SUMMARYThe fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO 4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO 4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

Opioid receptors regulate neuronal activity by both pre- and postsynaptic mechanisms. We recently reported that the cloned delta- opioid receptor (DOR1) is primarily targeted to axons, suggesting a presynaptic role. In the present study we have studied the distribution and targeting of another opioid receptor, the mu-opioid receptor (MOR1), by raising anti-peptide antisera to the C-terminal peptide of MOR1. The specificity of the antisera was determined by analysis of transfected cells, Western blots, and immunoisolation studies. Immunohistochemistry showed that MOR1 immunoreactivity was enriched in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus, and the superficial laminae of the dorsal horn. Moreover, MOR1-expressing neurons seem to target this receptor preferentially to their somatodendritic domain as determined by double- labeling experiments with MAP2. However, discrete populations of neurons target MOR1 to their axons, including some primary afferent neurons that express DOR1. In many regions enkephalin-containing axons were complementary to MOR1, suggesting by their proximity that enkephalins may be physiologically relevant ligands for this receptor. Thus, these results provide a morphological basis for understanding pre- and postsynaptic functions mediated by MOR1.

We have recently developed antisera which recognize epitopes of the cloned delta-opioid receptor (DOR; Dado et al., 1993). In the present report we have further characterized these antisera, and raised additional antisera in rats. We used these antisera to determine the distribution of DOR-like immunoreactivity (-Ll) in rat spinal cord and brainstem in relation to serotoninergic, noradrenergic, and enkephalinergic neurons. We found DOR-Ll in fibers and varicosities distributed throughout the spinal cord gray matter, with highest densities in the superficial dorsal horn, in autonomic regions, around the central canal as well as in the ventral horn motor nuclei. In the brainstem a dense innervation of DOR-immunoreactive (-IR) fibers was found in several nuclei such as spinal trigeminal nuclei, midline raphe nuclei, parabrachial nuclei, periaqueductal gray matter (PAG), interpeduncular nucleus, ans substantia nigra. A group of DOR-positive cells was seen in the laterodorsal tegmental nucleus. In addition, a few DOR-IR cell bodies were demonstrated in the parabrachial nuclei, interpeduncular nucleus, PAG, and superior and inferior colliculi as well as around the central canal in the spinal cord. All DOR-positive cells showed a punctuate staining pattern within the cytoplasm of the cell body and in primary dendrites. No plasma membrane staining of cells or dendrites could be demonstrated using the DOR antisera. Double-labeling experiments for DOR and 5-hydroxytryptamine (5HT, serotonin) revealed that some 5HT-IR neurons in the raphe complex were surrounded by DOR-IR fibers. In the spinal cord a high degree of coexistence was found between DOR and 5HT in nerve fibers and varicosities in the neuropil around the motoneurons and in lamina V of the dorsal horn. In autonomic regions of the spinal cord, a low degree of colocalization was seen between DOR and 5HT; in the superficial dorsal horn no coexistence was found. Tyrosine hydroxylase (TH)-positive neurons in the brainstem (in the A5 area, locus coeruleus, and A7 area) were apposed by DOR-positive fibers. However, no coexistence could be seen between DOR and TH in any part of the spinal cord. A close relation, but no coexistence, was observed between DOR- and enkephalin (ENK)-IR fibers in the spinal cord ventral horn; in the intermediolateral nucleus a low degree of colocalization was observed. Thus, a delta-opioid receptor may affect the activity of descending serotoninergic and noradrenergic neurons by means of modulating the release of neurotransmitters from afferents to these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

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