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In vitro autoradiography using 125I-alpha-bungarotoxin (alpha-BGTx) and anti-alpha7 immunohistochemistry were performed on the dorsal motor nucleus of the vagus (DMV) of sham and chronically vagotomized rats to determine whether the alpha7-nicotinic acetylcholine receptor (nAChR) is located postsynaptically on DMV neurons whose axons contribute to the vagus nerve. Intense bilateral 125I-alpha-BGTx binding and anti-alpha7 immunostaining were observed in coronal brain sections containing the DMV of sham-vagotomized animals. Unilateral cervical vagotomy resulted in ipsilateral losses of 125I-alpha-BGTx binding and anti-alpha7 immunostaining from the DMV. Simultaneous staining of rat brainstem sections with anti-alpha7 and anti-choline acetyltransferase (ChAT) antibodies (to identify cholinergic DMV neurons that project into the vagus nerve) revealed that every DMV neuron that was stained for ChAT showed alpha7-staining as well. In vagotomized animals, no ChAT-positive neurons expressing alpha7-nAChRs remained in the ipsilateral DMV. We conclude that the alpha7-nAChR subtype is located postsynaptically on DMV neurons. To test whether the alpha7-nAChR is similar to the alpha7-homomeric nAChR, experiments were performed in anesthetized rats, and compounds were microinjected into the DMV while monitoring intragastric pressure (IGP). alpha-BGTx and strychnine antagonized nicotine-induced increases in IGP; no antagonism was observed with methyllycaconitine, a compound known to block the homomeric alpha7-nAChR subtype. Recovery from alpha-BGTx-induced antagonism of the nicotine response was observed. We conclude that there is a nAChR containing the alpha7-subunit in the DMV that is different from the homomeric alpha7-nAChR subtype.

Recent evidence has linked striatal N-methyl-D-aspartate (NMDA) receptor function to the adverse effects of long-term dopaminergic treatment in Parkinson's disease. We have studied the abundance, composition, and phosphorylation of NMDA receptor subunits (NRs) in the rat 6-hydroxydopamine lesion model of parkinsonism. In lesioned striatum, the abundance of NR1 and NR2B in striatal membranes was decreased to 68 +/- 3.2 and 62 +/- 4.4%, respectively, relative to the unlesioned striata, whereas the abundance of NR2A was unchanged. Coimmunoprecipitation of NMDA receptors under nondenaturing conditions revealed that these changes reflected a selective depletion of receptors composed of NR1/NR2B, without alteration in receptors composed of NR1/NR2A. However, the abundance and composition of striatal NMDA receptors in extracts containing both cytoplasmic and membrane proteins were not altered in lesioned rats, suggesting that the changes in the membrane fraction resulted from intracellular redistribution of receptors. The phosphorylation of NR1 protein at serine 890 and serine 896, but not at serine 897, and the tyrosine phosphorylation of NR2B but not NR2A were decreased in the membrane fraction of the lesioned striatum. Chronic treatment of lesioned rats with L-dopa normalized the alterations in the abundance and subunit composition of the NMDA receptors in striatal membranes, and produced striking hyperphosphorylation, both of NR1 at serine residues, and NR2A and NR2B at tyrosine residues. These findings suggest that the adverse motor effects of chronic L-dopa therapy may result from alterations in regulatory phosphorylation sites on NMDA receptors.

The N-methyl-D-aspartate (NMDA) receptor is assembled using proteins from two gene families, NR1 and NR2. Although a few studies have examined the composition of NMDA receptors containing NR1, NR2A, and NR2B, the composition of native NMDA receptors that incorporate the NR2D subunit is not known. The goal of the current study was to examine the subunit composition of native NMDA receptors that contain the NR2D subunit in the rat central nervous system by immunoprecipitation of assembled NMDA receptors from rat brain tissues using specific antibodies against NR1, NR2A, NR2B, and NR2D subunits. NMDA receptors were solubilized using either nondenaturing (native) conditions, in which the subunits remain assembled in complexes, or denaturing conditions, in which the NMDA subunits are dissociated from one another. Each of the antibodies selectively and quantitatively immunoprecipitated only the corresponding subunit when the subunits were solubilized using denaturing conditions. In contrast, when NMDA receptors were solubilized under nondenaturing conditions, immunoprecipitation followed by quantitative immunoblot analysis of the resulting pellets show that the majority of the NR2D protein is associated with the NR1 subunit. In addition, the NR2D subunit forms a heteromeric assembly with NR1, as well as with NR2A and/or NR2B subunits, reflecting ternary complex formation. Finally, a binary complex composed of only NR1/NR2D subunits was found in the thalamus but not in the midbrain, where the complexes always contained either NR2A or NR2B, demonstrating that in the central nervous system, different subtypes of NR2D-containing NMDA receptors are present that vary in spatial expression, perhaps indicating distinct physiological and behavioral roles.

