Pioneering new frontiers in circadian medicine chronotherapies for cardiovascular health.

Cardiovascular disease (CVD) is a global health concern. Circadian medicine improves cardiovascular care by aligning treatments with our body's daily rhythms and their underlying cellular circadian mechanisms. Time-based therapies, or chronotherapies, show special promise in clinical cardiology. They optimize treatment schedules for better outcomes with fewer side effects by recognizing the profound influence of rhythmic body cycles. In this review, we focus on three chronotherapy areas (medication, light, and meal timing) with potential to enhance cardiovascular care. We also highlight pioneering research in the new field of rest, the gut microbiome, novel chronotherapies for hypertension, pain management, and small molecules that targeting the circadian mechanism.

Reactive Astrocytes: Critical Players in the Development of Chronic Pain.

Chronic pain is associated with long term plasticity of nociceptive pathways in the central nervous system. Astrocytes can profoundly affect synaptic function and increasing evidence has highlighted how altered astrocyte activity may contribute to the pathogenesis of chronic pain. In response to injury, astrocytes undergo a shift in form and function known as reactive astrogliosis, which affects their release of cytokines and gliotransmitters. These neuromodulatory substances have been implicated in driving the persistent changes in central nociceptive activity. Astrocytes also release lactate which neurons can use to produce energy during synaptic plasticity. Furthermore, recent research has provided insight into lactate's emerging role as a signaling molecule in the central nervous system, which may be involved in directly modulating neuronal and astrocytic activity. In this review, we present evidence for the involvement of astrocyte-derived tumor necrosis factor alpha in pain-associated plasticity, in addition to research suggesting the potential involvement of gliotransmitters D-serine and adenosine-5'-triphosphate. We also discuss work implicating astrocyte-neuron metabolic coupling, and the possible role of lactate, which has been sparsely studied in the context of chronic pain, in supporting pathological changes in central nociceptive activity.

CIM6P/IGF-2 Receptor Ligands Reverse Deficits in Angelman Syndrome Model Mice.

Angelman syndrome (AS), a genetic disorder that primarily affects the nervous system, is characterized by delayed development, intellectual disability, severe speech impairment, and problems with movement and balance (ataxia). Most affected children also have recurrent seizures (epilepsy). No existing therapies are capable of comprehensively treating the deficits in AS; hence, there is an urgent need to identify new treatments. Here we show that insulin-like growth factor 2 (IGF-2) and mannose-6-phosphate (M6P), ligands of two independent binding sites of the cation-independent M6P/IGF-2 receptor (CIM6P/IGF-2R), reverse most major deficits of AS modeled in mice. Subcutaneous injection of IGF-2 or M6P in mice modeling AS restored cognitive impairments as assessed by measurements of contextual and recognition memories, motor deficits assessed by rotarod and hindlimb clasping, and working memory/flexibility measured by Y-maze. IGF-2 also corrected deficits in marble burying and significantly attenuated acoustically induced seizures. An observational battery of tests confirmed that neither ligand changed basic functions including physical characteristics, general behavioral responses, and sensory reflexes, indicating that they are relatively safe. Our data provide strong preclinical evidence that targeting CIM6P/IGF-2R is a promising approach for developing novel therapeutics for AS. LAY SUMMARY: There is no effective treatment for the neurodevelopmental disorder Angelman syndrome (AS). Using a validated AS mouse model, the Ube3am-/p+ , in this study we show that systemic administration of ligands of the cation independent mannose-6-phosphate receptor, also known as insulin-like growth factor 2 receptor (CIM6P/IGF-2R) reverses cognitive impairment, motor deficits, as well as seizures associated with AS. Thus, ligands that activate the CIM6P/IGF-2R may represent novel, potential therapeutic targets for AS.

Lactate from astrocytes fuels learning-induced mRNA translation in excitatory and inhibitory neurons.

Glycogenolysis and lactate transport from astrocytes to neurons is required for long-term memory formation, but the role of this lactate is poorly understood. Here we show that the Krebs cycle substrates pyruvate and ketone body B3HB can functionally replace lactate in rescuing memory impairment caused by inhibition of glycogenolysis or expression knockdown of glia monocarboxylate transporters (MCTs) 1 and 4 in the dorsal hippocampus of rats. In contrast, either metabolite is unable to rescue memory impairment produced by expression knockdown of MCT2, which is selectively expressed by neurons, indicating that a critical role of astrocytic lactate is to provide energy for neuronal responses required for long-term memory. These responses include learning-induced mRNA translation in both excitatory and inhibitory neurons, as well as expression of Arc/Arg3.1. Thus, astrocytic lactate acts as an energy substrate to fuel learning-induced de novo neuronal translation critical for long-term memory.

