Assessment of the olfactory function in Italian patients with type 3 von Willebrand disease caused by a homozygous 253 Kb deletion involving VWF and TMEM16B/ANO2.

Type 3 Von Willebrand disease is an autosomal recessive disease caused by the virtual absence of the von Willebrand factor (VWF). A rare 253 kb gene deletion on chromosome 12, identified only in Italian and German families, involves both the VWF gene and the N-terminus of the neighbouring TMEM16B/ANO2 gene, a member of the family named transmembrane 16 (TMEM16) or anoctamin (ANO). TMEM16B is a calcium-activated chloride channel expressed in the olfactory epithelium. As a patient homozygous for the 253 kb deletion has been reported to have an olfactory impairment possibly related to the partial deletion of TMEM16B, we assessed the olfactory function in other patients using the University of Pennsylvania Smell Identification Test (UPSIT). The average UPSIT score of 4 homozygous patients was significantly lower than that of 5 healthy subjects with similar sex, age and education. However, 4 other members of the same family, 3 heterozygous for the deletion and 1 wild type, had a slightly reduced olfactory function indicating that socio-cultural or other factors were likely to be responsible for the observed difference. These results show that the ability to identify odorants of the homozygous patients for the deletion was not significantly different from that of the other members of the family, showing that the 253 kb deletion does not affect the olfactory performance. As other genes may compensate for the lack of TMEM16B, we identified some predicted functional partners from in silico studies of the protein-protein network of TMEM16B. Calculation of diversity for the corresponding genes for individuals of the 1000 Genomes Project showed that TMEM16B has the highest level of diversity among all genes of the network, indicating that TMEM16B may not be under purifying selection and suggesting that other genes in the network could compensate for its function for olfactory ability

Flash photolysis of caged compounds in the cilia of olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds(1). Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels(2). Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium(3) and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP(4) or caged Ca(5). We use a flash lamp(6,7) to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration(8-11)

ENDOCANNABINOID-MEDIATED PLASTICITY AT INHIBITORY SYNAPSES ONTO MIDBRAIN DOPAMINE NEURONS AS A POSSIBLE MARKER OF VOLUNTARY ALCOHOL PREFERENCE

Alcoholism is a psychiatric disorder, whose aetiology involves inherited predispositions and environmental factors. Alcohol activates the brain reward circuitry, which stems from the ventral tegmental area (VTA) where dopamine (DA) cells are located. Among synaptic inputs that regulate DA neuron impulse activity, those arising from the newly identified rostromedial tegmental nucleus (RMTg) play a major role. DA neurons can escape their afferent control by releasing endocannabinoids, which serve as retrograde signaling molecules at many synapses in the brain. In this study we took advantage of significant differences between pairs of lines of rats selectively bred for their voluntary alcohol preference or aversion, that is the Sardinian alcohol-preferring (sP) or nonpreferring (sNP) rat line and investigated their electrophysiological properties and synaptic plasticity both in vivo and in vitro. Extracellular single unit recordings in anesthetized rats revealed a difference in baseline firing activity of DA neurons between sP and sNP rats consistent with our previous study (Melis et al., 2009). More particularly, sP rats showed an increased spontaneous neuronal activity that was paralleled by a reduced strength of RMTg inputs. This enhanced firing activity of DA cells in sP rats negatively correlated with the duration of inhibition elicited by electrical stimulation of the RMTg. We next examined one form of short-term synaptic plasticity mediated by endocannabinoids that is depolarization-induced suppression of inhibition (DSI), at inhibitory synapses of DA cells. We have found that DSI is differently expressed by two discrete sets of inhibitory synapses arising from rostral and caudal afferents onto VTA DA neurons. This phenomenon is selectively mediated by the endocannabinoid 2-arachidonoylglycerol (2-AG), which activates presynaptic type 1-cannabinoid (CB1) receptors. However, the two discrete DSI do not seem to depend upon differences in CB1 number and/or function, but upon the rate 2-AG is degraded. Thus, 2-AG by differently depressing inhibitory synapses arising from either rostral or caudal afferents might indirectly alter DA neuron functional state, and enhance the responsiveness of the reward pathway to phasic DA. Given that sP rats are vulnerable phenotype, and that they possess this endocannabinoid-mediated DSI, our results suggest that differences in the equipment of the endocannabinoid system machinery might control specific sources of vulnerability