36 results found
Kopanitsa M, Lehtimaki KK, Forsman M, et al., 2020, Cognitive disturbances in the cuprizone model of multiple sclerosis, Genes, Brain and Behavior, ISSN: 1601-183X
Cognitive problems frequently accompany neurological manifestations of multiple sclerosis (MS). However, during screening of preclinical candidates, assessments of behaviour in mouse models of MS typically focus on locomotor activity. In the present study, we analysed cognitive behaviour of 9 to 10‐week‐old female C57Bl/6J mice orally administered with the toxin cuprizone that induces demyelination, a characteristic feature of MS. Animals received 400 mg/kg cuprizone daily for 2 or 4 weeks, and their performance was compared with that of vehicle‐treated mice. Cuprizone‐treated animals showed multiple deficits in short touchscreen‐based operant tasks: they responded more slowly to visual stimuli, rewards and made more errors in a simple rule‐learning task. In contextual/cued fear conditioning experiments, cuprizone‐treated mice showed significantly lower levels of contextual freezing than vehicle‐treated mice. Diffusion tensor imaging showed treatment‐dependent changes in fractional anisotropy as well as in axial and mean diffusivities in different white matter areas. Lower values of fractional anisotropy and axial diffusivity in cuprizone‐treated mice indicated developing demyelination and/or axonal damage. Several diffusion tensor imaging measurements correlated with learning parameters. Our results show that translational touchscreen operant tests and fear conditioning paradigms can reliably detect cognitive consequences of cuprizone treatment. The suggested experimental approach enables screening novel MS drug candidates in longitudinal experiments for their ability to improve pathological changes in brain structure and reverse cognitive deficits.
Synapses are found in vast numbers in the brain and contain complex proteomes. We developed genetic labeling and imaging methods to examine synaptic proteins in individual excitatory synapses across all regions of the mouse brain. Synapse catalogs were generated from the molecular and morphological features of a billion synapses. Each synapse subtype showed a unique anatomical distribution and each brain region showed a distinct signature of synapse subtypes. Whole brain synaptome cartography revealed spatial architecture from dendritic to global systems levels and previously unknown anatomical features. Synaptome mapping of circuits showed correspondence between synapse diversity and structural and functional connectomes. Behaviorally relevant patterns of neuronal activity trigger spatio-temporal postsynaptic responses sensitive to the structure of synaptome maps. Areas controlling higher cognitive function contain greatest synapse diversity and mutations causing cognitive disorders reorganized synaptome maps. Synaptome technology and resources have wide-ranging application in studies of the normal and diseased brain.
Bach A, Clausen BH, Kristensen LK, et al., 2019, Selectivity, efficacy and toxicity studies of UCCB01-144, a dimeric neuroprotective PSD-95 inhibitor, Neuropharmacology, Vol: 150, Pages: 100-111, ISSN: 0028-3908
Inhibition of postsynaptic density protein-95 (PSD-95) decouples N-methyl-d-aspartate (NMDA) receptor downstream signaling and results in neuroprotection after focal cerebral ischemia. We have previously developed UCCB01-144, a dimeric PSD-95 inhibitor, which binds PSD-95 with high affinity and is neuroprotective in experimental stroke. Here, we investigate the selectivity, efficacy and toxicity of UCCB01-144 and compare with the monomeric drug candidate Tat-NR2B9c. Fluorescence polarization using purified proteins and pull-downs of mouse brain lysates showed that UCCB01-144 potently binds all four PSD-95-like membrane-associated guanylate kinases (MAGUKs). In addition, UCCB01-144 affected NMDA receptor signaling pathways in ischemic brain tissue. UCCB01-144 reduced infarct size in young and aged male mice at various doses when administered 30 min after permanent middle cerebral artery occlusion, but UCCB01-144 was not effective in young male mice when administered 1 h post-ischemia or in female mice. Furthermore, UCCB01-144 was neuroprotective in a transient stroke model in rats, and in contrast to Tat-NR2B9c, high dose of UCCB01-144 did not lead to significant changes in mean arterial blood pressure or heart rate. Overall, UCCB01-144 is a potent MAGUK inhibitor that reduces neurotoxic PSD-95-mediated signaling and improves neuronal survival following focal brain ischemia in rodents under various conditions and without causing cardiovascular side effects, which encourages further studies towards clinical stroke trials.
