Hippocampal Calcium Research Articles

Searching for cognitive specializations of neurons using mutual information framework

Of particular interest for researching the cognitive specializations of neurons is their correlation with environmental variables and animal behavior. Mutual information (MI) is a preferable method for measuring such correlations, as it allows for the assessment of non-linear relationships between variables, detects synchronization, and provides both significance and strength quantification. However, calculating MI for real data is significantly challenging. In this study, we used updated MI calculation techniques to analyze the connection between calcium fluorescence signaling and behavioral variables. Our approach encompasses novel strategies which we compiled into a software program known as INTENS (Information-Theoretic Evaluation of Neuronal Specializations), and it enabled to identify specialized neurons in mice hippocampal calcium activity data while they explored the arena with varying levels of novelty. Numerous methods exist for analyzing the relationship between neuron spikes and behavioral variables, including information-theoretical approaches [1]. Extracting information about the relationship between calcium fluorescent signals and behavior is of particular interest due to the signal’s ability to provide crucial information about subthreshold activations of the neuron. In this study, we use the GCMI Gaussian copula entropy method to calculate mutual information [2]. This method relies on the fact that mutual information between two random variables is independent of their marginal distributions and only depends on the type of copula used (a multidimensional distribution where each marginal distribution is uniform). The actual MI was compared to its corresponding values computed on the time-shifted signals for assessing the statistical significance of the computed information association between the calcium signal and the behavioral variable. Additionally, we devised a technique for gauging the strength of the coupling effect. This involved normalizing the mutual information between the fluorescence signal and the behavior with the entropy value of both variables, previously calculated as random variables. Importantly, the approach outlined earlier is effective for analyzing continuous variables such as calcium signal and animal speed, as well as pairs of continuous and discrete variables such as calcium signal and the presence or absence of grooming. The analysis of calcium signals recorded from the CA1 region of the hippocampus revealed neuronal specializations related to the animal’s external environment, such as place cells, and specializations related to its behavioral activities, including neurons activated during running, rearing, and freezing. Some neurons selectively activated in response to discrete parameters included the animal’s location within the arena (center, walls, and corners) and its speed (rest, slow, and fast). A total of 781 specializations were detected across 472 neurons throughout all four sessions of the experiment. Notably, a single neuron could have several specializations. However, more than half (55%) of the neurons were found to have only one specialization.

Neurophysiology of creativity and machine learning applications for creative process’ stages differentiation through assessment of EEG/VP signals

Of particular interest for researching the cognitive specializations of neurons is their correlation with environmental variables and animal behavior. Mutual information (MI) is a preferable method for measuring such correlations, as it allows for the assessment of non-linear relationships between variables, detects synchronization, and provides both significance and strength quantification. However, calculating MI for real data is significantly challenging. In this study, we used updated MI calculation techniques to analyze the connection between calcium fluorescence signaling and behavioral variables. Our approach encompasses novel strategies which we compiled into a software program known as INTENS (Information-Theoretic Evaluation of Neuronal Specializations), and it enabled to identify specialized neurons in mice hippocampal calcium activity data while they explored the arena with varying levels of novelty. Numerous methods exist for analyzing the relationship between neuron spikes and behavioral variables, including information-theoretical approaches [1]. Extracting information about the relationship between calcium fluorescent signals and behavior is of particular interest due to the signal’s ability to provide crucial information about subthreshold activations of the neuron. In this study, we use the GCMI Gaussian copula entropy method to calculate mutual information [2]. This method relies on the fact that mutual information between two random variables is independent of their marginal distributions and only depends on the type of copula used (a multidimensional distribution where each marginal distribution is uniform). The actual MI was compared to its corresponding values computed on the time-shifted signals for assessing the statistical significance of the computed information association between the calcium signal and the behavioral variable. Additionally, we devised a technique for gauging the strength of the coupling effect. This involved normalizing the mutual information between the fluorescence signal and the behavior with the entropy value of both variables, previously calculated as random variables. Importantly, the approach outlined earlier is effective for analyzing continuous variables such as calcium signal and animal speed, as well as pairs of continuous and discrete variables such as calcium signal and the presence or absence of grooming. The analysis of calcium signals recorded from the CA1 region of the hippocampus revealed neuronal specializations related to the animal’s external environment, such as place cells, and specializations related to its behavioral activities, including neurons activated during running, rearing, and freezing. Some neurons selectively activated in response to discrete parameters included the animal’s location within the arena (center, walls, and corners) and its speed (rest, slow, and fast). A total of 781 specializations were detected across 472 neurons throughout all four sessions of the experiment. Notably, a single neuron could have several specializations. However, more than half (55%) of the neurons were found to have only one specialization.

