Correction of amygdalar dysfunction in a rat model of fragile X syndrome.

Fragile X syndrome (FXS), a commonly inherited form of autism and intellectual disability, is associated with emotional symptoms that implicate dysfunction of the amygdala. However, current understanding of the pathogenesis of the disease is based primarily on studies in the hippocampus and neocortex, where FXS defects have been corrected by inhibiting group I metabotropic glutamate receptors (mGluRs). Here, we observe that activation, rather than inhibition, of mGluRs in the basolateral amygdala reverses impairments in a rat model of FXS. FXS rats exhibit deficient recall of auditory conditioned fear, which is accompanied by a range of in vitro and in vivo deficits in synaptic transmission and plasticity. We find presynaptic mGluR5 in the amygdala, activation of which reverses deficient synaptic transmission and plasticity, thereby restoring normal fear learning in FXS rats. This highlights the importance of modifying the prevailing mGluR-based framework for therapeutic strategies to include circuit-specific differences in FXS pathophysiology.

Early Seizure Activity Accelerates Depletion of Hippocampal Neural Stem Cells and Impairs Spatial Discrimination in an Alzheimer's Disease Model.

Adult hippocampal neurogenesis has been reported to be decreased, increased, or not changed in Alzheimer's disease (AD) patients and related transgenic mouse models. These disparate findings may relate to differences in disease stage, or the presence of seizures, which are associated with AD and can stimulate neurogenesis. In this study, we investigate a transgenic mouse model of AD that exhibits seizures similarly to AD patients and find that neurogenesis is increased in early stages of disease, as spontaneous seizures became evident, but is decreased below control levels as seizures recur. Treatment with the antiseizure drug levetiracetam restores neurogenesis and improves performance in a neurogenesis-associated spatial discrimination task. Our results suggest that seizures stimulate, and later accelerate the depletion of, the hippocampal neural stem cell pool. These results have implications for AD as well as any disorder accompanied by recurrent seizures, such as epilepsy.

On-Probe Neural Interface ASIC for Combined Electrical Recording and Optogenetic Stimulation.

Neuromodulation technologies are progressing from pacemaking and sensory operations to full closed-loop control. In particular, optogenetics-the genetic modification of light sensitivity into neural tissue allows for simultaneous optical stimulation and electronic recording. This paper presents a neural interface application-specified integrated circuit (ASIC) for intelligent optoelectronic probes. The architecture is designed to enable simultaneous optical neural stimulation and electronic recording. It provides four low noise (2.08  µV) recording channels optimized for recording local field potentials (LFPs) (0.1-300 Hz bandwidth, 5 mV range, sampled 10-bit@4 kHz), which are more stable for chronic applications. For stimulation, it provides six independently addressable optical driver circuits, which can provide both intensity (8-bit resolution across a 1.1 mA range) and pulse-width modulation for high-radiance light emitting diodes (LEDs). The system includes a fully digital interface using a serial peripheral interface (SPI) protocol to allow for use with embedded controllers. The SPI interface is embedded within a finite state machine (FSM), which implements a command interpreter that can send out LFP data whilst receiving instructions to control LED emission. The circuit has been implemented in a commercially available 0.35  µm CMOS technology occupying a 1.95 mm 1.10 mm footprint for mounting onto the head of a silicon probe. Measured results are given for a variety of bench-top, in vitro and in vivo experiments, quantifying system performance and also demonstrating concurrent recording and stimulation within relevant experimental models.

ΔFosB Regulates Gene Expression and Cognitive Dysfunction in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by cognitive decline and 5- to 10-fold increased seizure incidence. How seizures contribute to cognitive decline in AD or other disorders is unclear. We show that spontaneous seizures increase expression of ΔFosB, a highly stable Fos-family transcription factor, in the hippocampus of an AD mouse model. ΔFosB suppressed expression of the immediate early gene c-Fos, which is critical for plasticity and cognition, by binding its promoter and triggering histone deacetylation. Acute histone deacetylase (HDAC) inhibition or inhibition of ΔFosB activity restored c-Fos induction and improved cognition in AD mice. Administration of seizure-inducing agents to nontransgenic mice also resulted in ΔFosB-mediated suppression of c-Fos, suggesting that this mechanism is not confined to AD mice. These results explain observations that c-Fos expression increases after acute neuronal activity but decreases with chronic activity. Moreover, these results indicate a general mechanism by which seizures contribute to persistent cognitive deficits, even during seizure-free periods.

