Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling.

Opioid tolerance is well-described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here, we delineate a cellular mechanism underlying this longer-term form of opioid tolerance in cultured rat medium spiny neurons. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the µ-opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptor's cytoplasmic tail, and that partial or biased agonist drugs with reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the δ-opioid receptor (DOR) conforms to the same general paradigm except that presynaptic endocytosis of DOR, in contrast to MOR, does not require phosphorylation of the receptor's cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.

Rab34 GTPase mediates ciliary membrane formation in the intracellular ciliogenesis pathway.

The primary cilium is an essential organizing center for signal transduction, and ciliary defects cause congenital disorders known collectively as ciliopathies.1-3 Primary cilia form by two pathways that are employed in a cell-type- and tissue-specific manner: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium first forms inside the cell.4-8 After exposure to the external environment, cilia formed via the intracellular pathway may have distinct functional properties, as they often remain recessed within a ciliary pocket.9,10 However, the precise mechanism of intracellular ciliogenesis and its relatedness to extracellular ciliogenesis remain poorly understood. Here we show that Rab34, a poorly characterized GTPase recently linked to cilia,11-13 is a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by polarized MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and its paralog Rab36. Together, these findings establish that different modes of ciliogenesis have distinct molecular requirements and reveal Rab34 as a new GTPase mediator of ciliary membrane biogenesis.

WAIS-III task perception and performance as a function of stopwatch display.

Examiner's instructions for the Wechsler Adult Intelligence Scale-Third Edition indicate that stopwatch display is optional. Since verbal instructions do not consistently inform examinees whether a subtest is timed, examinees may rely upon the visual cue of the stopwatch. Furthermore, examinees' understanding of the actual subtest demands may affect their performances. In the present study, 58 volunteers were randomly assigned to two groups, one in which the examiner displayed the stopwatch and one in which the stopwatch was concealed. Participants were administered the first five subtests. Predictions were that (1) participants to whom the stopwatch was displayed would more accurately perceive whether the subtests were timed, and (2) participants who accurately understood the demands of subtests would score higher than those who did not. Results supported the first hypothesis for three subtests out of five, one remaining inconclusive due to a ceiling effect. The second hypothesis was not supported. Both the implications and limitations of the present study are discussed.

Wais-III matrix reasoning: instruction effects on task perception and performance.

Instructions for the Matrix Reasoning subtest of the WAIS-III do not communicate to examinees that the subtest is untimed. The present study examined what percentage of participants (34 women, 26 men, M age = 20.1; 55 Caucasian, 3 African American, 2 Hispanic) made the assumption that Matrix Reasoning was timed, and its effect on examinees' scores. 55% of participants receiving standard instructions retrospectively reported assuming the subtest was timed, and those who did not assume Matrix Reasoning was timed scored significantly higher than participants who did. Participants receiving additional instructions that clarified the untimed nature of Matrix Reasoning scored significantly higher than those receiving standard instructions who believed the subtest was timed.