PV-specific loss of the transcriptional coactivator PGC-1α slows down the evolution of epileptic activity in an acute ictogenic model.

The transcriptional coactivator, PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), plays a key role in coordinating energy requirement within cells. Its importance is reflected in the growing number of psychiatric and neurological conditions that have been associated with reduced PGC-1α levels. In cortical networks, PGC-1α is required for the induction of parvalbumin (PV) expression in interneurons, and PGC-1α deficiency affects synchronous GABAergic release. It is unknown, however, how this affects cortical excitability. We show here that knocking down PGC-1α specifically in the PV-expressing cells (PGC-1αPV-/-) blocks the activity-dependent regulation of the synaptic proteins, SYT2 and CPLX1. More surprisingly, this cell class-specific knockout of PGC-1α appears to have a novel antiepileptic effect, as assayed in brain slices bathed in 0 Mg2+ media. The rate of occurrence of preictal discharges developed approximately equivalently in wild-type and PGC-1αPV-/- brain slices, but the intensity of these discharges was lower in PGC-1αPV-/- slices, as evident from the reduced power in the γ range and reduced firing rates in both PV interneurons and pyramidal cells during these discharges. Reflecting this reduced intensity in the preictal discharges, the PGC-1αPV-/- brain slices experienced many more discharges before transitioning into a seizure-like event. Consequently, there was a large increase in the latency to the first seizure-like event in brain slices lacking PGC-1α in PV interneurons. We conclude that knocking down PGC-1α limits the range of PV interneuron firing and this slows the pathophysiological escalation during ictogenesis.NEW & NOTEWORTHY Parvalbumin expressing interneurons are considered to play an important role in regulating cortical activity. We were surprised, therefore, to find that knocking down the transcriptional coactivator, PGC-1α, specifically in this class of interneurons appears to slow ictogenesis. This anti-ictogenic effect is associated with reduced activity in preictal discharges, but with a far longer period of these discharges before the first seizure-like events finally start. Thus, PGC-1α knockdown may promote schizophrenia while reducing epileptic tendencies.

Pyramidal cell activity levels affect the polarity of activity-induced gene transcription changes in interneurons.

Changes in gene expression are an important mechanism by which activity levels are regulated in the nervous system. It is not known, however, how network activity influences gene expression in interneurons; since they themselves provide negative feedback in the form of synaptic inhibition, there exists a potential conflict between their cellular homeostatic tendencies and those of the network. We present a means of examining this issue, utilizing simple in vitro models showing different patterns of intense network activity. We found that the degree of concurrent pyramidal activation changed the polarity of the induced gene transcription. When pyramidal cells were quiescent, interneuronal activation led to an upregulation of glutamate decarboxylase 1 ( GAD1) and parvalbumin ( Pvalb) gene transcriptions, mediated by activation of the Ras/extracellular signal-related kinase mitogen-activated protein kinase (Ras/ERK MAPK) pathway. In contrast, coactivation of pyramidal cells led to an ionotropic glutamate receptor N-methyl-d-aspartate 2B-dependent decrease in transcription. Our results demonstrate a hitherto unrecognized complexity in how activity-dependent gene expression changes are manifest in cortical networks. NEW & NOTEWORTHY We demonstrate a novel feedback mechanism in cortical networks, by which glutamatergic drive, mediated through the Ras/ERK MAPK pathway, regulates gene transcription in interneurons. Using a unique feature of certain in vitro epilepsy models, we show that without this glutamatergic feedback, intense activation of interneurons causes parvalbumin and glutamate decarboxylase 1 mRNA expression to increase. If, on the other hand, pyramidal cells are coactivated with interneurons, this leads to a downregulation of these genes.

Activity-Dependent Neuroplasticity Induced by an Enriched Environment Reverses Cognitive Deficits in Scribble Deficient Mouse.

