Inner ear epithelia of mature birds regenerate hair cells after ototoxic or acoustic insult. The lack of markers that selectively label cells in regenerating epithelia and of culture systems composed primarily of progenitor cells has hampered the identification of cellular and molecular interactions that regulate hair cell regeneration. In control basilar papillae, we identified two markers that selectively label hair cells (calmodulin and TUJ1 ␤ tubulin antibodies) and one marker unique for support cells (cytokeratin antibodies). Examination of regenerating epithelia demonstrated that calmodulin and ␤ tubulin are also expressed in early differentiating hair cells, and cytokeratins are retained in proliferative support cells. Enzymatic and mechanical methods were used to isolate sensory epithelia from mature chick basilar papillae, and epithelia were cultured in different conditions. In control cultures, hair cells are morphologically stable for up to 6 d, because calmodulin immunoreactivity and phalloidin labeling of filamentous actin are retained. The addition of an ototoxic antibiotic to cultures, however, causes complete hair cell loss by 2 d in vitro and generates cultures composed of calmodulinnegative, cytokeratin-positive support cells. These cells are highly proliferative for the first 2-7 d after plating, but stop dividing by 9 d. Calmodulin-or TUJ1-positive cells reemerge in cultures treated with antibiotic for 5 d and maintained for an additional 5 d without antibiotic. A subset of calmodulinpositive cells was also labeled with BrdU when it was continuously present in cultures, suggesting that some cells generated in culture begin to differentiate into hair cells. Key words: hair cells; regeneration; chick; basilar papilla; cell culture; differentiationHair cells are sensory receptors for hearing, equilibrium, and motion detection. Some animals demonstrate the capacity to generate hair cells throughout their lifetime (Popper and Hoxter, 1984;Corwin, 1985; Jörgenson and Mathiessen, 1989;Roberson et al., 1992) or to initiate hair cell regeneration in the event of their loss (Corwin and Cotanche, 1988;Ryals and Rubel, 1988). The progenitors of hair cells seem to be a subset of support cells that reside adjacent to hair cells in the sensory epithelia (Girod et al., 1989;Balak et al., 1990;Raphael, 1992;Hashino and Salvi, 1993;Weisleder and Rubel, 1993;Stone and Cotanche, 1994;Tsue et al., 1994a;Roberson et al., 1996). Although mature mammals normally do not generate new hair cells, recent in vivo and in vitro studies have documented mitotic activity and immature-looking hair cells in mammalian vestibular epithelia after exposure to ototoxic drugs Warchol et al., 1993;Rubel et al., 1995), suggesting that hair cell regeneration in mammals may be inducible. The development of culture methods for mature cochlear and vestibular end organs has been initiated to identify molecules that regulate cell proliferation and differentiation in avian and mammalian hair cell epithelia. Co-culture experiments suggest that...

