GABAAR Modulator for Postpartum Depression.

During the postpartum period, the brain's inhibitory GABAA receptors may not recover in time following their reduced numbers during pregnancy. This is likely the cause of postpartum depression prevalent in ∼12% of childbearing women. A new therapy for this condition consists of administering a synthetic neurosteroid during the postpartum period to alleviate the mood disorder. To view this Bench to Bedside, open or download the PDF.

Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model.

Alzheimer's disease (AD) results in cognitive decline and altered network activity, but the mechanisms are unknown. We studied human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Electroencephalographic recordings in hAPP mice revealed spontaneous epileptiform discharges, indicating network hypersynchrony, primarily during reduced gamma oscillatory activity. Because this oscillatory rhythm is generated by inhibitory parvalbumin (PV) cells, network dysfunction in hAPP mice might arise from impaired PV cells. Supporting this hypothesis, hAPP mice and AD patients had decreased levels of the interneuron-specific and PV cell-predominant voltage-gated sodium channel subunit Nav1.1. Restoring Nav1.1 levels in hAPP mice by Nav1.1-BAC expression increased inhibitory synaptic activity and gamma oscillations and reduced hypersynchrony, memory deficits, and premature mortality. We conclude that reduced Nav1.1 levels and PV cell dysfunction critically contribute to abnormalities in oscillatory rhythms, network synchrony, and memory in hAPP mice and possibly in AD.

Motor Activity-Induced White Matter Repair in White Matter Stroke.

Subcortical white matter stroke (WMS) is a progressive disorder which is demarcated by the formation of small ischemic lesions along white matter tracts in the CNS. As lesions accumulate, patients begin to experience severe motor and cognitive decline. Despite its high rate of incidence in the human population, our understanding of the cause and outcome of WMS is extremely limited. As such, viable therapies for WMS remain to be seen. This study characterizes myelin recovery following stroke and motor learning-based rehabilitation in a mouse model of subcortical WMS. Following WMS, a transient increase in differentiating oligodendrocytes occurs within the peri-infarct in young male adult mice, which is completely abolished in male aged mice. Compound action potential recording demonstrates a decrease in conduction velocity of myelinated axons at the peri-infarct. Animals were then tested on one of three distinct motor learning-based rehabilitation strategies (skilled reach, restricted access to a complex running wheel, and unrestricted access to a complex running wheel) for their capacity to induce repair. These studies determined that unrestricted access to a complex running wheel alone increases the density of differentiating oligodendrocytes in infarcted white matter in young adult male mice, which is abolished in aged male mice. Unrestricted access to a complex running wheel was also able to enhance conduction velocity of myelinated axons at the peri-infarct to a speed comparable to naive controls suggesting functional recovery. However, there was no evidence of motor rehabilitation-induced remyelination or myelin protection.SIGNIFICANCE STATEMENT White matter stroke is a common disease with no medical therapy. A form of motor rehabilitation improves some aspects of white matter repair and recovery.

A dark quencher genetically encodable voltage indicator (dqGEVI) exhibits high fidelity and speed.

Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol-enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.

Neurofibromin regulation of ERK signaling modulates GABA release and learning.

We uncovered a role for ERK signaling in GABA release, long-term potentiation (LTP), and learning, and show that disruption of this mechanism accounts for the learning deficits in a mouse model for learning disabilities in neurofibromatosis type I (NF1). Our results demonstrate that neurofibromin modulates ERK/synapsin I-dependent GABA release, which in turn modulates hippocampal LTP and learning. An Nf1 heterozygous null mutation, which results in enhanced ERK and synapsin I phosphorylation, increased GABA release in the hippocampus, and this was reversed by pharmacological downregulation of ERK signaling. Importantly, the learning deficits associated with the Nf1 mutation were rescued by a subthreshold dose of a GABA(A) antagonist. Accordingly, Cre deletions of Nf1 showed that only those deletions involving inhibitory neurons caused hippocampal inhibition, LTP, and learning abnormalities. Importantly, our results also revealed lasting increases in GABA release triggered by learning, indicating that the mechanisms uncovered here are of general importance for learning.

