Unbiased classification of spatial strategies in the Barnes maze.

MOTIVATION: Spatial learning is one of the most widely studied cognitive domains in neuroscience. The Morris water maze and the Barnes maze are the most commonly used techniques to assess spatial learning and memory in rodents. Despite the fact that these tasks are well-validated paradigms for testing spatial learning abilities, manual categorization of performance into behavioral strategies is subject to individual interpretation, and thus to bias. We have previously described an unbiased machine-learning algorithm to classify spatial strategies in the Morris water maze. RESULTS: Here, we offer a support vector machine-based, automated, Barnes-maze unbiased strategy (BUNS) classification algorithm, as well as a cognitive score scale that can be used for memory acquisition, reversal training and probe trials. The BUNS algorithm can greatly benefit Barnes maze users as it provides a standardized method of strategy classification and cognitive scoring scale, which cannot be derived from typical Barnes maze data analysis. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://okunlab.wix.com/okunlab as a MATLAB application. CONTACT: eitan.okun@biu.ac.ilSupplementary information: Supplementary data are available at Bioinformatics online.

Unraveling cognitive traits using the Morris water maze unbiased strategy classification (MUST-C) algorithm.

The assessment of spatial cognitive learning in rodents is a central approach in neuroscience, as it enables one to assess and quantify the effects of treatments and genetic manipulations from a broad perspective. Although the Morris water maze (MWM) is a well-validated paradigm for testing spatial learning abilities, manual categorization of performance in the MWM into behavioral strategies is subject to individual interpretation, and thus to biases. Here we offer a support vector machine (SVM) - based, automated, MWM unbiased strategy classification (MUST-C) algorithm, as well as a cognitive score scale. This model was examined and validated by analyzing data obtained from five MWM experiments with changing platform sizes, revealing a limitation in the spatial capacity of the hippocampus. We have further employed this algorithm to extract novel mechanistic insights on the impact of members of the Toll-like receptor pathway on cognitive spatial learning and memory. The MUST-C algorithm can greatly benefit MWM users as it provides a standardized method of strategy classification as well as a cognitive scoring scale, which cannot be derived from typical analysis of MWM data.

Toll-like receptors 2 and 4 modulate autonomic control of heart rate and energy metabolism.

Toll-like receptors (TLR) are innate immune receptors typically activated by microbial-associated molecular patterns (MAMPs) during infection or damage-associated molecular patterns (DAMPs) as a result of tissue injury. Recent findings suggest that TLR2 and TLR4 signaling play important roles in developmental and adult neuroplasticity, and in learning and memory. In addition, activation of TLR2 and TLR4 worsens ischemic injury to the heart and brain in animal models of myocardial infarction and stroke. TLR activation is also implicated in thermoregulation and fever in response to infection. However, it is not known whether TLRs participate in the regulation of the sympathetic and/or parasympathetic components of the autonomic nervous system (ANS). Here we provide evidence that TLR2 and TLR4 influence autonomic regulation of heart rate (HR) body temperature and energy metabolism in mice. We show that mice lacking TLR2 or TLR4 exhibit reduced basal HR, which results from an increase of parasympathetic tone. In addition, thermoregulatory responses to stress are altered in TLR2-/- and TLR4-/- mice, and brown fat-dependent thermoregulation is altered in TLR4-/- mice. Moreover, TLR2-/- and TLR4-/- mice consume less food and exhibit a greater mass compared to wild type mice. Collectively, our findings suggest important roles for TLR2 and TLR4 in the ANS regulation of cardiovascular function, thermoregulation, and energy metabolism.

TLR2 activation inhibits embryonic neural progenitor cell proliferation.