Planar cell polarity (PCP) signaling is well known to play a critical role during prenatal brain development; whether it plays specific roles at postnatal stages remains rather unknown. Here, we investigated the role of a key PCP-associated gene scrib in CA1 hippocampal structure and function at postnatal stages. We found that Scrib is required for learning and memory consolidation in the Morris water maze as well as synaptic maturation and NMDAR-dependent bidirectional plasticity. Furthermore, we unveiled a direct molecular interaction between Scrib and PP1/PP2A phosphatases whose levels were decreased in postsynaptic density of conditional knock-out mice. Remarkably, exposure to enriched environment (EE) preserved memory formation in CaMK-Scrib-/- mice by recovering synaptic plasticity and maturation. Thus, Scrib is required for synaptic function involved in memory formation and EE has beneficiary therapeutic effects. Our results demonstrate a distinct new role for a PCP-associated protein, beyond embryonic development, in cognitive functions during adulthood.

Rabphilin 3A: A novel target for the treatment of levodopa-induced dyskinesias.

N-methyl-d-aspartate receptor (NMDAR) subunit composition strictly commands receptor function and pharmacological responses. Changes in NMDAR subunit composition have been documented in brain disorders such as Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesias (LIDs), where an increase of NMDAR GluN2A/GluN2B subunit ratio at striatal synapses has been observed. A therapeutic approach aimed at rebalancing NMDAR synaptic composition represents a valuable strategy for PD and LIDs. To this, the comprehension of the molecular mechanisms regulating the synaptic localization of different NMDAR subtypes is required. We have recently demonstrated that Rabphilin 3A (Rph3A) is a new binding partner of NMDARs containing the GluN2A subunit and that it plays a crucial function in the synaptic stabilization of these receptors. Considering that protein-protein interactions govern the synaptic retention of NMDARs, the purpose of this work was to analyse the role of Rph3A and Rph3A/NMDAR complex in PD and LIDs, and to modulate Rph3A/GluN2A interaction to counteract the aberrant motor behaviour associated to chronic L-DOPA administration. Thus, an array of biochemical, immunohistochemical and pharmacological tools together with electron microscopy were applied in this study. Here we found that Rph3A is localized at the striatal postsynaptic density where it interacts with GluN2A. Notably, Rph3A expression at the synapse and its interaction with GluN2A-containing NMDARs were increased in parkinsonian rats displaying a dyskinetic profile. Acute treatment of dyskinetic animals with a cell-permeable peptide able to interfere with Rph3A/GluN2A binding significantly reduced their abnormal motor behaviour. Altogether, our findings indicate that Rph3A activity is linked to the aberrant synaptic localization of GluN2A-expressing NMDARs characterizing LIDs. Thus, we suggest that Rph3A/GluN2A complex could represent an innovative therapeutic target for those pathological conditions where NMDAR composition is significantly altered.

The contribution of raised intraneuronal chloride to epileptic network activity.

Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory if intraneuronal chloride rises. However, it has proved difficult to separate the role of raised chloride from other contributory factors in complex network phenomena, such as epileptic pathology. Therefore, we asked what patterns of activity are associated with chloride dysregulation by making novel use of Halorhodopsin to load clusters of mouse pyramidal cells artificially with Cl(-). Brief (1-10 s) activation of Halorhodopsin caused substantial positive shifts in the GABAergic reversal potential that were proportional to the charge transfer during the illumination and in adult neocortical pyramidal neurons decayed with a time constant of τ = 8.0 ± 2.8s. At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high-frequency oscillations with evidence of out-of-phase firing (Ibarz et al., 2010). We show how such firing patterns can arise from quite small shifts in the mean intracellular Cl(-) level, within heterogeneous neuronal populations. Notably, however, chloride loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, subepileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that chloride loading has at most an adjunctive role in ictogenesis. Our simulations also show how chloride loading can affect the jitter of action potential timing associated with imminent recruitment to an ictal event (Netoff and Schiff, 2002).

Mitochondrial abnormality associates with type-specific neuronal loss and cell morphology changes in the pedunculopontine nucleus in Parkinson disease.

