Adenosine STEPs on synaptic function: An Editorial for 'The activity of the STriatal-enriched protein tyrosine phosphatase in neuronal cells is modulated by adenosine A2A receptor on' page 284.

This is an Editorial Highlight of a manuscript by Mallozzi et al. (2019) in the current issue of the Journal of Neurochemistry, in which the authors detail the biochemical pathway that leads to synaptic depression by cocaine. This pathway requires the adenosine A2A receptor and STEP phosphatases. Activation of the adenosine A2A receptor leads to an increase in intracellular calcium, activation of STEP by dephosphorylation, inhibition of excitatory ionotropic glutamate receptors by dephosphorylation of phospho-tyrosine residues and subsequent internalization of the ionotropic glutamate receptors. This adenosine A2A receptor pathway could lead to potential drug targets for neurologic and neuropsychiatric disorders.

The (α4)3(ß2)2 Stoichiometry of the Nicotinic Acetylcholine Receptor Predominates in the Rat Motor Cortex.

The α4ß2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(ß2)3 and (α4)3(ß2)2, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and ß2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4ß2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of α4 or ß2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological "two-concentration" acetylcholine or carbachol paradigm was developed and validated to determine α4/ß2 subunit stoichiometry. This paradigm was then used in layers I-IV of slices of the rat motor cortex to determine the percent contribution of HS and LS α4ß2 receptors in this brain region. We report that the majority of α4ß2 nAChRs in this brain region possess a stoichiometry of the (α4)3(ß2)2 LS subtype.

Chronic sazetidine-A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain.

Chronic nicotine administration increases the density of brain α4ß2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4ß2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4ß2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.

Ligand bias at metabotropic glutamate 1a receptors: molecular determinants that distinguish ß-arrestin-mediated from G protein-mediated signaling.

The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ß-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the ß-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for ß-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward ß-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate-induced PI hydrolysis. Taken together, these data indicate that ligand bias at mGlu1 receptors is attributable to different modes of receptor-glutamate interactions, which are differentially coupled to PI hydrolysis and ß-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.

Endogenously expressed muscarinic receptors in HEK293 cells augment up-regulation of stably expressed α4ß2 nicotinic receptors.

Nicotine-induced up-regulation of neuronal nicotinic receptors (nAChRs) has been known and studied for more than 25 years. Other nAChR ligands can also up-regulate nAChRs, but it is not known if these ligands induce up-regulation by mechanisms similar to that of nicotine. In this study, we compared up-regulation by three different nicotinic agonists and a competitive antagonist of several different nAChR subtypes expressed in HEK293 cells. Nicotine markedly increased α4ß2 nAChR binding site density and ß2 subunit protein. Carbachol, a known nAChR and muscarinic receptor agonist, up-regulated both α4ß2 nAChR binding sites and subunit protein 2-fold more than did nicotine. This increased up-regulation was shown pharmacologically to involve endogenously expressed muscarinic receptors, and stimulation of these muscarinic receptors also correlated with a 2-fold increase in α4 and ß2 mRNA. Muscarinic receptor activation in these cells appears to affect CMV promoter activity only minimally (∼1.2 fold), suggesting that the increase in α4 and ß2 nAChR mRNA may not be dependent on enhanced transcription. Instead, other mechanisms may contribute to the increase in mRNA and a consequent increase in receptor subunits and binding site density. These studies demonstrate the possibility of augmenting nAChR expression in a cell model through mechanisms and targets other than the nAChR receptor itself.

Chronic sazetidine-A at behaviorally active doses does not increase nicotinic cholinergic receptors in rodent brain.

