Faculty Bibliography

Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels

The wasp Nasonia vitripennis is emerging as a useful model organism in which to address a variety of biological questions, due, in part, to its ease of laboratory use, unique aspects of its biology and the sequencing of its genome. In order to take full advantage of the potential of this organism, methods for manipulating gene function are needed. To this end, a protocol for parental RNA interference (pRNAi) in N. vitripennis is described. pRNAi entails injecting pupae with double-stranded RNA, allowing the injected wasps to eclose and examining the progeny for developmental defects. This basic protocol is described in the context of the life cycle of N. vitripennis. This technique has been useful in elucidating the function of most, although not all, genes tested to date, and has potential applications beyond embryonic patterning. pRNAi experiments in Nasonia can be completed in as little as 2 weeks.

The ability to form tenable hypotheses regarding the neurobiological basis of normative functions as well as mechanisms underlying neurodegenerative and neuropsychiatric disorders is often limited by the highly complex brain circuitry and the cellular and molecular mosaics therein. The brain is an intricate structure with heterogeneous neuronal and nonneuronal cell populations dispersed throughout the central nervous system. Varied and diverse brain functions are mediated through gene expression, and ultimately protein expression, within these cell types and interconnected circuits. Large-scale high-throughput analysis of gene expression in brain regions and individual cell populations using modern functional genomics technologies has enabled the simultaneous quantitative assessment of dozens to hundreds to thousands of genes. Technical and experimental advances in the accession of tissues, RNA amplification technologies, and the refinement of downstream genetic methodologies including microarray analysis and real-time quantitative PCR have generated a wellspring of informative studies pertinent to understanding brain structure and function. In this review, we outline the advantages as well as some of the potential challenges of applying high throughput functional genomics technologies toward a better understanding of brain tissues and diseases using animal models as well as human postmortem tissues

We consider quantitative measures of behavioral and neuronal dynamics as a means of characterizing phenotypes. Such measures are important from a scientific perspective; because understanding brain function is contingent on understanding the link between the dynamics of the nervous system and behavioral dynamics. They are also important from a biomedical perspective because they provide a contrast to purely psychological characterizations of phenotype or characterizations via static brain images or maps, and are a potential means for differential diagnoses of neuropsychiatric illnesses. After a brief presentation of background work and some current advances, we suggest that more attention needs to be paid to dynamic characterizations of phenotypes. We will discuss some of the relevant time series analysis tools

This study investigated whether age and ADHD symptoms affected choice preferences in children and adolescents when they chose between (1) small immediate rewards and larger delayed rewards and (2) small certain rewards and larger probabilistic uncertain rewards. A temporal discounting (TD) task and a probabilistic discounting (PD) task were used to measure the degree to which the subjective value of a large reward decreased as one had to wait longer for it (TD), and as the probability of obtaining it decreased (PD). Rewards used were small amounts of money. In the TD task, the large reward (10 cents) was delayed by between 0 and 30s, and the immediate reward varied in magnitude (0-10 cents). In the PD task, receipt of the large reward (10 cents) varied in likelihood, with probabilities of 0, 0.25, 0.5, 0.75, and 1.0 used, and the certain reward varied in magnitude (0-10 cents). Age and diagnostic group did not affect the degree of PD of rewards: All participants made choices so that total gains were maximized. As predicted, young children, aged 6-11 years (n=25) demonstrated steeper TD of rewards than adolescents, aged 12-17 years (n=21). This effect remained significant even when choosing the immediate reward did not shorten overall task duration. This, together with the lack of interaction between TD task version and age, suggests that steeper discounting in young children is driven by reward immediacy and not by delay aversion. Contrary to our predictions, participants with ADHD (n=22) did not demonstrate steeper TD of rewards than controls (n=24). These results raise the possibility that strong preferences for small immediate rewards in ADHD, as found in previous research, depend on factors such as total maximum gain and the use of fixed versus varied delay durations. The decrease in TD as observed in adolescents compared to children may be related to developmental changes in the (dorsolateral) prefrontal cortex. Future research needs to investigate these possibilities

Pacemaker cells in the heart generate periodic electrical signals that are conducted to the working myocardium via the specialized conduction system. Effective cell-to-cell communication is critical for rapid, uniform conduction of cardiac action potentials-- a prerequisite for effective, synchronized cardiac contraction. Local circuit currents form the basis of the depolarization wave front in the working myocardium. These currents flow from cell to cell via gap junction channels. In this chapter, we trace the path of the action potential from its generation in the sinus node to propagation through the working myocardium, with a detailed discussion of the role of gap junctions. First, we review the transmembrane ionic currents and the basic principles of conduction of the action potential to the working myocardium via the specialized tissues of the heart. Next, we consider the relative contribution of cell geometry, size, and gap junction conductance. These factors are examined in terms of their source-to-sink relationships. Lastly, we will discuss new insights into the importance of gap junctions in cardiac conduction in health and disease which have been gained from high resolution optical mapping in connexin-deficient mice

Intracellular management of cholesterol is a critical process in the brain. Deficits with cholesterol transport and storage are linked to neurodegenerative disorders such as Neimann-Pick disease type C and Alzheimer's disease. One protein putatively involved in cholesterol transport is metastatic lymph node 64 (MLN64). MLN64 localizes to late endosomes which are part of the cholesterol internalization pathway. However, a detailed pattern of MLN64 expression in the brain is unclear. Using immunocytochemical and immunoblot analyses, we demonstrated the presence of MLN64 in several tissue types and various regions within the brain. MLN64 immunostaining in the CNS was heterogeneous, indicating selective expression in discrete specific cell populations and regions. MLN64 immunoreactivity was detected in glia and neurons, which displayed intracellular labeling consistent with an endosomal localization. Although previous studies suggested that MLN64 may promote steroid production in the brain, MLN64 immunoreactivity did not colocalize with steroidogenic cells in the CNS. These results demonstrate that MLN64 is produced in the mouse and human CNS in a restricted pattern of expression, suggesting that MLN64 serves a cell-specific function in cholesterol transport.