Cholinergic neuronal loss in the pedunculopontine nucleus (PPN) associates with abnormal functions, including certain motor and nonmotor symptoms. This realization has led to low-frequency stimulation of the PPN for treating patients with Parkinson disease (PD) who are refractory to other treatment modalities. However, the molecular mechanisms underlying PPN neuronal loss and the therapeutic substrate for the clinical benefits following PPN stimulation remain poorly characterized, hampering progress toward designing more efficient therapies aimed at restoring the PPN's normal functions during progressive parkinsonism. Here, we investigated postmortem pathological changes in the PPN of PD cases. Our study detected a loss of neurons producing gamma-aminobutyric acid (GABA) as their output and glycinergic neurons, along with the pronounced loss of cholinergic neurons. These losses were accompanied by altered somatic cell size that affected the remaining neurons of all neuronal subtypes studied here. Because studies showed that mitochondrial dysfunction exists in sporadic PD and in PD animal models, we investigated whether altered mitochondrial composition exists in the PPN. A significant up-regulation of several mitochondrial proteins was seen in GABAergic and glycinergic neurons; however, cholinergic neurons indicated down-regulation of the same proteins. Our findings suggest an imbalance in the activity of key neuronal subgroups of the PPN in PD, potentially because of abnormal inhibitory activity and altered cholinergic outflow.

The planar polarity protein Scribble1 is essential for neuronal plasticity and brain function.

Scribble (Scrib) is a key regulator of apicobasal polarity, presynaptic architecture, and short-term synaptic plasticity in Drosophila. In mammals, its homolog Scrib1 has been implicated in cancer, neural tube closure, and planar cell polarity (PCP), but its specific role in the developing and adult nervous system is unclear. Here, we used the circletail mutant, a mouse model for PCP defects, to show that Scrib1 is located in spines where it influences actin cytoskeleton and spine morphing. In the hippocampus of these mutants, we observed an increased synapse pruning associated with an increased number of enlarged spines and postsynaptic density, and a decreased number of perforated synapses. This phenotype was associated with a mislocalization of the signaling pathway downstream of Scrib1, leading to an overall activation of Rac1 and defects in actin dynamic reorganization. Finally, Scrib1-deficient mice exhibit enhanced learning and memory abilities and impaired social behavior, two features relevant to autistic spectrum disorders. Our data identify Scrib1 as a crucial regulator of brain development and spine morphology, and suggest that Scrib1(crc/+) mice might be a model for studying synaptic dysfunction and human psychiatric disorders.

NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex.

NMDA (N-methyl-D-aspartate) receptors (NMDARs) are targeted to dendrites and anchored at the post-synaptic density (PSD) through interactions with PDZ proteins. However, little is known about how these receptors are sorted from the endoplasmic reticulum and Golgi apparatus to the synapse. Here, we find that synapse-associated protein 102 (SAP102) interacts with the PDZ-binding domain of Sec8, a member of the exocyst complex. Our results show that interactions between SAP102 and Sec8 are involved in the delivery of NMDARs to the cell surface in heterologous cells and neurons. Furthermore, they suggest that an exocyst-SAP102-NMDAR complex is an important component of NMDAR trafficking.

Vangl2 in the Dentate Network Modulates Pattern Separation and Pattern Completion.

The organization of spatial information, including pattern completion and pattern separation processes, relies on the hippocampal circuits, yet the molecular and cellular mechanisms underlying these two processes are elusive. Here, we find that loss of Vangl2, a core PCP gene, results in opposite effects on pattern completion and pattern separation processes. Mechanistically, we show that Vangl2 loss maintains young postmitotic granule cells in an immature state, providing increased cellular input for pattern separation. The genetic ablation of Vangl2 disrupts granule cell morpho-functional maturation and further prevents CaMKII and GluA1 phosphorylation, disrupting the stabilization of AMPA receptors. As a functional consequence, LTP at lateral perforant path-GC synapses is impaired, leading to defects in pattern completion behavior. In conclusion, we show that Vangl2 exerts a bimodal regulation on young and mature GCs, and its disruption leads to an imbalance in hippocampus-dependent pattern completion and separation processes.

mGluR7 is a metaplastic switch controlling bidirectional plasticity of feedforward inhibition.