McKay S, Ryan TJ, McQueen J, et al., 2018, The developmental shift of NMDA receptor composition proceeds independently of the GluN2B CaMKII interaction site and distinct 2A/2B C-termini-directed events, Cell Reports, Vol: 25, Pages: 841-851.e4, ISSN: 2211-1247
The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This “switch” is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition.
Piiponniemi TO, Parkkari T, Heikkinen T, et al., 2018, Impaired performance of the Q175 mouse model of Huntington’s disease in the touch screen paired associates learning task, Frontiers in Behavioral Neuroscience, Vol: 12, ISSN: 1662-5153
Cognitive disturbances often predate characteristic motor dysfunction in individuals with Huntington’s disease (HD) and place an increasing burden on the HD patients and caregivers with the progression of the disorder. Therefore, application of maximally translational cognitive tests to animal models of HD is imperative for the development of treatments that could alleviate cognitive decline in human patients. Here, we examined the performance of the Q175 mouse knock-in model of HD in the touch screen version of the paired associates learning (PAL) task. We found that 10–11-month-old heterozygous Q175 mice had severely attenuated learning curve in the PAL task, which was conceptually similar to previously documented impaired performance of individuals with HD in the PAL task of the Cambridge Neuropsychological Test Automated Battery (CANTAB). Besides high rate of errors in PAL task, Q175 mice exhibited considerably lower responding rate than age-matched wild-type (WT) animals. Our examination of effortful operant responding during fixed ratio (FR) and progressive ratio (PR) reinforcement schedules in a separate cohort of similar age confirmed slower and unselective performance of mutant animals, as observed during PAL task, but suggested that motivation to work for nutritional reward in the touch screen setting was similar in Q175 and WT mice. We also demonstrated that pronounced sensorimotor disturbances in Q175 mice can be detected at early touch screen testing stages, (e.g., during “Punish Incorrect” phase of operant pretraining), so we propose that shorter test routines may be utilised for more expedient studies of treatments aimed at the rescue of HD-related phenotype.
Kopanitsa M, Gou G, Afinowi N, et al., 2018, Chronic treatment with the MEK inhibitor PD-0325901 reverses enhanced I-O relationships of excitatory field potentials in Syngap1+/− mice, Pharmacological Reports, Vol: 70, Pages: 777-783, ISSN: 1734-1140
Background: Synaptic Ras-GTPase-activating protein 1 (SYNGAP1) is an abundant brain-specific protein localized at the postsynaptic density of mammalian excitatory synapses. SYNGAP1 functions as a crucial regulator of downstream intracellular signaling triggered by N-methyl-D-aspartate receptor activation. One of the most important signaling pathways regulated by SYNGAP1 is the Ras-Raf-MEK-ERK pathway. SYNGAP1 deficiency is associated with hyperphosphorylation of MEK and ERK kinases and with altered synaptic function in Syngap1+/− mice. Loss-of-function mutations in the SYNGAP1 gene have been documented in many human cognitive and neurological disorders. However, there are currently no approaches that reverse the phenotypes of SYNGAP1 deficiency. Methods: Using electrophysiological recordings of field responses in hippocampal slices, we examined if disturbances of synaptic physiology in the hippocampus of 7–8-month old Syngap1+/− mice were sensitive to the effect of the MEK inhibitor PD-0325901 given orally for 6 days. Results: We found that in hippocampal slices from vehicle-treated Syngap1+/− mice, basal synaptic responses were higher and their long-term potentiation (LTP) was lower than in slices from wild-type littermates. Chronic administration of PD-0325901 normalized the basal synaptic responses, but did not reverse the LTP deficit.Conclusions: The differential sensitivity of basal synaptic transmission and LTP to MEK inhibition indicates that the effects of SYNGAP1 deficiency on these synaptic parameters are mediated by distinct pathways. Our findings also suggest that at least some physiological phenotypes of the germline Syngap1 mutation can be ameliorated by pharmacological treatment of adult animals.