Toll-like receptors (TLRs) play essential roles in innate immunity, and increasing evidence indicates that these receptors are expressed in neurons, astrocytes, and microglia in the brain, where they mediate responses to infection, stress, and injury. To address the possibility that TLR2 heterodimer activation could affect progenitor cells in the developing brain, we analyzed the expression of TLR2 throughout mouse cortical development, and assessed the role of TLR2 heterodimer activation in neuronal progenitor cell (NPC) proliferation. TLR2 mRNA and protein was expressed in the cortex in embryonic and early postnatal stages of development, and in cultured cortical NPC. While NPC from TLR2-deficient and wild type embryos had the same proliferative capacity, TLR2 activation by the synthetic bacterial lipopeptides Pam(3)CSK(4) and FSL1, or low molecular weight hyaluronan, an endogenous ligand for TLR2, inhibited neurosphere formation in vitro. Intracerebral in utero administration of TLR2 ligands resulted in ventricular dysgenesis characterized by increased ventricle size, reduced proliferative area around the ventricles, increased cell density, an increase in phospho-histone 3 cells, and a decrease in BrdU(+) cells in the sub-ventricular zone. Our findings indicate that loss of TLR2 does not result in defects in cerebral development. However, TLR2 is expressed and functional in the developing telencephalon from early embryonic stages and infectious agent-related activation of TLR2 inhibits NPC proliferation. TLR2-mediated inhibition of NPC proliferation may therefore be a mechanism by which infection, ischemia, and inflammation adversely affect brain development.

Toll-like receptors in neurodegeneration.

The key roles of toll-like receptors (TLRs) as mediators of the detection and responses of immune cells to invading pathogens are well known. There are at least 13 mammalian TLRs which are integral membrane proteins with a leucine-rich extracellular domain and a cytoplasmic domain similar to that of the interleukin-1 receptor which initiates downstream signaling through kinases to activate transcription factors such as AP-1 and NFkappaB. TLRs are activated in glial cells (microglia, astrocytes and oligodendrocytes) and lymphocytes that infiltrate the nervous system in response to inflammation caused by infectious agents, tissue injury or autoimmune conditions. By inducing the production of pro-inflammatory cytokines and cell adhesion molecules in immune cells, TLRs may indirectly damage neurons in conditions such as ischemic stroke and multiple sclerosis. Recent findings suggest that neurons also express a subset of TLRs and that their activation promotes neuronal degeneration in experimental models of stroke and Alzheimer's disease. TLRs may also play roles in regulating the processes of neurogenesis and neurite outgrowth, suggesting roles in neuronal plasticity. A better understanding of the molecular and cellular biology of TLRs in the normal and diseased nervous system, may lead to novel approaches for preventing neuronal degeneration and promoting recovery of function in an array of neurodegenerative conditions.

Differential control of central cardiorespiratory interactions by hypercapnia and the effect of prenatal nicotine.

Hypercapnia evokes a strong cardiorespiratory response including gasping and a pronounced bradycardia; however, the mechanism responsible for these survival responses initiated in the brainstem is unknown. To examine the effects of hypercapnia on the central cardiorespiratory network, we used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and inhibitory synaptic neurotransmission to cardioinhibitory vagal neurons (CVNs). Hypercapnia differentially modulated inhibitory neurotransmission to CVNs; whereas hypercapnia selectively depressed spontaneous glycinergic IPSCs in CVNs without altering respiratory-related increases in glycinergic neurotransmission, it decreased both spontaneous and inspiratory-associated GABAergic IPSCs. Because maternal smoking is the highest risk factor for sudden infant death syndrome (SIDS) and prenatal nicotine exposure is proposed to be the link between maternal smoking and SIDS, we examined the cardiorespiratory responses to hypercapnia in animals exposed to nicotine in the prenatal and perinatal period. In animals exposed to prenatal nicotine, hypercapnia evoked an exaggerated depression of GABAergic IPSCs in CVNs with no significant change in glycinergic neurotransmission. Hypercapnia altered inhibitory neurotransmission to CVNs at both presynaptic and postsynaptic sites. Although the results obtained in this study in vitro cannot be extrapolated with certainty to in vivo responses, the results of this study provide a likely neurochemical mechanism for hypercapnia-evoked bradycardia and the dysregulation of this response with exposure to prenatal nicotine, creating a higher risk for SIDS.

Peri-nuclear clustering of mitochondria is triggered during aluminum maltolate induced apoptosis.