Chronic nicotine administration increases α4ß2 neuronal nicotinic acetylcholine receptor (nAChR) density in brain. This up-regulation probably contributes to the development and/or maintenance of nicotine dependence. nAChR up-regulation is believed to be triggered at the ligand binding site, so it is not surprising that other nicotinic ligands also up-regulate nAChRs in the brain. These other ligands include varenicline, which is currently used for smoking cessation therapy. Sazetidine-A (saz-A) is a newer nicotinic ligand that binds with high affinity and selectivity at α4ß2* nAChRs. In behavioral studies, saz-A decreases nicotine self-administration and increases performance on tasks of attention. We report here that, unlike nicotine and varenicline, chronic administration of saz-A at behaviorally active and even higher doses does not up-regulate nAChRs in rodent brains. We used a newly developed method involving radioligand binding to measure the concentrations and nAChR occupancy of saz-A, nicotine, and varenicline in brains from chronically treated rats. Our results indicate that saz-A reached concentrations in the brain that were ∼150 times its affinity for α4ß2* nAChRs and occupied at least 75% of nAChRs. Thus, chronic administration of saz-A did not up-regulate nAChRs despite it reaching brain concentrations that are known to bind and desensitize virtually all α4ß2* nAChRs in brain. These findings reinforce a model of nicotine addiction based on desensitization of up-regulated nAChRs and introduce a potential new strategy for smoking cessation therapy in which drugs such as saz-A can promote smoking cessation without maintaining nAChR up-regulation, thereby potentially increasing the rate of long-term abstinence from nicotine.

Effects of chronic nicotine on heteromeric neuronal nicotinic receptors in rat primary cultured neurons.

Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [¹²5I]epibatidine ([¹²5I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 µM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, ß2 and α5 subunits, but α2, α3, α6 and ß4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the ß2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.

Quantitative analysis of the heteromeric neuronal nicotinic receptors in the rat hippocampus.

The objective of this study was to identify and quantify the heteromeric neuronal nicotinic receptors (nAChRs) in the rat hippocampus. The density of nAChR subtypes was assessed by labeling them with [(3)H]epibatidine ([(3)H]EB) followed by immunoprecipitation with subunit-selective antibodies. Sequential immunoprecipitation assays were used to establish associations between two different subunits, which then allowed the full subunit composition of the receptors to be deduced. Our results show that most of the hippocampal heteromeric nAChRs contain α4 and ß2 subunits. In fact, we identified two populations containing these two predominant subunits, the α4ß2 and α4ß2α5 subtypes which account for ∼ 40% and ∼ 35%, respectively, of the total [(3)H]EB-labeled receptors. An additional heteromeric subtype with the subunit composition of α4ß2α3 represented ∼ 10% of the total nAChRs, and another 10% of the immunoprecipitated receptors contained α4 and ß4 subunits, with or without the α3 subunit. To determine if α4ß2 and α4ß2α5 nAChR subtypes differ in their ligand binding affinities, the α3- and ß4-containing receptors were first removed by immunoprecipitation and then, competition studies with acetylcholine, nicotine, cytisine and sazetidine-A against [(3)H]EB were carried out on the remaining α4ß2 and α4ß2α5 subtypes. Results suggested these subtypes have comparable binding affinities for the nicotinic ligands used here.

The alpha4beta2alpha5 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo.

We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the alpha5 subunit in the adult rat brain. Among the regions of brain we surveyed, the alpha5 subunit is associated in approximately 37% of the nAChRs in the hippocampus, approximately 24% of the nAChRs in striatum, and 11-16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the alpha5 subunit is associated with alpha4beta2* nAChRs exclusively. Importantly, in contrast to alpha4beta2 nAChRs, which are increased by 37-85% after chronic administration of nicotine, the alpha4beta2alpha5 receptors are not increased by nicotine treatment. These data thus indicate that the alpha4beta2alpha5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo, which suggests an important regulatory role for the alpha5 subunit. To the extent that nicotine-induced up-regulation of alpha4beta2 nAChRs is involved in nicotine addiction, the resistance of the alpha4beta2alpha5 subtype to up-regulation may have important implications for nicotine addiction.