Disruption of hippocampal neuronal circuit function depends upon behavioral state in the APP/PS1 mouse model of Alzheimer’s disease

The Alzheimer’s disease-associated peptide amyloid-beta (Aβ) has been associated with neuronal hyperactivity under anesthesia, but clinical trials of anticonvulsants or neural system suppressors have, so far, failed to improve symptoms in AD. Using simultaneous hippocampal calcium imaging and electrophysiology in freely moving mice expressing human Aβ, here we show that Aβ aggregates perturbed neural systems in a state-dependent fashion, driving neuronal hyperactivity in exploratory behavior and slow wave sleep (SWS), yet suppressing activity in quiet wakefulness (QW) and REM sleep. In exploratory behavior and REM sleep, Aβ impaired hippocampal theta–gamma phase-amplitude coupling and altered neuronal synchronization with theta. In SWS, Aβ reduced cortical slow oscillation (SO) power, the coordination of hippocampal sharp wave-ripples with both the SO and thalamocortical spindles, and the coordination of calcium transients with the sharp wave-ripple. Physostigmine improved Aβ-associated hyperactivity in exploratory behavior and hypoactivity in QW and expanded the range of gamma that coupled with theta phase, but exacerbated hypoactivity in exploratory behavior. Together, these findings show that the effects of Aβ alone on hippocampal circuit function are profoundly state dependent and suggest a reformulation of therapeutic strategies aimed at Aβ induced hyperexcitability.

0300 Early Life Sleep Fragmentation Impairs Hippocampal-Dependent Learning and Sleep-Dependency in Hippocampal Calcium Transients

Abstract Introduction Sleep deprivation impairs hippocampal-dependent memory, and hippocampal-dependent memory impairments occur in some dementias, including Alzheimer’s disease. As our population continues to age, understanding the molecular basis for memory impairments is increasingly important. We hypothesized that early life sleep fragmentation would result in lasting increases in hippocampal calcium transient activity. Methods B6 mice were randomized to 12wk of sleep fragmentation or rested control conditions at age 8wk. Mice were microinjected with AAV9-CamKII-GCamp6F into the hippocampus and later implanted with a GRIN Lens into CA1 secured to a baseplate along with chronic EEG/EMG electrodes and recording connector. Calcium recordings were obtained two to three months after injection and recordings were obtained across sleep-wake cycles>4mins of wake and NREM sleep. Individual cells across animal were combined into sleep fragmented (n = 521 cells) or rested (n = 443 cells) groups during wake or sleep. Average FFx was analyzed by group and condition by T-tests, paired for within and unpaired across groups. A spatial object recognition assay was also performed on all mice (n=16 for both groups) and performance across group was analyzed by paired T-tests. Results Rested mice showed normal spatial object recognition (n = 16, p<0.05). In contrast, SF mice showed impaired spatial object recognition (n = 16, N.S.). There were no differences across sleep conditions in calcium transient FFx for waking (p>0.05). However, in sleep, cells in SF mice had significantly higher average FFx values than cells in rested mice (p<0.0001). Conclusion Early-life sleep fragmentation has long-lasting impacts on memory. Since spatial memory is dependent on hippocampal function, the calcium transient FFx data suggests that a driver of this hippocampal memory impairment may be higher firing rates in sleep and/or greater calcium exposure in hippocampal CamKII neurons in sleep, both of which may perturb microglial maintenance of synapses. Understanding the molecular drivers behind this calcium dysfunction will be essential in our understanding of neurodegeneration, dementia, and Alzheimer’s disease. Support (If Any) NIH AG054104; AG064231

The memory modulatory effect of agmatine in passive avoidance task coincides with alterations of hippocampal CaMKII-α and ERK signaling in mice

