Protocol for two models of behavioral transition from action to no-action when facing prolonged uncontrollable experience in mice.

Uncontrollability could lead to behavioral adjustment or even giving up when facing repeated failure. Here, we detail a protocol to study the behavioral transition from action to no-action induced by prolonged uncontrollable experiences in mice. We describe the behavioral devices, video analysis, and the exponential learning curve fitting for mathematical assessment. We perform further validation experiments evaluating locomotor, social, and anxiety-/depression-like behaviors. This approach helps study neural mechanisms underlying adaptive decision-making when facing repeated failure. For complete details on the use and execution of this protocol, please refer to Li et al.1.

TRPM7 kinase activity induces amyloid-ß degradation to reverse synaptic and cognitive deficits in mouse models of Alzheimer's disease.

Altered abundance or activity of the dual-function transient receptor potential melastatin-like 7 (TRPM7) protein is implicated in neurodegenerative disorders, including Alzheimer's disease (AD). Toxic aggregation of amyloid-ß (Aß) in neurons is implicated in AD pathology. Here, we found that the kinase activity of TRPM7 is important to stimulate the degradation of Aß. TRPM7 expression was decreased in hippocampal tissue samples from patients with AD and two mouse models of AD (APP/PS1 and 5XFAD). In cultures of hippocampal neurons from mice, overexpression of full-length TRPM7 or of its functional kinase domain M7CK prevented synapse loss induced by exogenous Aß. In contrast, this neuroprotection was not afforded by overexpression of either the functional ion channel portion alone or a TRPM7 mutant lacking kinase activity. M7CK overexpression in the hippocampus of young and old 5XFAD mice prevented and reversed, respectively, memory deficits, synapse loss, and Aß plaque accumulation. In both neurons and mice, M7CK interacted with and activated the metalloprotease MMP14 to promote Aß degradation. Thus, TRPM7 loss in patients with AD may contribute to the associated Aß pathology.

A neural circuit for regulating a behavioral switch in response to prolonged uncontrollability in mice.

Persistence in the face of failure helps to overcome challenges. But the ability to adjust behavior or even give up when the task is uncontrollable has advantages. How the mammalian brain switches behavior when facing uncontrollability remains an open question. We generated two mouse models of behavioral transition from action to no-action during exposure to a prolonged experience with an uncontrollable outcome. The transition was not caused by pain desensitization or muscle fatigue and was not a depression-/learned-helplessness-like behavior. Noradrenergic neurons projecting to GABAergic neurons within the orbitofrontal cortex (OFC) are key regulators of this behavior. Fiber photometry, microdialysis, mini-two-photon microscopy, and tetrode/optrode in vivo recording in freely behaving mice revealed that the reduction of norepinephrine and downregulation of alpha 1 receptor in the OFC reduced the number and activity of GABAergic neurons necessary for driving action behavior resulting in behavioral transition. These findings define a circuit governing behavioral switch in response to prolonged uncontrollability.

Characteristic Features of Deep Brain Lymphatic Vessels and Their Regulation by Chronic Stress.

Studies have demonstrated that a functional network of meningeal lymphatic vessels exists in the brain. However, it is unknown whether lymphatic vessels could also extend deep into the brain parenchyma and whether the vessels could be regulated by stressful life events. We used tissue clearing techniques, immunostaining, light-sheet whole-brain imaging, confocal imaging in thick brain sections and flow cytometry to demonstrate the existence of lymphatic vessels deep in the brain parenchyma. Chronic unpredictable mild stress or chronic corticosterone treatment was used to examine the regulation of brain lymphatic vessels by stressful events. Western blotting and coimmunoprecipitation were used to provide mechanistic insights. We demonstrated the existence of lymphatic vessels deep in the brain parenchyma and characterized their features in the cortex, cerebellum, hippocampus, midbrain, and brainstem. Furthermore, we showed that deep brain lymphatic vessels can be regulated by stressful life events. Chronic stress reduced the length and areas of lymphatic vessels in the hippocampus and thalamus but increased the diameter of lymphatic vessels in the amygdala. No changes were observed in prefrontal cortex, lateral habenula, or dorsal raphe nucleus. Chronic corticosterone treatment reduced lymphatic endothelial cell markers in the hippocampus. Mechanistically, chronic stress might reduce hippocampal lymphatic vessels by down-regulating vascular endothelial growth factor C receptors and up-regulating vascular endothelial growth factor C neutralization mechanisms. Our results provide new insights into the characteristic features of deep brain lymphatic vessels, as well as their regulation by stressful life events.

