SUMMARYThe main goal of this study was to develop a better light microscopic procedure for quantitative study of the cellular co-localization of neuropeptides in adult human brain tissue. To reach this goal, we opted for a method (proved to be optimal on rat brain) in which sections were double immunolabeled with two different fluorophore-conjugated secondary antibodies and analyzed with a confocal laser scanning fluorescence microscope. One of our main problems faced was a strong autofluorescence of the sections, mainly caused by lipofuscin granules normally present in adult human brain tissue, which made any analysis of specific fluorescence impossible. This problem could be solved by staining the sections after immunolabeling with the dye Sudan Black B, which completely blocked this autofluorescence. The complete optimized procedure that we eventually developed can be summarized as follows. After a relatively short fixation time (6-14 days) in 4% freshly depolymerized paraformaldehyde, the resected brain tissue can best be stored in a 30% sucrose solution supplemented with 0.05% NaN 3 at 4C. Stored under these conditions, cryosections from the tissue still reveal good histology and allow successful immunocytochemical staining after a period of 6 months. Double immunolabeling is done by incubating cryo-or paraffin sections in a mixture of two primary antibodies directed against the targeted antigens, followed by incubation with two different fluorophore-conjugated secondary antibodies. Amplification with a biotinylated secondary antibody followed by fluorophore-conjugated streptavidin is possible. Finally, the sections are stained with Sudan Black B, mounted in plain 80% Tris-buffered glycerol, and studied by confocal laser scanning fluorescence microscopy. Sections processed in this way are well suited for qualitative and quantitative analyses of co-localized neuropeptides in human brain tissue.

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On-line in vivo microdialysis was used to determine the effects of a 16-mm handling period on release of dopamine (DA) in the nucleus accumbens and of DA and noradrenaline (NA) in the medial prefrontal cortex of awake, freely moving rats. DA and NA were determined in one HPLC run. Handling resulted in an immediate and strong increase of both catecholamines in the prefrontal cortex. Maximal values for DA were 295%, and for NA 225%, of controls. DA in the nucleus accumbens was also increased (to 135% of controls) but only after a short delay. Local inhibition of ionotropic glutamate receptors by continuous reversed dialysis of the drugs 6-cyano-7-nitroquinoxaline, D-2-amino-5-phosphonopentanoic acid, or dizocilpine did not significantly affect handling-induced increases in cortical DA and NA release. Neither did the agonist of metabotropic glutamate receptors, trans-(1 S,3R)-1 -aminocyclopentane-1 ,3-dicarboxylic acid (ACPD), or the GABA-B agonist baclofen. Reversed dialysis of dizocilpine in the nucleus accumbens was equally ineffective, but ACPD inhibited the increase in DA release in this area. Stimulation of metabotropic glutamate receptors in the nucleus accumbens was previously reported to inhibit activation of DA release in that area after stimulation of glutamatergic or dopaminergic afferents. It is concluded that metabotropic receptors in the nucleus accumbens are important for the control of activation of DA release in the accumbens by physiological stimuli but that a similar mechanism is lacking in the prefrontal cortex.

The prefrontal cortex (PFC) and the amygdala are involved in a number of common functions, such as emotional and social behavior, stress, visceral functions, ingestive behavior, self-stimulation, and certain aspects of learning and memory. The amygdala massively projects to the PFC and may play a role in the developmental plasticity reported for several of these functions. We have studied the normal postnatal development of the amygdaloid projections to the rat prefrontal cortex by using the retrogradely transported fluorescent dye fast blue and the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Shortly after birth some fibers were observed in the frontal pole of the rat brain. These fibers were scattered throughout all prefrontal cortical areas. The majority of the amygdaloid cells contributing to this pattern at that stage of development were located in the anterior and ventral basolateral nuclei, whereas a minority were located in the posterior basolateral nucleus. The transition from a diffuse fiber distribution to a characteristic bilaminar pattern occurred around postnatal day 12 in the lateral and rostral medial PFC. The PHA-L injections confirmed the existence of a topographical organization of the amygdalo-prefrontocortical projections. Our observations suggest that the development of amygdala innervation of the PFC parallels the emergence of PFC cytoarchitectural organization.

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