Horner AE, McLaughlin CL, Afinowi NO, et al., 2018, Enhanced cognition and dysregulated hippocampal synaptic physiology in mice with a heterozygous deletion of PSD-95, European Journal of Neuroscience, Vol: 47, Pages: 164-176, ISSN: 0953-816X
PSD-95 is one of the most abundant proteins of the postsynaptic density of excitatory synapses. It functions as the backbone of protein supercomplexes that mediate signalling between membrane glutamate receptors and intracellular pathways. Homozygous deletion of the Dlg4 gene encoding PSD-95 was previously found to cause a profound impairment in operant and Pavlovian conditioning in Dlg4 −/− mice studied in touch screen chambers that precluded evaluation of PSD-95's role in shaping more subtle forms of learning and memory. In this study, using a battery of touch screen tests, we investigated cognitive behaviour of mice with a heterozygous Dlg4 mutation. We found that in contrast to learning deficits of Dlg4 −/− mice, Dlg4 +/− animals demonstrated enhanced performance in the Visual Discrimination, Visual Discrimination Reversal and Paired-Associates Learning touch screen tasks. The divergent directions of learning phenotypes observed in Dlg4 −/− and Dlg4 +/− mice also contrasted with qualitatively similar changes in the amplitude and plasticity of field excitatory postsynaptic potentials recorded in the CA1 area of hippocampal slices from both mutants. Our results have important repercussions for the studies of genetic models of human diseases, because they demonstrate that reliance on phenotypes observed solely in homozygous mice may obscure qualitatively different changes in heterozygous animals and potentially weaken the validity of translational comparisons with symptoms seen in heterozygous human carriers.
Fernandez E, Collins MO, Frank RAW, et al., 2017, Arc requires PSD95 for assembly into postsynaptic complexes involved with neural dysfunction and intelligence, Cell Reports, Vol: 21, Pages: 679-691, ISSN: 2211-1247
Arc is an activity-regulated neuronal protein, but little is known about its interactions, assembly into multiprotein complexes, and role in human disease and cognition. We applied an integrated proteomic and genetic strategy by targeting a tandem affinity purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice. This allowed biochemical and proteomic characterization of native complexes in wild-type and knockout mice. We identified many Arc-interacting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5-MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed that Arc-PSD95 complexes are enriched in schizophrenia, intellectual disability, autism, and epilepsy mutations and normal variants in intelligence. We propose that Arc-PSD95 postsynaptic complexes potentially affect human cognitive function.
Arbogast T, Iacono G, Chevalier C, et al., 2017, Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition, PLoS Genetics, Vol: 13, ISSN: 1553-7390
Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.
Piiponniemi TO, Bragge T, Vauhkonen EE, et al., 2017, Acquisition and reversal of visual discrimination learning in APPSwDI/Nos2-/-(CVN) mice., Neuroscience Letters, Vol: 650, Pages: 126-133, ISSN: 0304-3940
Studies of cognitive behavior in rodent models of Alzheimer's disease (AD) are the mainstay of academic and industrial efforts to find effective treatments for this disorder. However, in the majority of such studies, the nature of rodent behavioral tests is considerably different from the setting associated with cognitive assessments of individuals with AD. The recently developed touchscreen technique provides a more translational way of rodent cognitive testing because the stimulus (images in different locations on the screen) and reaction (touch) are similar to those employed in human test routines, such as the Cambridge Neuropsychological Test Automated Battery. Here, we used Visual Discrimination and Reversal of Visual Discrimination touchscreen tasks to assess cognitive performance of APPSwDI/Nos2-/-(CVN) mice, which express mutated human APP and have a homozygous deletion of the Nos2 gene. We revealed that CVN mice made more first-time errors and received more correction trials than WT mice across both discrimination and reversal phases, although mutation effect size was larger during the latter phase. These results indicate sensitivity of touchscreen-based measurements to AD-relevant mutations in CVN mice and warrant future touchscreen experiments aimed at evaluating other cognitive and motivational phenotypes in this AD mouse model.
