Adult Single-nucleus Neuronal Transcriptomes of Insulin Signaling Mutants Reveal Regulators of Behavior and Learning.

The insulin/insulin-like signaling (IIS) pathway regulates many of C. elegans' adult functions, including learning and memory 1 . While whole-worm and tissue-specific transcriptomic analyses have identified IIS targets 2,3 , a higher-resolution single-cell approach is required to identify changes that confer neuron-specific improvements in the long-lived insulin receptor mutant, daf-2 . To understand how behaviors that are controlled by a small number of neurons change in daf-2 mutants, we used the deep resolution of single-nucleus RNA sequencing to define each neuron type's transcriptome in adult wild-type and daf-2 mutants. First, we found surprising differences between wild-type L4 larval neurons and young adult neurons in chemoreceptor expression, synaptic genes, and learning and memory genes. These Day 1 adult neuron transcriptomes allowed us to identify adult AWC-specific regulators of chemosensory function and to predict neuron-to-neuron peptide/receptor pairs. We then identified gene expression changes that correlate with daf-2's improved cognitive functions, particularly in the AWC sensory neuron that controls learning and associative memory 4 , and used behavioral assays to test their roles in cognitive function. Combining deep single-neuron transcriptomics, genetic manipulation, and behavioral analyses enabled us to identify genes that may function in a single adult neuron to control behavior, including conserved genes that function in learning and memory. One-Sentence Summary: Single-nucleus sequencing of adult wild-type and daf-2 C. elegans neurons reveals functionally relevant transcriptional changes, including regulators of chemosensation, learning, and memory.

Neuronal activation of Gαq EGL-30/GNAQ late in life rejuvenates cognition across species.

Loss of cognitive function with age is devastating. EGL-30/GNAQ and Gαq signaling pathways are highly conserved between C. elegans and mammals, and murine Gnaq is enriched in hippocampal neurons and declines with age. We found that activation of EGL-30 in aged worms triples memory span, and GNAQ gain of function significantly improved memory in aged mice: GNAQ(gf) in hippocampal neurons of 24-month-old mice (equivalent to 70- to 80-year-old humans) rescued age-related impairments in well-being and memory. Single-nucleus RNA sequencing revealed increased expression of genes regulating synaptic function, axon guidance, and memory in GNAQ-treated mice, and worm orthologs of these genes were required for long-term memory extension in worms. These experiments demonstrate that C. elegans is a powerful model to identify mammalian regulators of memory, leading to the identification of a pathway that improves memory in extremely old mice. To our knowledge, this is the oldest age at which an intervention has improved age-related cognitive decline.

Motor learning rapidly increases synaptogenesis and astrocytic structural plasticity in the rat cerebellum.

Motor-skill learning is associated with cerebellar synaptogenesis and astrocytic hypertrophy, but most of these assessments of cerebellar ultrastructure have been completed after one month of training. After one month of training, the motor-skills necessary to complete these tasks have been acquired for weeks. This experiment aimed to characterize cerebellar ultrastructure during the acquisition phase of motor-skill learning, at a point when performance is still improving. Male and female rats trained for four days on the acrobatic motor learning task, which involved traversing challenging obstacles such as narrow beams and ladders. Concurrently, rats in the motor control condition walked a flat alleyway requiring no skilled movements. After training was complete, all rats were euthanized, and tissue was prepared for electron microscopy. Unbiased stereology techniques were used to assess synaptic and astrocytic plasticity. Results indicated that during the initial days of training, female rats made fewer errors and had shorter latencies on the acrobatic course compared to male rats. However, there were no sex differences in cerebellar ultrastructure. Male and female rats that completed four days of acrobatic training displayed an increase in the density of parallel fiber-Purkinje cell synapses per Purkinje cell and an increase in astrocytic volume, relative to rats in the motor control group.

Aerobic exercise increases sprouting angiogenesis in the male rat motor cortex.

