Rescue of behavioral and EEG deficits in male and female Mecp2-deficient mice by delayed Mecp2 gene reactivation.

Mutations of the X-linked gene encoding methyl CpG binding protein type 2 (MECP2) are the predominant cause of Rett syndrome, a severe neurodevelopmental condition that affects primarily females. Previous studies have shown that major phenotypic deficits arising from MeCP2-deficiency may be reversible, as the delayed reactivation of the Mecp2 gene in Mecp2-deficient mice improved aspects of their Rett-like phenotype. While encouraging for prospective gene replacement treatments, it remains unclear whether additional Rett syndrome co-morbidities recapitulated in Mecp2-deficient mice will be similarly responsive to the delayed reintroduction of functional Mecp2. Here, we show that the delayed reactivation of Mecp2 in both male and female Mecp2-deficient mice rescues established deficits in motor and anxiety-like behavior, epileptiform activity, cortical and hippocampal electroencephalogram patterning and thermoregulation. These findings indicate that neural circuitry deficits arising from the deficiency in Mecp2 are not engrained, and provide further evidence that delayed restoration of Mecp2 function can improve a wide spectrum of the Rett-like deficits recapitulated by Mecp2-deficient mice.

Selective preservation of MeCP2 in catecholaminergic cells is sufficient to improve the behavioral phenotype of male and female Mecp2-deficient mice.

Rett syndrome (RTT) is a neurodevelopmental disorder caused primarily by mutations of the X-linked MECP2 gene. Although the loss of MeCP2 function affects many neural systems, impairments of catecholaminergic function have been hypothesized to underlie several of the cardinal behavioral deficits of RTT patients and Mecp2-deficient mice. Although recent Mecp2 reactivation studies indicate that RTT may be a reversible condition, it remains unclear whether specifically preserving Mecp2 function within a specific system will be sufficient to convey beneficial effects. Here, we test whether the selective preservation of Mecp2 within catecholaminergic cells will improve the phenotype of Mecp2-deficient mice. Our results show that this targeted preservation of Mecp2 significantly improves the lifespan, phenotypic severity and cortical epileptiform discharge activity of both male and female Mecp2-deficient mice. Further, we found that the catecholaminergic preservation of Mecp2 also improves the ambulatory rate, rearing activity, motor coordination, anxiety and nest-building performances of Mecp2-deficient mice of each gender. Interestingly, our results also revealed a gender-specific improvement, as specific cortical and hippocampal electroencephalographic abnormalities were significantly improved in male, but not female, rescue mice. Collectively, these results support the role of the catecholaminergic system in the pathogenesis of RTT and provide proof-of-principle that restoring MeCP2 function within this specific system could represent a treatment strategy for RTT.

Role of anti-inflammatory compounds in human immunodeficiency virus-1 glycoprotein120-mediated brain inflammation.

BACKGROUND: Neuroinflammation is a common immune response associated with brain human immunodeficiency virus-1 (HIV-1) infection. Identifying therapeutic compounds that exhibit better brain permeability and can target signaling pathways involved in inflammation may benefit treatment of HIV-associated neurological complications. The objective of this study was to implement an in vivo model of brain inflammation by intracerebroventricular administration of the HIV-1 viral coat protein gp120 in rats and to examine anti-inflammatory properties of HIV adjuvant therapies such as minocycline, chloroquine and simvastatin. METHODS: Male Wistar rats were administered a single dose of gp120ADA (500 ng) daily for seven consecutive days, intracerebroventricularly, with or without prior intraperitoneal administration of minocycline, chloroquine or simvastatin. Maraviroc, a CCR5 antagonist, was administered intracerebroventricularly prior to gp120 administration for seven days as control. Real-time qPCR was used to assess gene expression of inflammatory markers in the frontal cortex, hippocampus and striatum. Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) secretion in cerebrospinal fluid (CSF) was measured applying ELISA. Protein expression of mitogen-activated protein kinases (MAPKs) (extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs) and P38 kinases (P38Ks)) was detected using immunoblot analysis. Student's t-test and ANOVA were applied to determine statistical significance. RESULTS: In gp120ADA-injected rats, mRNA transcripts of interleukin-1ß (IL-1ß) and inducible nitric oxide synthase (iNOS) were significantly elevated in the frontal cortex, striatum and hippocampus compared to saline or heat-inactivated gp120-injected controls. In CSF, a significant increase in TNF-α and IL-1ß was detected. Maraviroc reduced upregulation of these markers suggesting that the interaction of R5-tropic gp120 to CCR5 chemokine receptor is critical for induction of an inflammatory response. Minocycline, chloroquine or simvastatin attenuated upregulation of IL-1ß and iNOS transcripts in different brain regions. In CSF, minocycline suppressed TNF-α and IL-1ß secretion, whereas chloroquine attenuated IL-1ß secretion. In gp120-injected animals, activation of ERK1/2 and JNKs was observed in the hippocampus and ERK1/2 activation was significantly reduced by the anti-inflammatory agents. CONCLUSIONS: Our data demonstrate that anti-inflammatory compounds can completely or partially reverse gp120-associated brain inflammation through an interaction with MAPK signaling pathways and suggest their potential role in contributing towards the prevention and treatment of HIV-associated neurological complications.