Insulin-Like Growth Factor II Targets the mTOR Pathway to Reverse Autism-Like Phenotypes in Mice.

Autism spectrum disorder (ASD) is a developmental disability characterized by impairments in social interaction and repetitive behavior, and is also associated with cognitive deficits. There is no current treatment that can ameliorate most of the ASD symptomatology; thus, identifying novel therapies is urgently needed. We used male BTBR T+Itpr3tf /J (BTBR) mice, a model that reproduces most of the core behavioral phenotypes of ASD, to test the effects of systemic administration of insulin-like growth factor II (IGF-II), a polypeptide that crosses the blood-brain barrier and acts as a cognitive enhancer. We show that systemic IGF-II treatments reverse the typical defects in social interaction, cognitive/executive functions, and repetitive behaviors reflective of ASD-like phenotypes. In BTBR mice, IGF-II, via IGF-II receptor, but not via IGF-I receptor, reverses the abnormal levels of the AMPK-mTOR-S6K pathway and of active translation at synapses. Thus, IGF-II may represent a novel potential therapy for ASD.SIGNIFICANCE STATEMENT Currently, there is no effective treatment for autism spectrum disorder (ASD), a developmental disability affecting a high number of children. Using a mouse model that expresses most of the key core as well as associated behavioral deficits of ASD, that are, social, cognitive, and repetitive behaviors, we report that a systemic administration of the polypeptide insulin-like growth factor II (IGF-II) reverses all these deficits. The effects of IGF-II occur via IGF-II receptors, and not IGF-I receptors, and target both basal and learning-dependent molecular abnormalities found in several ASD mice models, including those of identified genetic mutations. We suggest that IGF-II represents a potential novel therapeutic target for ASD.

Astrocyte glycogen and lactate: New insights into learning and memory mechanisms.

Memory, the ability to retain learned information, is necessary for survival. Thus far, molecular and cellular investigations of memory formation and storage have mainly focused on neuronal mechanisms. In addition to neurons, however, the brain comprises other types of cells and systems, including glia and vasculature. Accordingly, recent experimental work has begun to ask questions about the roles of non-neuronal cells in memory formation. These studies provide evidence that all types of glial cells (astrocytes, oligodendrocytes, and microglia) make important contributions to the processing of encoded information and storing memories. In this review, we summarize and discuss recent findings on the critical role of astrocytes as providers of energy for the long-lasting neuronal changes that are necessary for long-term memory formation. We focus on three main findings: first, the role of glucose metabolism and the learning- and activity-dependent metabolic coupling between astrocytes and neurons in the service of long-term memory formation; second, the role of astrocytic glucose metabolism in arousal, a state that contributes to the formation of very long-lasting and detailed memories; and finally, in light of the high energy demands of the brain during early development, we will discuss the possible role of astrocytic and neuronal glucose metabolisms in the formation of early-life memories. We conclude by proposing future directions and discussing the implications of these findings for brain health and disease. Astrocyte glycogenolysis and lactate play a critical role in memory formation. Emotionally salient experiences form strong memories by recruiting astrocytic ß2 adrenergic receptors and astrocyte-generated lactate. Glycogenolysis and astrocyte-neuron metabolic coupling may also play critical roles in memory formation during development, when the energy requirements of brain metabolism are at their peak.

Transdermal light neuromodulation: Optogenetics in the murine urinary tract.

BACKGROUND: Optogenetics is a biologic technique that uses light to control living neurons, which have been genetically modified to express light-sensitive ion-channels. OBJECTIVES: Using an adenovirus to modify the sciatic nerves of mice, we aim to demonstrate peripheral neuromodulation of bladder pain using transdermal light. STUDY DESIGN: This pilot study is divided into: A) Confirmation and Application and B) Behavioral Step. A) Six mice were injected with AAV6-hSyn-ChR2(H134R)-eYFP virus into their sciatic nerves. This encoded an excitatory opsin, enabling light-inducible stimulation. At 4-6 weeks after injection, we induced foot pain responses with an activating blue 475 nm wavelength of light. B) Two optogenetically primed mice and two control mice underwent anesthesia and capsaicin was instilled into their bladders via catheter. The catheters were removed and the mice awoke in a chamber that exposed them to either blue 475 nm light or no light. Groin licking was scored in a binary fashion by two blinded observers. RESULTS: A) All six mice exhibited pain response to 475 nm blue light either by licking of foot or avoidance of light. B) The optogenetically primed mice had a 48% reduction in bladder pain behavior when exposed to blue 475 nm light whereas the control mice had a 18% reduction. CONCLUSION: To our knowledge this is the first demonstration of the application of optogenetics to modulate sensation in the lower urinary tract. It suggests that the process of priming peripheral nerves for optogenetic modulation is possible and can be used to study bladder pain response in mice.