Mossy Cells in the Dorsal and Ventral Dentate Gyrus Differ in Their Patterns of Axonal Projections.

Mossy cells (MCs) of the dentate gyrus (DG) are a major group of excitatory hilar neurons that are important for regulating activity of dentate granule cells. MCs are particularly intriguing because of their extensive longitudinal connections within the DG. It has generally been assumed that MCs in the dorsal and ventral DG have similar patterns of termination in the inner one-third of the dentate molecular layer. Here, we demonstrate that axonal projections of MCs in these two regions are considerably different. MCs in dorsal and ventral regions were labeled selectively with Cre-dependent eYFP or mCherry, using two transgenic mouse lines (including both sexes) that express Cre-recombinase in MCs. At four to six weeks following unilateral labeling of MCs in the ventral DG, a dense band of fibers was present in the inner one-fourth of the molecular layer and extended bilaterally throughout the rostral-caudal extent of the DG, replicating the expected distribution of MC axons. In contrast, following labeling of MCs in the dorsal DG, the projections were more diffusely distributed. At the level of transfection, fibers were present in the inner molecular layer, but they progressively expanded into the middle molecular layer and, most ventrally, formed a distinct band in this region. Optical stimulation of these caudal fibers expressing ChR2 demonstrated robust EPSCs in ipsilateral granule cells and enhanced the effects of perforant path stimulation in the ventral DG. These findings suggest that MCs in the dorsal and ventral DG differ in the distribution of their axonal projections and possibly their function.SIGNIFICANCE STATEMENT Mossy cells (MCs), a major cell type in the hilus of the dentate gyrus (DG), are unique in providing extensive longitudinal and commissural projections throughout the DG. Although it has been assumed that all MCs have similar patterns of termination in the inner molecular layer of the DG, we discovered that the axonal projections of dorsal and ventral MCs differ. While ventral MC projections exhibit the classical pattern, with dense innervation in the inner molecular layer, dorsal MCs have a more diffuse distribution and expand into the middle molecular layer where they overlap and interact with innervation from the perforant path. These distinct locations and patterns of axonal projections suggest that dorsal and ventral MCs may have different functional roles.

The Rapid Effect of Bisphenol-A on Long-Term Potentiation in Hippocampus Involves Estrogen Receptors and ERK Activation.

Bisphenol-A (BPA), a widely used synthetic compound in plastics, disrupts endocrine function and interferes with physiological actions of endogenous gonadal hormones. Chronic effects of BPA on reproductive function, learning and memory, brain structure, and social behavior have been intensively investigated. However, less is known about the influence of BPA on long-term potentiation (LTP), one of the major cellular mechanisms that underlie learning and memory. In the present study, for the first time we investigated the effect of different doses of BPA on hippocampal LTP in rat brain slices. We found a biphasic effect of BPA on LTP in the dentate gyrus: exposure to BPA at a low dose (100 nM) enhanced LTP and exposure to BPA at a high dose (1000 nM) inhibited LTP compared with vehicle controls. The rapid facilitatory effect of low-dose BPA on hippocampal LTP required membrane-associated estrogen receptor (ER) and involved activation of the extracellular signal-regulated kinase (ERK) signaling pathway. Coadministration of 17ß-estradiol (E2, the primary estrogen hormone) and BPA (100 nM) abolished both the BPA-induced enhancement of LTP and the E2-induced enhancement of baseline fEPSP, suggesting a complex interaction between BPA- and E2-mediated signaling pathways. Our investigation implies that even nanomolar levels of endocrine disrupters (e.g., BPA) can induce significant effects on hippocampal LTP.

Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke.