A reliable method for intracranial electrode implantation and chronic electrical stimulation in the mouse brain.

BACKGROUND: Electrical stimulation of brain structures has been widely used in rodent models for kindling or modeling deep brain stimulation used clinically. This requires surgical implantation of intracranial electrodes and subsequent chronic stimulation in individual animals for several weeks. Anchoring screws and dental acrylic have long been used to secure implanted intracranial electrodes in rats. However, such an approach is limited when carried out in mouse models as the thin mouse skull may not be strong enough to accommodate the anchoring screws. We describe here a screw-free, glue-based method for implanting bipolar stimulating electrodes in the mouse brain and validate this method in a mouse model of hippocampal electrical kindling. METHODS: Male C57 black mice (initial ages of 6-8 months) were used in the present experiments. Bipolar electrodes were implanted bilaterally in the hippocampal CA3 area for electrical stimulation and electroencephalographic recordings. The electrodes were secured onto the skull via glue and dental acrylic but without anchoring screws. A daily stimulation protocol was used to induce electrographic discharges and motor seizures. The locations of implanted electrodes were verified by hippocampal electrographic activities and later histological assessments. RESULTS: Using the glue-based implantation method, we implanted bilateral bipolar electrodes in 25 mice. Electrographic discharges and motor seizures were successfully induced via hippocampal electrical kindling. Importantly, no animal encountered infection in the implanted area or a loss of implanted electrodes after 4-6 months of repetitive stimulation/recording. CONCLUSION: We suggest that the glue-based, screw-free method is reliable for chronic brain stimulation and high-quality electroencephalographic recordings in mice. The technical aspects described this study may help future studies in mouse models.

Acute postischemic seizures are associated with increased mortality and brain damage in adult mice.

Postischemic seizures are associated with worsened outcome following stroke, but the underlying pathophysiology is poorly understood. Here we examined acute seizures in adult mice following hypoxia-ischemia (HI) via combined behavioral, electrophysiological, and histological assessments. C57BL/6 mice aged 4-9 months received a permanent occlusion of the right common carotid artery and then underwent a systemic hypoxic episode. Generalized motor seizures were observed within 72 h following HI. These seizures occurred nearly exclusively in animals with extensive brain injury in the hemisphere ipsilateral to the carotid occlusion, but their generation was not associated with electroencephalographic discharges in bilateral hippocampal and neocortical recordings. Animals exhibiting these seizures had a high rate of mortality, and post-HI treatments with diazepam and phenytoin greatly suppressed these behavioral seizures and improved post-HI animal survival. Based on these data, we conclude that these seizures are a consequence of HI brain injury, contribute to worsened outcome following HI, and that they originate from deep subcortical structures.

Electrographic Features of Spontaneous Recurrent Seizures in a Mouse Model of Extended Hippocampal Kindling.