Neuropathic pain promotes adaptive changes in gene expression in brain networks involved in stress and depression.

Neuropathic pain is a complex chronic condition characterized by various sensory, cognitive, and affective symptoms. A large percentage of patients with neuropathic pain are also afflicted with depression and anxiety disorders, a pattern that is also seen in animal models. Furthermore, clinical and preclinical studies indicate that chronic pain corresponds with adaptations in several brain networks involved in mood, motivation, and reward. Chronic stress is also a major risk factor for depression. We investigated whether chronic pain and stress affect similar molecular mechanisms and whether chronic pain can affect gene expression patterns that are involved in depression. Using two mouse models of neuropathic pain and depression [spared nerve injury (SNI) and chronic unpredictable stress (CUS)], we performed next-generation RNA sequencing and pathway analysis to monitor changes in gene expression in the nucleus accumbens (NAc), the medial prefrontal cortex (mPFC), and the periaqueductal gray (PAG). In addition to finding unique transcriptome profiles across these regions, we identified a substantial number of signaling pathway-associated genes with similar changes in expression in both SNI and CUS mice. Many of these genes have been implicated in depression, anxiety, and chronic pain in patients. Our study provides a resource of the changes in gene expression induced by long-term neuropathic pain in three distinct brain regions and reveals molecular connections between pain and chronic stress.

Epigenetic mechanisms of chronic pain.

Neuropathic and inflammatory pain promote a large number of persisting adaptations at the cellular and molecular level, allowing even transient tissue or nerve damage to elicit changes in cells that contribute to the development of chronic pain and associated symptoms. There is evidence that injury-induced changes in chromatin structure drive stable changes in gene expression and neural function, which may cause several symptoms, including allodynia, hyperalgesia, anxiety, and depression. Recent findings on epigenetic changes in the spinal cord and brain during chronic pain may guide fundamental advances in new treatments. Here, we provide a brief overview of epigenetic regulation in the nervous system and then discuss the still-limited literature that directly implicates epigenetic modifications in chronic pain syndromes.

Coexistence of two forms of LTP in ACC provides a synaptic mechanism for the interactions between anxiety and chronic pain.

Chronic pain can lead to anxiety and anxiety can enhance the sensation of pain. Unfortunately, little is known about the synaptic mechanisms that mediate these re-enforcing interactions. Here we characterized two forms of long-term potentiation (LTP) in the anterior cingulate cortex (ACC); a presynaptic form (pre-LTP) that requires kainate receptors and a postsynaptic form (post-LTP) that requires N-methyl-D-aspartate receptors. Pre-LTP also involves adenylyl cyclase and protein kinase A and is expressed via a mechanism involving hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Interestingly, chronic pain and anxiety both result in selective occlusion of pre-LTP. Significantly, microinjection of the HCN blocker ZD7288 into the ACC in vivo produces both anxiolytic and analgesic effects. Our results provide a mechanism by which two forms of LTP in the ACC may converge to mediate the interaction between anxiety and chronic pain.

RGS9-2 modulates sensory and mood related symptoms of neuropathic pain.