Plasticity of feedforward inhibition in the hippocampal mossy fiber (MF) pathway can dramatically influence dentate gyrus-CA3 dialog. Interestingly, MF inputs to CA3 stratum lucidum interneurons (SLINs) undergo long-term depression (LTD) following high-frequency stimulation (HFS), in contrast to MF-pyramid (PYR) synapses, where long-term potentiation (LTP) occurs. Furthermore, activity-induced potentiation of MF-SLIN transmission has not previously been observed. Here we report that metabotropic glutamate receptor subtype 7 (mGluR7) is a metaplastic switch at MF-SLIN synapses, whose activation and surface expression governs the direction of plasticity. In naive slices, mGluR7 activation during HFS generates MF-SLIN LTD, depressing presynaptic release through a PKC-dependent mechanism. Following agonist exposure, mGluR7 undergoes internalization, unmasking the ability of MF-SLIN synapses to undergo presynaptic potentiation in response to the same HFS that induces LTD in naive slices. Thus, selective mGluR7 targeting to MF terminals contacting SLINs and not PYRs provides cell target-specific plasticity and bidirectional control of feedforward inhibition.

RNA editing produces glycine receptor alpha3(P185L), resulting in high agonist potency.

The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 microM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyR alpha3(P185L), a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 approximately 5 microM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyR alpha3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyR alpha3(P185L) is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

Linking NMDA Receptor Synaptic Retention to Synaptic Plasticity and Cognition.

NMDA receptor (NMDAR) subunit composition plays a pivotal role in synaptic plasticity at excitatory synapses. Still, the mechanisms responsible for the synaptic retention of NMDARs following induction of plasticity need to be fully elucidated. Rabphilin3A (Rph3A) is involved in the stabilization of NMDARs at synapses through the formation of a complex with GluN2A and PSD-95. Here we used different protocols to induce synaptic plasticity in the presence or absence of agents modulating Rph3A function. The use of Forskolin/Rolipram/Picrotoxin cocktail to induce chemical LTP led to synaptic accumulation of Rph3A and formation of synaptic GluN2A/Rph3A complex. Notably, Rph3A silencing or use of peptides interfering with the GluN2A/Rph3A complex blocked LTP induction. Moreover, in vivo disruption of GluN2A/Rph3A complex led to a profound alteration of spatial memory. Overall, our results demonstrate a molecular mechanism needed for NMDAR stabilization at synapses after plasticity induction and to trigger downstream signaling events necessary for cognitive behavior.

Region-specific changes in gamma and beta2 rhythms in NMDA receptor dysfunction models of schizophrenia.

Cognitive disruption in schizophrenia is associated with altered patterns of spatiotemporal interaction associated with multiple electroencephalogram (EEG) frequency bands in cortex. In particular, changes in the generation of gamma (30-80 Hz) and beta2 (20-29 Hz) rhythms correlate with observed deficits in communication between different cortical areas. Aspects of these changes can be reproduced in animal models, most notably those involving acute or chronic reduction in glutamatergic synaptic communication mediated by N-methyl D-aspartate (NMDA) receptors. In vitro electrophysiological and immunocytochemical approaches afforded by such animal models continue to reveal a great deal about the mechanisms underlying EEG rhythm generation and are beginning to uncover which basic molecular, cellular, and network phenomena may underlie their disruption in schizophrenia. Here we briefly review the evidence for changes in gamma-aminobutyric acidergic (GABAergic) and glutamatergic function and address the problem of region specificity of changes with quantitative comparisons of effects of ketamine on gamma and beta2 rhythms in vitro. We conclude, from available evidence, that many observed changes in markers for GABAergic function in schizophrenia may be secondary to deficits in NMDA receptor-mediated excitatory synaptic activity. Furthermore, the broad range of changes in cortical dynamics seen in schizophrenia -- with contrasting effects seen in different brain regions and for different frequency bands -- may be more directly attributable to underlying deficits in glutamatergic neuronal communication rather than GABAergic inhibition alone.

