Faculty Bibliography

Background/Purpose: Interferon (IFN)-alpha contributes to susceptibility and severe manifestations in systemic lupus erythematosus (SLE). The PRKG1 rs7897633 variant has been previously identified as the top hit in European ancestry patients with SLE and high IFN-alpha compared to those with low circulating IFN activity. However, the mechanisms by which PRKG1 polymorphisms impact the immune system remain unknown. PRKG1 codes for the cGMP-dependent protein kinase I (PKGI). Activation of PKGI leads to VASP phosphorylation, and the inhibition of RhoA and Rho-associated kinases (ROCK). A subgroup of patients with SLE exhibit higher ROCK activity in circulating immune cells compared to healthy controls. ROCK inhibition decreases IFN-alpha production and ameliorates disease in murine models of lupus. Accordingly, we aimed to assess whether the PRKG1 gene variant was associated with decreased gene expression and activity of PRKG1, hyperactivation of the ROCK pathway, and altered response to type I IFN.
Method(s): We used B lymphoblastoid cell lines (LCL) derived from healthy subjects of European ancestry (Coriell repositories) homozygous (AA or CC) and heterozygous (AC) at the rs7897633 SNP for all experiments. Gene expression of PRKG1, RHOA, and ROCK was assessed by RT-qPCR using gene-specific primers, normalized by GAPDH, and measured by the 2-DELTADELTACT method. IFN score at baseline and after treatment of LCL with increasing doses of IFN-alpha was measured by quantifying 3 canonical IFN-stimulated genes (IFIT1, MX1 and PKR) and summing to generate a score reflecting the degree of IFN-induced gene expression in the cells. Abundance of PKGI and VASP phosphorylation (as a surrogate of PKGI activity) were determined by Western blotting at baseline and after treatment with a PKGI agonist. ROCK2 enzymatic activity was performed by a colorimetric assay (Cell Biolabs). Unstimulated and stimulated cells were compared among PRKG1 genotype categories. Statistically significant differences were determined by Mann Whitney U test or sum-of-squares F test, as appropriate.
Result(s): PRKG1 expression was lower in the homozygous AA genotype as compared with the homozygous CC genotype in LCL (p< 0.05). In contrast, ROCK expression was higher in LCL with the AA rs7897633 genotype (p< 0.05). Compared to LCL with the homozygous AA variant of rs7897633, homozygous CC LCL have greater abundance of PKGI, phosphorylated VASP/total VASP ratio in response to a PKGI agonist (indicating increased PKGI activity), and lower baseline ROCK activity (sum-of-squares F test, p< 0.05). The IFN score was significantly higher in the homozygous CC allele LCL, both at baseline and with increasing doses of IFN-alpha, suggesting increased sensitivity to type I IFN (p< 0.05).
Conclusion(s): PRKG1 AA genotype associates with lower PRKG1 and higher ROCK mRNA expression, decreased PKGI abundance and activity, and greater ROCK baseline activity. In contrast, the rs7897633 CC genotype is associated with increased response to IFNalpha. Overall, these findings suggest an important role of genetic variation at PRKG1 in modulating the RhoA-ROCK pathway and regulating response to type I IFNs in LCL, which may have therapeutic implications in patients with SLE

OBJECTIVE:The combined spatiotemporal dynamics underlying sign language production remains largely unknown. To investigate these dynamics as compared to speech production we utilized intracranial electrocorticography during a battery of language tasks. METHODS:We report a unique case of direct cortical surface recordings obtained from a neurosurgical patient with intact hearing and bilingual in English and American Sign Language. We designed a battery of cognitive tasks to capture multiple modalities of language processing and production. RESULTS:We identified two spatially distinct cortical networks: ventral for speech and dorsal for sign production. Sign production recruited peri-rolandic, parietal and posterior temporal regions, while speech production recruited frontal, peri-sylvian and peri-rolandic regions. Electrical cortical stimulation confirmed this spatial segregation, identifying mouth areas for speech production and limb areas for sign production. The temporal dynamics revealed superior parietal cortex activity immediately before sign production, suggesting its role in planning and producing sign language. CONCLUSIONS:Our findings reveal a distinct network for sign language and detail the temporal propagation supporting sign production.

PURPOSE/OBJECTIVE:A phase I feasibility study to determine the accuracy of identifying seizures based on audio recordings. METHODS:We systematically generated 166 audio clips of 30 s duration from 83 patients admitted to an epilepsy monitoring unit between 1/2015 and 12/2016, with one clip during a seizure period and one clip during a non-seizure control period for each patient. Five epileptologists performed a blinded review of the audio clips and rated whether a seizure occurred or not, and indicated the confidence level (low or high) of their rating. The accuracy of individual and consensus ratings were calculated. RESULTS:The overall performance of the consensus rating between the five epileptologists showed a positive predictive value (PPV) of 0.91 and a negative predictive value (NPV) of 0.66. The performance improved when confidence was high (PPV of 0.96, NPV of 0.70). The agreement between the epileptologists was moderate with a kappa of 0.584. Hyperkinetic (PPV 0.92, NPV 0.86) and tonic-clonic (PPV and NPV 1.00) seizures were most accurately identified. Seizures with automatisms only and non-motor seizures could not be accurately identified. Specific seizure-related sounds associated with accurate identification included disordered breathing (PPV and NPV 1.00), rhythmic sounds (PPV 0.93, NPV 0.80), and ictal vocalizations (PPV 1.00, NPV 0.97). CONCLUSION/CONCLUSIONS:This phase I feasibility study shows that epileptologists are able to accurately identify certain seizure types from audio recordings when the seizures produce sounds. This provides guidance for the development of audio-based seizure detection devices and demonstrate which seizure types could potentially be detected.