The signal transduction modulator Rgs9-2 (Regulator of G protein signaling 9-2) plays a key role in dopaminergic and opioidergic transmission in the striatum. Rgs9-2 is a potent modulator of opiate reward and analgesia, but its role in chronic pain remains unknown. Here, we use the spared nerve injury model (SNI), to evaluate the influence of Rgs9-2 in sensory symptoms, as well as in anxiety and depression-like behaviors observed under neuropathic pain conditions. Our data demonstrate that knockout of the Rgs9 gene reduces the intensity of thermal hyperalgesia and mechanical allodynia the first few days after nerve injury. This small, but significant effect is only observed at early time points after nerve injury, whereas after the first week of SNI, Rgs9 knockout (Rgs9KO) and Rgs9 wildtype (Rgs9WT) mice show similar levels of mechanical allodynia and thermal hyperalgesia. Furthermore, Rgs9-2 deletion exacerbates anxiety and depression like behaviors several weeks after the emergence of the neuropathic pain symptoms. Our findings also reveal a temporal and regional regulation of Rgs9-2 protein expression by neuropathic pain, as Rgs9-2 levels are reduced in the spinal cord a few days after nerve injury, whereas decreased Rgs9-2 levels in the Nucleus Accumbens (NAc) are only observed several weeks after nerve injury. Thus, adaptations in Rgs9-2 activity in the spinal cord and in the NAc may contribute to sensory and affective components of neuropathic pain.

Postsynaptic potentiation of corticospinal projecting neurons in the anterior cingulate cortex after nerve injury.

Long-term potentiation (LTP) is the key cellular mechanism for physiological learning and pathological chronic pain. In the anterior cingulate cortex (ACC), postsynaptic recruitment or modification of AMPA receptor (AMPAR) GluA1 contribute to the expression of LTP. Here we report that pyramidal cells in the deep layers of the ACC send direct descending projecting terminals to the dorsal horn of the spinal cord (lamina I-III). After peripheral nerve injury, these projection cells are activated, and postsynaptic excitatory responses of these descending projecting neurons were significantly enhanced. Newly recruited AMPARs contribute to the potentiated synaptic transmission of cingulate neurons. PKA-dependent phosphorylation of GluA1 is important, since enhanced synaptic transmission was abolished in GluA1 phosphorylation site serine-845 mutant mice. Our findings provide strong evidence that peripheral nerve injury induce long-term enhancement of cortical-spinal projecting cells in the ACC. Direct top-down projection system provides rapid and profound modulation of spinal sensory transmission, including painful information. Inhibiting cortical top-down descending facilitation may serve as a novel target for treating neuropathic pain.

Pharmacological rescue of cortical synaptic and network potentiation in a mouse model for fragile X syndrome.

Fragile X syndrome, caused by the mutation of the Fmr1 gene, is characterized by deficits of attention and learning ability. In the hippocampus of Fmr1 knockout mice (KO), long-term depression is enhanced whereas long-term potentiation (LTP) including late-phase LTP (L-LTP) is reduced or unaffected. Here we examined L-LTP in the anterior cingulate cortex (ACC) in Fmr1 KO mice by using a 64-electrode array recording system. In wild-type mice, theta-burst stimulation induced L-LTP that does not occur in all active electrodes/channels within the cingulate circuit and is typically detected in ∼75% of active channels. Furthermore, L-LTP recruited new responses from previous inactive channels. Both L-LTP and the recruitment of inactive responses were blocked in the ACC slices of Fmr1 KO mice. Bath application of metabotropic glutamate receptor 5 (mGluR5) antagonist or glycogen synthase kinase-3 (GSK3) inhibitors rescued the L-LTP and network recruitment. Our results demonstrate that loss of FMRP will greatly impair L-LTP and recruitment of cortical network in the ACC that can be rescued by pharmacological inhibition of mGluR5 or GSK3. This study is the first report of the network properties of L-LTP in the ACC, and provides basic mechanisms for future treatment of cortex-related cognitive defects in fragile X patients.

Cortical GluK1 kainate receptors modulate scratching in adult mice.

Recent investigations into the mechanisms mediating itch transmission have focused on spinal mechanisms, whereas few studies have investigated the role of the cerebral cortex in itch-related behaviors. Human imaging studies show that several cortical regions are active in correspondence with itch, including the anterior cingulate cortex (ACC). We present here evidence of cortical modulation of pruritogen-induced scratching behavior. We combine pharmacological, genetic, and electrophysiological approaches to show that cortical GluK1-containing kainate (KA) receptors are involved in scratching induced by histamine and non-histamine-dependent itching stimuli. We further show that scratching corresponds with enhanced excitatory transmission in the ACC through KA receptor modulation of inhibitory circuitry. In addition, we found that inhibiting GluK1-containing KA receptors in the ACC also reduced behavioral nociceptive responses induced by formalin. Our results reveal a new role of the cortex in pruritogen-induced scratching.

An increase in synaptic NMDA receptors in the insular cortex contributes to neuropathic pain.