A monoclonal antibody (R1JHL) against the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor has been developed that recognizes an epitope in the region of the amino-terminal amino acids 341-561 (a region common to all splice variants of NR1). This monoclonal antibody identifies a broad band at 115 kDa in immunoblots using membranes from NR1-transfected cells and from rat brain tissue. No cross-reactivity with any NR2 subunit is seen. With the goal to determine quantitatively the subunit composition of cortical NMDA receptors, we used the monoclonal antibody to NR1 and polyclonal antibodies against the NR2A and NR2B subunits to perform immunoprecipitations of receptor subunits from solubilized adult rat cortical membranes. Solubilization of the receptor subunits was accomplished under both nondenaturing (native) conditions, under which the subunits seem to remain associated with one another, and denaturing conditions, under which the subunits are associated from each other. Although each of these antibodies selectively immunoprecipitates only its corresponding (cognate) subunit when the subunits have been solubilized under denaturing conditions, each of the antibodies immunoprecipitates a sizable fraction of the other two NMDA receptor subunits when membranes are solubilized under nondenaturing conditions, indicating an interaction in situ. Using quantitative immunoblot analysis of the three subunits in both the pellets and supernatants from the immunoprecipitations, we found 1) the dominant NMDA receptor complex in adult rat cortex contains at least three subunits, NR1/NR2A/NR2B; 2) a smaller fraction of NMDA receptors are composed of only two subunits, NR1/NR2B or NR1/NR2A; 3) there are no complexes that contain NR2A/NR2B that do not contain NR1; 4) only a small fraction of each subunit is not associated with any other NMDA receptor subunit; 5) no coimmunoprecipitation of noncognate subunits occurs unless the subunits are assembled with each other in situ; and 6) there is no physical interaction between these NMDA receptor subunits and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluR2 or GluR3 subunits. These results suggest that functional studies with recombinant receptors composed of at least three subunits may be the most physiologically meaningful.

A portion of the cDNA sequence corresponding to the third intracellular loop of either the m4 or m5 muscarinic cholinergic receptor was ligated into the pRIT23 or pET-3a expression vector, respectively. The expressed fusion proteins were purified and used to develop selective polyclonal antisera to the m4 and m5 muscarinic receptors. These antisera were used in an immunoprecipitation protocol to examine quantitatively the distribution of receptor subtypes in regions of rat brain. The density of m4 receptors in rat brain increased in the caudal to rostral direction. The highest levels of m4 receptors were detected in the striatum (1280 fmol/mg) and olfactory tubercle (750 fmol/mg). Low levels of m5 receptors were detected in several brain regions (< 25 fmol/mg). By combining the previously determined receptor densities for the m1, m2, and m3 receptors and results obtained with the newly developed antisera to m4 and m5 receptors, it was determined that 86-99% of the [3H]quinuclidinyl benzilate binding sites in several brain regions were immunoprecipitated. In addition to measuring receptor densities in rat brain, the immunoprecipitation protocol was used to quantify muscarinic receptor levels in tissues reported to express mRNA encoding the m4 receptor. Thus, although only m4 mRNA has been detected in rabbit lung, NG108-15 cells, and N1E-115 cells, both rabbit lung and NG108-15 cells possess both m2 (rabbit lung, 27%; NG108-15 cells, 31%) and m4 (rabbit lung, 55%; NG108-15, 42%) receptors, whereas N1E-115 cells were found to have both m1 (15%) and m4 (65%) receptors. These antisera will be useful in studies of receptor regulation and in determining alterations in density that may occur after pharmacological or physiological manipulations and in various disease states.

