Dopamine axon varicosities in the prelimbic division of the rat prefrontal cortex exhibit sparse immunoreactivity for the dopamine transporter - PubMed (original) (raw)

Comparative Study

Dopamine axon varicosities in the prelimbic division of the rat prefrontal cortex exhibit sparse immunoreactivity for the dopamine transporter

S R Sesack et al. J Neurosci. 1998.

Abstract

The dopamine transporter (DAT) critically regulates the duration of the cellular actions of dopamine and the extent to which dopamine diffuses in the extracellular space. We sought to determine whether the reportedly greater diffusion of dopamine in the rat prefrontal cortex (PFC) as compared with the striatum is associated with a more restricted axonal distribution of the cortical DAT protein. By light microscopy, avidin-biotin-peroxidase immunostaining for DAT was visualized in fibers that were densely distributed within the dorsolateral striatum and the superficial layers of the dorsal anterior cingulate cortex. In contrast, DAT-labeled axons were distributed only sparsely to the deep layers of the prelimbic cortex. By electron microscopy, DAT-immunoreactive profiles in the striatum and cingulate cortex included both varicose and intervaricose segments of axons. However, DAT-labeled processes in the prelimbic cortex were almost exclusively intervaricose axon segments. Immunolabeling for tyrosine hydroxylase in adjacent sections of the prelimbic cortex was localized to both varicosities and intervaricose segments of axons. These qualitative observations were supported by a quantitative assessment in which the diameter of immunoreactive profiles was used as a relative measure of whether varicose or intervaricose axon segments were labeled. These results suggest that considerable extracellular diffusion of dopamine in the prelimbic PFC may result, at least in part, from a paucity of DAT content in mesocortical dopamine axons, as well as a distribution of the DAT protein at a distance from synaptic release sites. The results further suggest that different populations of dopamine neurons selectively target the DAT to different subcellular locations.

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Figures

Fig. 1.

Schematic drawings illustrating the regions sampled in coronal sections of rat forebrain. _Shading_indicates the approximate density of the dopamine innervation to the prelimbic division of the PFC (PL), which is heaviest in layers V–VI, the rostral anterior cingulate cortex (CING), which is most dense in layers I–III, and the corpus striatum (STR), which receives a dense dopamine input throughout its dorsoventral extent. The black trapezoids indicate the regions sampled by electron microscopy. These regions also are illustrated by light microscopy in Figure 3.ac, Anterior commissure; cc, corpus callosum.

Fig. 2.

Schematic drawing illustrating the measurement of diameter after the perimeter of immunolabeled profiles has been traced. The representative profiles include (a) a varicosity cut in cross section, (b) a longitudinally sectioned axon, and (c) an eccentrically shaped profile representing both varicose and intervaricose segments of an axon. In each case the number of pixel layers from the perimeter is counted until each counted pixel is bounded by other counted pixels. This represents the pixel radius, which is doubled to obtain the pixel diameter (arrows) and then converted to micrometers with a calibration. This dimension represents the maximal diameter through the short axis of each profile.

Fig. 3.

Light micrographs illustrating peroxidase immunoreactivity for DAT in the rat forebrain. A, In the dorsolateral striatum, dense peroxidase product for DAT is localized to the neuropil immediately beneath the corpus callosum (cc). Perikarya (asterisks) and bundles of myelinated axons (m) are unlabeled.B, No DAT immunoreactivity is detected in the same striatal region of sections incubated in primary antibody preadsorbed with the DAT antigen. C, In the rostral portion of the anterior cingulate cortex, a dense cluster of DAT-immunoreactive fibers is visualized in layer III. These presumed axons exhibit the branching (small arrows) and beading (large arrows) that are characteristic of terminal fibers. D, In layer VI of the prelimbic cortex from the same section as that shown in_C_, sparse fibers immunoreactive for DAT are observed. Although some are beaded or branched, others appear to be fibers of passage (open arrows) exiting the white matter. In_A–D_, up is dorsal and_left_ is medial. Scale bar, 150 μm.

Fig. 4.

Electron micrographs illustrating peroxidase immunoreactivity for DAT in the rat forebrain. A, In the dorsolateral striatum, immunoreactivity for DAT is localized to axon varicosities (DAT-v) that contain numerous synaptic vesicles surrounded by dense peroxidase product. One immunoreactive varicosity appears to form a small symmetric synapse on a dendritic process (thick arrow). B, In the cingulate cortex, DAT immunoreactivity is observed both in a varicose structure and in small axon-like profiles (DAT-a, thin arrows), some of which contain synaptic vesicles.C, In the prelimbic cortex, immunoreactivity for DAT is seen almost exclusively in small axon-like profiles (thin arrows). Immunonegative structures that exhibit similar size and morphology are indicated at the open arrows.D, DAT immunolabeling in the prelimbic cortex is visualized clearly in an axon cut longitudinally (thin arrow), whereas the contiguous varicosity forms a synapse on a dendritic spine (thick arrow) but otherwise is unlabeled for DAT. Scale bar, 0.5 μm.

Fig. 5.

Electron micrographs illustrating peroxidase immunoreactivity for TH in the rat prelimbic PFC. In the deep layers of the prelimbic cortex, peroxidase immunoreactivity for TH was localized in axon varicosities (TH-v) as well as in intervaricose axon segments (TH-a, thin arrow). The varicosities sometimes formed synapses on small dendrites (thick arrows). Scale bar, 0.5 μm.

Fig. 6.

Histogram illustrating the frequency of diameters measured for profiles immunoreactive for DAT or TH in the rat forebrain. Each bar represents the total number (frequency) of profiles having a particular diameter that were immunoreactive for DAT in the prelimbic cortex (PL,black), anterior cingulate cortex (CING,hatched), or dorsolateral striatum (STR,gray) or for tyrosine hydroxylase in the prelimbic cortex (TH, white). For each region 30 profiles for each of six animals are represented in the case of DAT and for each of three animals in the case of TH. The histogram includes all of the raw data from which means and SD were calculated; see Results for the statistical analysis.

Fig. 7.

Schematic diagrams illustrating the putative distribution of DAT proteins in the dorsolateral striatum and prelimbic cortex. In the striatum, dopamine released at synaptic sites is subject to extensive re-uptake by DAT localized to the plasma membrane adjacent to the presynaptic zone and along preterminal portions of these axons. In the prelimbic PFC, evidence for perisynaptic DAT is lacking, although a low density of DAT protein is observed along preterminal portions of presumed dopamine axons. Thus, synaptically released dopamine is subject to less extensive uptake and greater extracellular diffusion. The functional impact of this extracellular dopamine may be limited by the low density of receptors for dopamine, which are substantially less abundant in the cortex than in the striatum.

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Dopamine axon varicosities in the prelimbic division of the rat prefrontal cortex exhibit sparse immunoreactivity for the dopamine transporter - PubMed (original) (raw)