Epilepsy is a chronic neurological disorder characterized by spontaneous recurrent seizures (SRS) and comorbidities. Kindling through repetitive brief stimulation of a limbic structure is a commonly used model of temporal lobe epilepsy. Particularly, extended kindling over a period up to a few months can induce SRS, which may simulate slowly evolving epileptogenesis of temporal lobe epilepsy. Currently, electroencephalographic (EEG) features of SRS in rodent models of extended kindling remain to be detailed. We explored this using a mouse model of extended hippocampal kindling. Intracranial EEG recordings were made from the kindled hippocampus and unstimulated hippocampal, neocortical, piriform, entorhinal, or thalamic area in individual mice. Spontaneous EEG discharges with concurrent low-voltage fast onsets were observed from the two corresponding areas in nearly all SRS detected, irrespective of associated motor seizures. Examined in brain slices, epileptiform discharges were induced by alkaline artificial cerebrospinal fluid in the hippocampal CA3, piriform and entorhinal cortical areas of extended kindled mice but not control mice. Together, these in vivo and in vitro observations suggest that the epileptic activity involving a macroscopic network may generate concurrent discharges in forebrain areas and initiate SRS in hippocampally kindled mice.

Hippocampal seizures alter the expression of the pannexin and connexin transcriptome.

Some forms of seizure activity can be stopped by gap junctional (GJ) blockade. Here, we found that GJ blockers attenuate hippocampal seizure activity induced by a novel seizuregenic protocol using Co(2+). We hypothesized that this activity may occur because of the altered expression of connexin (Cx) and/or pannexin (Panx) mRNAs and protein. We found a 1.5-, 1.4-, and 2-fold increase in Panx1, Panx2, and Cx43 mRNAs, respectively. Significant post-translational modifications of the proteins Cx43 and Panx1 were also observed after the Co(2+) treatment. No changes were observed in the presence of tetrodotoxin, indicating that seizure activity is required for these alterations in expression, rather than the Co(2+) treatment itself. Further analysis of the QPCR data showed that the Cx and Panx transcriptome becomes remarkably re-organized. Pannexin (Panxs 1 and 2) and glial connexin mRNA became highly correlated to one another; suggesting that these genes formed a transcriptomic network of coordinated gene expression, perhaps facilitating seizure induction. These data show that seizure activity up-regulates the expression of both glial and neuronal GJ mRNAs and protein while inducing a high degree of coordinate expression of the GJ transcriptome.

Alterations of cortical and hippocampal EEG activity in MeCP2-deficient mice.

Rett syndrome is a pediatric neurological condition caused by mutations of the gene encoding the transcriptional regulator MECP2. In this study, we examined cortical and hippocampal electroencephalographic (EEG) activity in male and female MeCP2-deficient mice at symptomatic stages during different behavioral states. During acute sleep, MeCP2-deficient mice displayed normal delta-like activity in cortex and sharp-wave activity in hippocampus. However, when the mice were awake but immobile, abnormal spontaneous, rhythmic EEG discharges of 6-9 Hz were readily detected in the somatosensory cortex. During exploratory activity, MeCP2-deficient mice displayed clear theta rhythm activity in hippocampus, but its peak frequency was significantly attenuated compared to wild type. Collectively, these findings indicate that a deficiency in MeCP2 function in mice leads to alterations in EEG activity with similarities to what has been observed clinically in Rett syndrome patients.

Adenosine as an endogenous regulating factor of hippocampal sharp waves.

The rodent hippocampus exhibits population activities called sharp waves (SPWs) during slow wave sleep and wake immobility. SPWs are important for hippocampal-cortical communication and memory consolidation, and abnormal sharp wave-ripple complexes are closely related to epileptic seizures. Although the SPWs are known to arise from the CA3 circuit, the local mechanisms underlying their generation are not fully understood. We hypothesize that endogenous adenosine is a local regulator of hippocampal SPWs. We tested this hypothesis in thick mouse hippocampal slices that encompass a relatively large hippocampal circuit and have a high propensity of generating spontaneous in vitro SPWs. We found that application of adenosine A1 receptor antagonists induced in vitro SPWs and that such induction was sensitive to blockade by NMDA receptor antagonists. By contrast, an increase in endogenous adenosine via pharmacological inhibition of adenosine transporters or adenosine degrading enzymes suppressed spontaneous in vitro SPWs. We thus suggest that the initiation and incidence of sharp wave-like population events are under tight control by the activity of endogenously stimulated A1 receptors.