Stroke is a leading cause of disability, but no pharmacological therapy is currently available for promoting recovery. The brain region adjacent to stroke damage-the peri-infarct zone-is critical for rehabilitation, as it shows heightened neuroplasticity, allowing sensorimotor functions to re-map from damaged areas. Thus, understanding the neuronal properties constraining this plasticity is important for the development of new treatments. Here we show that after a stroke in mice, tonic neuronal inhibition is increased in the peri-infarct zone. This increased tonic inhibition is mediated by extrasynaptic GABA(A) receptors and is caused by an impairment in GABA (γ-aminobutyric acid) transporter (GAT-3/GAT-4) function. To counteract the heightened inhibition, we administered in vivo a benzodiazepine inverse agonist specific for α5-subunit-containing extrasynaptic GABA(A) receptors at a delay after stroke. This treatment produced an early and sustained recovery of motor function. Genetically lowering the number of α5- or δ-subunit-containing GABA(A) receptors responsible for tonic inhibition also proved beneficial for recovery after stroke, consistent with the therapeutic potential of diminishing extrasynaptic GABA(A) receptor function. Together, our results identify new pharmacological targets and provide the rationale for a novel strategy to promote recovery after stroke and possibly other brain injuries.

Behavioral Deficits in Juveniles Mediated by Maternal Stress Hormones in Mice.

Maternal depression has been shown to negatively impact offspring development. Investigation into the impact of maternal depression and offspring behavior has relied on correlative studies in humans. Further investigation into the underlying mechanisms has been hindered by the lack of useful animal models. We previously characterized a mouse model which exhibits depression-like behaviors restricted to the postpartum period and abnormal/fragmented maternal care (Gabrd (-/-) mice). Here we utilized this unique mouse model to investigate the mechanism(s) through which maternal depression-like behaviors adversely impact offspring development. Cross-fostering experiments reveal increased anxiety-like and depression-like behaviors in mice reared by Gabrd (-/-) mothers. Wild type and Gabrd (-/-) mice subjected to unpredictable stress during late pregnancy exhibit decreased pup survival and depression-like behavior in the postpartum period. Exogenous corticosterone treatment in wild type mice during late pregnancy is sufficient to decrease pup survival and induce anxiety-like and depression-like behaviors in the offspring. Further, the abnormal behaviors in juvenile mice reared by Gabrd (-/-) mice are alleviated by treatment of the mothers with the corticotropin-releasing hormone (CRH) antagonist, Antalarmin. These studies suggest that hyperresponsiveness of the HPA axis is associated with postpartum depression and may mediate the adverse effects of maternal depression on offspring behavior.

Intracellular bicarbonate regulates action potential generation via KCNQ channel modulation.

Bicarbonate (HCO3(-)) is an abundant anion that regulates extracellular and intracellular pH. Here, we use patch-clamp techniques to assess regulation of hippocampal CA3 pyramidal cell excitability by HCO3(-) in acute brain slices from C57BL/6 mice. We found that increasing HCO3(-) levels enhances action potential (AP) generation in both the soma and axon initial segment (AIS) by reducing Kv7/KCNQ channel activity, independent of pH (i.e., at a constant pH of 7.3). Conversely, decreasing intracellular HCO3(-) leads to attenuation of AP firing. We show that HCO3(-) interferes with Kv7/KCNQ channel activation by phosphatidylinositol-4,5-biphosphate. Consequently, we propose that, even in the presence of a local depolarizing Cl(-) gradient, HCO3(-) efflux through GABAA receptors may ensure the inhibitory effect of axoaxonic cells at the AIS due to activation of Kv7/KCNQ channels.

Evolution of temporal and spectral dynamics of pathologic high-frequency oscillations (pHFOs) during epileptogenesis.