Animals collect sensory information from the world and make adaptive choices about how to respond to it. Here, we reveal a network motif in the brain for one of the most fundamental behavioral choices made by bilaterally symmetric animals: whether to respond to a sensory stimulus by moving to the left or to the right. We define network connectivity in the hindbrain important for the lateralized escape behavior of zebrafish and then test the role of neurons by using laser ablations and behavioral studies. Key inhibitory neurons in the circuit lie in a column of morphologically similar cells that is one of a series of such columns that form a developmental and functional ground plan for building hindbrain networks. Repetition within the columns of the network motif we defined may therefore lie at the foundation of other lateralized behavioral choices.

Is there a distinct area within the human visual system that has a preferential response to numerals, as there is for faces, words, or scenes? We addressed this question using intracranial electrophysiological recordings and observed a significantly higher response in the high-frequency broadband range (high gamma, 65-150 Hz) to visually presented numerals, compared with morphologically similar (i.e., letters and false fonts) or semantically and phonologically similar stimuli (i.e., number words and non-number words). Anatomically, this preferential response was consistently localized in the inferior temporal gyrus and anterior to the temporo-occipital incisure. This region lies within or close to the fMRI signal-dropout zone produced by the nearby auditory canal and venous sinus artifacts, an observation that may account for negative findings in previous fMRI studies of preferential response to numerals. Because visual numerals are culturally dependent symbols that are only learned through education, our novel finding of anatomically localized preferential response to such symbols provides a new example of acquired category-specific responses in the human visual system.

Optogenetic approaches allow the manipulation of neuronal activity patterns in space and time by light, particularly in small animals such as zebrafish. However, most techniques cannot control neuronal activity independently at different locations. Here we describe equipment and provide a protocol for single-photon patterned optical stimulation of neurons using a digital micromirror device (DMD). This method can create arbitrary spatiotemporal light patterns with spatial and temporal resolutions in the micrometer and submillisecond range, respectively. Different options to integrate a DMD into a multiphoton microscope are presented and compared. We also describe an ex vivo preparation of the adult zebrafish head that greatly facilitates optogenetic and other experiments. After assembly, the initial alignment takes about one day and the zebrafish preparation takes <30 min. The method has previously been used to activate channelrhodopsin-2 and manipulate oscillatory synchrony among spatially distributed neurons in the zebrafish olfactory bulb. It can be adapted easily to a wide range of other species, optogenetic probes and scientific applications.

Dopamine (DA) is an important modulator of synaptic transmission and plasticity that is causally involved in fundamental brain functions and dysfunctions. We examined the dopaminergic modulation of synaptic transmission and sensory responses in telencephalic area Dp of zebrafish, the homolog of olfactory cortex. By combining anatomical tracing and immunohistochemistry, we detected no DA neurons in Dp itself but long-range dopaminergic input from multiple other brain areas. Whole-cell recordings revealed no obvious effects of DA on membrane potential or input resistance in the majority of Dp neurons. Electrical stimulation of the olfactory tracts produced a complex sequence of synaptic currents in Dp neurons. DA selectively decreased inhibitory currents with little or no effect on excitatory components. Multiphoton calcium imaging showed that population responses of Dp neurons to olfactory tract stimulation or odor application were enhanced by DA, consistent with its effect on inhibitory synaptic transmission. These effects of DA were blocked by an antagonist of D2-like receptors. DA therefore disinhibits and reorganizes sensory responses in Dp. This modulation may affect sensory perception and could be involved in the experience-dependent modification of odor representations.

Neuronal activity patterns contain information in their temporal structure, indicating that information transfer between neurons may be optimized by temporal filtering. In the zebrafish olfactory bulb, subsets of output neurons (mitral cells) engage in synchronized oscillations during odour responses, but information about odour identity is contained mostly in non-oscillatory firing rate patterns. Using optogenetic manipulations and odour stimulation, we found that firing rate responses of neurons in the posterior zone of the dorsal telencephalon (Dp), a target area homologous to olfactory cortex, were largely insensitive to oscillatory synchrony of mitral cells because passive membrane properties and synaptic currents act as low-pass filters. Nevertheless, synchrony influenced spike timing. Moreover, Dp neurons responded primarily during the decorrelated steady state of mitral cell activity patterns. Temporal filtering therefore tunes Dp neurons to components of mitral cell activity patterns that are particularly informative about precise odour identity. These results demonstrate how temporal filtering can extract specific information from multiplexed neuronal codes.