In vivo mapping of a GPCR interactome using knockin mice.

With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

α2* Nicotinic acetylcholine receptors influence hippocampus-dependent learning and memory in adolescent mice.

The absence of α2* nicotinic acetylcholine receptors (nAChRs) in oriens lacunosum moleculare (OLM) GABAergic interneurons ablate the facilitation of nicotine-induced hippocampal CA1 long-term potentiation and impair memory. The current study delineated whether genetic mutations of α2* nAChRs (Chrna2L9'S/L9'S and Chrna2KO) influence hippocampus-dependent learning and memory and CA1 synaptic plasticity. We substituted a serine for a leucine (L9'S) in the α2 subunit (encoded by the Chrna2 gene) to make a hypersensitive nAChR. Using a dorsal hippocampus-dependent task of preexposure-dependent contextual fear conditioning, adolescent hypersensitive Chrna2L9'S/L9'S male mice exhibited impaired learning and memory. The deficit was rescued by low-dose nicotine exposure. Electrophysiological studies demonstrated that hypersensitive α2 nAChRs potentiate acetylcholine-induced ion channel flux in oocytes and acute nicotine-induced facilitation of dorsal/intermediate CA1 hippocampal long-term potentiation in Chrna2L9'S/L9'S mice. Adolescent male mice null for the α2 nAChR subunit exhibited a baseline deficit in learning that was not reversed by an acute dose of nicotine. These effects were not influenced by locomotor, sensory or anxiety-related measures. Our results demonstrated that α2* nAChRs influenced hippocampus-dependent learning and memory, as well as nicotine-facilitated CA1 hippocampal synaptic plasticity.

Targeted deletion of the mouse α2 nicotinic acetylcholine receptor subunit gene (Chrna2) potentiates nicotine-modulated behaviors.

Baseline and nicotine-modulated behaviors were assessed in mice harboring a null mutant allele of the nicotinic acetylcholine receptor (nAChR) subunit gene α2 (Chrna2). Homozygous Chrna2(-/-) mice are viable, show expected sex and Mendelian genotype ratios, and exhibit no gross neuroanatomical abnormalities. A broad range of behavioral tests designed to assess genotype-dependent effects on anxiety (elevated plus maze and light/dark box), motor coordination (narrow bean traverse and gait), and locomotor activity revealed no significant differences between mutant mice and age-matched wild-type littermates. Furthermore, a panel of tests measuring traits, such as body position, spontaneous activity, respiration, tremors, body tone, and startle response, revealed normal responses for Chrna2-null mutant mice. However, Chrna2(-/-) mice do exhibit a mild motor or coordination phenotype (a decreased latency to fall during the accelerating rotarod test) and possess an increased sensitivity to nicotine-induced analgesia in the hotplate assay. Relative to wild-type, Chrna2(-/-) mice show potentiated nicotine self-administration and withdrawal behaviors and exhibit a sex-dependent enhancement of nicotine-facilitated cued, but not trace or contextual, fear conditioning. Overall, our results suggest that loss of the mouse nAChR α2 subunit has very limited effects on baseline behavior but does lead to the potentiation of several nicotine-modulated behaviors.

A point mutation in the hair cell nicotinic cholinergic receptor prolongs cochlear inhibition and enhances noise protection.

The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T) at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T) mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.

Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice.

In humans, tobacco withdrawal produces symptoms that contribute to the difficulty associated with smoking cessation. Nicotine withdrawal symptoms can also be observed in rodents. A major standing question is which nicotinic receptor subtypes and which areas of the brain are necessary for nicotine withdrawal to occur. Using knock-out mice, we previously showed that the beta4, but not the beta2 subunit of nicotinic acetylcholine receptors, is necessary for the somatic manifestations of nicotine withdrawal. Since the beta4 subunit is highly expressed in the medial habenula, we focused our studies on the medial habenula and its primary target, the interpeduncular nucleus. In particular, we studied nicotine withdrawal in mice lacking the alpha2 or the alpha5 nicotinic receptor subunits, which are highly expressed in the interpeduncular nucleus. We precipitated withdrawal by systemically injecting the nicotinic antagonist mecamylamine in mice chronically treated with nicotine. Both the alpha2 and the alpha5 null mutations abolished the somatic manifestations of nicotine withdrawal. In addition, in wild-type mice chronically treated with nicotine, mecamylamine precipitated withdrawal when microinjected into the habenula or the interpeduncular nucleus, but not into the cortex, ventral tegmental area or hippocampus. Our results demonstrate a major role for the habenulo-interpeduncular system and the nicotinic receptor subunits expressed therein, in nicotine withdrawal symptoms. Our data suggest that the efforts to develop new smoking cessation therapies should concentrate on these areas and receptor types.