OBJECTIVE: In temporal lobe epilepsy (TLE), pathologic high frequency oscillations (pHFOs, 200-600 Hz) are present in the hippocampus, especially the dentate gyrus (DG). The pHFOs emerge during a latent period prior to the onset of spontaneous generalized seizures. We used a unilateral suprahippocampal injection of kainic acid (KA) mouse model of TLE to characterize the properties of hippocampal pHFOs during epileptogenesis. METHODS: In awake head-fixed mice, 4-14 days after KA-induced status epilepticus (SE), we recorded local field potentials (LFPs) with 64-channel silicon probes spanning from CA1 alveus to the DG hilus, or with glass pipettes in the DC mode in the CA1 str radiatum. RESULTS: The pHFOs, are observed simultaneously in the CA1 and the DG, or in the DG alone, as early as 4 days post-SE. The pHFOs ride on top of DC deflections, occur during motionless periods, persist through the onset of TLE, and are generated in bursts. Burst parameters remain remarkably constant during epileptogenesis, with a random number of pHFOs generated per burst. In contrast, pHFO duration and spectral dynamics evolve from short events at 4 days post-SE to prolonged discharges with complex spectral characteristics by 14 days post-SE. Simultaneous dural EEG recordings were exceedingly unreliable for detecting hippocampal pHFOs; therefore, such recordings may deceptively indicate a "silent" period even when massive hippocampal activity is present. SIGNIFICANCE: Our results demonstrate that hippocampal pHFOs exhibit a dynamic evolution during the epileptogenic period following SE, consistent with their role in transitioning to the chronic stage of TLE.

WONOEP appraisal: molecular and cellular imaging in epilepsy.

Great advancements have been made in understanding the basic mechanisms of ictogenesis using single-cell electrophysiology (e.g., patch clamp, sharp electrode), large-scale electrophysiology (e.g., electroencephalography [EEG], field potential recording), and large-scale imaging (magnetic resonance imaging [MRI], positron emission tomography [PET], calcium imaging of acetoxymethyl ester [AM] dye-loaded tissue). Until recently, it has been challenging to study experimentally how population rhythms emerge from cellular activity. Newly developed optical imaging technologies hold promise for bridging this gap by making it possible to simultaneously record the many cellular elements that comprise a neural circuit. Furthermore, easily accessible genetic technologies for targeting expression of fluorescent protein-based indicators make it possible to study, in animal models of epilepsy, epileptogenic changes to neural circuits over long periods. In this review, we summarize some of the latest imaging tools (fluorescent probes, gene delivery methods, and microscopy techniques) that can lead to the advancement of cell- and circuit-level understanding of epilepsy, which in turn may inform and improve development of next generation antiepileptic and antiepileptogenic drugs.

Microglia contribute to neuronal synchrony despite endogenous ATP-related phenotypic transformation in acute mouse brain slices.

Acute brain slices represent a workhorse model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue damage, it is unclear how microglia, the main immune cells and damage sensors of the CNS react to this injury and shape neuronal activity ex vivo. To this end, we investigated microglial phenotypes and contribution to network organization and functioning in acute brain slices. We reveal time-dependent microglial phenotype changes influenced by complex extracellular ATP dynamics through P2Y12R and CX3CR1 signalling, which is sustained for hours in ex vivo mouse brain slices. Downregulation of P2Y12R and changes of microglia-neuron interactions occur in line with alterations in the number of excitatory and inhibitory synapses over time. Importantly, functional microglia modulate synapse sprouting, while microglial dysfunction results in markedly impaired ripple activity both ex vivo and in vivo. Collectively, our data suggest that microglia are modulators of complex neuronal networks with important roles to maintain neuronal network integrity and activity. We suggest that slice preparation can be used to model time-dependent changes of microglia-neuron interactions to reveal how microglia shape neuronal circuits in physiological and pathological conditions.

Measuring the kinetics of calcium binding proteins with flash photolysis.