Corticothalamic network dysfunction and behavioral deficits in a mouse model of Alzheimer's disease.

Alzheimer's disease is associated with cognitive decline and seizures. Growing evidence indicates that seizures contribute to cognitive deficits early in disease, but how they develop and impact cognition are unclear. To investigate potential mechanisms, we studied a mouse model that overexpresses mutant human amyloid precursor protein with high levels of amyloid beta (Aß). These mice develop generalized epileptiform activity, including nonconvulsive seizures, consistent with alterations in corticothalamic network activity. Amyloid precursor protein mice exhibited reduced activity marker expression in the reticular thalamic nucleus, a key inhibitory regulatory nucleus, and increased activity marker expression in downstream thalamic relay targets that project to cortex and limbic structures. Slice recordings revealed impaired cortical inputs to the reticular thalamic nucleus that may contribute to corticothalamic dysfunction. These results are consistent with our findings of impaired sleep maintenance in amyloid precursor protein mice. Finally, the severity of sleep impairments predicted the severity of deficits in Morris water maze, suggesting corticothalamic dysfunction may relate to hippocampal dysfunction, and may be a pathophysiological mechanism underlying multiple behavioral and cognitive alterations in Alzheimer's disease.

Delayed thalamic astrocytosis and disrupted sleep-wake patterns in a preclinical model of traumatic brain injury.

Traumatic brain injury (TBI) involves diffuse axonal injury and induces subtle but persistent changes in brain tissue and function and poses challenges for early detection of neurological injury. The present study uses an automated behavioral analysis system to assess alterations in rodent behavior in the subacute phase in a preclinical mouse model of TBI, controlled cortical impact (CCI) injury. In the first few weeks following CCI, mice demonstrated normal exploratory behaviors and other typical home-cage behaviors. However, beginning 4 weeks post-injury, CCI mice developed disruptions in sleep-wake patterns, including an increased number of awakenings from sleep. Such impaired sleep maintenance was accompanied by an increased latency to reach peak sleep in CCI mice. These sleep disruptions implicate involvement of the thalamocortical network, the activity of which must be tightly regulated to control sleep maintenance. After injury, there was an increase in reactive microglia in thalamic regions as well as delayed reactive astrocytosis that was evident in the thalamic reticular nucleus, which preceded the development of sleep disruptions. These data suggest that cortical injury may trigger inflammatory responses in deeper neuroanatomical structures, including the thalamic reticular nucleus. Such engagement of the thalamus may perturb the thalamocortical network that regulates sleep/awake patterns and contribute to sleep disruptions observed in this model as well as those documented in patients with TBI.

Oxygen and seizure dynamics: I. Experiments.

We utilized a novel ratiometric nanoquantum dot fluorescence resonance energy transfer (NQD-FRET) optical sensor to quantitatively measure oxygen dynamics from single cell microdomains during hypoxic episodes as well as during 4-aminopyridine (4-AP)-induced spontaneous seizure-like events in rat hippocampal slices. Coupling oxygen sensing with electrical recordings, we found the greatest reduction in the O2 concentration ([O2]) in the densely packed cell body stratum (st.) pyramidale layer of the CA1 and differential layer-specific O2 dynamics between the st. pyramidale and st. oriens layers. These hypoxic decrements occurred up to several seconds before seizure onset could be electrically measured extracellularly. Without 4-AP, we quantified a narrow range of [O2], similar to the endogenous hypoxia found before epileptiform activity, which permits a quiescent network to enter into a seizure-like state. We demonstrated layer-specific patterns of O2 utilization accompanying layer-specific neuronal interplay in seizure. None of the oxygen overshoot artifacts seen with polarographic measurement techniques were observed. We therefore conclude that endogenously generated hypoxia may be more than just a consequence of increased cellular excitability but an influential and critical factor for orchestrating network dynamics associated with epileptiform activity.