Heise C, Taha E, Murru L, et al., 2017, eEF2K/eEF2 Pathway Controls the Excitation/Inhibition Balance and Susceptibility to Epileptic Seizures, CEREBRAL CORTEX, Vol: 27, Pages: 2226-2248, ISSN: 1047-3211
Benevento M, Oomen CA, Horner AE, et al., 2017, Haploinsufficiency of EHMT1 improves pattern separation and increases hippocampal cell proliferation., Scientific Reports, Vol: 7, ISSN: 2045-2322
Heterozygous mutations or deletions of the human Euchromatin Histone Methyltransferase 1 (EHMT1) gene are the main causes of Kleefstra syndrome, a neurodevelopmental disorder that is characterized by impaired memory, autistic features and mostly severe intellectual disability. Previously, Ehmt1+/-heterozygous knockout mice were found to exhibit cranial abnormalities and decreased sociability, phenotypes similar to those observed in Kleefstra syndrome patients. In addition, Ehmt1+/-knockout mice were impaired at fear extinction and novel- and spatial object recognition. In this study, Ehmt1+/-and wild-type mice were tested on several cognitive tests in a touchscreen-equipped operant chamber to further investigate the nature of learning and memory changes. Performance of Ehmt1+/-mice in the Visual Discrimination &Reversal learning, object-location Paired-Associates learning- and Extinction learning tasks was found to be unimpaired. Remarkably, Ehmt1+/-mice showed enhanced performance on the Location Discrimination test of pattern separation. In line with improved Location Discrimination ability, an increase in BrdU-labelled cells in the subgranular zone of the dentate gyrus was observed. In conclusion, reduced levels of EHMT1 protein in Ehmt1+/-mice does not result in general learning deficits in a touchscreen-based battery, but leads to increased adult cell proliferation in the hippocampus and enhanced pattern separation ability.
Broadhead MJ, Horrocks MH, Zhu F, et al., 2016, PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits., Scientific Reports, Vol: 6, ISSN: 2045-2322
The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity.
Arbogast T, Ouagazzal A-M, Chevalier C, et al., 2016, Reciprocal effects on neurocognitive and metabolic phenotypes in mouse models of 16p11.2 deletion and duplication syndromes, PLoS Genetics, Vol: 12, ISSN: 1553-7390
The 16p11.2 600 kb BP4-BP5 deletion and duplication syndromes have been associated with developmental delay; autism spectrum disorders; and reciprocal effects on the body mass index, head circumference and brain volumes. Here, we explored these relationships using novel engineered mouse models carrying a deletion (Del/+) or a duplication (Dup/+) of the Sult1a1-Spn region homologous to the human 16p11.2 BP4-BP5 locus. On a C57BL/6N inbred genetic background, Del/+ mice exhibited reduced weight and impaired adipogenesis, hyperactivity, repetitive behaviors, and recognition memory deficits. In contrast, Dup/+ mice showed largely opposite phenotypes. On a F1 C57BL/6N × C3B hybrid genetic background, we also observed alterations in social interaction in the Del/+ and the Dup/+ animals, with other robust phenotypes affecting recognition memory and weight. To explore the dosage effect of the 16p11.2 genes on metabolism, Del/+ and Dup/+ models were challenged with high fat and high sugar diet, which revealed opposite energy imbalance. Transcriptomic analysis revealed that the majority of the genes located in the Sult1a1-Spn region were sensitive to dosage with a major effect on several pathways associated with neurocognitive and metabolic phenotypes. Whereas the behavioral consequence of the 16p11 region genetic dosage was similar in mice and humans with activity and memory alterations, the metabolic defects were opposite: adult Del/+ mice are lean in comparison to the human obese phenotype and the Dup/+ mice are overweight in comparison to the human underweight phenotype. Together, these data indicate that the dosage imbalance at the 16p11.2 locus perturbs the expression of modifiers outside the CNV that can modulate the penetrance, expressivity and direction of effects in both humans and mice.
Balemans MCM, Kasri NN, Kopanitsa MV, et al., 2013, Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome., Hum Mol Genet, Vol: 22, Pages: 852-866
Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS.
Ryan TJ, Kopanitsa MV, Indersmitten T, et al., 2013, Evolution of GluN2A/B cytoplasmic domains diversified vertebrate synaptic plasticity and behavior., Nat Neurosci, Vol: 16, Pages: 25-32
Two genome duplications early in the vertebrate lineage expanded gene families, including GluN2 subunits of the NMDA receptor. Diversification between the four mammalian GluN2 proteins occurred primarily at their intracellular C-terminal domains (CTDs). To identify shared ancestral functions and diversified subunit-specific functions, we exchanged the exons encoding the GluN2A (also known as Grin2a) and GluN2B (also known as Grin2b) CTDs in two knock-in mice and analyzed the mice's biochemistry, synaptic physiology, and multiple learned and innate behaviors. The eight behaviors were genetically separated into four groups, including one group comprising three types of learning linked to conserved GluN2A/B regions. In contrast, the remaining five behaviors exhibited subunit-specific regulation. GluN2A/B CTD diversification conferred differential binding to cytoplasmic MAGUK proteins and differential forms of long-term potentiation. These data indicate that vertebrate behavior and synaptic signaling acquired increased complexity from the duplication and diversification of ancestral GluN2 genes.
