C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.

The ability to control osmotic balance is essential for cellular life. Cellular osmotic homeostasis is maintained by accumulation and loss of inorganic ions and organic osmolytes. Although osmoregulation has been studied extensively in many cell types, major gaps exist in our molecular understanding of this essential process. Because of its numerous experimental advantages, the nematode Caenorhabditis elegans provides a powerful model system to characterize the genetic basis of animal cell osmoregulation. We therefore characterized the ability of worms to adapt to extreme osmotic stress. Exposure of worms to high-salt growth agar causes rapid shrinkage. Survival is normal on agar containing up to 200 mM NaCl. When grown on 200 mM NaCl for 2 wk, worms are able to survive well on agar containing up to 500 mM NaCl. HPLC analysis demonstrated that levels of the organic osmolyte glycerol increase 15- to 20-fold in nematodes grown on 200 mM NaCl agar. Accumulation of glycerol begins 3 h after exposure to hypertonic stress and peaks by 24 h. Glycerol accumulation is mediated primarily by synthesis from metabolic precursors. Consistent with this finding, hypertonicity increases transcriptional expression of glycerol 3-phosphate dehydrogenase, an enzyme that is rate limiting for hypertonicity-induced glycerol synthesis in yeast. Worms adapted to high salt swell and then return to their initial body volume when exposed to low-salt agar. During recovery from hypertonic stress, glycerol levels fall rapidly and glycerol excretion increases approximately fivefold. Our studies provide the first description of osmotic adaptation in C. elegans and provide the foundation for genetic and functional genomic analysis of animal cell osmoregulation.

The inwardly rectifying Cl(-) current in C. elegans oocytes is due to the activity of a ClC channel encoded by clh-3. Functional and structural similarities suggest that CLH-3 and mammalian ClC-2 are orthologs. CLH-3 is activated during oocyte meiotic maturation and functions in part to modulate ovulatory contractions of gap junction-coupled gonadal sheath cells.

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7α and GLC-7β. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle– and cell volume–regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl− content, cell volume, and epithelial fluid secretion.

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.

This study of continuing care for substance dependent patients compared a telephone-based monitoring and brief counseling intervention (TEL) with 2 face-to-face interventions, relapse prevention (RP) and standard 12-step group counseling (STND). The participants were graduates of intensive outpatient programs who had current dependence on alcohol and/or cocaine. Self-report, collateral, and biological measures of alcohol and cocaine use were obtained over a 12-month follow-up. The treatment groups did not differ on abstinence-related outcomes in the complete sample (N = 359) or on cocaine use outcomes in participants with cocaine dependence (n = 268). However, in participants with alcohol dependence only (n = 91), TEL produced better alcohol use outcomes than STND on all measures examined and better outcomes than RP on some of the measures.

Psychological distress among college students represents a serious health concern. The aim of this research was to investigate, for the first time, whether interactions between rumination and different measures of stress could differentially predict components of psychological distress, within a diathesis-stress framework. This self-report study employed a longitudinal design, spanning a period of 6 months. One hundred sixty-one undergraduate college students completed selected measures of psychological distress, rumination, and stress at two time points 6 months apart. Both independent and interaction effects were examined through hierarchical regression analyses. Rumination and stress were found to interact significantly to predict the social dysfunction components of psychological distress. Other main effects are reported. The evidence supported the proposed diathesis-stress model and extended previous research by relating rumination to different components of psychological distress prospectively.

Rumination has been persistently implicated in the etiology of hopelessness and depression: proximal predictors of suicidality. As a result, research has started to examine the role of rumination in suicidality. This systematic review aims to: (i) provide a concise synopsis of the current progress in examining the relationship between rumination and suicidality; and (ii) highlight areas for future research. To this end, a search of the international literature was conducted using the three main psychological and medical databases (Psych Info [1887-October 2007, Medline [1966[ -October 2007 and Web of Knowledge [1981-October 2007).Eleven studies were identified providing evidence, with one exception, of a relationship between rumination and suicidality. This systematic review has highlighted a considerable dearth of studies, specifically of case-control and prospective, clinical studies, in the worldwide literature. Key areas for future research are discussed.