Faculty Bibliography

In the hippocampus, extracellular carbonic anhydrase (Car) speeds the buffering of an activity-generated rise in extracellular pH that impacts H(+)-sensitive NMDA receptors (NMDARs). We studied the role of Car14 in this brain structure, in which it is expressed solely on neurons. Current-clamp responses were recorded from CA1 pyramidal neurons in wild-type (WT) versus Car14 knock-out (KO) mice 2 s before (control) and after (test) a 10 pulse, 100 Hz afferent train. In both WT and KO, the half-width (HW) of the test response, and its number of spikes, were augmented relative to the control. An increase in presynaptic release was not involved, because AMPAR-mediated EPSCs were depressed after a train. The increases in HW and spike number were both greater in the Car14 KO. In 0 Mg(2+) saline with picrotoxin (using a 20 Hz train), the HW measures were still greater in the KO. The Car inhibitor benzolamide (BZ) enhanced the test response HW in the WT but had no effect on the already-prolonged HW in the KO. With intracellular MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]-cyclohepten-5,10-imine maleate], the curtailed WT and KO responses were indistinguishable, and BZ caused no change. In contrast, the extracellular alkaline changes evoked by the train were not different between WT and KO, and BZ amplified these alkalinizations similarly. These data suggest that Car14 regulates pH transients in the perisynaptic microenvironment and govern their impact on NMDARs but plays little role in buffering pH shifts in the broader, macroscopic, extracellular space.

Locomotion requires coordinated motor activity throughout an animal's body. In both vertebrates and invertebrates, chains of coupled central pattern generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement.

The integration of an organic electrochemical transistor with human barrier tissue cells provides a novel method for assessing toxicology of compounds in vitro. Minute variations in paracellular ionic flux induced by toxic compounds are measured in real time, with unprecedented temporal resolution and extreme sensitivity.

BACKGROUND: Our brains are capable of remarkably stable stimulus representations despite time-varying neural activity. For instance, during delay periods in working memory tasks, while stimuli are represented in working memory, neurons in the prefrontal cortex, thought to support the memory representation, exhibit time-varying neuronal activity. Since neuronal activity encodes the stimulus, its time-varying dynamics appears to be paradoxical and incompatible with stable network stimulus representations. Indeed, this finding raises a fundamental question: can stable representations only be encoded with stable neural activity, or, its corollary, is every change in activity a sign of change in stimulus representation? RESULTS: Here we explain how different time-varying representations offered by individual neurons can be woven together to form a coherent, time-invariant, representation. Motivated by two ubiquitous features of the neocortex-redundancy of neural representation and sparse intracortical connections-we derive a network architecture that resolves the apparent contradiction between representation stability and changing neural activity. Unexpectedly, this network architecture exhibits many structural properties that have been measured in cortical sensory areas. In particular, we can account for few-neuron motifs, synapse weight distribution, and the relations between neuronal functional properties and connection probability. CONCLUSIONS: We show that the intuition regarding network stimulus representation, typically derived from considering single neurons, may be misleading and that time-varying activity of distributed representation in cortical circuits does not necessarily imply that the network explicitly encodes time-varying properties.

The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination.

Sensory neurons have been hypothesized to efficiently encode signals from the natural environment subject to resource constraints. The predictions of this efficient coding hypothesis regarding the spatial filtering properties of the visual system have been found consistent with human perception, but they have not been compared directly with neural responses. Here, we analyze the information that retinal ganglion cells transmit to the brain about the spatial information in natural images subject to three resource constraints: the number of retinal ganglion cells, their total response variances, and their total synaptic strengths. We derive a model that optimizes the transmitted information and compare it directly with measurements of complete functional connectivity between cone photoreceptors and the four major types of ganglion cells in the primate retina, obtained at single-cell resolution. We find that the ganglion cell population exhibited 80% efficiency in transmitting spatial information relative to the model. Both the retina and the model exhibited high redundancy (~30%) among ganglion cells of the same cell type. A novel and unique prediction of efficient coding, the relationships between projection patterns of individual cones to all ganglion cells, was consistent with the observed projection patterns in the retina. These results indicate a high level of efficiency with near-optimal redundancy in visual signaling by the retina.