The regulation of individual muscarinic receptor subtypes in rat cerebral cortex/dorsal hippocampus was examined following 14-day administration of the nonselective antagonist atropine. Total muscarinic receptor density increased 24%, from 2196 fmol/mg to 2722 fmol/mg. The nature of this increase was examined using a panel of antisera selective for the m1 to m5 muscarinic receptors. Thus, 97% of all cortical/hippocampal receptors were accounted for by immunoprecipitation. Three subtypes were observed to increase significantly in density: m1 receptor from 824 to 982 fmol/mg (19%, P less than .05); m2 receptor from 476 to 519 fmol/mg (9%, N.S.); m3 receptor from 259 to 438 fmol/mg (69%, P less than .001); m4 receptor from 574 to 638 fmol/mg (11%, P less than .05); and the m5 receptor from 23 to 38 fmol/mg (65%, N.S.). Receptors coupled to the hydrolysis of phosphoinositides (m1, m3, m5) appeared to be preferentially up-regulated (32% over control levels, P less than .001) compared with those coupled to the inhibition of adenylyl cyclase (m2, m4; 10% over control levels, P less than .05). The absolute density of the molecularly defined m1 and m4 receptors, which reportedly possess the highest affinity for pirenzepine (M1), increased 16% (P less than .05), whereas the density of receptors having the lowest affinity for pirenzepine (m2, m3 and m5) increased 31% (P less than .001) with atropine treatment. When the increase in total receptor density was examined with [3H]pirenzepine, the 16% elevation in high-affinity (m1 and m4) sites was not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic oligopeptide (QCDKRKRRKQQYQQRQSV) corresponding to a carboxyl-terminal sequence of the rat m3 receptor (amino acids 561-578) was coupled to carrier proteins and used to generate a polyclonal antiserum. This serum selectively immunoprecipitates at least 90% of the m3 receptors expressed by A9 cells transfected with the cDNA encoding the m3 muscarinic receptor but does not precipitate receptors from cells transfected with cDNA encoding m1, m2, m4, or m5 receptors. Using this m3 antiserum, the density of m3 receptors in various regions of rat brain was quantified. Areas expressing the highest density of m3 receptors are the cortex, hippocampus, striatum, and olfactory tubercle, with 232 fmol/mg, 197 fmol/mg, 140 fmol/mg, and 130 fmol/mg, respectively. Hindbrain regions (i.e., cerebellum, thalamus/hypothalamus, and pons/medulla) expressed fewer m3 receptors, both as a percentage of total muscarinic receptors (5-6%) and in terms of absolute receptor density (12-70 fmol/mg). A panel of subtype-selective antisera (m1, m2, and m3) was used to determine receptor distribution in several peripheral tissues of the rat (lung, ileum, and bladder). The m2 receptor subtype constitutes the majority of total receptors in the bladder (86%), lung (91%), and ileum (69%). The m3 receptor was found at lower densities in these tissues (5-11%), whereas the m1 receptor is present in highest amounts in the ileum (17%). Human clonal cell lines, in which regulation of muscarinic receptors has been commonly studied, were also examined. The SK-N-SH neuroblastoma line, which has been reported to express M3 receptors, on the basis of pharmacology and molecular size, was found to express a mixture of subtypes (m1 = 31%, m2 = 21%, m3 = 43%). Interestingly, SH-SY-5Y and SH-IN cells, both derived from SK-N-SH cells, exhibit predominantly m3 receptors (74% for SH-SY-5Y; 58% for SH-IN), with lower levels of m1 and m2 receptors (5% and 8% for SH-SY-5Y; 4% and 23% for SH-IN, respectively.) Another commonly studied cell line, 132-1-N1 astrocytoma cells, reportedly expressing M3 receptors, based upon mRNA measurements and second messenger linkage, also expresses a predominance of m3 receptors (91% of total). This m3-selective antiserum should prove useful not only for localizing and quantifying m3 muscarinic receptors but also for examining mechanisms involved in the regulation of receptor expression in human tissues or animal models of disease, as well as in cell culture.