Cellular mechanisms of cobalt-induced hippocampal epileptiform discharges.

PURPOSE: To explore the cellular mechanisms of cobalt-induced epileptiform discharges in mouse hippocampal slices. METHODS: Hippocampal slices were prepared from adult mice and briefly exposed to a CoCl(2)-containing external solution. Population and single cell activities were examined via extracellular and whole-cell patch recordings. RESULTS: Brief cobalt exposure induced spontaneous, ictal-like discharges originating from the CA3 area. These discharges were suppressed by anticonvulsants, gap junction blockers, or by raising extracellular Ca(2+), but their generation was not associated with overall hyperexcitability or impairment in GABAergic inhibition in the CA3 circuit. Electroencephalographic ictal discharges of similar waveforms were observed in behaving rats following intrahippocampal cobalt infusion. DISCUSSION: Mechanisms involving activity-dependent facilitation of gap junctional communication may play a major role in cobalt-induced epileptiform discharges.

Increased seizure susceptibility in a mouse model of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder linked to higher rates of epilepsy as compared with the general population. Although some epilepsy cases in NF1 are related to intracranial lesions, epileptogenic lesions are not always identified. It is unknown whether the genetic mutation itself, which leads to lower levels of the tumor suppressor protein neurofibromin, alters seizure susceptibility. The purpose of this research was to determine whether Nf1+/- mice have altered seizure susceptibility to the chemical convulsants kainic acid and pilocarpine. Young adult Nf1+/- or WT control (Nf1+/+) mice were injected with either 20 mg/kg kainic acid or scopolamine 1 mg/kg and pilocarpine 300 mg/kg and assessed for various behavioral seizure parameters. Another subset of mice were implanted with intracranial electrodes and injected with 10 mg/kg kainic acid for electrographic seizure testing. Histological analyses were performed one week after kainic acid challenge to assess hippocampal damage. A higher proportion of Nf1+/- mice had behavioral seizures after kainic acid or pilocarpine challenge, with shorter seizure latency, longer seizure duration, and higher Racine scores compared to WT mice. Nf1+/- and WT mice with severe behavioral seizures demonstrated similar levels of hippocampal damage. EEG recordings confirmed decreased seizure latency and longer seizure duration in response to KA in the Nf1+/- group. These data demonstrate increased seizure susceptibility in a mouse model of NF1 and support the use of the Nf1+/- mouse for further investigations into the mechanistic link between NF1 and seizures.

Impaired Spatial Learning and Memory in Middle-Aged Mice with Kindling-Induced Spontaneous Recurrent Seizures.

Temporal lobe epilepsy is the most common and often drug-resistant type of epilepsy in the adult and aging populations and has great diversity in etiology, electro-clinical manifestations, and comorbidities. Kindling through repeated brief stimulation of limbic structures is a commonly used model of temporal lobe epilepsy. Particularly, extended kindling can induce spontaneous recurrent seizures in several animal species. However, kindling studies in middle-aged, aging, or aged animals remain scarce, and currently, little is known about kindling-induced behavioral changes in middle-aged/aging animals. We therefore attempted to provide more information in this area using a mouse model of extended hippocampal kindling. We conducted experiments in middle-aged mice (C57BL/6, male, 12-14 months of age) to model new-onset epilepsy in adult/aging populations. Mice experienced twice daily hippocampal stimulations or handling manipulations for 60-70 days and then underwent continuous electroencephalogram (EEG)-video monitoring to detect spontaneous recurrent seizures. Extended kindled mice consistently exhibited spontaneous recurrent seizures with mean incidences of 6-7 events per day, and these seizures featured EEG discharges and corresponding convulsions. The handling control mice showed neither seizure nor aberrant EEG activity. The two groups of mice underwent the Morris water maze test of spatial learning and memory 1-2 weeks after termination of the kindling stimulation or handling manipulation. During visible platform trials, the kindled mice took a longer distance and required more time than the control mice to find the platform. During hidden platform trials, the kindled mice showed no improvement over 5-day trials in finding the platform whereas the control mice improved significantly. During probe tests in which the hidden platform was removed, the kindled mice spent less time than the controls searching in the correct platform location. There were no significant differences between the kindled and control mice with respect to swim speed or total locomotor activity in an open-field test. Together, these observations indicate that the extended kindled mice with spontaneous recurrent seizures are impaired in spatial learning and memory as assessed by the Morris water maze test. We postulate that the extended hippocampal kindling in middle-aged mice may help explore epileptogenic mechanisms and comorbidities potentially relevant to new-onset temporal lobe epilepsy in adult and aging patients. Limitations and confounds of our present experiments are discussed to improve future examinations of epileptic comorbidities in extended kindled mice.