Effect of truncation on TRPM7 channel activity.

Transient receptor potential melastatin-like 7 (TRPM7) is a key player in various physiological and pathological processes. TRPM7 channel activity is regulated by different factors. The effects of cleavage of different domains on channel activity remain unknown. Here, we constructed several TRPM7 clones and explored the effects of truncating the mouse TRPM7 at different locations on the ion channel activity in two cell lines. We compared the clones' activity with the full-length TRPM7 and the native TRPM7 in transfected and untransfected cells. We also expressed fluorescently tagged truncated clones to examine their protein stability and membrane targeting. We found that truncating the kinase domain induced reduction in TRPM7 channel activity. Further truncations beyond the kinase (serine/threonine rich domain and/or coiled-coil domain) did not result in further reductions in channel activity. Two truncated clones lacking the TRP domain or the melastatin homology domain had a completely nonfunctional channel apparently due to disruption of protein stability. We identified the shortest structure of TRPM7 with measurable channel activity. We found that the truncated TRPM7 containing only S5 and S6 domains retained some channel activity. Adding the TRP domain to the S5-S6 resulted in a significant increase in channel activity. Finally, our analysis showed that TRPM7 outward currents are more sensitive to truncations than inward currents. Our data provide insights on the effects of truncating TRPM7 at different locations on the channel functions, highlighting the importance of different domains in impacting channel activity, protein stability, and/or membrane targeting.

Neuroprotective Effects of TRPM7 Deletion in Parvalbumin GABAergic vs. Glutamatergic Neurons following Ischemia.

Oxidative stress induced by brain ischemia upregulates transient receptor potential melastatin-like-7 (TRPM7) expression and currents, which could contribute to neurotoxicity and cell death. Accordingly, suppression of TRPM7 reduces neuronal death, tissue damage and motor deficits. However, the neuroprotective effects of TRPM7 suppression in different cell types have not been investigated. Here, we found that induction of ischemia resulted in loss of parvalbumin (PV) gamma-aminobutyric acid (GABAergic) neurons more than Ca2+/calmodulin-kinase II (CaMKII) glutamatergic neurons in the mouse cortex. Furthermore, brain ischemia increased TRPM7 expression in PV neurons more than that in CaMKII neurons. We generated two lines of conditional knockout mice of TRPM7 in GABAergic PV neurons (PV-TRPM7-/-) and in glutamatergic neurons (CaMKII-TRPM7-/-). Following exposure to brain ischemia, we found that deleting TRPM7 reduced the infarct volume in both lines of transgenic mice. However, the volume in PV-TRPM7-/- mice was more significantly lower than that in the control group. Neuronal survival of both GABAergic and glutamatergic neurons was increased in PV-TRPM7-/- mice; meanwhile, only glutamatergic neurons were protected in CaMKII-TRPM7-/-. At the behavioral level, only PV-TRPM7-/- mice exhibited significant reductions in neurological and motor deficits. Inflammatory mediators such as GFAP, Iba1 and TNF-α were suppressed in PV-TRPM7-/- more than in CaMKII-TRPM7-/-. Mechanistically, p53 and cleaved caspase-3 were reduced in both groups, but the reduction in PV-TRPM7-/- mice was more than that in CaMKII-TRPM7-/- following ischemia. Upstream from these signaling molecules, the Akt anti-oxidative stress signaling was activated only in PV-TRPM7-/- mice. Therefore, deleting TRPM7 in GABAergic PV neurons might have stronger neuroprotective effects against ischemia pathologies than doing so in glutamatergic neurons.