Acute desensitization of olfactory signaling is a critical property of the olfactory system that allows animals to detect and respond to odorants. Correspondingly, an important feature of odorant-stimulated cAMP increases is their transient nature, a phenomenon that may be attributable to the unique regulatory properties of the olfactory adenylyl cyclase (AC3). AC3 is stimulated by receptor activation and inhibited by Ca2+ through Ca2+/calmodulin kinase II (CaMKII) phosphorylation at Ser-1076. Since odorant-stimulated cAMP increases are accompanied by elevated intracellular Ca2+, CaMKII inhibition of AC3 may contribute to termination of olfactory signaling. To test this hypothesis, we generated a polyclonal antibody specific for AC3 phosphorylated at Ser-1076. A brief exposure of mouse olfactory cilia or primary olfactory neurons to odorants stimulated phosphorylation of AC3 at Ser-1076. This phosphorylation was blocked by inhibitors of CaMKII, which also ablated cAMP decreases associated with odorant-stimulated cAMP transients. These data define a novel mechanism for termination of olfactory signaling that may be important in olfactory responses.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT 3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT 6 and 5-HT 7 couple to stimulation of adenylyl cyclases through G s and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT 6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT 7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT 6 or 5-HT 7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT 6 receptor functioned as a typical G s -coupled receptor in that it stimulated AC5, a G s -sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by G s -coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT 7A stimulated AC1 and AC8 by increasing intracellular Ca 2؉ . 5-HT also increased intracellular Ca 2؉ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin. Serotonin (5-hydroxytryptamine, 5-HT)1 is a ubiquitous neurotransmitter that elicits a variety of physiological effects peripherally and centrally (1-3). A growing family of plasma membrane receptors bind 5-HT and mediate its cellular effects (4). All of the 5-HT receptors except 5-HT 3 belong to the superfamily of G protein-coupled receptors. The recently cloned 5-HT 6 (5, 6) and 5-HT 7 (7-10) receptors activate adenylyl cyclase(s) through the heterotrimeric G protein G s , and expression of these receptors in cultured cells couples serotonin to increases in cAMP (6,8). 5-HT 6 and 5-HT 7 display high affinities for antipsychotic and antidepressant drugs including clozapine, amoxapine, and amitryptiline (5-8), suggesting a role for these receptors in affective function. In the brain, the 5-HT 6 receptor is most highly expressed in the hippocampus, nucleus accumbens, striatum, and limbic regions, and the 5-HT 7 receptor is found in the hypothalamus, hippocampus, and cortex. The distribution of these receptors in brain is consistent with the hypothesis that they play a role in mood and affect (8,(11)(12)(13). In addition, the expression of the 5-HT 7 receptor in the suprachiasmatic nucleus (SCN) and the ability of serotonin and cAMP to advance the mammalian circadian rhythm indicates that 5-HT 7 plays an important role in circadian physiology (10, 14, 15). 5-HT 7 receptors are also expressed in glial cells (16,17) and at lower levels in peripheral tissues including the spleen, intestine, and vascular smooth muscle (7-9, 18).Many psychotherapeutic agents modulate the cAMP signaling pathway (19) and perturbation of this signal transduction system may contribute to some affective disorders. To date, nine distinct cDNA clones for mammalian...

Globalization of ADHD and the rise of cognitive enhancement have raised fresh concerns about the validity of ADHD diagnosis and the ethics of stimulant drug treatment. We review the literature on these two emerging phenomena, with a focus on the corresponding social, scientific and ethical debates over the universality of ADHD and the use of stimulant drug treatments in a global population of children and adolescents. Drawing on this literature, we reflect on the importance of ethically informed, ecologically sensitive clinical practices in relation to ADHD diagnosis and treatment.

Arterial smooth muscle cell (SMC) proliferation contributes to a number of vascular pathologies. Prostaglandin E 2 (PGE 2 ), produced by the endothelium and by SMCs themselves, acts as a potent SMC growth inhibitor. The growth-inhibitory effects of PGE 2 are mediated through activation of G-protein-coupled membrane receptors, activation of adenylyl cyclases (ACs), formation of cAMP, and subsequent inhibition of mitogenic signal transduction pathways in SMCs. Of the 10 different mammalian AC isoforms known today, seven isoforms (AC2-7 and AC9) are expressed in SMCs from various species. We show that, despite the presence of several different AC isoforms, the principal AC isoform activated by PGE 2 in human arterial SMCs is a calmodulin kinase II-inhibited AC with characteristics similar to those of AC3. AC3 is expressed in isolated human arterial SMCs and in intact aorta. We further show that arterial SMCs isolated from AC3-deficient mice are resistant to PGE 2 -induced growth inhibition. In summary, AC3 is the principal AC isoform activated by PGE 2 in arterial SMCs, and AC3 mediates the growth-inhibitory effects of PGE 2 . Because AC3 activity is inhibited by intracellular calcium through calmodulin kinase II, AC3 may serve as an important integrator of growth-inhibitory signals that stimulate cAMP formation and growth factors that increase intracellular calcium.