Quantification of the dopamine innervation in adult rat neostriatum

Abstract

Conditions leading to specific and integral visualization of dopamine axon terminals (varicosities) were tested in adult rat cerebral hemisphere slices incubated with [³H]dopamine and processed for high resolution radioautography. Specific visualization of the dopamine endings was achieved after incubation with 10⁻⁶ M [³H]dopamine in the presence of a monoamine oxidase inhibitor (pargyline 10⁻⁴ M), and of desipramine (5 × 10⁻⁶M), an inhibitor of catecholamine uptake by noradrenaline and serotonin neurons. [³H]Dopamine varicosity labeling was eliminated by the addition of 5 × 10⁻⁵M benztropine (an inhibitor of catecholamine uptake by catecholamine neurons), and was almost absent when dopamine nerve cell bodies of the midbrain had been previously destroyed with 6-hydroxydopamine. In dopamine-denervated neostriatum incubated without desipramine, a second set of labeled terminals was also visible. These were identified as serotoninergic, since their labeling was suppressed by citalopram, an inhibitor of monoamine uptake highly specific for serotonin neurons. There was no desipramine-sensitive but citalopram-resistant varicosity labeling suggestive of neostriatal noradrenaline innervation. In normal striatum, incubation at 35°C always resulted in a labeling of dopamine varicosities restricted to a narrow band which followed the contours and cut surfaces of this anatomical region. This unusual distribution was the result of an uptake barrier generated by the tightly packed dopamine varicosities. Indeed, the striatal dopamine varicosity labeling was more widespread after partial 6-hydroxydopamine denervation or in normal tissue incubated either with a higher [³H]dopamine concentration (5 × 10⁻⁶M), in the presence of relatively low benztropine concentrations (10⁻⁵ M), or at lower temperature (15°C). Material incubated at 15°C for 90 min was suitable for purposes of quantification: labeled varicosities were then visualized throughout the striatum and across the full thickness of the slices; moreover, the number of labeled varicosities plotted against radioautographic exposure time increased in parallel and reached a plateau at the same time in neostriatal sectors with widely different innervation densities. At a rostral transverse level across neostriatum, the dorsolateral quadrant showed hyperdense “patches” of labeled terminals distinguishable from an already dense surrounding “matrix”, whereas, ventromedially, the dopamine innervation appeared more uniform and somewhat less dense. Counts within these sectors and measurement of the average diameter of the labeled profiles in electron microscope radioautographs allowed to extrapolate the absolute number of dopamine varicosities to 1.0 × 10⁸ and 1.7 × 10⁸ per mm³ of tissue in the dorsolateral matrix and patches, respectively, and 0.6 × 10⁸ in the ventromedial sector. Correlation of such quantitated data with measurements of dopamine content, receptors, associated secondary messengers, recognition sites for uptake and/or other neuronochemical, hodological and cytological parameters, should eventually lead to a better understanding of the functional role of the dopamine input in the various compartments of neostriatum.