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

Therapeutic Potential of Neurotrophins for Repair After Brain Injury: A Helping Hand From Biomaterials.

Stroke remains the leading cause of long-term disability with limited options available to aid in recovery. Significant effort has been made to try and minimize neuronal damage following stroke with use of neuroprotective agents, however, these treatments have yet to show clinical efficacy. Regenerative interventions have since become of huge interest as they provide the potential to restore damaged neural tissue without being limited by a narrow therapeutic window. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), and their high affinity receptors are actively produced throughout the brain and are involved in regulating neuronal activity and normal day-to-day function. Furthermore, neurotrophins are known to play a significant role in both protection and recovery of function following neurodegenerative diseases such as stroke and traumatic brain injury (TBI). Unfortunately, exogenous administration of these neurotrophins is limited by a lack of blood-brain-barrier (BBB) permeability, poor half-life, and rapid degradation. Therefore, we have focused this review on approaches that provide a direct and sustained neurotrophic support using pharmacological therapies and mimetics, physical activity, and potential drug delivery systems, including discussion around advantages and limitations for use of each of these systems. Finally, we discuss future directions of biomaterial drug-delivery systems, including the incorporation of heparan sulfate (HS) in conjunction with neurotrophin-based interventions.

Role of the chanzyme TRPM7 in the nervous system in health and disease.

The channel kinase (chanzyme) transient receptor potential melastatin-like 7 (TRPM7) has a unique dual protein structure composed of an ion channel with an α-kinase domain on its C-terminus. In the nervous system, under physiological conditions, TRPM7 contributes to critical neurobiological processes ranging from synaptic transmission to cognitive functions. Following certain pathological triggers, TRPM7 mediates neurotoxicity, neuro-injuries, and neuronal death. Here, we summarize the current knowledge of TRPM7 functions in neuronal systems in health and disease. The molecular mechanisms by which this chanzyme might regulate synaptic and cognitive functions are discussed. We also discuss the lack of knowledge regarding the molecular mechanisms responsible for turning TRPM7 into "a vicious tool" that mediates neuronal death following certain pathological triggers. Some synthetic and natural pharmacological modulators of the TRPM7 channel and its α-kinase are reviewed. We suggest that based on current knowledge, we should reshape our thinking regarding the implications of TRPM7 in neurological and neurodegenerative disorders. Moreover, we propose a paradigm shift concerning the targeting of TRPM7 as a therapeutic approach for treating certain neurological diseases. We agree that TRPM7 overexpression or overactivation may mediate neurodegenerative processes following certain triggers. However, TRPM7 dysfunction and/or downregulation might also be among the pathological changes leading to neurodegeneration. Consequently, further investigations are required before we decide whether blocking or activating the chanzyme is the correct therapeutic approach to treat certain neurological and/or neurodegenerative diseases.

TRPM7 Is Required for Normal Synapse Density, Learning, and Memory at Different Developmental Stages.

The TRPM7 chanzyme contributes to several biological and pathological processes in different tissues. However, its role in the CNS under physiological conditions remains unclear. Here, we show that TRPM7 knockdown in hippocampal neurons reduces structural synapse density. The synapse density is rescued by the α-kinase domain in the C terminus but not by the ion channel region of TRPM7 or by increasing extracellular concentrations of Mg2+ or Zn2+. Early postnatal conditional knockout of TRPM7 in mice impairs learning and memory and reduces synapse density and plasticity. TRPM7 knockdown in the hippocampus of adult rats also impairs learning and memory and reduces synapse density and synaptic plasticity. In knockout mice, restoring expression of the α-kinase domain in the brain rescues synapse density/plasticity and memory, probably by interacting with and phosphorylating cofilin. These results suggest that brain TRPM7 is important for having normal synaptic and cognitive functions under physiological, non-pathological conditions.

TRPM7 functions in non-neuronal and neuronal systems: Perspectives on its role in the adult brain.