Deletion of the GABA(A) receptor alpha1 subunit increases tonic GABA(A) receptor current: a role for GABA uptake transporters.

The loss of more than half the number of GABA(A) receptors yet lack of pronounced phenotype in mice lacking the gene for the GABA(A) alpha1 subunit is somewhat paradoxical. We explored the role of tonic GABA(A) receptor-mediated current as a target of compensatory regulation in the alpha1 knock-out (-/-) mice. A 62% increase of tonic current was observed in the cerebellar granule cells (CGCs) of alpha1-/- compared with wild-type (+/+) mice along with a 67% increase of baseline current variance. Examination of whole-cell currents evoked by low concentrations of GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol suggested no upregulation of alpha6 and delta subunit-containing GABA(A) receptors in the alpha1-/-, confirming previous biochemical studies. Single-channel current openings were on average 32% shorter in the alpha1-/- neurons. Single-channel conductance and frequency of opening were not different between genotypes. Tonic current induced by application of the GABA transporter GAT-1 blocker NO711 (1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride) was significantly larger in the alpha1-/-, suggesting an increase of ambient GABA concentration. Experiments done with a known concentration of extracellular GABA complemented by a series of biochemical experiments revealed a reduction of GAT activity in alpha1-/- without an identifiable reduction of GAT-1 or GAT-3 protein. We report increased tonic GABA(A) receptor-mediated current in the alpha1-/- CGCs as a novel compensatory mechanism. Our data establish a role for GABA transporters as regulators of neuronal excitability in this and relevant models and examine other tonic conductance-regulating mechanisms responsible for the adaptive response of the cerebellar network to a deletion of a major synaptic GABA(A) receptor subunit.

Ethanol withdrawal is accompanied by downregulation of calcium channel alpha 1B subunit in rat inferior colliculus neurons.

Ethanol withdrawal enhances the current density of calcium (Ca(2+)) channels in inferior colliculus (IC) neurons. The present report shows that ethanol withdrawal markedly enhanced the susceptibility to seizures as it decreased significantly the protein levels of alpha(1B) subunit associated with N-type Ca(2+) channel in IC neurons of animals not tested for seizures. Thus, remodeling of N-type Ca(2+) channels may play an important role in neuronal hyperexcitability that leads to ethanol withdrawal seizures.

Glutamate receptor subunit expression after spinal cord injury in young rats.

To investigate the possibility that glutamate receptor levels in the spinal cord are altered following injury to young rats, we used a previously characterized model of spinal cord contusion that produces a reliable injury in rats at postnatal day 14-15. Quantitative Western blot analysis was used to measure relative amounts of protein for several glutamate receptor subunits acutely (24 h) and chronically (28 days) after spinal cord injury (SCI). Acutely after injury significant decreases were observed in the GluR1, GluR2, and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor, and the NR2A and NR2B subunits, but not the NR1 subunit, of the N-methyl-d-aspartate (NMDA) receptor. However, 28 days after injury only one subunit (GluR4) was shown to be altered. These widespread changes that occur acutely in receptor subunit expression may be an attempt to protect cells from glutamate-induced death. The injured spinal cord in these young animals, however, appears to have the capacity to regulate receptor subunit levels to normal within a month of injury.

Quantitative measurement of glutamate receptor subunit protein expression in the postnatal rat spinal cord.

Glutamate is the major excitatory neurotransmitter in the CNS and its effects on neurons are dependent on the type and composition of glutamate receptors with which it interacts. In this study, the protein expression levels of several ionotropic glutamate receptor subunits (N-methyl-D-aspartate (NMDA) subunits NR1, NR2A, NR2B, and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits GluR1, GluR2, GluR4) were quantified in particulate preparations from rat spinal cord at various ages after birth. We found that all six subunits showed high expression in the early postnatal period, followed by a subsequent decline as the rats matured to adults. The levels of two subunits (NR2A and GluR4) were found to initially increase during the first postnatal week prior to the decline to adult levels. The high levels of expression observed of these subunits in the early postnatal period may have implications for mechanisms of neural injury and cell death in the immature nervous system that involve cation influx through ionotropic glutamate receptors.