Coba MP, Komiyama NH, Nithianantharajah J, et al., 2012, TNiK Is Required for Postsynaptic and Nuclear Signaling Pathways and Cognitive Function, JOURNAL OF NEUROSCIENCE, Vol: 32, Pages: 13987-13999, ISSN: 0270-6474
Fernandez E, Collins MO, Uren RT, et al., 2009, Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins, Molecular Systems Biology, Vol: 5, ISSN: 1744-4292
The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K+ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K+ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.
Coba MP, Pocklington AJ, Collins MO, et al., 2009, Neurotransmitters Drive Combinatorial Multistate Postsynaptic Density Networks, SCIENCE SIGNALING, Vol: 2, ISSN: 1945-0877
Coba MP, Valor LM, Kopanitsa MV, et al., 2008, Kinase networks integrate profiles of N-methyl-D-aspartate receptor-mediated gene expression in hippocampus., J Biol Chem, Vol: 283, Pages: 34101-34107, ISSN: 0021-9258
The postsynaptic N-methyl-d-aspartate (NMDA) receptor activates multiple kinases and changes the phosphorylation of many postsynaptic proteins organized in signaling networks. Because the NMDA receptor is known to regulate gene expression, it is important to examine whether networks of kinases control signaling to gene expression. We examined the requirement of multiple kinases and NMDA receptor-interacting proteins for gene expression in mouse hippocampal slices. Protocols that induce long-term depression (LTD) and long-term potentiation (LTP) activated common kinases and overlapping gene expression profiles. Combinations of kinases were required for induction of each gene. Distinct combinations of kinases were required to up-regulate Arc, Npas4, Egr2, and Egr4 following either LTP or LTD protocols. Consistent with the combinatorial data, a mouse mutant model of the human cognition disease gene SAP102, which couples ERK kinase to the NMDA receptor, showed deregulated expression of specific genes. These data support a network model of postsynaptic integration where kinase signaling networks are recruited by differential synaptic activity and control both local synaptic events and activity-dependent gene expression.
Kopanitsa MV, Afinowi NO, Grant SGN, 2006, Recording long-term potentiation of synaptic transmission by three-dimensional multi-electrode arrays., BMC Neurosci, Vol: 7
BACKGROUND: Multi-electrode arrays (MEAs) have become popular tools for recording spontaneous and evoked electrical activity of excitable tissues. The majority of previous studies of synaptic transmission in brain slices employed MEAs with planar electrodes that had limited ability to detect signals coming from deeper, healthier layers of the slice. To overcome this limitation, we used three-dimensional (3D) MEAs with tip-shaped electrodes to probe plasticity of field excitatory synaptic potentials (fEPSPs) in the CA1 area of hippocampal slices of 129S5/SvEvBrd and C57BL/6J-TyrC-Brd mice. RESULTS: Using 3D MEAs, we were able to record larger fEPSPs compared to signals measured by planar MEAs. Several stimulation protocols were used to induce long-term potentiation (LTP) of synaptic responses in the CA1 area recorded following excitation of Schäffer collateral/commissural fibres. Either two trains of high frequency tetanic stimulation or three trains of theta-burst stimulation caused a persistent, pathway specific enhancement of fEPSPs that remained significantly elevated for at least 60 min. A third LTP induction protocol that comprised 150 pulses delivered at 5 Hz, evoked moderate LTP if excitation strength was increased to 1.5x of the baseline stimulus. In all cases, we observed a clear spatial plasticity gradient with maximum LTP levels detected in proximal apical dendrites of pyramidal neurones. No significant differences in the manifestation of LTP were observed between 129S5/SvEvBrd and C57BL/6J-TyrC-Brd mice with the three protocols used. All forms of plasticity were sensitive to inhibition of N-methyl-D-aspartate (NMDA) receptors. CONCLUSION: Principal features of LTP (magnitude, pathway specificity, NMDA receptor dependence) recorded in the hippocampal slices using MEAs were very similar to those seen in conventional glass electrode experiments. Advantages of using MEAs are the ability to record from different regions of the slice and the ease of condu
Kopanytsia MV, 2003, [Effect of phenazepam metabolite, 2-amino-5-bromo-2'-chlorobenzophenone, on glycine and glutamate NMDA receptors of rat hippocampal pyramidal neurones]., Fiziol Zh, Vol: 49, Pages: 23-27, ISSN: 2522-9028
Action of 2-amino-5-bromo-2'-chlorobenzophenone (ABPH), a metabolite of phenazepam, on currents evoked by the activation of glycine and glutamate NMDA receptors has been investigated in enzymatically isolated hippocampal rat pyramidal neurones. It is demonstrated that 3-10 microM ABPH caused an inhibitory effect on glycine receptors which was more prominent after pre-incubation with the drug. Peak amplitude of NMDA receptor-mediated currents was weakly potentiated after prolonged (4-6 min) incubation with 10 microM ABPH. Thus, the effects of the benzophenone metabolites should be taken into account when analysing the action of phenazepam and other benzodiazepine drugs.