A glue-based, screw-free method for implantation of intra-cranial electrodes in young mice.

Intra-cranial electroencephalographic recordings are increasingly employed in mice because of the availability of genetically manipulated mouse models. Currently, dental acrylic and anchoring screws are used to cement implanted electrodes. This technique works well for adult animals but often encounters difficulty when employed in young mice because their skulls are not strong enough to bear the anchoring screws. Here we describe a novel method favorable for implantation of intra-cranial electrodes in mice as young as postnatal 18 days and suitable for long-term intra-cranial electroencephalographic recordings. Our approach is to construct a multi-electrode assembly according to the desired stereotaxic coordinates of intra-cranial recordings and to secure the implanted electrode assembly to the skull via glue rather than dental acrylic/anchoring screws. The surgical operation for such electrode implantation is relatively quick and rarely associated with complications such as infection, bleeding, neurological deficits, spontaneous seizures or behavioral disturbances. The implanted electrodes are stable, allowing repeated monitoring for several months. Data obtained by simultaneous intra-hippocampal and intra-cortical recordings indicate that our method is suitable for the examination of behaviorally related electroencephalographic activities and experimentally induced seizures. Technical aspects of our methods are discussed, and the procedures for constructing the electrode assembly are presented in detail.

Contributions of the Hippocampal CA3 Circuitry to Acute Seizures and Hyperexcitability Responses in Mouse Models of Brain Ischemia.

The hippocampal circuitry is widely recognized as susceptible to ischemic injury and seizure generation. However, hippocampal contribution to acute non-convulsive seizures (NCS) in models involving middle cerebral artery occlusion (MCAO) remains to be determined. To address this, we occluded the middle cerebral artery in adult C57 black mice and monitored electroencephalographic (EEG) discharges from hippocampal and neocortical areas. Electrographic discharges in the absence of convulsive motor behaviors were observed within 90 min following occlusion of the middle cerebral artery. Hippocampal discharges were more robust than corresponding cortical discharges in all seizure events examined, and hippocampal discharges alone or with minimal cortical involvement were also observed in some seizure events. Seizure development was associated with ipsilateral hippocampal injuries as determined by subsequent histological examinations. We also introduced hypoxia-hypoglycemia episodes in mouse brain slices and examined regional hyperexcitable responses ex vivo. Extracellular recordings showed that the hippocampal CA3 region had a greater propensity for exhibiting single/multiunit activities or epileptiform field potentials following hypoxic-hypoglycemic (HH) episodes compared to the CA1, dentate gyrus, entorhinal cortical (EC) or neocortical regions. Whole-cell recordings revealed that CA3 pyramidal neurons exhibited excessive excitatory postsynaptic currents, attenuated inhibitory postsynaptic currents and intermittent or repetitive spikes in response to HH challenge. Together, these observations suggest that hippocampal discharges, possibly as a result of CA3 circuitry hyperexcitability, are a major component of acute NCS in a mouse model of MCAO.

Effects of Antiepileptic Drugs on Spontaneous Recurrent Seizures in a Novel Model of Extended Hippocampal Kindling in Mice.