Transient receptor potential melastatin-like 7 (TRPM7) has a unique dual protein structure. It is an ion channel that has biophysical characteristics enabling divalent cations transport and a kinase domain involved in molecular events starting from modulating signaling pathways to inducing chromatin remodeling. Over the past 15 years, significant progress in the molecular and functional characterization of TRPM7 has been made in peripheral tissue and/or cell lines. TRPM7 appears to be involved in a plethora of physiological and pathological processes including embryonic development, organogenesis, cell proliferation and survival, and cell death following certain triggers. In the post-mitotic neuronal cells, however, the functional role of TRPM7 remains unclear. Majority of the progress in this area of research has focused on the potential role of TRPM7 in mediating neuronal death following ischemia-like and neuronal injuries-like conditions. Here, we summarize major progress on the biological roles of the TRPM7 during development and in mitotic systems (cell lines). Then, we address the recent developments made in neuronal systems. Besides its role in neuronal death, we emphasize on direct and indirect evidences that could link TRPM7 to fundamental neurobiological processes such as synaptic transmission, synapse remodeling, plasticity, cognitive functions as well as to some mental disorders. Therefore, we propose that an equivalent effort is demanded to systematically characterize the role of TRPM7 in healthy neural system before presenting it as a potential molecular target to treat neurodegenerative disorders or to prevent neuronal death following ischemia and/or neuronal injuries.

Profiling and Co-expression Network Analysis of Learned Helplessness Regulated mRNAs and lncRNAs in the Mouse Hippocampus.

Although studies provide insights into the neurobiology of stress and depression, the exact molecular mechanisms underlying their pathologies remain largely unknown. Long non-coding RNA (lncRNA) has been implicated in brain functions and behavior. A potential link between lncRNA and psychiatric disorders has been proposed. However, it remains undetermined whether IncRNA regulation, in the brain, contributes to stress or depression pathologies. In this study, we used a valid animal model of depression-like symptoms; namely learned helplessness, RNA-seq, Gene Ontology and co-expression network analyses to profile the expression pattern of lncRNA and mRNA in the hippocampus of mice. We identified 6346 differentially expressed transcripts. Among them, 340 lncRNAs and 3559 protein coding mRNAs were differentially expressed in helpless mice in comparison with control and/or non-helpless mice (inescapable stress resilient mice). Gene Ontology and pathway enrichment analyses indicated that induction of helplessness altered expression of mRNAs enriched in fundamental biological functions implicated in stress/depression neurobiology such as synaptic, metabolic, cell survival and proliferation, developmental and chromatin modification functions. To explore the possible regulatory roles of the altered lncRNAs, we constructed co-expression networks composed of the lncRNAs and mRNAs. Among our differentially expressed lncRNAs, 17% showed significant correlation with genes. Functional co-expression analysis linked the identified lncRNAs to several cellular mechanisms implicated in stress/depression neurobiology. Importantly, 57% of the identified regulatory lncRNAs significantly correlated with 18 different synapse-related functions. Thus, the current study identifies for the first time distinct groups of lncRNAs regulated by induction of learned helplessness in the mouse brain. Our results suggest that lncRNA-directed regulatory mechanisms might contribute to stress-induced pathologies; in particular, to inescapable stress-induced synaptic modifications.

Elevation of brain magnesium prevents synaptic loss and reverses cognitive deficits in Alzheimer's disease mouse model.