Purinergic control of hippocampal circuit hyperexcitability in Dravet syndrome.

OBJECTIVE: Severe myoclonic epilepsy in infancy (SMEI) or Dravet syndrome is one of the most devastating childhood epilepsies. Children with SMEI have febrile and afebrile seizures (FS and aFS), ataxia, and social and cognitive dysfunctions. SMEI is pharmacologically intractable and can be fatal in 10-20% of patients. It remains to be elucidated how channelopathies that cause SMEI impact synaptic activities in key neural circuits, and there is an ongoing critical need for alternative methods of controlling seizures in SMEI. Using the SCN1A gene knock-in mouse model of SMEI (mSMEI), we studied hippocampal cell and circuit excitability, particularly during hyperthermia, and tested whether an adenosine A1 receptor (A1R) agonist can reliably control hippocampal circuit hyperexcitability. METHODS: Using a combination of electrophysiology (extracellular and whole-cell voltage clamp) and fast voltage-sensitive dye imaging (VSDI), we quantified synaptic excitation and inhibition, spatiotemporal characteristics of neural circuit activity, and hyperthermia-induced febrile seizure-like events (FSLEs) in juvenile mouse hippocampal slices. We used hyperthermia to elicit FSLEs in hippocampal slices, while making use of adenosine A1R agonist N6-cyclopentyladenosine (CPA) to control abnormally widespread neural activity and FSLEs. RESULTS: We discovered a significant excitation/inhibition (E/I) imbalance in mSMEI hippocampi, in which inhibition was decreased and excitation increased. This imbalance was associated with an increased spatial extent of evoked neural circuit activation and a lowered FSLE threshold. We found that a low concentration (50 nm) of CPA blocked FSLEs and reduced the spatial extent of abnormal neural activity spread while preserving basal levels of excitatory synaptic transmission. SIGNIFICANCE: Our study reveals significant hippocampal synapse and circuit dysfunctions in mSMEI and demonstrates that the A1R agonist CPA can reliably control hippocampal hyperexcitability and FSLEs in vitro. These findings may warrant further investigations of purinergic agonists as part of the development of new therapeutic approaches for Dravet syndrome.

Inhibitory neuron and hippocampal circuit dysfunction in an aged mouse model of Alzheimer's disease.

In Alzheimer's disease (AD), a decline in explicit memory is one of the earliest signs of disease and is associated with hippocampal dysfunction. Amyloid protein exerts a disruptive impact on neuronal function, but the specific effects on hippocampal network activity are not well known. In this study, fast voltage-sensitive dye imaging and extracellular and whole-cell electrophysiology were used on entorhinal cortical-hippocampal slice preparations to characterize hippocampal network activity in 12-16 month old female APPswe/PSEN1DeltaE9 (APdE9 mice) mice. Aged APdE9 mice exhibited profound disruptions in dentate gyrus circuit activation. High frequency stimulation of the perforant pathway in the dentate gyrus (DG) area of APdE9 mouse tissue evoked abnormally large field potential responses corresponding to the wider neural activation maps. Whole-cell patch clamp recordings of the identified inhibitory interneurons in the molecular layer of DG revealed that they fail to reliably fire action potentials. Taken together, abnormal DG excitability and an inhibitory neuron failure to generate action potentials are suggested to be important contributors to the underlying cellular mechanisms of early-stage Alzheimer's disease pathophysiology.

Sodium channel cleavage is associated with aberrant neuronal activity and cognitive deficits in a mouse model of Alzheimer's disease.

