Abstract:The vesicular contents in bovine chromaffin cells are maintained at high levels owing to the strong association of its contents, which is promoted by the low vesicular pH. The association is among the catecholamines, Ca 2ϩ , ATP, and vesicular proteins. It was found that transient application of a weak base, methylamine (30 mM), amphetamine (10 M), or tyramine (10 M), induced exocytotic release. Exposure to these agents was also found to increase both cytosolic catecholamine and intracellular Ca 2ϩ concentration, as measured by amperometry and fura-2 fluorescence. Amphetamine, the most potent amine with respect to evoking exocytosis, was found to be effective even in buffer without external Ca 2ϩ ; however, the occurrence of spikes was suppressed when BAPTA-acetoxymethyl ester was used to complex intracellular Ca 2ϩ . Amphetamine-induced spikes in Ca 2ϩ -free medium were not suppressed by thapsigargin or ruthenium red, inhibitors of the sarco-(endo)plasmic reticulum Ca 2ϩ -ATPase and mitochondrial Ca 2ϩ stores. Atomic absorption measurements of amphetamine-and methylamine-treated vesicles reveal that intravesicular Ca 2ϩ stores are decreased after a 15-min incubation. Taken together, these data indicate that amphetamine and methylamine can disrupt vesicular stores to a sufficient degree that Ca 2ϩ can escape and trigger exocytosis.

Monitoring the release and uptake of catecholamines from terminals in weakly innervated brain regions is an important step in understanding their importance in normal brain function. To that end, we have labeled brain slices from transgenic mice that synthesize placental alkaline phosphatase (PLAP) on neurons containing tyrosine hydroxylase with antibody±¯uorochrome conjugate, PLAP-Cy5. Excitation of the¯uorochrome enables catecholamine neurons to be visualized in living tissue. Immunohistochemical¯uorescence with antibodies to tyrosine hydroxylase and dopamine b-hydroxylase revealed that the PLAP labeling was speci®c to catecholamine neurons. In the prefrontal cortex (PFC), immunohistochemical¯uorescence of the PLAP along with staining for dopamine transporter (DAT) and norepinephrine transporter (NET) revealed that all three exhibit remarkable spatial overlap. Fluorescence from the PLAP antibody was used to position carbon-®ber microelectrodes adjacent to catecholamine neurons in the PFC. Following incubation with L-DOPA, catecholamine release and subsequent uptake was measured and the effect of uptake inhibitors examined. Release and uptake in NET and DAT knockout mice were also monitored. Uptake rates in the cingulate and prelimbic cortex are so slow that catecholamines can exist in the extracellular¯uid for suf®cient time to travel ,100 mm. The results support heterologous uptake of catecholamines and volume transmission in the PFC of mice.

Effects of vesicular monoamine transporter inhibitors on catecholamine release from bovine chromaffin cells have been examined at the level of individual exocytotic events. As expected for a depletion of vesicular stores, release evoked by depolarizing agents was decreased following 15-min incubations with reserpine and tetrabenazine, as evidenced by a decrease in exocytotic frequency and amount released per event. In contrast, two reserpine derivatives, methyl reserpate and reserpic acid, were much less effective. Surprisingly, the incubations also decreased the accompanying rise in intracel-

The release and clearance of electrically evoked catecholamine (CA) in the ventral portion of the bed nucleus of the stria terminalis (BSTV) in mouse brain slices was evaluated with fast-scan cyclic voltammetry at carbon-fiber microelectrodes (CFME). Uptake in this region was observed to be markedly slower than in the caudate putamen (CPu). Clearance rates were reduced in the BSTV in both norepinephrine transporter knockout (NET KO) and dopamine transporter knockout (DAT KO) mice when compared to results in wild-type (WT) mice. However, uptake was faster in the BSTV in both the DAT and NET KO mice than in the CPu of DAT KO mice. This indicates that both transporters play a role in CA clearance in the BSTV. The transporters restrict extracellular CA to the general area of the BSTV, as revealed by the diminished signal as the CFME is moved sequentially further and further from the site where CA release is evoked. However, in slices from the DAT KOs and NET KOs, CA release could be observed outside of the BSTV region during such experiments. These results show that the low rate uptake in the BSTV facilitates extrasynaptic diffusion of catecholamine, but that uptake still regulates and limits the range of the transmitter to the region. Slower clearance from the extracellular fluid allows the released CA to act as a volume transmitter and diffuse to distant sites within the region to exert its neurochemical action.

The oxidation of catecholamines with a carbon-fiber electrode can be used to monitor exocytosis at the single cell level at a variety of different types of cells. These measurements allow release to be followed from individual vesicles and have revealed several unique aspects concerning the coupling between release and storage. The strong association of the vesicular components in chromaffin cells dictates the time course of extrusion of the vesicle contents. Furthermore, liberation of the Ca(2+) normally stored within the vesicles can promote exocytosis without an external Ca(2+) source.