Simultaneous profiling of activity patterns in multiple neuronal subclasses.

BACKGROUND: Neuronal networks typically comprise heterogeneous populations of neurons. A core objective when seeking to understand such networks, therefore, is to identify what roles these different neuronal classes play. Acquiring single cell electrophysiology data for multiple cell classes can prove to be a large and daunting task. Alternatively, Ca2+ network imaging provides activity profiles of large numbers of neurons simultaneously, but without distinguishing between cell classes. NEW METHOD: We therefore developed a strategy for combining cellular electrophysiology, Ca2+ network imaging, and immunohistochemistry to provide activity profiles for multiple cell classes at once. This involves cross-referencing easily identifiable landmarks between imaging of the live and fixed tissue, and then using custom MATLAB functions to realign the two imaging data sets, to correct for distortions of the tissue introduced by the fixation or immunohistochemical processing. RESULTS: We illustrate the methodology for analyses of activity profiles during epileptiform events recorded in mouse brain slices. We further demonstrate the activity profile of a population of parvalbumin-positive interneurons prior, during, and following a seizure-like event. COMPARISON WITH EXISTING METHODS: Current approaches to Ca2+ network imaging analyses are severely limited in their ability to subclassify neurons, and often rely on transgenic approaches to identify cell classes. In contrast, our methodology is a generic, affordable, and flexible technique to characterize neuronal behaviour with respect to classification based on morphological and neurochemical identity. CONCLUSIONS: We present a new approach for analysing Ca2+ network imaging datasets, and use this to explore the parvalbumin-positive interneuron activity during epileptiform events.

Graphical user interface for simultaneous profiling of activity patterns in multiple neuronal subclasses.

We provide notes on how to use a graphical user interface (GUI), implemented with MATLAB, for aligning imaging datasets of biological tissue. The original use was for matching two imaging data sets, where one set was taken of the living preparation and another was taken post-fixation and following immunohistochemical processing. This technique is described in detail in an accompanying paper (Parrish et al., [1], where we also include information about the experimental procedures, and examples of the usage of the GUI.

Region-specific reduction in entorhinal gamma oscillations and parvalbumin-immunoreactive neurons in animal models of psychiatric illness.

Psychiatric illnesses, particularly schizophrenia, are associated with disrupted markers for interneuronal function and interneuron-mediated brain rhythms such as gamma frequency oscillations. Here we investigate a possible link between these two observations in the entorhinal cortex and hippocampus by using a genetic and an acute model of psychiatric illness. Lysophosphatidic acid 1 receptor-deficient (LPA1-deficient) mice show psychomotor-gating deficits and neurochemical changes resembling those seen in postmortem schizophrenia studies. Similar deficits are seen acutely with antagonism of the NMDA subtype of glutamate receptor. Neither model induced any change in power or frequency of gamma rhythms generated by kainate in hippocampal slices. In contrast, a dramatic decrease in the power of gamma oscillations was seen in superficial, but not deep, medial entorhinal cortex layers in both models. Immunolabeling for GABA, parvalbumin, and calretinin in medial entorhinal cortex from LPA1-deficient mice showed an approximately 40% reduction in total GABA- and parvalbumin-containing neurons, but no change in the number of calretinin-positive neurons. This deficit was specific for layer II (LII). No change in the number of neurons positive for these markers was seen in the hippocampus. Acute NMDA receptor blockade, which selectively reduces synaptic drive to LII entorhinal interneurons, also disrupted gamma rhythms in a similar manner in superficial entorhinal cortex, but not in hippocampus. These data demonstrate an area-specific deficit in gamma rhythmogenesis in animal models of psychiatric illness and suggest that loss, or reduction in function, of interneurons having a large NMDA receptor expression may underlie the network dysfunction that is seen.

The role of inhibition in epileptic networks.

