphorylation and ATP changes. Selective increases in cortical expression of TNF-α primarily circumscribed to neurons emerged. Consequently, sleepiness and cognitive dysfunction were absent in TNF-α double receptor knockout mice subjected to sleep fragmentation, and similarly, treatment with a TNF-α neutralizing antibody abrogated sleep fragmentation-induced learning deficits and increases in sleep propensity. CONCLUSIONS: Taken together, our findings show that recurrent arousals during sleep, as happens during sleep apnea, induce excessive sleepiness via activation of inflammatory mechanisms, and more specifically TNF-α-dependent pathways, despite preserved sleep duration.

Human apolipoprotein E4 targeted replacement in mice reveals increased susceptibility to sleep disruption and intermittent hypoxia.

Intermittent hypoxia (IH) and sleep fragmentation (SF) are major manifestations of sleep apnea, a frequent condition in aging humans. Sleep perturbations are frequent in Alzheimer's disease (AD) and may underlie the progression of disease. We hypothesized that acute short-term IH, SF, and their combination (IH+SF) may reveal unique susceptibility in sleep integrity in a murine model of AD. The effects of acute IH, SF, and IH+SF on sleep architecture, delta power, sleep latency, and core body temperature were assessed in adult male human ApoE4-targeted replacement mice (hApoE4) and wild-type (WT) controls. Slow wave sleep (SWS) was significantly reduced, and rapid eye movement (REM) sleep was almost abolished during acute exposure to IH alone and IH+SF for 6 h in hApoE4, with milder effects in WT controls. Decreased delta power during SWS did not show postexposure rebound in hApoE4 unlike WT controls. IH and IH+SF induced hypothermia, which was more prominent in hApoE4 than WT controls. Mice subjected to SF also showed sleep deficits but without hypothermia. hApoE4 mice, unlike WT controls, exhibited increased sleep propensity, especially following IH and IH+SF, suggesting limited ability for sleep recovery in hApoE4 mice. These findings substantiate the potential impact of IH and SF in modulating sleep architecture and sleep homeostasis including maintenance of body temperature. Furthermore, the increased susceptibility and limited recovery ability of hApoE4 mice to sleep apnea suggests that early recognition and treatment of the latter in AD patients may restrict the progression and clinical manifestations of this frequent neurodegenerative disorder.

Socially isolated mice exhibit a blunted homeostatic sleep response to acute sleep deprivation compared to socially paired mice.

Sleep is an important physiological process underlying maintenance of physical, mental and emotional health. Consequently, sleep deprivation (SD) is associated with adverse consequences and increases the risk for anxiety, immune, and cognitive disorders. SD is characterized by increased energy expenditure responses and sleep rebound upon recovery that are regulated by homeostatic processes, which in turn are influenced by stress. Since all previous studies on SD were conducted in a setting of social isolation, the impact of the social contextual setting is unknown. Therefore, we used a relatively stress-free SD paradigm in mice to assess the impact of social isolation on sleep, wakefulness and delta electroencephalogram (EEG) power during non-rapid eye movement (NREM) sleep. Paired or isolated C57BL/6J adult chronically-implanted male mice were exposed to SD for 6h and telemetric polygraphic recordings were conducted, including 18 h recovery. Recovery from SD in the paired group showed a significant decrease in wake and significant increase in NREM sleep and rapid eye movement (REM), and a similar, albeit less robust response occurred in the isolated mice. Delta power during NREM sleep was increased in both groups immediately following SD, but paired mice exhibited significantly higher delta power throughout the dark period. The increase in body temperature and gross motor activity observed during the SD procedure was decreased during the dark period. In both open field and elevated plus maze tests, socially isolated mice showed significantly higher anxiety than paired mice. The homeostatic processes altered by SD are differentially affected in paired and isolated mice, suggesting that the social context of isolation stress may adversely affect the quantity and quality of sleep in mice.

Intermittent hypoxia activates temporally coordinated transcriptional programs in visceral adipose tissue.