Synapse loss and neuronal death are key features of Alzheimer's disease pathology. Disrupted axonal transport of mitochondria is a potential mechanism that could contribute to both. As the major producer of ATP in the cell, transport of mitochondria to the synapse is required for synapse maintenance. However, mitochondria also play an important role in the regulation of apoptosis. Investigation of aluminum (Al) maltolate induced apoptosis in human NT2 cells led us to explore the relationship between apoptosis related changes and the disruption of mitochondrial transport. Similar to that observed with tau over expression, NT2 cells exhibit peri-nuclear clustering of mitochondria following treatment with Al maltolate. Neuritic processes largely lacked mitochondria, except in axonal swellings. Similar, but more rapid results were observed following staurosporine administration, indicating that the clustering effect was not specific to Al maltolate. Organelle clustering and transport disruption preceded apoptosis. Incubation with the caspase inhibitor zVAD-FMK effectively blocked apoptosis, however failed to prevent organelle clustering. Thus, transport disruption is associated with the initiation, but not necessarily the completion of apoptosis. These results, together with observed transport defects and apoptosis related changes in Alzheimer disease brain suggest that mitochondrial transport disruption may play a significant role in synapse loss and thus the pathogenesis or Alzheimer's disease.

Fentanyl inhibits GABAergic neurotransmission to cardiac vagal neurons in the nucleus ambiguus.

Fentanyl citrate is a synthetic opiate analgesic often used clinically for neonatal anesthesia. Although fentanyl significantly depresses heart rate, the mechanism of inducing bradycardia remains unclear. One possible site of action is the cardioinhibitory parasympathetic vagal neurons in the nucleus ambiguus (NA), from which originates control of heart rate and cardiac function. Inhibitory synaptic activity to cardiac vagal neurons is a major determinant of their activity. Therefore, the effect of fentanyl on GABAergic neurotransmission to parasympathetic cardiac vagal neurons was studied using whole-cell patch clamp electrophysiology. Application of fentanyl induced a reduction in both the frequency and amplitude of GABAergic IPSCs in cardiac vagal neurons. This inhibition was mediated at both pre- and postsynaptic sites as evidenced by a dual decrease in the frequency and amplitude of spontaneous miniature IPSCs. Application of the selective micro-antagonist CTOP abolished the fentanyl-mediated inhibition of GABAergic IPSCs. These results demonstrate that fentanyl acts on micro-opioid receptors on cardiac vagal neurons and neurons preceding them to reduce GABAergic neurotransmission and increase parasympathetic activity. The inhibition of GABAergic effects may be one mechanism by which fentanyl induces bradycardia.

Aluminum maltolate-induced toxicity in NT2 cells occurs through apoptosis and includes cytochrome c release.

Aluminum (Al) compounds are neurotoxic and have been shown to induce experimental neurodegeneration although the mechanism of this effect is unclear. In order to study this neurotoxic effect of Al, we have developed an in vitro model system using Al maltolate and human NT2 cells. Al maltolate at 500 microM caused significant cell death with a 24-h incubation and this toxicity was even more evident after 48 h. Lower doses of Al maltolate were also effective, but required a longer incubation for cell death. Nuclear fragmentation suggestive of apoptosis was observed as early as three hours and increased substantially through 24 h. Chromatin condensation and nuclear fragmentation were confirmed by electron microscopy. In addition, TUNEL positive nuclei were also observed. The release of cytochrome c was demonstrated with Western blot analysis. This in vitro model using human cells adds to our understanding of Al neurotoxicity and could provide insight into the neurodegenerative processes in human disease.

Metabolic abnormalities and hypoleptinemia in α-synuclein A53T mutant mice.