BACKGROUND: Calcium-binding proteins (CBPs) are instrumental in the control of Ca2+ signaling. They are the fastest players within the Ca2+ toolkit responding within microseconds to [Ca2+] changes. The CBPs compete for Ca2+ which plays a direct role in modulating Ca2+ transients and the resulting biochemical message. The kinetic properties of the CBPs have to be known to have a good understanding of Ca2+ signaling. SCOPE OF REVIEW: Most techniques used to measure binding kinetics are too slow to accurately determine the fast kinetics of most CBP. Furthermore, many CBPs bind Ca2+ in a cooperative way, which should be incorporated in the kinetic modeling. Here we will review a new ultra-fast in vitro technique for measuring Ca2+ binding properties of CBPs following flash photolysis of caged Ca2+. Compartmental modeling is used to resolve the kinetics of fast cooperative Ca2+ binding to CBPs. MAJOR CONCLUSIONS: Currently this technique has only been used to quantify the kinetics of three CBPs (calbindin, calretinin and calmodulin), but has already provided remarkable insights into the specific role that these kinetics in Ca2+ signaling. GENERAL SIGNIFICANCE: The potential to gain novel insights into Ca2+ signaling by quantifying kinetics of other CBPs using this technique is very promising. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.

Connectomics and epilepsy.

PURPOSE OF REVIEW: Tremendous advances have occurred in recent years in elucidating basic mechanisms of epilepsy at the level of ion channels and neurotransmitters. Epilepsy, however, is ultimately a disease of functionally and/or structurally aberrant connections between neurons and groups of neurons at the systems level. Recent advances in neuroimaging and electrophysiology now make it possible to investigate structural and functional connectivity of the entire brain, and these techniques are currently being used to investigate diseases that manifest as global disturbances of brain function. Epilepsy is such a disease, and our understanding of the mechanisms underlying the development of epilepsy and the generation of epileptic seizures will undoubtedly benefit from research utilizing these connectomic approaches. RECENT FINDINGS: MRI using diffusion tensor imaging provides structural information, whereas functional MRI and electroencephalography provide functional information about connectivity at the whole brain level. Optogenetics, tracers, electrophysiological approaches, and calcium imaging provide connectivity information at the level of local circuits. These approaches are revealing important neuronal network disturbances underlying epileptic abnormalities. SUMMARY: An understanding of the fundamental mechanisms underlying the development of epilepsy and the generation of epileptic seizures will require delineation of the aberrant functional and structural connections of the whole brain. The field of connectomics now provides approaches to accomplish this.

Issues related to development of antiepileptogenic therapies.

Several preclinical proof-of-concept studies have provided evidence for positive treatment effects on epileptogenesis. However, none of these hypothetical treatments has advanced to the clinic. The experience in other fields of neurology such as stroke, Alzheimer's disease, or amyotrophic lateral sclerosis has indicated several problems in the design of preclinical studies, which likely contribute to failures in translating the positive preclinical data to the clinic. The Working Group on "Issues related to development of antiepileptogenic therapies" of the International League Against Epilepsy (ILAE) and the American Epilepsy Society (AES) has considered the possible problems that arise when moving from proof-of-concept antiepileptogenesis (AEG) studies to preclinical AEG trials, and eventually to clinical AEG trials. This article summarizes the discussions and provides recommendations on how to design a preclinical AEG monotherapy trial in adult animals. We specifically address study design, animal and model selection, number of studies needed, issues related to administration of the treatment, outcome measures, statistics, and reporting. In addition, we give recommendations for future actions to advance the preclinical AEG testing.

Epilepsy biomarkers.

A biomarker is defined as an objectively measured characteristic of a normal or pathologic biologic process. Identification and proper validation of biomarkers of epileptogenesis (the development of epilepsy) and ictogenesis (the propensity to generate spontaneous seizures) might predict the development of an epilepsy condition; identify the presence and severity of tissue capable of generating spontaneous seizures; measure progression after the condition is established; and determine pharmacoresistance. Such biomarkers could be used to create animal models for more cost-effective screening of potential antiepileptogenic and antiseizure drugs and devices, and to reduce the cost of clinical trials by enriching the trial population, and acting as surrogate markers to shorten trial duration. The objectives of the biomarker subgroup for the London Workshop were to define approaches for identifying possible biomarkers for these purposes. Research to identify reliable biomarkers may also reveal underlying mechanisms that could serve as therapeutic targets for the development of new antiepileptogenic and antiseizure compounds.

