The nucleus tractus solitarii, the first central relay for gustatory and a variety of visceral afferents, is also an integrative center for numerous functions. Its interstitial subdivision is involved in swallowing and respiratory reflexes. The ultrastructural characteristics of this subdivision and of its laryngeal afferents were investigated in adult rat by a serial-section study and by application of wheat germ agglutinin-horseradish peroxidase conjugate to the peripheral afferent fibers. The interstitial subnucleus contained scattered small neuronal cell bodies with such ultrastructural features as a large nucleus with deep indentations and an organelle-poor cytoplasm. On the basis of their size and vesicular content, the axon terminals were classified into three categories. Group I and group II terminals were small or large, respectively, and contained mainly small, round, and clear synaptic vesicles. Group III terminals were also small but contained small, pleomorphic, and clear vesicles. Axodendritic synapses were the most numerous. They were either asymmetrical, comprised of group I and II terminals, or symmetrical, comprised of group III terminals. More than 50% were part of complex synaptic arrangements in the form of rosettes or glomeruli. Axosomatic contacts involved both group I and group III terminals and were always symmetrical. A high frequency of axoaxonic synapses was found. They were symmetrical, comprised of group III terminals on group I or II terminals. Different types of symmetrical synaptic contacts made by dendrites were also found. This study indicates also that the ipsilateral interstitial subdivision constitutes the preferential site of termination for superior laryngeal afferents. The labeled axon terminals belonged exclusively to groups I and II and were involved in both axodendritic and axoaxonic synapses. Some of the axodendritic synapses were part of rosettes or glomeruli. All these synaptic arrangements may be considered a morphological substrate for important processing of afferent information in the nucleus tractus solitarii. They may account for some of the integrative functions of the interstitial subnucleus such as physiological processes triggered from the superior laryngeal nerve.

WIN 35428 and GBR 12935, two uptake blocker ligands of the membrane transporter for dopamine (DA), were evaluated as quantitative markers of DA innervation density in CNS tissue. From alternate rat brain slices respectively processed for either light microscope or film autoradiography, counts of DA axon terminals (varicosities) labeled by uptake/storage of [3H]DA were matched with densitometric measurements of the specific binding of [3H]WIN 35428 and [3H]GBR 12935 in the same anatomical areas. The relation between the two parameters was examined in 1) the normal cingulate cortex; 2) the neostriatum severely DA-denervated by unilateral intramesencephalic injections of 6-hydroxydopamine; and 3) the neostriatum, partly DA-reinnervated by an intrastriatal graft of fetal mesencephalic neurons after prior 6-hydroxydopamine lesion. For technical reasons, the hyperdense DA innervation of normal striatum was not amenable to such correlative testing. Data were subjected to multilevel analysis. Specific [3H]WIN binding at 37 degrees C was tightly and linearly correlated with the number of DA varicosities over the full range of DA innervation densities tested. The regression lines for intact cortex and for DA-denervated as well as DA-reinnervated neostriatum had the same slope and crossed the ordinate near zero. In contrast, [3H]GBR 12935 binding at 37 degrees C showed no correlation with the number of DA varicosities. A linear correlation could be obtained after incubation with [3H]GBR 12935 at 4 degrees C in the presence of ZnSO4, but the intercept of this regression line remained significantly above zero at origin, indicating extraneous binding to non-DA transporter sites. Providing that the hyperdense DA innervation of the normal neostriatum does not generate a particular problem in vivo as it does in vitro. WIN 35428, but not GBR 12935, might satisfy the selectivity and sensitivity requirements of a quantitative marker of DA innervation density for eventual use in positron emission tomographic studies.

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Optimal ultrastructural preservation of brain tissue for electron microscopy is best achieved with fixatives containing high concentrations of glutaraldehyde, which is generally considered detrimental to the immunogenicity of most protein antigens. We tested seventeen mono- or polyclonal antibodies against peptide or protein antigens, including a majority for which immunoreactivity had previously been reported to be sensitive to glutaraldehyde fixation. Forebrain sections of rats or mice fixed by perfusion with 3.5% glutaraldehyde were processed for pre-embedding immunocytochemistry by the avidin-biotin method. The resulting immunostaining was in most cases at least similar to that obtained in sections fixed with paraformaldehyde. Immunoreactivity against the mouse or human neurofilament protein NF-L was even improved, being similar to that previously reported for unfixed brain tissue. Of all antigens tested, only choline acetyltransferase, phenylethanolamine-N-methyl transferase, and neuropeptide Y were detected with lower sensitivity than after paraformaldehyde fixation, which was attributed to a rather restricted penetration of the primary antibody into glutaraldehyde-fixed tissue sections. These results indicate that glutaraldehyde may be envisaged as a possible fixative for optimal immunocytochemical detection of any tissue antigen at the electron microscopic level, including antigens which, on the basis of results obtained after fixation with paraformaldehyde-glutaraldehyde mixtures, were considered highly sensitive to glutaraldehyde fixation.

Direct counting of axon terminals (varicosities) labeled by uptake/storage of a tritiated monoamine provides a means to test radioligands of the corresponding membrane transporter as quantitative markers of regional monoamine innervation density in brain tissue. In autoradiographs from alternate rat brain slices, counts of [3H]5-HT-labeled axon terminals were matched with densitometric measurements of the specific binding of tritiated cyanoimipramine (CYI), citalopram (CITAL), and 6-nitroquipazine (6-NTQ), under conditions of hypo-, normo-, or hyper-5-HT innervation of the neostriatum. A total of 267 pairs of data were subjected to a multilevel analysis (iterative generalized least square procedure). With all three ligands, there was a linear relationship between the density of 5-HT innervation and the density of specific binding and no change in the slope of the regression lines as a function of 5-HT innervation density. Thus, none of these ligands gave any sign of down- or up-regulation of the 5-HT transporter consequent to 5-HT hypo- or hyper-innervation. The regression lines for CYI and CITAL were not significantly different from one another and crossed the ordinate near zero, whereas the regression line for 6-NTQ was less steep and had a higher intercept with the ordinate. In addition, the dispersion of values around the regression line (residuals) was lower with CYI and CITAL than 6-NTQ. It was concluded that both CYI and CITAL may serve as quantitative markers of 5-HT innervation density, at least in vitro, whereas 6-NTQ demonstrates a certain lack of specificity and sensitivity. Further work will be needed to assess the potential of CYI and CITAL for positron emission tomographic studies of living brain. Such empirical testing should also be applicable for screening radioligands of the dopamine or the noradrenaline transporters.