BACKGROUND: Profound synapse loss is one of the major pathological hallmarks associated with Alzheimer's disease, which might underlie memory impairment. Our previous work demonstrates that magnesium ion is a critical factor in controlling synapse density/plasticity. Here, we tested whether elevation of brain magnesium, using a recently developed compound (magnesium-L-threonate, MgT), can ameliorate the AD-like pathologies and cognitive deficits in the APPswe/PS1dE9 mice, a transgenic mouse model of Alzheimer's disease. RESULTS: MgT treatment reduced Aß-plaque, prevented synapse loss and memory decline in the transgenic mice. Strikingly, MgT treatment was effective even when the treatment was given to the mice at the end-stage of their Alzheimer's disease-like pathological progression. To explore how elevation of brain magnesium ameliorates the AD-like pathologies in the brain of transgenic mice, we studied molecules critical for APP metabolism and signaling pathways implicated in synaptic plasticity/density. In the transgenic mice, the NMDAR signaling pathway was downregulated, while the BACE1 expression were upregulated. MgT treatment prevented the impairment of these signaling pathways, stabilized BACE1 expression and reduced sAPPß and ß-CTF in the transgenic mice. At the molecular level, elevation of extracellular magnesium prevented the high Aß-induced reductions in synaptic NMDARs by preventing calcineurin overactivation in hippocampal slices. CONCLUSIONS: Our results suggest that elevation of brain magnesium exerts substantial synaptoprotective effects in a mouse model of Alzheimer's disease, and hence it might have therapeutic potential for treating Alzheimer's disease.

Magnesium supplement enhances spatial-context pattern separation and prevents fear overgeneralization.

Enhancement of pattern separation could be helpful in improving the quality of normal daily learning and in treating individuals with cognitive impairment and certain psychiatric disorders. Previously, we have shown that elevating brain magnesium, by a novel magnesium compound (magnesium-L-threonate; MgT), enhances extinction of fear memory without enhancing amygdala-dependent fear memory. Here, we investigated the effects of MgT treatment on contextual-fear memory and subsequent pattern separation. Sprague-Dawley male rats were treated with MgT for 4 weeks and memory was evaluated using a spatial-context fear conditioning task. The pattern separation ability of MgT-treated rats was assessed using a spatial-context-discrimination task. MgT treatment did not enhance the retention of contextual-fear memory. Interestingly, the ability to discriminate between two, more or less distinct, contexts was enhanced in MgT-treated rats. Our results suggest that elevation of brain magnesium might be helpful in enhancing spatial-context discrimination and/or pattern separation besides preventing aversive-event-induced overgeneralization of fear.

Elevation of brain magnesium prevents and reverses cognitive deficits and synaptic loss in Alzheimer's disease mouse model.

Profound synapse loss is one of the major pathological hallmarks associated with Alzheimer's disease (AD) and might underlie memory impairment. Our previous work demonstrated that the magnesium ion is a critical factor in controlling synapse density/plasticity. Here, we investigated whether elevation of brain magnesium by the use of a recently developed compound, magnesium-l-threonate (MgT), can ameliorate the AD-like pathologies and cognitive deficits in the APPswe/PS1dE9 mice, a transgenic (Tg) mouse model of AD. MgT treatment reduced Aß plaque and prevented synapse loss and memory decline in the Tg mice. Strikingly, MgT treatment was effective even when given to the mice at the end stage of their AD-like pathological progression. To explore how elevation of brain magnesium ameliorates the AD-like pathologies in the brains of Tg mice, we studied molecules critical for APP metabolism and signaling pathways implicated in synaptic plasticity/density. In the Tg mice, the NMDAR/CREB/BDNF signaling was downregulated, whereas calpain/calcineurin/Cdk5 neurodegenerative signaling and ß-secretase (BACE1) expression were upregulated. MgT treatment prevented the impairment of these signaling pathways, stabilized BACE1 expression, and reduced soluble APPß and ß-C-terminal fragments in the Tg mice. At the molecular level, elevation of extracellular magnesium prevented the high-Aß-induced reductions in synaptic NMDARs by preventing calcineurin overactivation in hippocampal slices. Correlation studies suggested that the protection of NMDAR signaling might underlie the stabilization of BACE1 expression. Our results suggest that elevation of brain magnesium exerts substantial synaptoprotective effects in a mouse model of AD and may have therapeutic potential for treating AD in humans.

Chronic psychosocial stress and citalopram modulate the expression of the glial proteins GFAP and NDRG2 in the hippocampus.