The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands.

Mutations in the DSL (Delta, Serrate, Lag2) Notch (N) ligand Delta-like (Dll) 3 cause skeletal abnormalities in spondylocostal dysostosis, which is consistent with a critical role for N signaling during somitogenesis. Understanding how Dll3 functions is complicated by reports that DSL ligands both activate and inhibit N signaling. In contrast to other DSL ligands, we show that Dll3 does not activate N signaling in multiple assays. Consistent with these findings, Dll3 does not bind to cells expressing any of the four N receptors, and N1 does not bind Dll3-expressing cells. However, in a cell-autonomous manner, Dll3 suppressed N signaling, as was found for other DSL ligands. Therefore, Dll3 functions not as an activator as previously reported but rather as a dedicated inhibitor of N signaling. As an N antagonist, Dll3 promoted Xenopus laevis neurogenesis and inhibited glial differentiation of mouse neural progenitors. Finally, together with the modulator lunatic fringe, Dll3 altered N signaling levels that were induced by other DSL ligands.

The alpha10 nicotinic acetylcholine receptor subunit is required for normal synaptic function and integrity of the olivocochlear system.

Although homomeric channels assembled from the alpha9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both alpha9 and alpha10 subunits. To gain insight into alpha10 subunit function in vivo, we examined olivo cochlear innervation and function in alpha10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in alpha10(+/+) and alpha10(+/-) mice, with no detectable responses to ACh in alpha10(-/-) mice. In contrast, a proportion of alpha10(-/-) outer hair cells showed small ACh-evoked currents. In alpha10(-/-) mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual alpha9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, alpha10(-/-) mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that alpha9(-/-) and alpha10(-/-) mice have overlapping but nonidentical phenotypes. Moreover, alpha10 nAChR subunits are required for normal olivocochlear activity because alpha9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in alpha10(-/-) mutant mice.

Pharmacological and immunochemical characterization of alpha2* nicotinic acetylcholine receptors (nAChRs) in mouse brain.

AIM: alpha2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of alpha2* nAChRs in these mouse brain regions. METHODS: alpha2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of alpha2 subunit-containing (alpha2*) nAChRs in these two regions. RESULTS: [(125)I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that alpha2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8+/-1.7 and 92+/-26 fmol mg(-1) protein, respectively). Pharmacological characterization using the beta2-selective drug A85380 to inhibit [(125)I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize alpha2* nAChRs. Protocols were established to immunoprecipitate beta2 and beta4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from beta2- and beta4-null mutant mice, and efficacy was good (>90% of beta2* and >80% of beta4* nAChRs were routinely recovered). CONCLUSION: Immunoprecipitation experiments indicated that interpeduncular nucleus alpha2* nAChRs predominantly contain beta2 subunits, while those in olfactory bulbs contain mainly beta4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both beta2 and beta4 subunits.

Properties of mutated murine alpha4beta2 nicotinic receptors linked to partial epilepsy.

We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.

CADASIL Notch3 mutant proteins localize to the cell surface and bind ligand.

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementia arising from abnormal arteriolar vascular smooth muscle cells. CADASIL results from mutations in Notch3 that alter the number of cysteine residues in the extracellular epidermal growth factor-like repeats, important for ligand binding. It is not known whether CADASIL mutations lead to loss or gain of Notch3 receptor function. To examine the functional consequences of CADASIL mutations, we engineered 4 CADASIL-like mutations into rat Notch3 and have shown that the presence of an unpaired cysteine does not impair cell-surface expression or ligand binding.

Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels.

The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGAT(flox/flox) mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT(-/-) mice were the same as Cx57-VGAT(+/+) controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABAA receptor agonist muscimol in Cx57-VGAT(-/-) mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT(-/-) mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors.

The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors.

Nigrostriatal dopaminergic neurons express many nicotinic acetylcholine receptor (nAChR) subunits capable of forming multiple nAChR subtypes. These subtypes are expressed differentially along the neuron and presumably mediate diverse responses. beta3 subunit mRNA has restricted expression but is abundant in the substantia nigra and ventral tegmental areas. To investigate the potential role(s) of nicotinic receptors containing the beta3 subunit in dopaminergic tracts, we generated mice with a null mutation in the beta3 gene. We were thereby able to identify a population of beta3-dependent alpha-conotoxin MII-binding nAChRs that modulate striatal dopamine release. Changes were also observed in locomotor activity and prepulse inhibition of acoustic startle, behaviors that are controlled, in part, by nigrostriatal and mesolimbic dopaminergic activity, respectively, suggesting that beta3-containing nAChRs modulate these behaviors.