Neurons in the insular cortex are activated by acute and chronic pain, and inhibition of neuronal activity in the insular cortex has analgesic effects. We found that in a mouse model in which peripheral nerve injury leads to the development of neuropathic pain, the insular cortex showed changes in synaptic plasticity, which were associated with a long-term increase in the amount of synaptic N-methyl-d-aspartate receptors (NMDARs), but not that of extrasynaptic NMDARs. Activation of cyclic adenosine monophosphate (cAMP)-dependent signaling enhanced the amount of synaptic NMDARs in acutely isolated insular cortical slices and increased the surface localization of NMDARs in cultured cortical neurons. We found that the increase in the amount of NMDARs required phosphorylation of the NMDAR subunit GluN2B at Tyr(1472) by a pathway involving adenylyl cyclase subtype 1 (AC1), protein kinase A (PKA), and Src family kinases. Finally, injecting NMDAR or GluN2B-specific antagonists into the insular cortex reduced behavioral responses to normally nonnoxious stimuli in the mouse model of neuropathic pain. Our results suggest that activity-dependent plasticity takes place in the insular cortex after nerve injury and that inhibiting the increase in NMDAR function may help to prevent or treat neuropathic pain.

Characterization of neuronal intrinsic properties and synaptic transmission in layer I of anterior cingulate cortex from adult mice.

The neurons in neocortex layer I (LI) provide inhibition to the cortical networks. Despite increasing use of mice for the study of brain functions, few studies were reported about mouse LI neurons. In the present study, we characterized intrinsic properties of LI neurons of the anterior cingulate cortex (ACC), a key cortical area for sensory and cognitive functions, by using whole-cell patch clamp recording approach. Seventy one neurons in LI and 12 pyramidal neurons in LII/III were recorded. Although all of the LI neurons expressed continuous adapting firing characteristics, the unsupervised clustering results revealed five groups in the ACC, including: Spontaneous firing neurons; Delay-sAHP neurons, Delay-fAHP neurons, and two groups of neurons with ADP, named ADP1 and ADP2, respectively. Using pharmacological approaches, we found that LI neurons received both excitatory (mediated by AMPA, kainate and NMDA receptors), and inhibitory inputs (which were mediated by GABA(A) receptors). Our studies provide the first report characterizing the electrophysiological properties of neurons in LI of the ACC from adult mice.

Translational investigation and treatment of neuropathic pain.

Neuropathic pain develops from a lesion or disease affecting the somatosensory system. Translational investigations of neuropathic pain by using different animal models reveal that peripheral sensitization, spinal and cortical plasticity may play critical roles in neuropathic pain. Furthermore, descending facilitatory or excitatory modulation may also act to enhance chronic pain. Current clinical therapy for neuropathic pain includes the use of pharmacological and nonpharmacological (psychological, physical, and surgical treatment) methods. However, there is substantial need to better medicine for treating neuropathic pain. Future translational researchers and clinicians will greatly facilitate the development of novel drugs for treating chronic pain including neuropathic pain.

Rapid synaptic potentiation within the anterior cingulate cortex mediates trace fear learning.

Although the cortex has been extensively studied in long-term memory storage, less emphasis has been placed on immediate cortical contributions to fear memory formation. AMPA receptor plasticity is strongly implicated in learning and memory, and studies have identified calcium permeable AMPA receptors (CP-AMPARs) as mediators of synaptic strengthening. Trace fear learning engages the anterior cingulate cortex (ACC), but whether plastic events occur within the ACC in response to trace fear learning, and whether GluN2B subunits are required remains unknown. Here we show that the ACC is necessary for trace fear learning, and shows a rapid 20% upregulation of membrane AMPA receptor GluA1 subunits that is evident immediately after conditioning. Inhibition of NMDA receptor GluN2B subunits during training prevented the upregulation, and disrupted trace fear memory retrieval 48 h later. Furthermore, intra-ACC injections of the CP-AMPAR channel antagonist, 1-naphthylacetyl spermine (NASPM) immediately following trace fear conditioning blocked 24 h fear memory retrieval. Accordingly, whole cell patch clamp recordings from c-fos positive and c-fos negative neurons within the ACC in response to trace fear learning revealed an increased sensitivity to NASPM in recently activated neurons that was reversed by reconsolidation update extinction. Our results suggest that trace fear learning is mediated through rapid GluN2B dependent trafficking of CP-AMPARs, and present in vivo evidence that CP-AMPAR activity within the ACC immediately after conditioning is necessary for subsequent memory consolidation processes.