Agmatine is a polyamine suggested to act as a supposed neurotransmitter in the brain. Evidence has indicated that acute agmatine administration might modulate memory. The present study aimed to investigate the effect of repeated agmatine treatment on passive avoidance memory, hippocampal calcium–calmodulin-dependent protein kinase II-alpha (CaMKII-α), and Extracellular Signal-Regulated Kinase (ERK) signaling pathways in naive mice. Adult male NMRI mice were treated with agmatine (10, 20, 30, 40, and 80 mg/kg/ip) or saline for 11 days. Acquisition and retention tests of passive avoidance memory were performed on days 10 and 11, respectively. Following the memory retention test, the hippocampi were assessed for the levels of CaMKII-α and ERK using the western blotting technique. The results revealed the dose-dependent effect of agmatine on the passive avoidance memory. Accordingly, the memory was impaired in lower doses, but was improved in higher ones. Agmatine in none of the doses affected the nociception of the mice in tail-flick test. Furthermore, agmatine increased the phosphorylation of CaMKII-α and ERK in the hippocampus at memory enhancing doses, while ERK phosphorylation decreased following the impairing doses of agmatine. Thus, the dose-dependent effect of agmatine on memory might be related to its modulatory effect on CaMKII-α and ERK signal transduction, eventually regulating the memory process.

The Role of Alcohol in Hippocampal Calcium Channel (Cav1.2) expression

Background and Hypothesis: 
 Voltage-gated L-type calcium channels (Cav1.2 and Cav1.3) in the hippocampus play important roles in glutamatergic neurotransmission underlying memory and learning. Their overexpression has been implicated in neuroexcitatory cell death and disease states including chronic alcoholism. While increases in Cav1.2 gene expression have been reported in the hippocampus after chronic ethanol exposure in rats, the regional distribution of Cav1.2 protein after voluntary ethanol (EtOH) drinking has not been reported. We hypothesize that the expression of Cav1.2 channels within the hippocampus is increased by EtOH drinking in a region-specific manner. 
 
 Methods: 
 Male Sprague Dawley rats were allowed 28 days of intermittent access to a 10% EtOH solution. At 24 hours after the last exposure to EtOH, brains were collected and processed for immunohistochemistry. Cav1.2 associated immunofluorescent signal from subregions of the hippocampus was quantified using ImageJ analysis software. 
 
 Results: 
 Immunohistochemical results indicate that Cav1.2 immunoreactivity in the hippocampal stratum granulosum layer within the Dentate Gyrus and the stratum pyramidale layer within CA1 and CA3 regions was increased in response to EtOH treatment. There was no significant change in Cav1.2 immunoreactivity for the CA2 region. 
 
 Conclusion: 
 This study suggests that calcium signaling in subregions of the hippocampus is differentially affected by EtOH consumption that may contribute to eventual calcium-mediated apoptosis. 
 
 Impact and Implications: 
 Understanding the process of EtOH-induced hippocampal calcium signaling presents opportunities for understanding the consequences of chronic alcohol exposure related to hippocampal function including memory and learning, and possible interventional therapies for alcohol damage.

Molecular mechanisms for the antidepressant-like effects of a low-dose ketamine treatment in a DFP-based rat model for Gulf War Illness

Exposure to organophosphates (OP) during the First Gulf War is among one of the factors for Gulf War Illness (GWI) development in veterans and it has been challenging to treat GWI symptoms with existing therapies. Ketamine produces a rapid-onset and sustained antidepressant response, but there is no evidence whether ketamine treatment is effective for GWI depression. Repeated, low-dose exposure to diisopropyl fluorophosphate (DFP) mimic Gulf War related OP exposures and produces a chronic depressive state in rats. In this study, DFP-exposed rats treated with ketamine (10 mg/kg, i.p.) exhibited antidepressant-like effect on the Forced Swim Test at 1-h. This effect persisted at 24-h post ketamine, a time-point by which it is eliminated from the brain suggesting involvement of mechanisms that affect long-term synaptic plasticity. Western blot analysis showed significantly lower Brain-Derived Neurotrophic Factor (BDNF) levels in DFP rat brains. Ketamine produced a nonsignificant increase in BDNF expression at 1-h but produced a larger, significant (2.2-fold) increase at 24-h in DFP rats. We previously reported chronic hippocampal calcium elevations ([Ca2+]i) in DFP rats. Ketamine-treated DFP rats exhibited significantly lower [Ca2+]i at 1-h but not at 24-h. Interestingly, treatment with ANA-12, a TrkB-BDNF receptor antagonist, in DFP rats blunted ketamine’s antidepressant-like effect at 24-h but not at 1-h. These experiments suggest that in a rat model of DFP-induced depression, inhibition of the NMDAR-Ca2+ contributes to the rapid-onset antidepressant effects of ketamine while the antidepressant actions that persisted at 24-h post ketamine administration involve upregulation of BDNF signaling.