Obstructive sleep apnea (OSA) is a prevalent disorder characterized by intermittent hypoxia (IH) during sleep. OSA is strongly associated with obesity and dysregulation of metabolism-yet the molecular pathways linking the effects of IH on adipocyte biology remain unknown. We hypothesized that exposure to IH would activate distinct, time-dependent transcriptional programs in visceral adipose tissue of mice. We exposed 36 mice to IH or normoxia for up to 13 days. We transcriptionally profiled visceral fat tissue harvested from the animals and performed functional enrichment and network analysis on differentially expressed genes. We identified over 3,000 genes with significant expression patterns during the time course of IH exposure. The most enriched pathways mapped to metabolic processes, mitochondrion, and oxidative stress responses. We confirmed the pathophysiological relevance of these findings by demonstrating that mice exposed to chronic IH developed dyslipidemia and underwent significant lipid and protein oxidation within their visceral adipose depots. We applied gene-gene interaction network analysis to identify critical controllers of IH-induced transcriptional programs in adipocytes-these network hubs represent putative targets to modulate the effects of chronic IH on adipose tissue. Our approach to integrate computational methods with gene expression profiling of visceral fat tissue during IH exposure shows promise in helping unravel the mechanistic links between OSA and adipocyte biology.

Sleep fragmentation induces cognitive deficits via nicotinamide adenine dinucleotide phosphate oxidase-dependent pathways in mouse.

RATIONALE: Sleep fragmentation (SF) is one of the major characteristics of sleep apnea, and has been implicated in its morbid consequences, which encompass excessive daytime sleepiness and neurocognitive impairments. We hypothesized that absence of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is neuroprotective in SF-induced cognitive impairments. OBJECTIVES: To examine whether increased NADPH oxidase activity may play a role in SF-induced central nervous system dysfunction. METHODS: The effect of chronic SF during the sleep-predominant period on sleep architecture, sleep latency, spatial memory, and oxidative stress parameters was assessed in mice lacking NADPH oxidase activity (gp91phox-(/Y)) and wild-type littermates. MEASUREMENTS AND MAIN RESULTS: SF for 15 days was not associated with differences in sleep duration, sleep state distribution, or sleep latency in both gp91phox-(/Y) and control mice. However, on a standard place training task, gp91phox-(/Y) mice displayed normal learning and were protected from the spatial learning deficits observed in wild-type littermates exposed to SF. Moreover, anxiety levels were increased in wild-type mice exposed to SF, whereas no changes emerged in gp91phox-(/Y) mice. Additionally, wild-type mice, but not gp91phox-(/Y) mice, had significantly elevated NADPH oxidase gene expression and activity, and in malondialdehyde and 8-oxo-2'-deoxyguanosine levels in cortical and hippocampal lysates after SF exposures. CONCLUSIONS: This work substantiates an important role for NADPH oxidase in hippocampal memory impairments induced by SF, modeling sleep apnea. Targeting NADPH oxidase, therefore, is expected to minimize hippocampal impairments from both intermittent hypoxia and SF associated with the disease.

Sleep deprivation upregulates A1 adenosine receptors in the rat basal forebrain.

Sleep deprivation increases the levels of extracellular adenosine and A1 receptor (A1R)mRNA in the cholinergic zone of the basal forebrain, a region involved in sleep homeostasis. To evaluate homeostatic control mechanisms, we examined the sleep deprivation-induced changes in the A1R density in rodent brain using [H]CPFPX receptor autoradiography. We also examined the role of nuclear factor-kappaB (NF-kappaB) in transcriptional upregulation of A1R mRNA by use of the inhibitor peptide SN50 to inhibit nuclear translocation of NF-kappaB. We found a significant increase in cholinergic basal forebrain A1R density following 24 h of sleep deprivation and evidence that the upregulation of A1R is mediated by NF-kappaB. The A1R increase may be important in sleep homeostasis, since the increase in A1R density would increase the inhibitory effect of given level of adenosine, thus increasing the gain of the homeostat.

Adenosine and sleep deprivation promote NF-kappaB nuclear translocation in cholinergic basal forebrain.