18F-ASEM, a radiolabeled antagonist for imaging the α7-nicotinic acetylcholine receptor with PET.

UNLABELLED: The α7-nicotinic cholinergic receptor (α7-nAChR) is a key mediator of brain communication and has been implicated in a wide variety of central nervous system disorders. None of the currently available PET radioligands for α7-nAChR are suitable for quantitative PET imaging, mostly because of insufficient specific binding. The goal of this study was to evaluate the potential of (18)F-ASEM ((18)F-JHU82132) as an α7-nAChR radioligand for PET. METHODS: The inhibition binding assay and receptor functional properties of ASEM were assessed in vitro. The brain regional distribution of (18)F-ASEM in baseline and blockade were evaluated in DISC1 mice (dissection) and baboons (PET). RESULTS: ASEM is an antagonist for the α7-nAChR with high binding affinity (Ki = 0.3 nM). (18)F-ASEM readily entered the baboon brain and specifically labeled α7-nAChR. The in vivo specific binding of (18)F-ASEM in the brain regions enriched with α7-nAChRs was 80%-90%. SSR180711, an α7-nAChR-selective partial agonist, blocked (18)F-ASEM binding in the baboon brain in a dose-dependent manner, suggesting that the binding of (18)F-ASEM was mediated by α7-nAChRs and the radioligand was suitable for drug evaluation studies. In the baboon baseline studies, the brain regional volume of distribution (VT) values for (18)F-ASEM were 23 (thalamus), 22 (insula), 18 (hippocampus), and 14 (cerebellum), whereas in the binding selectivity (blockade) scan, all regional VT values were reduced to less than 4. The range of regional binding potential values in the baboon brain was from 3.9 to 6.6. In vivo cerebral binding of (18)F-ASEM and α7-nAChR expression in mutant DISC1 mice, a rodent model of schizophrenia, was significantly lower than in control animals, which is in agreement with previous postmortem human data. CONCLUSION: (18)F-ASEM holds promise as a radiotracer with suitable imaging properties for quantification of α7-nAChR in the human brain.

Derivatives of dibenzothiophene for positron emission tomography imaging of α7-nicotinic acetylcholine receptors.

A new series of derivatives of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)dibenzo[b,d]thiophene 5,5-dioxide with high binding affinities and selectivity for α7-nicotinic acetylcholine receptors (α7-nAChRs) (Ki = 0.4-20 nM) has been synthesized for positron emission tomography (PET) imaging of α7-nAChRs. Two radiolabeled members of the series [(18)F]7a (Ki = 0.4 nM) and [(18)F]7c (Ki = 1.3 nM) were synthesized. [(18)F]7a and [(18)F]7c readily entered the mouse brain and specifically labeled α7-nAChRs. The α7-nAChR selective ligand 1 (SSR180711) blocked the binding of [(18)F]7a in the mouse brain in a dose-dependent manner. The mouse blocking studies with non-α7-nAChR central nervous system drugs demonstrated that [(18)F]7a is highly α7-nAChR selective. In agreement with its binding affinity the binding potential of [(18)F]7a (BPND = 5.3-8.0) in control mice is superior to previous α7-nAChR PET radioligands. Thus, [(18)F]7a displays excellent imaging properties in mice and has been chosen for further evaluation as a potential PET radioligand for imaging of α7-nAChR in non-human primates.

GABA(A) Receptor ß3 Subunit Expression Regulates Tonic Current in Developing Striatopallidal Medium Spiny Neurons.