Exercise is beneficial to brain health, and historically, the advantageous effects of exercise on the brain have been attributed to neuronal plasticity. However, it has also become clear that the brain vascular system also exhibits plasticity in response to exercise. This plasticity occurs in areas involved in movement, such as the motor cortex. This experiment aimed to further characterize the effects of exercise on structural vascular plasticity in the male rat motor cortex, by specifically identifying whether features of angiogenesis, the growth of new capillaries, or changes in vessel diameter were present. Male rats in the exercise group engaged in a 5-week bout of voluntary wheel running, while a second group of rats remained sedentary. After the exercise regimen, vascular corrosion casts, resin replicas of the brain vasculature, were made for all animals and imaged using a scanning electron microscope. Results indicate sprouting angiogenesis was the primary form of structural vascular plasticity detected in the motor cortex under these aerobic exercise parameters. Additionally, exercised rats displayed a slight increase in capillary diameter and expanded endothelial cell nuclei diameters in this region.

Wheel running for 26 weeks is associated with sustained vascular plasticity in the rat motor cortex.

Vascular pathologies represent the leading causes of mortality worldwide. The nervous system has evolved mechanisms to compensate for the cerebral hypoxia caused by many of these conditions. Vessel dilation and growth of new vessels are two prominent responses to hypoxia, both of which play a critical role in maintaining cerebral homeostasis. One way to facilitate cerebrovascular plasticity, and develop neuroprotection against vascular pathologies, is through aerobic exercise. The present study explored the long-term consequences of aerobic exercise on vascular structure and function in the motor cortex. Rats were assigned to a sedentary condition or were provided access to running wheels for 26 weeks. Rats were then anesthetized, and angiograms were captured using spectral domain optical coherence tomography (SD-OCT) to explore cerebrovascular reactivity in response to altered oxygen and carbon dioxide status. Following this procedure, all rats were euthanized, and unbiased stereological quantification of blood vessel density was collected from sections of the primary motor cortex infused with India ink. Results demonstrated that chronic exercise increased capillary and arteriole surface area densities and enhanced arteriole reactivity in response to hypercapnia-hypoxia, as displayed by increased vasodilation within the motor cortex of exercised animals.

Optimized Bexarotene Aerosol Formulation Inhibits Major Subtypes of Lung Cancer in Mice.

Bexarotene has shown inhibition of lung and mammary gland tumorigenesis in preclinical models and in clinical trials. The main side effects of orally administered bexarotene are hypertriglyceridemia and hypercholesterolemia. We previously demonstrated that aerosolized bexarotene administered by nasal inhalation has potent chemopreventive activity in a lung adenoma preclinical model without causing hypertriglyceridemia. To facilitate its future clinical translation, we modified the formula of the aerosolized bexarotene with a clinically relevant solvent system. This optimized aerosolized bexarotene formulation was tested against lung squamous cell carcinoma mouse model and lung adenocarcinoma mouse model and showed significant chemopreventive effect. This new formula did not cause visible signs of toxicity and did not increase plasma triglycerides or cholesterol. This aerosolized bexarotene was evenly distributed to the mouse lung parenchyma, and it modulated the microenvironment in vivo by increasing the tumor-infiltrating T cell population. RNA sequencing of the lung cancer cell lines demonstrated that multiple pathways are altered by bexarotene. For the first time, these studies demonstrate a new, clinically relevant aerosolized bexarotene formulation that exhibits preventive efficacy against the major subtypes of lung cancer. This approach could be a major advancement in lung cancer prevention for high risk populations, including former and present smokers.

Astrocytes and radial glia-like cells, but not neurons, display a nonapoptotic increase in caspase-3 expression following exercise.

