Dopamine-mediated volume transmission in midbrain is regulated by distinct extracellular geometry and uptake.

Somatodendritic release of dopamine (DA) in midbrain is, at least in part, nonsynaptic; moreover, midbrain DA receptors are predominantly extrasynaptic. Thus somatodendritic DA mediates volume transmission, with an efficacy regulated by the diffusion and uptake characteristics of the local extracellular microenvironment. Here, we quantitatively evaluated diffusion and uptake in substantia nigra pars compacta (SNc) and reticulata (SNr), ventral tegmental area (VTA), and cerebral cortex in guinea pig brain slices. The geometric parameters that govern diffusion, extracellular volume fraction (alpha) and tortuosity (lambda), together with linear uptake (k'), were determined for tetramethylammonium (TMA(+)), and for DA, using point-source diffusion combined with ion-selective and carbon-fiber microelectrodes. TMA(+)-diffusion measurements revealed a large alpha of 30% in SNc, SNr, and VTA, which was significantly higher than the 22% in cortex. Values for lambda and k' for TMA(+) were similar among regions. Point-source DA-diffusion curves fitted theory well with linear uptake, with significantly higher values of k' for DA in SNc and VTA (0.08--0.09 s(-1)) than in SNr (0.006 s(-1)), where DA processes are sparser. Inhibition of DA uptake by GBR-12909 caused a greater decrease in k' in SNc than in VTA. In addition, DA uptake was slightly decreased by the norepinephrine transport inhibitor, desipramine in both regions, although this was statistically significant only in VTA. We used these data to model the radius of influence of DA in midbrain. Simulated release from a 20-vesicle point source produced DA concentrations sufficient for receptor activation up to 20 microm away with a DA half-life at this distance of several hundred milliseconds. Most importantly, this model showed that diffusion rather than uptake was the most important determinant of DA time course in midbrain, which contrasts strikingly with the striatum where uptake dominates. The issues considered here, while specific for DA in midbrain, illustrate fundamental biophysical properties relevant for all extracellular communication

Three-dimensional confocal morphometry – a new approach for studying dynamic changes in cell morphology in brain slices

Pathological states in the central nervous system lead to dramatic changes in the activity of neuroactive substances in the extracellular space, to changes in ionic homeostasis and often to cell swelling. To quantify changes in cell morphology over a certain period of time, we employed a new technique, three-dimensional confocal morphometry. In our experiments, performed on enhanced green fluorescent protein/glial fibrillary acidic protein astrocytes in brain slices in situ and thus preserving the extracellular microenvironment, confocal morphometry revealed that the application of hypotonic solution evoked two types of volume change. In one population of astrocytes, hypotonic stress evoked small cell volume changes followed by a regulatory volume decrease, while in the second population volume changes were significantly larger without subsequent volume regulation. Three-dimensional cell reconstruction revealed that even though the total astrocyte volume increased during hypotonic stress, the morphological changes in various cell compartments and processes were more complex than have been previously shown, including swelling, shrinking and structural rearrangement. Our data show that astrocytes in brain slices in situ during hypotonic stress display complex behaviour. One population of astrocytes is highly capable of cell volume regulation, while the second population is characterized by prominent cell swelling, accompanied by plastic changes in morphology. It is possible to speculate that these two astrocyte populations play different roles during physiological and pathological states