The striatum is a key structure for movement control, but the mechanisms that dictate the output of distinct subpopulations of medium spiny projection neurons (MSNs), striatonigral projecting and dopamine D1 receptor- (D1+) or striatopallidal projecting and dopamine D2 receptor- (D2+) expressing neurons, remains poorly understood. GABA-mediated tonic inhibition largely controls neuronal excitability and action potential firing rates, and we previously suggested with pharmacological analysis that the GABA(A) receptor ß3 subunit plays a large role in the basal tonic current seen in D2+ MSNs from young mice (Ade et al., 2008; Janssen et al., 2009). In this study, we demonstrated the essential role of the ß3 GABA(A) receptor subunit in mediating MSN tonic currents using conditional ß3 subunit knock-out (ß3f/f(Drd2)) mice. Cre-lox genetics were used to generate mice where Cre recombinase was expressed under the D2 receptor (Drd2) promoter. We show that while the wild-type MSN tonic current pattern demonstrates a high degree of variability, tonic current patterns from ß3f/f(Drd2) mice are narrow, suggesting that the ß3 subunit is essential to striatal MSN GABA-mediated tonic current. Our data also suggest that a distinct population of synaptic receptors upregulate due to ß3 subunit removal. Further, deletion of this subunit significantly decreases the D2+ MSN excitability. These results offer insight for target mechanisms in Parkinson's disease, where symptoms arise due to the imbalance in striatal D1+ and D2+ MSN excitability and output.

Protein expression of small conductance calcium-activated potassium channels is altered in inferior colliculus neurons of the genetically epilepsy-prone rat.

The genetically epilepsy-prone rat (GEPR) exhibits inherited predisposition to sound stimuli-induced generalized tonic-clonic seizures (audiogenic reflex seizures) and is a valid model to study the physiopathology of epilepsy. In this model, the inferior colliculus (IC) exhibits enhanced neuronal firing that is critical in the initiation of reflex audiogenic seizures. The mechanisms underlying IC neuronal hyperexcitability that leads to seizure susceptibility are not as yet fully understood. The present report shows that the levels of protein expression of SK1 and SK3 subtypes of the small conductance Ca2+-activated K+ channels were significantly decreased, while SK2 channel proteins were increased in IC neurons of seizure-naive GEPR-3s (SN-GEPR-3), as compared to control Sprague-Dawley rats. No significant change was found in the expression of BK channel proteins in IC neurons of SN-GEPR-3s. Single episode of reflex audiogenic seizures in the GEPR-3s did not significantly alter the protein expression of SK1-3 and BK channels in IC neurons compared to SN-GEPR-3s. Thus, downregulation of SK1 and SK3 channels and upregulation of SK2 channels provide direct evidence that these Ca2+-activated K+ channels play important roles in IC neuronal hyperexcitability that leads to inherited seizure susceptibility in the GEPR.

Heterogeneity of nicotinic cholinergic receptors in rat superior cervical and nodose Ganglia.

Nicotinic cholinergic receptors (nAChRs) are present in ganglia in the peripheral nervous system. In autonomic ganglia, they are responsible for fast synaptic transmission, whereas in the sensory ganglia and sensory neurons, they may be involved in modulation of neurotransmission. The present study measured nAChRs in several rat autonomic ganglia: the superior cervical ganglia (SCG), sensory nodose ganglia, stellate ganglia, and pelvic ganglia. The densities of the heteromeric nAChRs determined by receptor binding assay in those four ganglia are 481, 45, 9, and 11 fmol/mg protein, respectively. Immunoprecipitation studies with subunit-specific antibodies showed that a majority of the nAChRs in the SCG and nodose ganglia contain the alpha3 and beta4 subunits, but a significant percentage of the nAChRs in these ganglia also contain alpha5 and beta2 subunits. A small percentage of the nAChRs in nodose ganglia also contain alpha2 and alpha4 subunits. Sequential immunoprecipitation assays indicated that in the SCG, all alpha5 subunits are associated with alpha3 and beta4 subunits, forming the mixed heteromeric alpha3beta4alpha5 subtype. A receptor composed of alpha3, beta2, and beta4 subunits in the SCG was also detected. In rat SCG, we found the following distribution of nAChRs subtypes: 55 to 60% simple alpha3beta4 subtype, 25 to 30% alpha3beta4alpha5 subtype, and 10 to 15% alpha3beta4beta2 subtype. These findings indicate that the nAChRs in SCG and nodose ganglia are heterogeneous, which suggests that different receptor subtypes may play different roles in these ganglia or may be activated under different conditions.