Epilepsy is a common neurological disorder characterized by naturally-occurring spontaneous recurrent seizures and comorbidities. Kindling has long been used to model epileptogenic mechanisms and to assess antiepileptic drugs. In particular, extended kindling can induce spontaneous recurrent seizures without gross brain lesions, as seen clinically. To date, the development of spontaneous recurrent seizures following extended kindling, and the effect of the antiepileptic drugs on these seizures are not well understood. In the present study we aim to develop a mouse model of extended hippocampal kindling for the first time. Once established, we plan to evaluate the effect of three different antiepileptic drugs on the development of the extended-hippocampal-kindled-induced spontaneous recurrent seizures. Male C57 black mice were used for chronic hippocampal stimulations or handling manipulations (twice daily for up to 70 days). Subsequently, animals underwent continuous video/EEG monitoring for seizure detection. Spontaneous recurrent seizures were consistently observed in extended kindled mice but no seizures were detected in the control animals. The aforementioned seizures were generalized events characterized by hippocampal ictal discharges and concurrent motor seizures. Incidence and severity of the seizures was relatively stable while monitored over a few months after termination of the hippocampal stimulation. Three antiepileptic drugs with distinct action mechanisms were tested: phenytoin, lorazepam and levetiracetam. They were applied via intra-peritoneal injections at anticonvulsive doses and their effects on the spontaneous recurrent seizures were analyzed 10-12 h post-injection. Phenytoin (25 mg/kg) and levetiracetam (400 mg/kg) abolished the spontaneous recurrent seizures. Lorazepam (1.5 mg/kg) decreased motor seizure severity but did not reduce the incidence and duration of corresponding hippocampal discharges, implicating its inhibitory effects on seizure spread. No gross brain lesions were observed in a set of extended hippocampal kindled mice submitted to histological evaluation. All these data suggests that our model could be considered as a novel mouse model of extended hippocampal kindling. Some limitations remain to be considered.

Continuous Monitoring via Tethered Electroencephalography of Spontaneous Recurrent Seizures in Mice.

We describe here a simple, cost-effective apparatus for continuous tethered electroencephalographic (EEG) monitoring of spontaneous recurrent seizures in mice. We used a small, low torque slip ring as an EEG commutator, mounted the slip ring onto a standard mouse cage and connected rotary wires of the slip ring directly to animal's implanted headset. Modifications were made in the cage to allow for a convenient installation of the slip ring and accommodation of animal ambient activity. We tested the apparatus for hippocampal EEG recordings in adult C57 black mice. Spontaneous recurrent seizures were induced using extended hippocampal kindling (≥95 daily stimulation). Control animals underwent similar hippocampal electrode implantations but no stimulations were given. Combined EEG and webcam monitoring were performed for 24 h daily for 5-9 consecutive days. During the monitoring periods, the animals moved and accessed water and food freely and showed no apparent restriction in ambient cage activities. Ictal-like hippocampal EEG discharges and concurrent convulsive behaviors that are characteristics of spontaneous recurrent seizures were reliably recorded in a majority of the monitoring experiments in extendedly kindled but not in control animals. However, 1-2 rotary wires were disconnected from the implanted headset in some animals after continuous recordings for ≥5 days. The key features and main limitations of our recording apparatus are discussed.

Susceptibility to hippocampal kindling seizures is increased in aging C57 black mice.

The incidence of seizures increases with old age. Stroke, dementia and brain tumors are recognized risk factors for new-onset seizures in the aging populations and the incidence of these conditions also increased with age. Whether aging is associated with higher seizure susceptibility in the absence of the above pathologies remains unclear. We used classic kindling to explore this issue as the kindling model is highly reproducible and allows close monitoring of electrographic and motor seizure activities in individual animals. We kindled male young and aging mice (C57BL/6 strain, 2-3 and 18-22 months of age) via daily hippocampal CA3 stimulation and monitored seizure activity via video and electroencephalographic recordings. The aging mice needed fewer stimuli to evoke stage-5 motor seizures and exhibited longer hippocampal afterdischarges and more frequent hippocampal spikes relative to the young mice, but afterdischarge thresholds and cumulative afterdischarge durations to stage 5 motor seizures were not different between the two age groups. While hippocampal injury and structural alterations at cellular and micro-circuitry levels remain to be examined in the kindled mice, our present observations suggest that susceptibility to hippocampal CA3 kindling seizures is increased with aging in male C57 black mice.