BACKGROUND: Exercise induces plasticity in the hippocampus, which includes increases in neurogenesis, the proliferation of new neurons, and angiogenesis, the sprouting of new capillaries from preexisting blood vessels. Following exercise, astrocytes also undergo morphological changes that parallel the events occurring in the neurovascular system. Interestingly, there have also been reports of apoptosis in the hippocampus following aerobic exercise. This experiment aimed to identify which population of hippocampal cells undergoes apoptosis after an acute bout of exercise. METHODS: Cleaved caspase-3, a terminal protein in the apoptotic cascade, was initially used to identify apoptotic cells in the hippocampus after rats completed an acute bout of exercise. Next, the proportion of immature neurons, adult neurons, astrocytes, or radial glia-like cells expressing cleaved caspase-3 was quantified. TUNEL staining was completed as a second measure of apoptosis. RESULTS: Following exercise, cleaved caspase-3 expression was increased in the CA1 and DG regions of the hippocampus. Cleaved caspase-3 was not highly expressed in neuronal populations, and expression was not increased in these cells postexercise. Instead, cleaved caspase-3 was predominantly expressed in astrocytes. Following exercise, there was an increased number of cleaved caspase-3 positive astrocytes in DG and CA1, and cleaved caspase-3 positive radial glia-like cells located in the subgranular zone. To determine whether cleaved caspase-3 expression in these glial cells was associated with apoptosis, a TUNEL assay was completed. TUNEL staining was negligible in all groups and did not mirror the pattern of caspase-3 labeling. CONCLUSIONS: Cleaved caspase-3 expression was detected largely in non-neuronal cell populations, and the pattern of cleaved caspase-3 expression did not match that of TUNEL. This suggests that after exercise, cleaved caspase-3 expression may serve a nonapoptotic role in these hippocampal astrocytes and radial glia-like cells. It will be important to identify the function of exercise-induced cleaved caspase-3 expression in the future experiments.

Inactivation of the cerebellar fastigial nuclei alters social behavior in the rat.

Research has implicated the deep cerebellar nuclei in autism. This study questioned whether fastigial nuclei abnormalities account for some of the irregular social behaviors seen in autism. Bilateral cannulation surgery was performed on 13 rats. An ABABAB reversal design was implemented. All animals received a microinfusion of saline during the baseline (A) phases. The experimental animal was placed in an open field with an unfamiliar confederate animal, and social interactions between the two animals were measured for 10 min. Seven animals received microinfusions of bupivacaine during the treatment phases (B), which temporarily inactivated the fastigial nuclei. Six control animals received saline again, and social interaction was retested. This sequence was executed 3 times over 6 days to achieve an ABABAB reversal design. Because the cerebellum is involved in motor behavior, the animals' locomotion was analyzed to ensure results were not because of locomotor deficits. A gait analysis and distance traveled in the open field were used to measure locomotion. No differences were found in locomotor behavior. Results of the social interaction analyses indicate animals with inactivated fastigial nuclei engage in less intense social interactions and engage in more behaviors to prevent social interaction. Knowledge that the fastigial nuclei mediate social interaction can further the understanding of pathology in the autistic brain and lead to breakthrough treatments. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Exercise pattern and distance differentially affect hippocampal and cerebellar expression of FLK-1 and FLT-1 receptors in astrocytes and blood vessels.

Aerobic exercise benefits the body and brain. In the brain, benefits include neuroprotection and improved cognition. These exercise-induced changes are attributed in part to angiogenesis: the growth of new capillaries from preexisting vessels. One critical factor involved in the regulation of angiogenesis is VEGF and its receptors Flk-1 and Flt-1. Although exercise is generally found to be beneficial, there are wide variations in exercise regimens across experiments. This study standardized some of these variations. Rats were assigned to a voluntary or a forced wheel running exercise condition. Within each condition, animals ran for either a long (1000m) or short distance (500m) for up to 24h. Additionally, one voluntary group had unrestricted access to the wheels for the full 24h. Exercising animals were then compared to inactive controls, based on unbiased stereological quantification of Flk-1 and Flt-1 immunohistochemical labeling in the hippocampus and cerebellum. Findings indicated that voluntary exercise, but not forced exercise, could significantly increase Flk-1 and Flt-1 expression in the hippocampus. Interestingly, Flk-1 expression was elevated in astrocytes and Flt-1 in vessels. In the cerebellum long distance forced exercise resulted in the least Flk-1 expression compared to other conditions, and Flt-1 expression in exercising animals either did not change or was suppressed relative to inactive controls.