Inhibition plays many roles in cortical circuits, including coordination of network activity in different brain rhythms and neuronal clusters, gating of activity, gain control, and dictating the manner in which activity flows through the network. This latter is particularly relevant to epileptic states, when extreme hypersynchronous discharges can spread across cortical territories. We review these different physiological and pathological roles and discuss how inhibition can be compromised and why this predisposes the network to seizures.

Rabphilin 3A retains NMDA receptors at synaptic sites through interaction with GluN2A/PSD-95 complex.

NMDA receptor (NMDAR) composition and synaptic retention represent pivotal features in the physiology and pathology of excitatory synapses. Here, we identify Rabphilin 3A (Rph3A) as a new GluN2A subunit-binding partner. Rph3A is known as a synaptic vesicle-associated protein involved in the regulation of exo- and endocytosis processes at presynaptic sites. We find that Rph3A is enriched at dendritic spines. Protein-protein interaction assays reveals that Rph3A N-terminal domain interacts with GluN2A(1349-1389) as well as with PSD-95(PDZ3) domains, creating a ternary complex. Rph3A silencing in neurons reduces the surface localization of synaptic GluN2A and NMDAR currents. Moreover, perturbing GluN2A/Rph3A interaction with interfering peptides in organotypic slices or in vivo induces a decrease of the amplitude of NMDAR-mediated currents and GluN2A density at dendritic spines. In conclusion, Rph3A interacts with GluN2A and PSD-95 forming a complex that regulates NMDARs stabilization at postsynaptic membranes.

Scribble1/AP2 complex coordinates NMDA receptor endocytic recycling.

The appropriate trafficking of glutamate receptors to synapses is crucial for basic synaptic function and synaptic plasticity. It is now accepted that NMDA receptors (NMDARs) internalize and are recycled at the plasma membrane but also exchange between synaptic and extrasynaptic pools; these NMDAR properties are also key to governing synaptic plasticity. Scribble1 is a large PDZ protein required for synaptogenesis and synaptic plasticity. Herein, we show that the level of Scribble1 is regulated in an activity-dependent manner and that Scribble1 controls the number of NMDARs at the plasma membrane. Notably, Scribble1 prevents GluN2A subunits from undergoing lysosomal trafficking and degradation by increasing their recycling to the plasma membrane following NMDAR activation. Finally, we show that a specific YxxR motif on Scribble1 controls these mechanisms through a direct interaction with AP2. Altogether, our findings define a molecular mechanism to control the levels of synaptic NMDARs via Scribble1 complex signaling.

Differential dendritic targeting of AMPA receptor subunit mRNAs in adult rat hippocampal principal neurons and interneurons.

In hippocampal neurons, AMPA receptors (AMPARs) mediate fast excitatory postsynaptic responses at glutamatergic synapses, and are involved in various forms of synaptic plasticity. Dendritic local protein synthesis of selected AMPAR subunit mRNAs is considered an additional mechanism to independently and rapidly control the strength of individual synapses. We have used fluorescent in situ hybridization and immunocytochemistry to analyze the localization of AMPAR subunit (GluA1-4) mRNAs and their relationship with the translation machinery in principal cells and interneurons of the adult rat hippocampus. The mRNAs encoding all four AMPAR subunits were detected in the somata and dendrites of CA3 and CA1 pyramidal cells and those of six classes of CA1 γ-aminobutyric acid (GABA)ergic interneurons. GluA1-4 subunit mRNAs were highly localized to the apical dendrites of pyramidal cells, whereas in interneurons they were present in multiple dendrites. In contrast, in the dentate gyrus, GluA1-4 subunit mRNAs were virtually restricted to the somata and were absent from the dendrites of granule cells. These different regional and cell type-specific labeling patterns also correlated with the localization of markers for components of the protein synthesis machinery. Our results support the local translation of GluA1-4 mRNAs in dendrites of hippocampal pyramidal cells and CA1 interneurons but not in granule cells of the dentate gyrus. Furthermore, the regional and cell type-specific differences we observed suggest that each cell type uses distinct ways of regulating the local translation of AMPAR subunits.