Effects of Early Life Adverse Experience on Contents of Essential Trace Elements Related to the Antioxidative System in the Adult Mouse Hippocampus

Neonatal maternal separation has been found to affect adult brain function. However, the molecular basis has not been clarified. The purpose of this study was to elucidate the changes in mineral levels related to the antioxidative system in the adult mouse hippocampus after neonatal maternal separation. Male mouse pups in the maternal separation group were separated from their dams for 3 hours daily during the first 2 postnatal weeks. The hippocampal selenium, calcium, zinc, copper, and manganese contents were analyzed with an inductively coupled plasma mass spectrometer at 13 postnatal weeks, and plasma protein carbonyl and 8-OHdG concentrations were examined by enzyme-linked immunosorbent assay. It was found that the hippocampal selenium content was about three times lower in the maternal separation group than that in the control group, representing a significant difference. The hippocampal calcium content was more than 2 times higher in the maternal separation group than that in the control group. The plasma protein carbonyl concentration was about seven-fold higher in the maternal separation group than that in the control group. However, no significant differences were found in the hippocampal calcium, zinc, copper, and manganese contents, or in plasma protein carbonyl and 8-OHdG concentrations between the maternal separation and control groups. Therefore, neonatal maternal separation affects the hippocampal selenium content of adult male mice, but does not affect other mineral contents, such as zinc, copper, manganese, and calcium in the hippocampus.

Global brain signal in awake rats

Although often used as a nuisance in resting-state functional magnetic resonance imaging (rsfMRI), the global brain signal in humans and anesthetized animals has important neural basis. However, our knowledge of the global signal in awake rodents is sparse. To bridge this gap, we systematically analyzed rsfMRI data acquired with a conventional single-echo (SE) echo planar imaging (EPI) sequence in awake rats. The spatial pattern of rsfMRI frames during peaks of the global signal exhibited prominent co-activations in the thalamo-cortical and hippocampo-cortical networks, as well as in the basal forebrain, hinting that these neural networks might contribute to the global brain signal in awake rodents. To validate this concept, we acquired rsfMRI data using a multi-echo (ME) EPI sequence and removed non-neural components in the rsfMRI signal. Consistent co-activation patterns were obtained in extensively de-noised ME-rsfMRI data, corroborating the finding from SE-rsfMRI data. Furthermore, during rsfMRI experiments, we simultaneously recorded neural spiking activities in the hippocampus using GCaMP-based fiber photometry. The hippocampal calcium activity exhibited significant correspondence with the global rsfMRI signal. These data collectively suggest that the global rsfMRI signal contains significant neural components that involve coordinated activities in the thalamo-cortical and hippocampo-cortical networks. These results provide important insight into the neural substrate of the global brain signal in awake rodents.

Cholinergic modulation of hippocampal calcium activity across the sleep-wake cycle.

Calcium is a critical second messenger in neurons that contributes to learning and memory, but how the coordination of action potentials of neuronal ensembles with the hippocampal local field potential (LFP) is reflected in dynamic calcium activity remains unclear. Here, we recorded hippocampal calcium activity with endoscopic imaging of the genetically encoded fluorophore GCaMP6 with concomitant LFP in freely behaving mice. Dynamic calcium activity was greater in exploratory behavior and REM sleep than in quiet wakefulness and slow wave sleep, behavioral states that differ with respect to theta and septal cholinergic activity, and modulated at sharp wave ripples (SWRs). Chemogenetic activation of septal cholinergic neurons expressing the excitatory hM3Dq DREADD increased calcium activity and reduced SWRs. Furthermore, inhibition of muscarinic acetylcholine receptors (mAChRs) reduced calcium activity while increasing SWRs. These results demonstrate that hippocampal dynamic calcium activity depends on behavioral and theta state as well as endogenous mAChR activation.

