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Exocytosis is an essential cellular trafficking process integral to the proper distribution and function of a plethora of molecules, including transporters, receptors, and enzymes. Moreover, incorrect protein targeting can lead to pathological conditions. Recently, the application of evanescent wave microscopy has allowed us to image the final steps of exocytosis. However, spatio-temporal analysis of fusion of constitutive vesicular traffic with the plasma membrane has not been systematically performed. Also, the spatial sites and times of vesicle fusion have not yet been analyzed together. In addition, more formal tests are required in testing biological hypotheses, rather than visual inspection combined with statistical descriptives. Ripley K-functions are used to examine the joint and marginal behavior of locations and fusion times. Semiautomatic detection and mapping of constitutive fusion sites reveals spatial and temporal clustering, but no dependency between the locations and times of fusion events. Our novel approach could be translated to other studies of membrane trafficking in health and diseases such as diabetes.

The lateral nucleus of the amygdala (LA) is a site of convergence for auditory (conditioned stimulus) and footshock (unconditioned stimulus) inputs during fear conditioning. The auditory pathways to LA are well characterized, but less is known about the pathways through which footshock is transmitted. Anatomical tracing and physiological recording studies suggest that the posterior intralaminar thalamic nucleus, which projects to LA, receives both auditory and somatosensory inputs. In the present study we examined the expression of the immediate-early gene c-fos in the LA in rats in response to footshock stimulation. We then determined the effects of posterior intralaminar thalamic lesions on footshock-induced c-Fos expression in the LA. Footshock stimulation led to an increase in the density of c-Fos-positive cells in all LA subnuclei in comparison to controls exposed to the conditioning box but not shocked. However, some differences among the dorsolateral, ventrolateral and ventromedial subnuclei were observed. The ventrolateral subnucleus showed a homogeneous activation throughout its antero-posterior extension. In contrast, only the rostral aspect of the ventromedial subnucleus and the central aspect of the dorsolateral subnucleus showed a significant increment in c-Fos expression. The density of c-Fos-labeled cells in all LA subnuclei was also increased in animals placed in the box in comparison to untreated animals. Unilateral electrolytic lesions of the posterior intralaminar thalamic nucleus and the medial division of the medial geniculate body reduced footshock-induced c-Fos activation in the LA ipsilateral to the lesion. The number of c-Fos labeled cells on the lesioned side was reduced to the levels observed in the animals exposed only to the box. These results indicate that the LA is involved in processing information about the footshock unconditioned stimulus and receives this kind of somatosensory information from the posterior intralaminar thalamic nucleus and the medial division of the medial geniculate body. KeywordsEmotional learning; fear conditioning; pain; freezing; electrolytic lesions; somatosensory thalamus Address NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn recent years classical fear conditioning has become a leading model for studying the neural mechanisms of learning and memory in mammals (LeDoux, 2000;Maren, 2005). In this behavioral paradigm, a neutral (innocuous) conditioned stimulus (CS), such as a tone, is presented in association with an aversive unconditioned stimulus (US), such as a mild footshock. After a few such paired presentations, the animal begins to respond defensively to the neutral stimulus. Considerable evidence suggests the lateral nucleus of the amygdala (LA) is a site of auditory CS and somatosensory US convergence (Romanski et al., 1993;Bordi and LeDoux, 1994b) and is crucial for the formation of the association between the CS and US (Blair et al, 2001;Fanselow and LeDoux, 1999;LeDoux, 2000;Maren, 2001Maren, , 2005; but...

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