Parkinson's disease (PD) patients frequently display loss of body fat mass and increased energy expenditure, and several studies have outlined a relationship between these metabolic abnormalities and disease severity, yet energy metabolism is largely unstudied in mouse models of PD. Here we characterize metabolic and physiologic responses to a high calorie diet (HCD) in mice expressing in neurons a mutant form of human α-synuclein (A53T) that causes dominantly inherited familial forms of the disease. A53T (SNCA) and wild type (WT) littermate mice were placed on a HCD for 12 weeks and evaluated for weight gain, food intake, body fat, blood plasma leptin, hunger, glucose tolerance, and energy expenditure. Results were compared with both SNCA and WT mice on a control diet. Despite consuming similar amounts of food, WT mice gained up to 66% of their original body weight on a HCD, whereas SNCA mice gained only 17%. Further, after 12 weeks on a HCD, magnetic resonance imaging analysis revealed that WT mice had significantly greater total and visceral body fat compared with SNCA mice (p < 0.007). At the age of 24 weeks SNCA mice displayed significantly increased hunger compared with WT (p < 0.03). At the age of 36 weeks, SNCA mice displayed significant hypoleptinemia compared with WT, both on a normal diet and a HCD (p < 0.03). The HCD induced insulin insensitivity in WT, but not SNCA mice, as indicated by an oral glucose tolerance test. Finally, SNCA mice displayed greater energy expenditure compared with WT, as measured in a Comprehensive Laboratory Animal Monitoring System, after 12 weeks on a HCD. Thus, SNCA mice are resistant to HCD-induced obesity and insulin resistance and display reduced body fat, increased hunger, hypoleptinemia and increased energy expenditure. Our findings reveal a profile of metabolic dysfunction in a mouse model of PD that is similar to that of human PD patients, thus providing evidence that α-synuclein pathology is sufficient to drive such metabolic abnormalities and providing an animal model for discovery of the underlying mechanisms and potential therapeutic interventions.

Neuronal expression of familial Parkinson's disease A53T α-synuclein causes early motor impairment, reduced anxiety and potential sleep disturbances in mice.

BACKGROUND: Mutations in the human α-synuclein gene lead to early-onset Parkinson's disease (PD); however, phenotypes of α-synuclein mutant mice vary depending upon the promoter driving transgene expression. OBJECTIVE: The goal of this study was to characterize behavior and neurochemical alterations in mice expressing mutant (A53T) human α-synuclein, controlled by a neuron-specific Thy-1 promoter. Our data provide important additional phenotypic and biochemical characterization of a previously generated model of PD. METHODS: A53T (SNCA) and wild type (WT) littermate mice were evaluated for motor function (rotarod and stride length) and anxiety (elevated plus maze and open field) every 2 weeks. At 24 weeks mice were evaluated in a Comprehensive Lab Animal Monitoring System (CLAMS). A separate cohort of mice were euthanized at 12, 24 and 36 weeks for immunoblot analysis of α-synuclein, dopamine transporter (DAT) and tyrosine hydroxylase (TH) in the striatum, and hypothalamic serotonin and metabolites were measured. RESULTS: SNCA mice display significant motor deficits at 14-18 weeks of age compared to WT mice, which progress over time. CLAMS analysis revealed an increase in activity during the dark phase and a reduction in overall estimated sleep time for SNCA mice compared to WT consistent with clinical reports of sleep abnormalities in PD. A transient change in the levels of DAT appeared at 12 weeks in the striatum and serotonin levels were also altered in the hypothalamus at this time point. CONCLUSIONS: This PD model displays consistent and clinically relevant motor and sleep phenotypes. Anxiety phenotypes are consistent with other α-synuclein based PD models yet incongruous with typical clinical symptoms. Early increases in serotonin levels potentially explain reductions in anxiety behaviors and sleep.

Dietary energy intake modifies brainstem autonomic dysfunction caused by mutant α-synuclein.

Parkinson's disease (PD) patients often exhibit impaired regulation of heart rate by the autonomic nervous system (ANS) that may precede motor symptoms in many cases. Results of autopsy studies suggest that brainstem pathology, including the accumulation of α-synuclein, precedes damage to dopaminergic neurons in the substantia nigra in PD. However, the molecular and cellular mechanisms responsible for the early dysfunction of brainstem autonomic neurons are unknown. Here we report that mice expressing a mutant form of α-synuclein that causes familial PD exhibit aberrant autonomic control of the heart characterized by elevated resting heart rate and an impaired cardiovascular stress response, associated with reduced parasympathetic activity and accumulation of α-synuclein in the brainstem. These ANS abnormalities occur early in the disease process. Adverse effects of α-synuclein on the control of heart rate are exacerbated by a high energy diet and ameliorated by intermittent energy restriction. Our findings establish a mouse model of early dysregulation of brainstem control of the cardiovascular system in PD, and further suggest the potential for energy restriction to attenuate ANS dysfunction, particularly in overweight individuals.