RATIONALE: It has been suggested that there are causal relationships between alterations in brain glia and major depression. OBJECTIVES: To investigate whether a depressive-like state induces changes in brain astrocytes, we used chronic social stress in male rats, an established preclinical model of depression. Expression of two astrocytic proteins, the intermediate filament component glial fibrillary acidic protein (GFAP) and the cytoplasmic protein N-myc downregulated gene 2 (NDRG2), was analyzed in the hippocampus. For comparison, expression of the neuronal protein syntaxin-1A was also determined. METHODS: Adult male rats were subjected to daily social defeat for 5 weeks and were concomitantly treated with citalopram (30 mg/kg/day, via the drinking water) for 4 weeks. RESULTS: Western blot analysis showed that the chronic stress downregulated GFAP but upregulated NDRG2 protein. Citalopram did not prevent these stress effects, but the antidepressant per se downregulated GFAP and upregulated NDRG2 in nonstressed rats. In contrast, citalopram prevented the stress-induced upregulation of the neuronal protein syntaxin-1A. CONCLUSIONS: These data suggest that chronic stress and citalopram differentially affect expression of astrocytic genes while the antidepressant drug does not prevent the stress effects. The inverse regulation of the cytoskeletal protein GFAP and the cytoplasmic protein NDRG2 indicates that the cells undergo profound metabolic changes during stress and citalopram treatment. Furthermore, the present findings indicate that a 4-week treatment with citalopram does not restore normal glial function in the hippocampus, although the behavior of the animals was normalized within this treatment period, as reported previously.

Effects of elevation of brain magnesium on fear conditioning, fear extinction, and synaptic plasticity in the infralimbic prefrontal cortex and lateral amygdala.

Anxiety disorders, such as phobias and posttraumatic stress disorder, are among the most common mental disorders. Cognitive therapy helps in treating these disorders; however, many cases relapse or resist the therapy, which justifies the search for cognitive enhancers that might augment the efficacy of cognitive therapy. Studies suggest that enhancement of plasticity in certain brain regions such as the prefrontal cortex (PFC) and/or hippocampus might enhance the efficacy of cognitive therapy. We found that elevation of brain magnesium, by a novel magnesium compound [magnesium-l-threonate (MgT)], enhances synaptic plasticity in the hippocampus and learning and memory in rats. Here, we show that MgT treatment enhances retention of the extinction of fear memory, without enhancing, impairing, or erasing the original fear memory. We then explored the molecular basis of the effects of MgT treatment on fear memory and extinction. In intact animals, elevation of brain magnesium increased NMDA receptors (NMDARs) signaling, BDNF expression, density of presynaptic puncta, and synaptic plasticity in the PFC but, interestingly, not in the basolateral amygdala. In vitro, elevation of extracellular magnesium concentration increased synaptic NMDAR current and plasticity in the infralimbic PFC, but not in the lateral amygdala, suggesting a difference in their sensitivity to elevation of brain magnesium. The current study suggests that elevation of brain magnesium might be a novel approach for enhancing synaptic plasticity in a regional-specific manner leading to enhancing the efficacy of extinction without enhancing or impairing fear memory formation.

Enhancement of learning and memory by elevating brain magnesium.

Learning and memory are fundamental brain functions affected by dietary and environmental factors. Here, we show that increasing brain magnesium using a newly developed magnesium compound (magnesium-L-threonate, MgT) leads to the enhancement of learning abilities, working memory, and short- and long-term memory in rats. The pattern completion ability was also improved in aged rats. MgT-treated rats had higher density of synaptophysin-/synaptobrevin-positive puncta in DG and CA1 subregions of hippocampus that were correlated with memory improvement. Functionally, magnesium increased the number of functional presynaptic release sites, while it reduced their release probability. The resultant synaptic reconfiguration enabled selective enhancement of synaptic transmission for burst inputs. Coupled with concurrent upregulation of NR2B-containing NMDA receptors and its downstream signaling, synaptic plasticity induced by correlated inputs was enhanced. Our findings suggest that an increase in brain magnesium enhances both short-term synaptic facilitation and long-term potentiation and improves learning and memory functions.