Hypothermia Reduces Mortality, Prevents the Calcium Plateau, and Is Neuroprotective Following Status Epilepticus in Rats.

Status Epilepticus (SE) is a major neurological emergency and is considered a leading cause of Acquired Epilepsy (AE). We have shown that SE produces neuronal injury and prolonged alterations in hippocampal calcium levels ([Ca2+]i) that may underlie the development of AE. Interventions preventing the SE-induced Ca2+ plateau could therefore prove to be beneficial in lowering the development of AE after SE. Hypothermia is used clinically to prevent neurological complications associated with Traumatic Brain Injury, cardiac arrest, and stroke. Here, we investigated whether hypothermia prevented the development of Ca2+ plateau following SE. SE was induced in hippocampal neuronal cultures (HNC) by exposing them to no added MgCl2 solution for 3 h. To terminate SE, low Mg2+ solution was washed off with 31°C (hypothermic) or 37°C (normothermic) physiological recording solution. [Ca2+]i was estimated with ratiometric Fura-2 imaging. HNCs washed with hypothermic solution exhibited [Ca2+]i ratios, which were significantly lower than ratios obtained from HNCs washed with normothermic solution. For in vivo SE, the rat pilocarpine (PILO) model was used. Moderate hypothermia (30–33°C) in rats was induced at 30-min post-SE using chilled ethanol spray in a cold room. Hypothermia following PILO-SE significantly reduced mortality. Hippocampal neurons isolated from hypothermia-treated PILO SE rats exhibited [Ca2+]i ratios which were significantly lower than ratios obtained from PILO SE rats. Hypothermia also provided significant neuroprotection against SE-induced delayed hippocampal injury as characterized by decreased FluoroJade C labeling in hypothermia-treated PILO SE rats. We previously demonstrated that hypothermia reduced Ca2+ entry via N-methyl-D-aspartate and ryanodine receptors in HNC. Together, our studies indicate that by targeting these two receptor systems hypothermia could interfere with epileptogenesis and prove to be an effective therapeutic intervention for reducing SE-induced AE.

Chronic Neurological Morbidities and Elevated Hippocampal Calcium Levels in a DFP-Based Rat Model of Gulf War Illness

Over 20 yr have elapsed since the end of the First Gulf War, yet approximately one-third of the veterans exhibit Gulf War Illness (GWI) symptoms, particularly depression and memory impairments. Exposure to organophosphate (OP) compounds is implicated for GWI development. The role of calcium (Ca2+) signaling in learning, memory, and mood is well established and disruptions in Ca2+ homeostasis are observed in many neurological disorders. However, the status of Ca2+ homeostasis in the development of GWI behavioral impairments is not known. Male Sprague-Dawley rats were exposed to OP agent diisopropyl fluorophosphate (DFP; 0.5 mg/kg, s.c. 5 days), and at 6 mo post-DFP exposure, rats were subjected to behavioral assays for the determination of GWI neurological morbidities. Fura-2AM loaded acutely isolated hippocampal neurons were used for [Ca2+]i estimations. We observed chronic depressive symptoms and cognitive deficits in rats exposed to repeated low-dose DFP. The GWI rats also manifested elevations in hippocampal [Ca2+]i along with a significant increase in the number of neurons displaying these elevations. As Ca2+ is a major second-messenger molecule, such sustained increases in its levels could activate multiple signaling cascades and alter gene expression of proteins involved in synaptic plasticity and possibly underlie the neuronal injury and chronic morbidities in GWI.

Role of the calcium plateau in neuronal injury and behavioral morbidities following organophosphate intoxication.

Organophosphate (OP) chemicals include nerve agents and pesticides, and there is a growing concern of OP-based chemical attacks against civilians. Current antidotes are essential in limiting immediate mortality associated with OP exposure. However, further research is needed to identify the molecular mechanisms underlying long-term neurological deficits following survival of OP toxicity in order to develop effective therapeutics. We have developed rat survival models of OP-induced status epilepticus (SE) that mimic chronic mortality and morbidity following OP intoxication. We have observed significant elevations in hippocampal calcium levels after OP SE that persisted for weeks following initial survival. Drugs inhibiting intracellular calcium-induced calcium release, such as dantrolene, levetiracetam, and carisbamate, lowered OP SE-mediated protracted calcium elevations. Given the critical role of calcium signaling in modulating behavior and cell death mechanisms, drugs targeted at preventing the development of the calcium plateau could enhance neuroprotection, help reduce morbidity, and improve outcomes following survival of OP SE.