In our investigations related to the homeostatic sleep factor adenosine (AD), we previously demonstrated that the DNA-binding activity of the transcription factor NF-kappaB in rat cholinergic basal forebrain increased following 3 h of sleep deprivation (SD). However, the neurotransmitter nature of the cells and the SD-induced stimuli responsible for NF-kappaB activation were not defined. In this report, we demonstrate, using double labeling immunohistochemistry, that nuclear translocation of NF-kappaB occurs almost exclusively in the cholinergic neurons of the basal forebrain following 3 h of SD. Furthermore, cholinergic basal forebrain microinjection of AD (25 nmol/L) or the A(1) receptor agonist N(6)-cyclo-hexyladenosine (100 nmol/L) induced nuclear translocation of NF-kappaB, thus suggesting that SD-induced increased extracellular concentrations of AD, acting via the A(1) AD receptor, may be responsible for the nuclear translocation of NF-kappaB in cholinergic neurons. Moreover, blocking the nuclear translocation of NF-kappaB by injection of inhibitor peptide, SN50, immediately prior to 6 h SD significantly reduced delta activity (1-4 Hz) during the first two hours of recovery sleep. Together, these data suggest a role in sleep homeostasis for the SD-induced activation of NF-kappaB in cholinergic basal forebrain, and that transcription factor NF-kappaB may code for factor(s) that play a role in sleep homeostasis.

Sleep deprivation-induced protein changes in basal forebrain: implications for synaptic plasticity.

The need to sleep is universal and lack of sleep often results in decreases in alertness and cognitive function. Data suggest that sleep-related mechanisms and deficits resulting from loss of sleep are associated anatomically with the basal forebrain. Long-term effects of sleep deprivation, those lasting a day or more, likely require transcriptional changes leading to changes in protein synthesis, whereas short-term effects may be mediated by rapid changes in the functional status of proteins. To understand sleep deprivation-induced changes in proteins in the wake-promoting area of the basal forebrain in rat, proteomic analysis was carried out by a combination of 2D gel electrophoresis to separate and visualize proteins and matrix-assisted laser desorption/ionization time-of flight mass spectrometry for protein identification. Among 969 protein spots that were compared, 89 spots showed more than a twofold difference between 6-hr sleep-deprived rats and undisturbed sleeping controls. We have identified 11 of these proteins to be either cytoskeletal or associated with synapses. The changes in four of these proteins were analyzed further by Western blots of 1D and 2D. Two proteins associated with the cytoskeleton, tubulin and GAP43, show posttranslational modifications. Increased tyrosination of alpha tubulin isoforms and increased phosphorylation of GAP43 was observed after 6-hr sleep deprivation when compared to that in sleeping controls. The synaptic protein synaptosomal-associated protein-25 (SNAP25) is decreased whereas amphiphysin is phosphorylated after sleep deprivation. These changes in proteins in the basal forebrain during short-term sleep deprivation are suggestive of changes in the substrate for neuronal transmission and plasticity.

Wakefulness-inducing effects of histamine in the basal forebrain of freely moving rats.

Histamine-containing neurons of the tuberomammillary nucleus (TMN) are implicated in facilitating wakefulness. They project to many brain areas, including the cholinergic basal forebrain (BF). The cholinergic magnocellular regions of the BF are important in the regulation of cortical arousal and wakefulness, and a role for histamine in this activity is suggested by in vitro data indicating histamine excites BF cholinergic and non-cholinergic neurons. To test the hypothesis that histamine induces wakefulness via actions in the BF, we performed microdialysis perfusion of different concentrations of histamine (100, 500 and 1000 microM) in the BF of Sprague-Dawley rats. A MANOVA analysis showed that histamine produced a highly statistically significant and dose-dependent increase in wakefulness and decrease in non-rapid eye movement (NREM) sleep compared with artificial cerebrospinal fluid perfusion. From a wakefulness baseline percentage time of about 12% with artificial cerebrospinal fluid, histamine perfusion increased this value to 26% (100 microM), 36% (500 microM), or 47% (1000 microM). There was no statistically significant change in rapid eye movement (REM) sleep. Histamine perfusion (500 microM) in a control site, the centromedian thalamic nucleus, did not produce any change in behavioral state. The results indicate a prominent role of histamine in wakefulness regulation via the BF and further support the hypothesis that the BF has an important role in EEG activation and wakefulness.