Capturing the power of seizures: an empirical mode decomposition analysis of epileptic activity in the mouse hippocampus.

Introduction: Various methods have been used to determine the frequency components of seizures in scalp electroencephalography (EEG) and in intracortical recordings. Most of these methods rely on subjective or trial-and-error criteria for choosing the appropriate bandwidth for filtering the EEG or local field potential (LFP) signals to establish the frequency components that contribute most to the initiation and maintenance of seizure activity. The empirical mode decomposition (EMD) with the Hilbert-Huang transform is an unbiased method to decompose a time and frequency variant signal into its component non-stationary frequencies. The resulting components, i.e., the intrinsic mode functions (IMFs) objectively reflect the various non-stationary frequencies making up the original signal. Materials and methods: We employed the EMD method to analyze the frequency components and relative power of spontaneous electrographic seizures recorded in the dentate gyri of mice during the epileptogenic period. Epilepsy was induced in mice following status epilepticus induced by suprahippocampal injection of kainic acid. The seizures were recorded as local field potentials (LFP) with electrodes implanted in the dentate gyrus. We analyzed recording segments that included a seizure (mean duration 28 s) and an equivalent time period both before and after the seizure. Each segment was divided into non-overlapping 1 s long epochs which were then analyzed to obtain their IMFs (usually 8-10), the center frequencies of the respective IMF and their spectral root-mean-squared (RMS) power. Results: Our analysis yielded unbiased identification of the spectral components of seizures, and the relative power of these components during this pathological brain activity. During seizures, the power of the mid frequency components increased while the center frequency of the first IMF (with the highest frequency) dramatically decreased, providing mechanistic insights into how local seizures are generated. Discussion: We expect this type of analysis to provide further insights into the mechanisms of seizure generation and potentially better seizure detection.

A therapeutic small molecule lead enhances γ-oscillations and improves cognition/memory in Alzheimer's disease model mice.

Brain rhythms provide the timing and concurrence of brain activity required for linking together neuronal ensembles engaged in specific tasks. In particular, the γ-oscillations (30-120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here we report on a potent brain permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a new class of therapeutics for AD. As a first in CNS pharmacotherapy, our lead candidate acts as a potent, efficacious, and selective negative allosteric modulator (NAM) of the γ-aminobutyric acid type A receptors (GABA A Rs) assembled from α1ß2δ subunits. We identified these receptors through anatomical and pharmacological means to mediate the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.

Changes in hippocampal neuronal activity during and after unilateral selective hippocampal ischemia in vivo.

The hippocampal formation is one of the brain regions most sensitive to ischemic damage. However, there are no studies about changes in hippocampal neuronal activity during and after a selective unilateral hippocampal ischemia. We developed a novel unilateral cerebrovascular ischemia model in mice that selectively shuts down blood supply to the ipsilateral hippocampal formation. Using a modified version of the photothrombotic method, we stereotaxically targeted the initial ascending part of the longitudinal hippocampal artery in urethane anesthetized and rose bengal-injected mice. To block blood flow in the targeted artery, we photoactivated the rose bengal by illuminating the longitudinal hippocampal artery through an optical fiber inserted into the brain. In vivo field potential recordings in the CA1 region of the hippocampus before, during and after the induction of ischemia demonstrated a high-frequency discharge (HFD) reaching frequencies of >300 Hz and lasting 7-24 s during the illumination consistent with a massive synchronous neuronal activity. The HFD was invariably followed by a DC voltage shift and a decreased activity at both low (30-57 Hz)- and high (63-119 Hz)-gamma frequencies. This decrease in gamma activity lasted for the entire duration of the recordings (∼160 min) following ischemia. The contralateral hippocampus displayed HFDs but with different frequency spectra and without DC voltage shifts or long-lasting decreases in gamma oscillations. Our findings reveal for the first time the acute effects of unilateral hippocampal ischemia on ensemble hippocampal neuronal activities.