Brain-derived neurotrophic factor as a regulator of systemic and brain energy metabolism and cardiovascular health.

Overweight sedentary individuals are at increased risk for cardiovascular disease, diabetes, and some neurological disorders. Beneficial effects of dietary energy restriction (DER) and exercise on brain structural plasticity and behaviors have been demonstrated in animal models of aging and acute (stroke and trauma) and chronic (Alzheimer's and Parkinson's diseases) neurological disorders. The findings described later, and evolutionary considerations, suggest brain-derived neurotrophic factor (BDNF) plays a critical role in the integration and optimization of behavioral and metabolic responses to environments with limited energy resources and intense competition. In particular, BDNF signaling mediates adaptive responses of the central, autonomic, and peripheral nervous systems from exercise and DER. In the hypothalamus, BDNF inhibits food intake and increases energy expenditure. By promoting synaptic plasticity and neurogenesis in the hippocampus, BDNF mediates exercise- and DER-induced improvements in cognitive function and neuroprotection. DER improves cardiovascular stress adaptation by a mechanism involving enhancement of brainstem cholinergic activity. Collectively, findings reviewed in this paper provide a rationale for targeting BDNF signaling for novel therapeutic interventions in a range of metabolic and neurological disorders.

Aberrant heart rate and brainstem brain-derived neurotrophic factor (BDNF) signaling in a mouse model of Huntington's disease.

Huntington's disease (HD) is associated with profound autonomic dysfunction including dysregulation of cardiovascular control often preceding cognitive or motor symptoms. Brain-derived neurotrophic factor (BDNF) levels are decreased in the brains of HD patients and HD mouse models, and restoring BDNF levels prevents neuronal loss and extends survival in HD mice. We reasoned that heart rate changes in HD may be associated with altered BDNF signaling in cardiovascular control nuclei in the brainstem. Here we show that heart rate is elevated in HD (N171-82Q) mice at presymptomatic and early disease stages, and heart rate responses to restraint stress are attenuated. BDNF levels were significantly reduced in brainstem regions containing cardiovascular nuclei in HD mice and human HD patients. Central administration of BDNF restored the heart rate to control levels. Our findings establish a link between diminished BDNF expression in brainstem cardiovascular nuclei and abnormal heart rates in HD mice, and suggest a novel therapeutic target for correcting cardiovascular dysfunction in HD.

Evidence for a developmental role for TLR4 in learning and memory.

Toll-like receptors (TLRs) play essential roles in innate immunity and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain where they mediate responses to infection, stress and injury. Very little is known about the roles of TLRs in cognition. To test the hypothesis that TLR4 has a role in hippocampus-dependent spatial learning and memory, we used mice deficient for TLR4 and mice receiving chronic TLR4 antagonist infusion to the lateral ventricles in the brain. We found that developmental TLR4 deficiency enhances spatial reference memory acquisition and memory retention, impairs contextual fear-learning and enhances motor functions, traits that were correlated with CREB up-regulation in the hippocampus. TLR4 antagonist infusion into the cerebral ventricles of adult mice did not affect cognitive behavior, but instead affected anxiety responses. Our findings indicate a developmental role for TLR4 in shaping spatial reference memory, and fear learning and memory. Moreover, we show that central TLR4 inhibition using a TLR4 antagonist has no discernible physiological role in regulating spatial and contextual hippocampus-dependent cognitive behavior.

Toll-like receptor signaling in neural plasticity and disease.