Pharmacological blockade of the calcium plateau provides neuroprotection following organophosphate paraoxon induced status epilepticus in rats

Organophosphate (OP) compounds which include nerve agents and pesticides are considered chemical threat agents. Currently approved antidotes are crucial in limiting OP mediated acute mortality. However, survivors of lethal OP exposure exhibit delayed neuronal injury and chronic behavioral morbidities. In this study, we investigated neuroprotective capabilities of dantrolene and carisbamate in a rat survival model of paraoxon (POX) induced status epilepticus (SE). Significant elevations in hippocampal calcium levels were observed 48-h post POX SE survival, and treatment with dantrolene (10mg/kg, i.m.) and carisbamate (90mg/kg, i.m.) lowered these protracted calcium elevations. POX SE induced delayed neuronal injury as characterized by Fluoro Jade C labeling was observed in critical brain areas including the dentate gyrus, parietal cortex, amygdala, and thalamus. Dantrolene and carisbamate treatment provided significant neuroprotection against delayed neuronal damage in these brain regions when administered one-hour after POX-SE. These results indicate that dantrolene or carisbamate could be effective adjuvant therapies to the existing countermeasures to reduce neuronal injury and behavioral morbidities post OP SE survival.

Role of hippocampal neuronal intracellular calcium overload in modulating cognitive dysfunction and the neuronprotective effect of mematine in a mouse model of chronic intermittent hypoxia

To investigate the role of hippocampal intracellular calcium overload in modulating cognitive dysfunction and the neuronprotective effect of mematine in a mouse model of chronic intermittent hypoxia. 45 ICR male mice were randomly divided into 3 groups: the unhandled control group (UC group, n = 15), the chronic intermittent hypoxia (CIH group, n = 15) and the pretreatment memantine group (MEM group, n = 15). CIH and MEM mice were subjected to intermittent hypoxia while UC mice to room air for 8 h per day during 4 weeks. Mice in the MEM group were pretreated with memantine (5 mg/kg) by intraperitoneal injection before the cycle started, and those in the UC group and the CIH group were treated with same volume of physiological saline. Neurobehavioral assessments were performed by Open filed and Morris water maze, [Ca²⁺]i in hippocampal neurons was evaluate by flow cytometry, and the expression of cleaved caspase-3, phospho-ERK1/2 in hippocampus were detected by Western blotting. Compared with the UC group, CIH mice displayed markedly more locomotor activity (P < 0.05) in Open filed test, longer mean escape latency (P < 0.05), less number of times of crossing the platform (P < 0.01) and less percentage of time in target quadrant (P < 0.01). Furthermore, exposure to CIH enhanced [Ca²⁺]i (vs. UC mice, 155 ± 12 vs. 92 ± 8, P < 0.01), and up-regulated the expression of cleaved caspase-3 (P < 0.01), but down-regulated the level of phospho-ERK1/2 (P < 0.05) in the hippocampus. Pre-treatment with memantine significantly decreased hyperlocomotion (P < 0.05), attenuated memory deficit (P < 0.05), mitigated [Ca²⁺]i (vs. CIH mice, 90 ± 8 vs. 155 ± 12, P < 0.01), decrease the expression of cleaved caspase-3 (P < 0.01), but increased the level of phospho-ERK1/2(P < 0.05) comparing to the CIH group. The neurobehavioral impairments induced by CIH are mediated, at least in part, by intracellular calcium concentration overload, neuron apoptosis, dephosphorylation of ERK1/2, which can be attenuated by memantine. Memantine may have a therapeutic effect in the neurocognitive impairment associated with OSAHS.

Enhancement of morphological plasticity in hippocampal neurons by a physically modified saline via phosphatidylinositol-3 kinase.

Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer’s disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias.

3,4-Methylenedioxymethamphetamine enhances kainic acid convulsive susceptibility.