Kopanitsa MV, Yakubovska LM, Rudenko OP, et al., 2002, Modulation of GABA(A) receptor-mediated currents by benzophenone derivatives in isolated rat Purkinje neurones., Neuropharmacology, Vol: 43, Pages: 764-777, ISSN: 0028-3908
We investigated modulation of GABA(A) receptor-mediated whole-cell currents in cerebellar Purkinje neurones by several derivatives of benzophenone. A metabolite of phenazepam, 5-bromo-2'-chloro-2-aminobenzophenone (I), caused dual modification of peak amplitudes of GABA-gated currents that depended upon the concentration of applied GABA and incubation time. Following short 10 s pre-incubations, 1-30 microM I facilitated activation and delayed deactivation of currents evoked by 500 ms pulses of 20 microM GABA. In addition, 10 microM I prominently enhanced desensitisation of currents during applications of 500 microM GABA mainly by decreasing the value of the fast time constant of the desensitisation. Continuous 6 min incubation with 10 microM I during GABA stimulation or its administration between but not during 1 s pulses of 500 microM GABA led to a gradual, partly reversible attenuation of GABA-activated currents. This inhibition was not observed when I was applied only during pulses of GABA, indicating that the blockade was not use-dependent. One of the possible mechanisms of this down-modulation could be an intracellular effect of I, because when applied intracellularly it caused slow inhibition of responses to consecutive GABA pulses. When 3-30 microM I was applied on the background of small 'plateau'-like current 5-7 s after application of 500 microM GABA, it was able to block open channels with on and off rates similar to those observed with 30 microM picrotoxin but much slower than in the case of 500 microM benzylpenicillin. At a concentration of 10 microM, 5-substituted benzophenones, but not 2-aminobenzophenone or benzophenone itself, exhibited modulatory properties similar to I and distinct from those of picrotoxin and benzylpenicillin. Therefore, we conclude that derivatives of benzophenone are a novel class of GABA(A) receptor modulators with a unique pharmacological profile.
Golovenko NY, Zhuk MS, Zin'kovskii VG, et al., 2001, Pharmacokinetics of ethanol in mice with different alcohol motivation., Bull Exp Biol Med, Vol: 132, Pages: 852-855, ISSN: 0007-4888
We studied the pharmacokinetics of (14)C-ethanol administered in various doses and via different routes to CBA, C57Bl/6, and (CBAxC57Bl/6)F1 mice. Kinetic scheme of ethanol distribution included its elimination by enzymatic (80-90% C(0)) and exponential (10-20% C(0)) mechanisms. Ethanol pharmacokinetics did not depend on the administration route and mouse strain. The kinetic scheme of ethanol distribution in mice was characterized by a dose-dependent linear increase in alcohol concentration in the plasma and brain and nonlinear (parabolic) increase in the area under its pharmacokinetic curve in the test tissue.
