Faculty Bibliography

Neuronal oscillations allow for temporal segmentation of neuronal spikes. Interdependent oscillators can integrate multiple layers of information. We examined phase-phase coupling of theta and gamma oscillators in the CA1 region of rat hippocampus during maze exploration and rapid eye movement sleep. Hippocampal theta waves were asymmetric, and estimation of the spatial position of the animal was improved by identifying the waveform-based phase of spiking, compared to traditional methods used for phase estimation. Using the waveform-based theta phase, three distinct gamma bands were identified: slow gamma(S) (gamma(S); 30-50 Hz), midfrequency gamma(M) (gamma(M); 50-90 Hz), and fast gamma(F) (gamma(F); 90-150 Hz or epsilon band). The amplitude of each sub-band was modulated by the theta phase. In addition, we found reliable phase-phase coupling between theta and both gamma(S) and gamma(M) but not gamma(F) oscillators. We suggest that cross-frequency phase coupling can support multiple time-scale control of neuronal spikes within and across structures.

Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately 'blind' neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric alpha3beta4 and alpha4beta2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.

The development of rapid volumetric imaging systems for functional multiphoton microscopy is essential for dynamical imaging of large-scale neuronal network activity. Here, we introduce a line-illuminating temporal-focusing microscope capable of rapid three-dimensional imaging at 10-20 volumes/sec, and study the system's characteristics both theoretically and experimentally. We demonstrate that our system is capable of functional volumetric calcium imaging of distributed neuronal activity patterns, and introduce a computational strategy for activity reconstruction in strongly scattering media.

RATIONALE: Previous work has demonstrated a profound effect of N-methyl-D: -aspartic acid receptor (NMDAR) antagonism in the infralimbic cortex (IL) to selectively elevate impulsive responding in a rodent reaction time paradigm. However, the mechanism underlying this effect is unclear. OBJECTIVES: This series of experiments investigated the pharmacological basis of this effect in terms of excitatory and inhibitory neurotransmission. We tested several pharmacological mechanisms that might produce the effect of NMDAR antagonism via disruption or dampening of IL output. METHODS: Drugs known to affect brain GABA or glutamate function were tested in rats pre-trained on a five-choice serial reaction time task (5-CSRTT) following either their systemic administration or direct administration into the IL. RESULTS: Systemic lamotrigine administration (15 mg/kg), which attenuates excess glutamate release, did not counteract the ability of the intra-IL NMDAR antagonist 3-((R)-2-carboxypiperazin-4-yl)-propyl-L: -phosphonic acid ((R)-CPP) to increase premature responding on the 5-CSRTT. Putative elevation of local extracellular glutamate via intra-IL infusions of the selective glutamate reuptake inhibitor DL: -threo-beta-benzyloxyaspartate as well as local alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonism also had no effect on this task. However, intra-IL infusions of the GABA(A) receptor agonist muscimol produced qualitatively but not quantitatively comparable increases in impulsive responding to those elicited by (R)-CPP. Moreover, the GABA(A) receptor antagonist bicuculline blocked the increase in impulsivity produced by (R)-CPP when infused in the IL. CONCLUSIONS: These findings implicate glutamatergic and GABAergic mechanisms in the IL in the expression of impulsivity and suggest that excessive glutamate release may not underlie increased impulsivity induced by local NMDA receptor antagonism.

HYPOTHESIS: Reactivation of herpes simplex virus type 1 (HSV-1) in geniculate ganglion neurons (GGNs) is an etiologic mechanism of Bell's palsy (BP) and delayed facial palsy (DFP) after otologic surgery. BACKGROUND: Several clinical studies, including temporal bone studies, antibody, titers, and intraoperative studies, suggest that reactivation of HSV-1 from latently infected GGNs may lead to both BP and DFP. However, it is difficult to study these processes in humans or live animals. METHODS: Primary cultures of GGNs were latently infected with Patton strain HSV-1 expressing a green fluorescent protein-late lytic gene chimera. Four days later, these cultures were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1 in other neurons. Cultures were monitored daily by fluorescent microscopy. Titers of media from lytic, latent, and latent/TSA treated GGN cultures were obtained using plaque assays on Vero cells. RNA was harvested from latently infected GGN cultures and examined for the presence of viral transcripts using reverse transcription-polymerase chain reaction. RESULTS: Latently infected GGN cultures displayed latency-associated transcripts only, whereas lytically infected and reactivated latent cultures produced other viral transcripts, as well. The GGN cultures displayed a reactivation rate of 65% after treatment with TSA. Media from latently infected cultures contained no detectable infectious HSV-1, whereas infectious virus was observed in both lytically and latently infected/TSA-treated culture media. CONCLUSION: We have shown that cultured GGNs can be latently infected with HSV-1, and HSV-1 in these latently infected neurons can be reactivated using TSA, yielding infectious virus. These results have implications for the cause of both BP and DFP

Diffusion-weighted imaging (DWI) involves data acquisitions at multiple b values. In this paper, we presented a method of selecting the b values that maximize estimation precision of the biexponential analysis of renal DWI data. We developed an error propagation factor for the biexponential model, and proposed to optimize the b-value samplings by minimizing the error propagation factor. A prospective study of four healthy human subjects (eight kidneys) was done to verify the feasibility of the proposed protocol and to assess the validity of predicted precision for DWI measures, followed by Monte Carlo simulations of DWI signals based on acquired data from renal lesions of 16 subjects. In healthy subjects, the proposed methods improved precision (P = 0.003) and accuracy (P < 0.001) significantly in region-of-interest based biexponential analysis. In Monte Carlo simulation of renal lesions, the b-sampling optimization lowered estimation error by at least 20-30% compared with uniformly distributed b values, and improved the differentiation between malignant and benign lesions significantly. In conclusion, the proposed method has the potential of maximizing the precision and accuracy of the biexponential analysis of renal DWI. Magn Reson Med, 2011. (c) 2011 Wiley-Liss, Inc

The longitudinal repeatability of proton MR spectroscopy ((1) H-MRS) in the healthy human brain at high fields over long periods is not established. Therefore, we assessed the inter- and intra-subject repeatability of (1) H-MRS in an approach suited for diffuse pathologies in 10 individuals, at 3T, annually for 3 years. Spectra from 480 voxels over 360 cm(3) ( approximately 30%) of the brain, were individually phased, frequency-aligned, and summed into one average spectrum. This dramatically increases metabolites' signal-to-noise-ratios while maintaining narrow linewidths that improve quantification precision. The resulting concentrations of the N-acetylaspartate, creatine, choline, and myo-inositol are: 8.9 +/- 0.8, 5.9 +/- 0.6, 1.4 +/- 0.1, and 4.5 +/- 0.5 mM (mean +/- standard-deviation). the inter-subject coefficients of variation are 8.7%, 10.2%, 10.7%, and 11.8%; and the longitudinal (intra-subject) coefficients of variation are lower still: 6.6%, 6.8%, 6.8%, and 10%, much better than the 35%, 44%, 55%, and 62% intra-voxel coefficients of variation. The biological and nonbiological components of the summed spectra coefficients of variation had similar contributions to the overall variance. Magn Reson Med, 2011. (c) 2011 Wiley-Liss, Inc