BACE1 is the rate-limiting enzyme that cleaves amyloid precursor protein (APP) to produce the amyloid ß peptides that accumulate in Alzheimer's disease (AD). BACE1, which is elevated in AD patients and APP transgenic mice, also cleaves the ß2-subunit of voltage-gated sodium channels (Navß2). Although increased BACE1 levels are associated with Navß2 cleavage in AD patients, whether Navß2 cleavage occurs in APP mice had not yet been examined. Such a finding would be of interest because of its potential impact on neuronal activity: previous studies demonstrated that BACE1-overexpressing mice exhibit excessive cleavage of Navß2 and reduced sodium current density, but the phenotype associated with loss of function mutations in either Navß-subunits or pore-forming α-subunits is epilepsy. Because mounting evidence suggests that epileptiform activity may play an important role in the development of AD-related cognitive deficits, we examined whether enhanced cleavage of Navß2 occurs in APP transgenic mice, and whether it is associated with aberrant neuronal activity and cognitive deficits. We found increased levels of BACE1 expression and Navß2 cleavage fragments in cortical lysates from APP transgenic mice, as well as associated alterations in Nav1.1α expression and localization. Both pyramidal neurons and inhibitory interneurons exhibited evidence of increased Navß2 cleavage. Moreover, the magnitude of alterations in sodium channel subunits was associated with aberrant EEG activity and impairments in the Morris water maze. Together, these results suggest that altered processing of voltage-gated sodium channels may contribute to aberrant neuronal activity and cognitive deficits in AD.

ß-Adrenergic modulation of spontaneous spatiotemporal activity patterns and synchrony in hyperexcitable hippocampal circuits.

A description of healthy and pathological brain dynamics requires an understanding of spatiotemporal patterns of neural activity and characteristics of its propagation between interconnected circuits. However, the structure and modulation of the neural activation maps underlying these patterns and their propagation remain elusive. We investigated effects of ß-adrenergic receptor (ß-AR) stimulation on the spatiotemporal characteristics of emergent activity in rat hippocampal circuits. Synchronized epileptiform-like activity, such as interictal bursts (IBs) and ictal-like events (ILEs), were evoked by 4-aminopyridine (4-AP), and their dynamics were studied using a combination of electrophysiology and fast voltage-sensitive dye imaging. Dynamic characterization of the spontaneous IBs showed that they originated in dentate gyrus/CA3 border and propagated toward CA1. To determine how ß-AR modulates spatiotemporal characteristics of the emergent IBs, we used the ß-AR agonist isoproterenol (ISO). ISO significantly reduced the spatiotemporal extent and propagation velocity of the IBs and significantly altered network activity in the 1- to 20-Hz range. Dual whole cell recordings of the IBs in CA3/CA1 pyramidal cells and optical analysis of those regions showed that ISO application reduced interpyramidal and interregional synchrony during the IBs. In addition, ISO significantly reduced duration not only of the shorter duration IBs but also the prolonged ILEs in 4-AP. To test whether the decrease in ILE duration was model dependent, we used a different hyperexcitability model, zero magnesium (0 Mg(2+)). Prolonged ILEs were readily formed in 0 Mg(2+), and addition of ISO significantly reduced their durations. Taken together, these novel results provide evidence that ß-AR activation dynamically reshapes the spatiotemporal activity patterns in hyperexcitable circuits by altering network rhythmogenesis, propagation velocity, and intercellular/regional synchronization.

Expression of foreign proteins in a Vibrio cholerae vaccine strain using the stationary phase hemagglutinin/protease promoter.

The use of the hemagglutinin(HA)/protease promoter and secretion signals to drive expression and secretion of a foreign antigen in a live genetically attenuated cholera vaccine candidate is demonstrated. A Vibrio cholerae vaccine strain, containing a HA/protease-tetanus toxin C fragment (TCF) fusion, produced soluble-and cell-associated TCF. The fraction of TCF secreted to the culture medium was degraded unless expressed in a HA/protease-defective vaccine strain. Comparison of the hapA promoter with the strong Tac promoter using quantitative real time PCR revealed that at least five times more TCF mRNA was produced when expressed from the hapA promoter.