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s. Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc and initiates the assembly of undifferentiated cells into ommatidia. The 'founder cell' R8 is specified first and recruits R1-6 and then R7. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes, including rhabdomere formation, synaptogenesis and eventually rh expression. In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Voltage-gated Na(+) channels in the brain are composed of a single pore-forming alpha subunit, one non-covalently linked beta subunit (beta1 or beta3), and one disulfide-linked beta subunit (beta2 or beta4). The final step in Na(+) channel biosynthesis in central neurons is concomitant alpha-beta2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding beta2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant beta2 cDNA constructs to investigate the cysteine residue(s) responsible for alpha-beta2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between alpha and beta2 subunits. Loss of alpha-beta2 covalent complex formation disrupts the targeting of beta2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with alpha is required for normal beta2 subcellular localization in vivo. WT beta2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant beta2 subunits, which cannot form disulfide bonds with alpha, are removed by detergent. Taken together, our results demonstrate that alpha-beta2 covalent association via a single, extracellular disulfide bond is required for beta2 targeting to specialized neuronal subcellular domains and for beta2 association with the neuronal cytoskeleton within those domains.

Cortical inhibitory circuits are formed by gamma-aminobutyric acid (GABA)-secreting interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how the number of cortical interneurons is ultimately determined. One possibility, suggested by the neurotrophic hypothesis, is that cortical interneurons are overproduced, and then after their migration into cortex the excess interneurons are eliminated through a competition for extrinsically derived trophic signals. Here we characterize the developmental cell death of mouse cortical interneurons in vivo, in vitro and after transplantation. We found that 40% of developing cortical interneurons were eliminated through Bax (Bcl-2-associated X)-dependent apoptosis during postnatal life. When cultured in vitro or transplanted into the cortex, interneuron precursors died at a cellular age similar to that at which endogenous interneurons died during normal development. Over transplant sizes that varied 200-fold, a constant fraction of the transplanted population underwent cell death. The death of transplanted neurons was not affected by the cell-autonomous disruption of TrkB (tropomyosin kinase receptor B), the main neurotrophin receptor expressed by neurons of the central nervous system. Transplantation expanded the cortical interneuron population by up to 35%, but the frequency of inhibitory synaptic events did not scale with the number of transplanted interneurons. Taken together, our findings indicate that interneuron cell death is determined intrinsically, either cell-autonomously or through a population-autonomous competition for survival signals derived from other interneurons.

BACKGROUND AND PURPOSE: Despite being the gold standard technique for stroke assessment, conventional diffusion MRI provides only partial information about tissue microstructure. Diffusional kurtosis imaging is an advanced diffusion MRI method that yields, in addition to conventional diffusion information, the diffusional kurtosis, which may help improve characterization of tissue microstructure. In particular, this additional information permits the description of white matter (WM) in terms of WM-specific diffusion metrics. The goal of this study is to elucidate possible biophysical mechanisms underlying ischemia using these new WM metrics. METHODS: We performed a retrospective review of clinical and diffusional kurtosis imaging data of 44 patients with acute/subacute ischemic stroke. Patients with a history of brain neoplasm or intracranial hemorrhages were excluded from this study. Region of interest analysis was performed to measure percent change of diffusion metrics in ischemic WM lesions compared with the contralateral hemisphere. RESULTS: Kurtosis maps exhibit distinct ischemic lesion heterogeneity that is not apparent on apparent diffusion coefficient maps. Kurtosis metrics also have significantly higher absolute percent change than complementary conventional diffusion metrics. Our WM metrics reveal an increase in axonal density and a larger decrease in the intra-axonal (Da) compared with extra-axonal diffusion microenvironment of the ischemic WM lesion. CONCLUSIONS: The well-known decrease in the apparent diffusion coefficient of WM after ischemia is found to be mainly driven by a significant drop in the intra-axonal diffusion microenvironment. Our results suggest that ischemia preferentially alters intra-axonal environment, consistent with a proposed mechanism of focal enlargement of axons known as axonal swelling or beading.