Cloning of the zebra finch androgen synthetic enzyme CYP17: a study of its neural expression throughout posthatch development.

Male zebra finches develop a robust neural song system that supports singing, but females have a minimal song circuit and do not sing. Estrogens masculinize the song circuit and are especially potent during the first 3 weeks of posthatch development. The gonads do not seem to supply the masculinizing steroids, implying that another tissue synthesizes steroids. Evidence suggests that the brain is capable of synthesizing neurosteroids, which in developing zebra finches may be required for song system differentiation. Aromatase, the enzyme that synthesizes estrogen from androgen, is equally abundant in male and female brains. To investigate further the potential for neurosteroidogenesis in the zebra finch brain, we cloned and examined the expression of 17alpha-hydroxylase/17,20 lyase (CYP17), the enzyme that synthesizes the androgenic substrate for aromatase. We used Northern blots, reverse transcription-polymerase chain reaction, and in situ hybridization to show that CYP17 is transcribed in developing and adult brains. CYP17 is transcribed at developmental stages and in brain areas potentially important to aspects of the developing song system, although no sex difference was detected in mRNA levels. Our results support the hypothesis that neurosteroids may act to influence brain organization and function in the zebra finch.

Nicotine gates long-term potentiation in the hippocampal CA1 region via the activation of alpha2* nicotinic ACh receptors.

Hippocampal CA1 pyramidal cells receive two major excitatory synaptic inputs via the Schaffer collateral (SC) and temporoammonic (TA) pathways. Nicotine promotes induction of long-term potentiation (LTP) in the SC path; however, it is not known whether the modulatory effect of nicotine on LTP induction is pathway-specific. Here we show that nicotine suppresses LTP induction in the TA path. Interestingly, these opposing effects of nicotine were absent or greatly reduced in alpha2 nicotinic acetylcholine receptor (nAChR)-knockout (KO) mice, suggesting that an alpha2-containing nAChR subtype mediates these effects. Optical imaging with a voltage-sensitive dye revealed significantly stronger membrane depolarization in the presence of nicotine in the SC path, facilitating spread of excitatory neural activity along both the somatodendritic and the CA1 proximodistal axes. These effects of nicotine were also absent in alpha2 nAChR-KO mice, suggesting that the enhanced optical signal is related to the nicotine-induced facilitation of LTP induction. In contrast, in the TA path nicotine terminated depolarization more quickly and increased the delayed hyperpolarization in the termination zone of the TA path input. These inhibitory effects of nicotine were absent in alpha2 nAChR-KO mice and, thus, most probably underlie the nicotine-induced suppression of LTP induction. Our results suggest that nicotine influences the local balance between excitation and inhibition, gates LTP, and directs information flow through the hippocampal circuits via the activation of alpha2* nAChRs. These effects of nicotine may represent the cellular basis of nicotine-mediated cognitive enhancement.

Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy.

Selected mutations in the human alpha4 or beta2 neuronal nicotinic acetylcholine receptor subunit genes cosegregate with a partial epilepsy syndrome known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To examine possible mechanisms underlying this inherited epilepsy, we engineered two ADNFLE mutations (Chrna4(S252F) and Chrna4(+L264)) in mice. Heterozygous ADNFLE mutant mice show persistent, abnormal cortical electroencephalograms with prominent delta and theta frequencies, exhibit frequent spontaneous seizures, and show an increased sensitivity to the proconvulsant action of nicotine. Relative to WT, electrophysiological recordings from ADNFLE mouse layer II/III cortical pyramidal cells reveal a >20-fold increase in nicotine-evoked inhibitory postsynaptic currents with no effect on excitatory postsynaptic currents. i.p. injection of a subthreshold dose of picrotoxin, a use-dependent gamma-aminobutyric acid receptor antagonist, reduces cortical electroencephalogram delta power and transiently inhibits spontaneous seizure activity in ADNFLE mutant mice. Our studies suggest that the mechanism underlying ADNFLE seizures may involve inhibitory synchronization of cortical networks via activation of mutant alpha4-containing nicotinic acetylcholine receptors located on the presynaptic terminals and somatodendritic compartments of cortical GABAergic interneurons.