Regulation of hippocampal sharp waves by Ca2+-dependent slow after hyperpolarization.

In rodent hippocampal pyramidal neurons, repetitive discharges are followed by a slow afterhyperpolarization (sAHP) as a result of activation of a Ca2+-dependent K+ current. The sAHP is sensitive to activation of several G-protein coupled neurotransmitter receptors and downstream signal cascades. Modulations of the sAHP have been shown to be closely associated with synaptic plasticity, learning, and aging processes. However, it is presently unclear whether the sAHP generation is involved in hippocampal network activities. We explored this issue using an in vitro (thick-slice) model of mouse hippocampal sharp waves. Our data show that the sAHP occurs in CA3 pyramidal neurons following each sharp wave event and sAHP suppression is associated with a large increase in occurrence frequency of spontaneous sharp waves. Considering that sharp waves are important for hippocampal-cortical communication and memory processes, we postulate that the sAHP serves as an intrinsic regulatory mechanism of sharp waves and plays a significant role in hippocampus-dependent cognitive functions.

The calcium-sensing receptor and integrins modulate cerebellar granule cell precursor differentiation and migration.

In the developing cerebellum granule cell precursors (GCPs) proliferate in the external granule cell layer before differentiating and migrating to the inner granule cell layer. Aberrant GCP proliferation leads to medulloblastoma, the most prevalent form of childhood brain cancer. Here, we demonstrate that the calcium-sensing receptor (CaSR), a homodimeric G-protein coupled receptor, functions in conjunction with cell adhesion proteins, the integrins, to enhance GCP migration and cell homing by promoting GCP differentiation. During the second postnatal week a robust peak in CaSR expression was observed in GCPs; reciprocal immunoprecipitation experiments conducted during this period established that the CaSR and ß1 integrins are present together in a macromolecular protein complex. Analysis of cell-surface proteins demonstrated that activation of the CaSR by positive allosteric modulators promoted plasma membrane expression of ß1 integrins via ERK2 and AKT phosphorylation and resulted in increased GCP migration toward an extracellular matrix protein. The results of in vivo experiments whereby CaSR modulators were injected i.c.v. revealed that CaSR activation promoted radial migration of GCPs by enhancing GCP differentiation, and conversely, a CaSR inhibitor disrupted GCP differentiation and promoted GCP proliferation. Our results demonstrate that an ion-sensing G-protein coupled receptor acts to promote neuronal differentiation and homing during cerebellar maturation. These findings together with those of others also suggest that CaSR/integrin complexes act to transduce extracellular calcium signals into cellular movement, and may function in this capacity as a universal cell migration/homing complex in the developing brain.

A Role for Diminished GABA Transporter Activity in the Cortical Discharge Phenotype of MeCP2-Deficient Mice.

Cortical network hyper-excitability is a common phenotype in mouse models lacking the transcriptional regulator methyl-CPG-binding protein 2 (MeCP2). Here, we implicate enhanced GABAB receptor activity stemming from diminished cortical expression of the GABA transporter GAT-1 in the genesis of this network hyper-excitability. We found that administering the activity-dependent GABAB receptor allosteric modulator GS-39783 to female Mecp2(+/-) mice at doses producing no effect in wild-type mice strongly potentiated their basal rates of spontaneous cortical discharge activity. Consistently, administering the GABAB receptor antagonist CGP-35348 significantly decreased basal discharge activity in these mice. Expression analysis revealed that while GABAB or extra-synaptic GABAA receptor prevalence is preserved in the MeCP2-deficient cortex, the expression of GAT-1 is significantly reduced from wild-type levels. This decrease in GAT-1 expression is consequential, as low doses of the GAT-1 inhibitor NO-711 that had no effects in wild-type mice strongly exacerbated cortical discharge activity in female Mecp2(+/-) mice. Taken together, these data indicate that the absence of MeCP2 leads to decreased cortical levels of the GAT-1 GABA transporter, which facilitates cortical network hyper-excitability in MeCP2-deficient mice by increasing the activity of cortical GABAB receptors.