Kainic acid (KA) causes seizures and neuronal loss in the hippocampus. The present study investigated whether a recreational schedule of 3,4-methylenedioxymethamphetamine (MDMA) favours the development of a seizure state in a model of KA-induced epilepsy and potentiates the toxicity profile of KA (20 or 30mg/kg). Adolescent male C57BL/6 mice received saline or MDMA t.i.d. (s.c. every 3h), on 1day a week, for 4 consecutive weeks. Twenty-four hours after the last MDMA exposure, the animals were injected with saline or KA (20 or 30mg/kg). After this injection, we evaluated seizures, hippocampal neuronal cell death, microgliosis, astrogliosis, and calcium binding proteins. MDMA pretreatment, by itself, did not induce neuronal damage but increased seizure susceptibility in all KA treatments and potentiated the presence of Fluoro-Jade-positive cells in CA1. Furthermore, MDMA, like KA, significantly decreased parvalbumin levels in CA1 and dentate gyrus, where it potentiated the effects of KA. The amphetamine derivative also promoted a transient decrease in calbindin and calretinin levels, indicative of an abnormal neuronal discharge. In addition, treatment of cortical neurons with MDMA (10-50μM) for 6 or 48h significantly increased basal Ca(2+), reduced basal Na(+) levels and potentiated kainate response. These results indicate that MDMA potentiates KA-induced neurodegeneration and also increases KA seizure susceptibility. The mechanism proposed includes changes in Calcium Binding Proteins expression, probably due to the disruption of intracellular ionic homeostasis, or/and an indirect effect through glutamate release.

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

Regional differences in hippocampal calcium handling provide a cellular mechanism for limiting plasticity

Although much is known about the mechanisms underlying synaptic plasticity, the cellular mechanisms that negatively regulate plasticity in some brain regions are considerably less studied. One region where neurons do not reliably express long-term potentiation (LTP) is the CA2 subfield of the hippocampus. Given the connection between synaptic plasticity and increases in postsynaptic [Ca(2+)], and that CA2 neurons express a large number of calcium-regulating proteins, we tested the hypothesis that the relative lack of LTP in CA2 results from differences in the calcium dynamics of these neurons. By measuring calcium-dependent fluorescence transients in dendritic spines, we show that CA2 neurons have smaller action potential-evoked intracellular Ca(2+) transients because of a higher endogenous Ca(2+)-buffering capacity and significantly higher rates of Ca(2+) extrusion when compared with CA1 and CA3 neurons. Perfusion with higher external [Ca(2+)] during induction restores LTP to CA2 neurons, suggesting that they possess the cellular machinery required for plasticity, but that the restriction of postsynaptic [Ca(2+)] limits its expression. Camstatin, an analogue of the calcium-modulating protein Pep-19 strongly expressed in CA2 neurons, blocked LTP and increased Ca(2+) extrusion in CA1 neurons, suggesting a role for extrusion in the regulation of plasticity in CA2. In agreement with this idea, we found that intracellular introduction of a PMCA pump inhibitor (carboxyeosin) allows for the induction of LTP in CA2 neurons. Our results indicate that regulation of postsynaptic [Ca(2+)] through modulation of extrusion and/or buffering regulates expression of LTP in CA2 and potentially other brain regions.

Hippocampal calcium dyshomeostasis and long-term potentiation in 2-week zinc deficiency

On the basis of abnormal neuropsychological behavior in the open-field test after 2-week zinc deprivation, neurochemical response was examined in young mice fed a zinc-deficient diet for 2 weeks. Serum corticosterone concentration was markedly higher in zinc-deficient mice than in the control mice. Basal signals of intracellular calcium (fluo-4 FF) were also significantly more in hippocampal slices from zinc-deficient mice. These results suggest that basal Ca2+ levels in hippocampal cells are increased by zinc deficiency. On the other hand, Schaffer collateral long-term potentiation (LTP) was unaffected by zinc deficiency; the averaged fEPSP after tetanic stimulation was 162±8% of baseline value in the control and 172±22% in zinc-deficient mice. In the Morris water maze, there was also no significant difference in learning behavior for the hidden platform task between the control and zinc-deficient mice. The present study indicates that Schaffer collateral LTP associated with spatial cognition performance are unaffected by calcium dyshomeostasis in the hippocampus elicited by 2-week zinc deprivation, which may be linked to the increased serum corticosterone concentration.