The DNA fragment encoding the third intracellular loop of rat m2 muscarinic receptor was fused to the gene for staphylococcal Protein A. The resultant fusion protein, expressed in bacteria, was purified via IgG affinity chromatography and used as an antigen to raise a polyclonal antiserum. Chinese hamster ovary cells transfected with cDNA coding for a single muscarinic receptor subtype were used as tissue sources to screen antisera. The antiserum was shown to immunoprecipitate quantitatively (greater than 90%) m2 receptors but not to precipitate m1, m3, m4, or m5 receptors. Additionally, immunoprecipitation by m2 antiserum could be inhibited by protein containing the third intracellular loop of the m2 receptor. This selective m2 antiserum was then used to study the distribution and density of m2 receptors in rat brain and heart. In agreement with previous studies, m2 receptors were found to be abundant in heart and comprise at least 92% of the total muscarinic receptor density. Hindbrain, brain stem, and midbrain regions such as cerebellum (75%), pons/medulla (70%), and thalamus/hypothalamus (43%) are also enriched in m2 receptors. In contrast, forebrain regions contain markedly lower percentages of m2 receptors, with cortex expressing 20%, hippocampus 19%, striatum 12%, and olfactory tubercle 20% of the total receptor density. Although the density of m2 receptors expressed as a percentage of total varied considerably from brain region to brain region, the absolute density of these receptors appeared relatively uniform throughout the brain. This study demonstrates that a gene fusion system can be used for efficient antibody production. The use of similar fusion protein antisera directed against other subtypes of muscarinic receptors should prove useful in future studies on regulation, function, and structure of muscarinic receptors.

A fragment of the cDNA encoding the third intracellular loop of the rat m1 muscarinic receptor was cloned, and the DNA was expressed in Escherichia coli as a fusion protein. The fusion protein was purified and utilized as an antigen to raise a polyclonal antiserum in rabbits. Chinese hamster ovary cells stably transfected with the cDNA encoding each of the five known subtypes of muscarinic receptor were used as tissue sources to test the antiserum. The antiserum was found to quantitatively immunoprecipitate m1 muscarinic receptors, while not precipitating m2, m3, m4, or m5 receptors. This selective antiserum was utilized to quantify the density of m1 muscarinic receptors in seven selected areas of the rat brain. Thus, cortex was found to contain approximately 0.8 pmol/mg of membrane protein, which represents 34% of the total density of muscarinic receptors. Similarly, hippocampus (1 pmol/mg; 47%), striatum (0.8 pmol/mg; 29%), and olfactory tubercule (0.9 pmol/mg; 35%) are rich in m1 receptors. In contrast, thalamus/hypothalamus contained only 0.15 pmol/mg, representing approximately 16% of the total density of muscarinic receptors, whereas pons/medulla (0.03 pmol/mg; 5%) and cerebellum (less than 0.01 pmol/mg; 2%) had very low levels of expression of m1 receptors. The development of a selective antiserum has provided a means for the quantification of a specific subtype of muscarinic receptor in tissues, such as the brain, that express multiple subtypes. This methodology will be applicable not only to the other subtypes of muscarinic receptor but also to the subtypes of several other neurotransmitter receptors that lack selective drugs with which to study them.

The purpose of this study was to test whether persistent changes consistent with behavioral sensitization occur in dopamine (DA) uptake, release or receptors following repeated cocaine administration. Our neurochemical experiments focused primarily on the striatum; however, quantitative autoradiography was used to measure D-1 and D-2 DA receptors in both cell body and terminal regions of the nigrostriatal and mesolimbic dopaminergic pathways. After receiving eight once-daily injections of cocaine (10 mg/kg, i.p.), rats remained behaviorally sensitized for 1 week. This repeated treatment with cocaine induced two changes consistent with increased dopaminergic transmission. Postsynaptic D-2 DA receptors were selectively increased in nucleus accumbens one day after termination of the repeated cocaine administration; however, these receptors returned to control levels one week after cocaine administration had been terminated. In contrast, amphetamine-stimulated [3H] DA release from striatal slices was increased in rats receiving repeated cocaine injections, but this increase was not apparent until 1 week after the drug administration had been terminated. While neither of these two changes is sufficient to explain cocaine-induced behavioral sensitization, both are consistent with increased dopaminergic responsiveness and may contribute to sensitization.