Dual immunocytochemistry using 125I-labeled protein A: a new electron microscopic technique applied to the investigation of chemical connectivity and axonal transmitter co-localization in the brain.

We have developed a double labeling immunocytochemical method utilizing peroxidase conjugated Fab fragments and 125I-labeled protein A to localize two neuronal markers on the same light or electron microscopic section with primary antibodies raised in the same animal species. The technique is applicable to the study of chemical connectivity in the brain, as illustrated by data obtained in the hypothalamus using rabbit polyclonal antisera against tyrosine hydroxylase (TH), phenylethanolamine-N-methyltransferase (PNMT), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP). Moreover, due to a high level of sensitivity and resolution, the technique offers considerable advantages over many previously developed dual labeling immunocytochemical methods for the demonstration of transmitter axonal co-localizations. Utilizing the peroxidase Fab/[125I]protein A method, we present here the first direct evidence that PNMT is present in many endings also containing NPY in the thalamic and hypothalamic paraventricular nuclei and in the arcuate nucleus. The method also may be combined as required with other labeling methods for localizing more than two neurochemical markers on one and the same electron microscopic section.

Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation.

Monoaminergic innervation of a histamine-producing cell group, the tuberomammillary nucleus in the posterior hypothalamus, was investigated in the rat by light and electron microscopic immunohistochemical techniques. Immunohistochemical staining of sections of the posterior hypothalamus was demonstrated afferent fibers immunoreactive to tyrosine hydroxylase in ventral and medial subgroups of the tuberomammillary nucleus afferent fibers immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenyletanolamine-N-methyltransferase (PNMT), and serotonin (5-HT). TH- and DBH-immunoreactive fibers were similar and were evenly and densely distributed throughout the tuberomammillary nucleus. Fibers stained with 5-HT antibodies were also present throughout the tuberomammillary nucleus but exhibited the densest labeling in the dendritic layer adjacent to the glia limitans in the ventral subgroup. Innervation by PNMT-immunoreactive axons was sparse. Electron microscopic analysis of TH-, DBH-, and 5-HT-immunoreactive fibers in the tuberomammillary nucleus revealed vesicle-containing terminal boutons, which formed synapses with dendrites of varying size. Synaptic contacts with nerve cell bodies were not found. Retrograde transport of the fluorescent dye Fast Blue injected into the tuberomammillary nucleus, combined with immunofluorescent staining with anti-TH, anti-DBH, anti-PNMT, and anti-5-HT antibodies, showed that monoaminergic input to the tuberomammillary nucleus originated mainly from the adrenergic and noradrenergic cell groups C1-C3 and A1-A2, respectively, and from the serotoninergic cell groups B5-B9 as designated by Dahlström and Fuxe ('65). Few double-labeled neurons were found in the nucleus locus coeruleus and the dopaminergic cell groups of the rostral brain stem. The present findings suggest that the activity of the histamine-producing neurons of the tuberomammillary nucleus is influenced by monoaminergic neurons in the ventrolateral and dorsomedial medulla oblongata and the raphe nuclei of the rostral brainstem.

Adrenaline synthesizing nerve cells in the medulla of normotensive and hypertensive rats.

1. We have studied the number and distribution of adrenaline synthesizing nerve cells in the medulla oblongata of the rat, using a combination of immunofluorescence to visualize the enzyme phenylethanolamine-N-methyltransferase (PNMT) and catecholamine fluorescence to detect central catecholamines. 2. The distribution of adrenaline synthesizing nerve cells was similar in normotensive (Wistar Kyoto) rats, spontaneous hypertensive rats, and stroke-prone rats. Few of the cells visualized by PNMT immunofluorescence were detected by the Faglu fluorescence method for catecholamines. The C1 (ventrolateral) and C2 (dorsomedial) groups of PNMT cells were anatomically distinct from the A1 and A2 groups of catecholamine fluorescent cells and lay rostral to these cells within the medulla. There was a third group of adrenaline synthesizing cells close to the midline in the rostral medulla, and we have called this the C3 group. 3. There was a 32% increase in the number of PNMT cells in the medulla of 4-week-old stroke-prone rats. 4. PNMT enzyme activity in a cross-segment of the medulla containing the adrenaline synthesizing cells was also increased by 30% in both spontaneous hypertensive rats and stroke-prone rats.