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.

Several P2x receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2xs) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2x2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2x1 [391][392][393][394][395][396][397][398][399] It has now become clear that P2x receptor subunits comprise a related family of proteins (9-12), but pharmacological ligands that could differentiate them in binding studies are not available. The first two members of the P2X family to be cloned were from rat vas deferens (11) (P2X1) and from rat pheochromocytoma (PC12) cells (9) (P2X2); we now describe the distribution of the P2Xl and P2X2 proteins using immunocytochemistry with receptor-specific antibodies. The purpose of the work was three-fold. First, when expressed in oocytes or mammalian cells, the two cDNAs result in ATP-gated cation channels with properties that correspond closely to those of the tissues of origin (9,11,13). This implies that P2X channels can form as homomultimers (or monomers); however, finding both proteins expressed in the membrane of the same cell would be consistent with heteropolymeric channels (10). Second, in situ hybridization detected the P2Xl and P2X2 RNAs in some tissues that were previously not known to have ATP-gated ion channels, such as the thymus and pituitary gland (9, 11). It is important to confirm, and perhaps extend, these observations at the protein level. Third, the determination of the subcellular distribution of the protein is critical to an understanding of its role in synaptic transmission. Thus, we sought to determine whether the receptors were concentrated at postsynaptic sites only or were also found presynaptically. METHODSGeneration of Antisera. Based upon our previous success in raising antisera that recognize receptors in aldehyde-fixed tissue, antisera against P2X1 and P2X2 were generated in New Zealand White rabbits (n = 12) against peptides corresponding to the C termini of P2X, (GLQENMRTS; residues 391-399) and P2X2 (DSTSTDPKGLAQL; residues 460-472) (14,15). Bleedings were initially screened on paraformaldehydefixed tissues known by in situ hybridization to express P2x1 and/or P2x2 receptors. Positive sera were further screened by immunostaining of paraformaldehyde-fixed HEK-293 cells transfected with either P2X1 or P2X2 (see below). Sera selected from these screenings were subjected to homologous and heterologous absorption controls on tissue and transfected cells (see below). Based on the above criteria, histochemically specific antisera were obtained from two rabbits immunized with the P2xl-derived peptide and from five rabbits immunized with the P2x2-derived peptide. Antisera against the P2x2 peptide were purified over an affinity support that consisted of CDSTSTDPKGLAQL coupled to Ultralink Iodoacetyl (Pierce) according to manufacturer's instructions.Immunocytochemistry. Tissues were obtained from male Sprague-Dawley rats (Harlan; body weight, 140-30...

Acetylcholine (ACh) is synthesized in nerve terminals from choline and acetyl coenzyme A by the cytoplasmic enzyme choline acetyltransferase (ChAT). The neurotransmitter is thereafter transported into synaptic vesicles, where it is stored until release. cDNA clones encoding a vesicular ACh transporter (VAChT) were recently isolated. In this paper, we report on the generation of highly specific goat polyclonal antisera to the rat VAChT protein by using a synthetic carboxy-terminal 20-amino-acid peptide sequence as an immunogen. Characterization of the antisera revealed recognition of VAChT, but not vesicular monoamine transporter (VMAT) protein, in transfected CV-1 cells. VAChT immunoreactivity was also detected in cells that endogenously express the protein, such as in PC12 cells and in primary cultures of spinal motoneurons. Absorption controls showed that the VAChT antisera could be completely blocked at the 10(-5) M concentration by cognate peptide used for immunization. The antisera cross-reacted with the VAChT protein in rat and mouse but not in guinea pig, rabbit, or cat. Immunohistochemistry and confocal laser microscopy, using the goat VAChT antisera, showed strong immunoreactivity in discrete fibers and neuronal cell bodies of the central and peripheral nervous systems. Within cell bodies and axonal nerve terminals, as well as in dendrites, the staining appeared granular, presumably representing labeling of synaptic vesicles containing ACh. In the rat central nervous system, VAChT-positive cell bodies were demonstrated in the cerebral cortex, striatum, septum, nucleus basalis, medial habenula, mesopontine complex, cranial, and autonomic and spinal motor nuclei and in the intermediomedial region near the central canal. High densities of VAChT-immunoreactive axonal fibers were encountered in areas such as the olfactory bulb, cerebral cortex, striatum, basal forebrain, amygdala, thalamus, hypothalamus including median eminence, hippocampal formation, superior colliculus, interpeduncular nucleus, and pedunculopontine and laterodorsal tegmental nuclei. In cranial and spinal motor nuclei, particularly large varicosities were seen in close proximity to the motoneuron cell somata and their proximal dendrites. In the peripheral nervous system, VAChT immunoreactivity was also detected in motor endplates of skeletal muscle as well as in fibers of sympathetic and parasympathetic abdominal ganglia, heart atrium, respiratory tract, gastrointestinal tract, pancreas, adrenal medulla, male genitourinary tract, and salivary and lacrimal glands. Direct double labeling revealed colocalization of VAChT and ChAT immunoreactivity in neurons. The results show that VAChT antisera represent novel and unique tools for the study of cholinergic neurons in the central and peripheral nervous systems.

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)

No abstract