Krishtal O, Lozovaya N, Fisunov A, et al., 2001, Modulation of ion channels in rat neurons by the constituents of Hypericum perforatum, 1st Biocenter Symposium on Drug Therapy, Publisher: GEORG THIEME VERLAG KG, Pages: S74-S82, ISSN: 0176-3679
Kopanitsa MV, Zhuk OV, Zinkovsky VG, et al., 2001, Modulation of GABAA receptor-mediated currents by phenazepam and its metabolites., Naunyn Schmiedebergs Arch Pharmacol, Vol: 364, Pages: 1-8, ISSN: 0028-1298
The effects of 7-bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (phenazepam, PNZ), a 1,4-benzodiazepine derivative, and its physiological metabolites on GABA-activated whole-cell currents were studied in enzymatically isolated rat Purkinje neurones. PNZ, its hydroxylated metabolite (HPNZ) and a reference benzodiazepine, diazepam, potently enhanced (up to 200% of control) peak amplitude of currents activated by 10 microM GABA with EC50s of 6.1 +/- 0.8, 10.3 +/- 1.4 and 13.5 +/- 1.9 nM respectively. Both PNZ and HPNZ caused a parallel leftwards shift of the concentration/effect relationship for GABA. Another metabolite, 6-bromo-(2-chlorophenyl) quinazoline-2-one (QNZ), augmented responses to 10 microM GABA with a maximal efficacy similar to that of the 1,4benzodiazepines tested, although its EC50 was 2.4 +/- 0.2 microM. A further metabolite, 5-bromo-(2-chlorophenyl)-2-aminobenzophenone (ABPH), had only minimal effects on the responses elicited by 10 microM GABA. Incubation with QNZ and ABPH had biphasic effects on the concentration/effect relationship for GABA. These compounds enhanced peak amplitudes of currents activated by low concentrations of GABA, but inhibited responses to saturating concentrations of the agonist. This effect could, in part, be explained by the acceleration of the desensitisation process by those substances. It is concluded that both PNZ and HPNZ can be referred to as full positive modulators of GABAA receptors and that they are primarily responsible for GABAergic effects of therapeutic doses of PNZ.
Kopanytsia MV, Vasylinin HB, Fedorova OA, et al., 2001, [Use of the acoustic startle response in the mouse to evaluate the pharmacodynamic action of ethanol]., Fiziol Zh, Vol: 47, Pages: 106-114, ISSN: 2522-9028
A method of the pharmacological screening of psychotropic drugs that is based on the estimation of the peak amplitude and latency of the acoustic startle response (ASR) is suggested. A linear relationship between the changes of the ASR parameters induced by acute i.p. 14C-ethanol administration and its contents in brain is demonstrated. It is proposed that changes of the ASR parameters are strictly dependent upon the concentration of a modulatory drug in the biophase of action (brain, serum) and therefore can be utilised for the effector analysis of pharmacokinetics and preliminary screening of psychotropic drugs.
Kirichok YV, Boychuk YA, Kopanitsa MV, et al., 2000, Effects of mibefradil on synaptic transmission in the hippocampus and on voltage-dependent currents in isolated hippocampal and thalamic neurons of the rat, Neurophysiology, Vol: 32, Pages: 12-16, ISSN: 0090-2977
Kopanitsa MV, Krishtal OA, Komissarov IV, 2000, Inhibitory action of ambocarb on voltage-operated sodium channels in rat isolated hippocampal pyramidal neurons., Clin Exp Pharmacol Physiol, Vol: 27, Pages: 46-54, ISSN: 0305-1870
1. A whole-cell patch-clamp study of the effects of ambocarb, a novel nootropic beta-carboline, on sodium currents in rat acutely isolated hippocampal pyramidal neurons was performed. 2. Ambocarb potently and reversibly suppressed sodium currents in a concentration range of 3-300 mumol/L. The amount of block was dependent on the holding potential, with half-maximal inhibition values being 26 and 94 mumol/L at -80 and -120 mV, respectively. 3. Ambocarb induced a hyperpolarizing shift in the steady state availability curve, which indicates an increase in the proportion of inactivated sodium channels. This action is presumably mediated by promoting the development of inactivation and slowing the recovery of sodium channels from inactivation. 4. Because many neuroprotective drugs were shown to inhibit sodium currents, down-modulation of voltage-operated sodium channels that complements the known positive interaction of ambocarb and other related beta-carbolines with GABAA receptors may provide a promising strategy in the treatment of brain disorders associated with trauma and ischaemia.
Kopanitsia MV, Boĭchuk IA, Lozova NO, et al., Interneuronal signaling mediated by the trans-synaptic diffusion of neurotransmitters, Fiziolohichnyi zhurnal (Kiev, Ukraine : 1994), Vol: 45, Pages: 132-147
A great number of modern neurophysiological studies give evidence about the possibility of the extrasynaptic diffusion of the released neurotransmitters. Such observations challenge the established view on synapses as isolated communication channels between pre- and postsynaptic neurones. In the present review article, we consider the latest experimental data on the interneuronal cross-talk mediated by extrasynaptic diffusion of L-glutamate (L-Glu) and gamma-amino-butyric acid (GABA). Possible physiological significance of this phenomenon is also discussed.
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