Toll-like receptors (TLRs) are a family of innate immune system receptors that respond to pathogen-derived and tissue damage-related ligands. TLR signaling in immune cells, glia and neurons can play roles in the pathogenesis of stroke, Alzheimer's disease (AD) and multiple sclerosis (MS). Recent findings suggest that TLR signaling also influences multiple dynamic processes in the developing and adult central nervous system including neurogenesis, axonal growth and structural plasticity. In addition, TLRs are implicated in the regulation of behaviors including learning, memory and anxiety. This review describes recently discovered and unexpected roles for TLRs in neuroplasticity, and the implications of these findings for future basic and translational research studies.

GLP-1 receptor stimulation depresses heart rate variability and inhibits neurotransmission to cardiac vagal neurons.

AIMS: glucagon-like peptide 1 (GLP-1) is an incretin hormone released from the gut in response to food intake. Whereas GLP-1 acts in the periphery to inhibit glucagon secretion and stimulate insulin release, it also acts in the central nervous system to mediate autonomic control of feeding, body temperature, and cardiovascular function. Because of its role as an incretin hormone, GLP-1 receptor analogs are used as a treatment for type 2 diabetes. Central or peripheral administration of GLP-1 increases blood pressure and heart rate, possibly by activating brainstem autonomic nuclei and increasing vagus nerve activity. However, the mechanism(s) by which GLP-1 receptor stimulation affects cardiovascular function are unknown. We used the long-lasting GLP-1 receptor agonist Exendin-4 (Ex-4) to test the hypothesis that GLP-1 signalling modulates central parasympathetic control of heart rate. METHODS AND RESULTS: using a telemetry system, we assessed heart rate in mice during central Ex-4 administration. Heart rate was increased by both acute and chronic central Ex-4 administration. Spectral analysis indicated that the high frequency and low frequency powers of heart rate variability were diminished by Ex-4 treatment. Finally, Ex-4 decreased both excitatory glutamatergic and inhibitory glycinergic neurotransmission to preganglionic parasympathetic cardiac vagal neurons. CONCLUSION: these data suggest that central GLP-1 receptor stimulation diminishes parasympathetic modulation of the heart thereby increasing heart rate.

Respiratory modulation of premotor cardiac vagal neurons in the brainstem.

The respiratory and cardiovascular systems are highly intertwined, both anatomically and physiologically. Respiratory and cardiovascular neurons are often co-localized in the same brainstem regions, and this is particularly evident in the ventral medulla which contains presympathetic neurons in the rostral ventrolateral medulla, premotor parasympathetic cardioinhibitory neurons in the nucleus ambiguus, and the ventral respiratory group, which includes the pre-Botzinger complex. Anatomical studies of respiratory and cardiovascular neurons have demonstrated that many of these neurons have projections and axon collateral processes which extend into their neighboring cardiorespiratory regions providing an anatomical substrate for cardiorespiratory interactions. As other reports in this Special Issue of Respiratory Physiology & Neurobiology focus on interactions between the respiratory network and baroreceptors, neurons in the nucleus tractus solitarius, presympathetic neurons and sympathetic activity, this report will focus on the respiratory modulation of parasympathetic activity and the neurons that generate parasympathetic activity to the heart, cardiac vagal neurons.

Purinergic P2X receptors mediate excitatory transmission to cardiac vagal neurons in the nucleus ambiguus after hypoxia.

Challenges such as hypoxia elicit a powerful response from both the central cardiovascular and respiratory neuronal networks. Recent work indicates that purinergic neurotransmission in the brain stem is an important modulator of central respiratory network responses to hypoxia. This study tests whether alterations in purinergic neurotransmission extend beyond respiratory responses to hypoxia and also mediates respiratory inputs to cardiac vagal neurons. To examine central cardiorespiratory responses to hypoxia, we used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and synaptic neurotransmission to cardioinhibitory vagal neurons. Here we show that P2X receptor activation mediates respiratory-related excitatory neurotransmission to parasympathetic cardiac vagal neurons, the dominant control of heart rate. These data demonstrate a critical functional role for adenosine 5'-triphosphate-mediated purinergic signaling in facilitating respiratory-related excitatory neurotransmission to cardiac vagal neurons after hypoxia.