NMDA receptor subtypes at autaptic synapses of cerebellar granule neurons.

We studied the action potential-evoked autaptic N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) using solitary cerebellar neurons cultured in microislands from wild-type (+/+), NR2A subunit knockout (NR2A-/-), and NR2C subunit knockout (NR2C-/-) mice. The peak amplitude of autaptic NMDA-EPSCs increased for all genotypes between days in vitro 8 (DIV8) and DIV13. Compared with +/+ cells at DIV13, NR2A-/- cells had smaller and NR2C-/- cells had larger NMDA-EPSCs. The decay time of these currents were all unexpectedly fast, except in NR2A-/- neurons, and showed small but significant shortening with development. Comparison of quantal parameters during development indicated an increase in quantal content in all genotypes. The synaptic portion of NMDA receptors measured using MK-801 blockade was roughly 50% in all genotypes at DIV8, and this percentage became slightly larger in NR2A-/- and NR2C-/- neurons at DIV12. The NR2B-selective antagonists Conantokin G and CP101,606 differed in their blocking actions with development, suggesting the presence of both heterodimeric NR1/NR2B and heterotrimeric NR1/NR2A/NR2B receptors. The most striking result we obtained was the significant increase of NMDA-EPSC peak amplitude and charge transfer in NR2C-/- mice. This was mainly the result of an increase in quantal size as estimated from miniature NMDA-EPSCs. The expression of NR2C subunit containing receptors was supported by the decreased Mg(2+) sensitivity of NMDA receptors at DIV13 in +/+ but not in NR2C-/- cells. Thus solitary cerebellar granule neurons provide a novel model to investigate the role of receptor subtypes in the developmental changes of synaptic NMDA receptors.

Nicotinic cholinergic receptors in the rat retina: simple and mixed heteromeric subtypes.

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in the rat retina to determine the heteromeric subtypes. We detected seven nicotinic receptor subunit mRNA transcripts, alpha2-alpha4, alpha6, and beta2-beta4, with RNase protection assays. The density of heteromeric nAChR binding sites is approximately 3 times higher in the retina than in the cerebral cortex. Moreover, the density of the sites in the retina measured with [3H]epibatidine ([3H]EB) is approximately 30% higher than with 125I-3-(2(S)-azetidinylmethoxy)pyridine (A-85380) and more than twice that measured with [3H]cytisine or [3H](-)nicotine. These data suggest that the retina expresses multiple subtypes of nAChRs, including a large fraction of receptors containing the beta2 subunit and a smaller fraction containing the beta4 subunit. Consistent with this, in binding competition studies, nicotinic ligands fit a model for two affinity classes of binding sites, with the higher affinity sites representing 70 to 80% of the nAChRs in the retina. To determine the specific subtypes of nAChRs in the rat retina, we used subunit-specific antibodies in immunoprecipitation assays. Immunoprecipitation of [3H]EB-labeled nAChRs with antibodies specific to the beta2 and beta4 subunits indicated that approximately 80% of the receptors contained beta2 subunits and approximately 25% contained beta4 receptors, consistent with the binding pharmacology results. Sequential immunoprecipitation assays indicated that the rat retina contains multiple subtypes of nAChRs. The majority of the receptors measured seemed to be simple heteromeric subtypes, composed of a single type of alpha and a single type of beta subunit; but a significant fraction are mixed heteromeric subtypes, composed of two or more alpha and/or beta subunits.