Tetraethylammonium ions block the nicotinic cholinergic receptors of cochlear outer hair cells

Cochlear outer hair cells (OHCs) express nicotinic acetylcholine receptors (nAChRs) whose calcium permeability allow the activation of co-localized Ca²⁺-sensitive K⁺ channels (SK-type). The large organic cation tetraethylammonium (TEA) is known to block at millimolar concentration voltage-gated and Ca²⁺-activated K⁺ currents in OHCs. In the present study, we show that extracellular TEA blocked much more efficiently, at micromolar concentrations, ACh-evoked K⁺ currents in isolated guinea pig OHCs. The dose–inhibition curve indicated an IC₅₀ of 60 μM, a value two orders of magnitude lower than the one reported on SK or BK channels. The site of the blocking action was on the extracellular side of the plasma membrane since 10 mM intracellular TEA did not prevent or change the characteristics of the ACh-evoked K⁺ current. The block of this K⁺ current in OHCs was mainly explained by a direct action of TEA at the nAChRs. Indeed, we demonstrated that extracellular TEA inhibited directly the ionotropic cation current flowing through the nAChRs (IC₅₀=30 μM). This study demonstrated for the first time that extracellular TEA is an effective blocker of the OHCs’ nAChRs.     

Fast cholinergic efferent inhibition in guinea pig outer hair cells

Hair cells of inner ear are suggested to be inhibited by the activation of the alpha9-containing nicotinic acetylcholine (ACh) receptors (alpha9-containing nAChRs). Several studies have suggested that the native nicotinic-like ACh receptors (nAChRs) in hair cells display a significant permeability of Ca(2+) ions and unusual pharmacological properties. The activation of native nAChRs will initiate the hyperpolarization of hair cells by activation of the small conductance, Ca(2+)-activated K(+) channels (SK). In this work, the properties of the ACh-sensitive potassium current (IK(ACh)) in outer hair cells (OHCs) of guinea pigs were investigated by employing whole-cell patch-clamp. Followed by perfusion of ACh, OHCs displayed a rapid desensitized current with an N-shaped current-voltage curve (I-V) and a reversal potential of - 66 +/- 7 mV. The IK(ACh) was still present during perfusion of either iberiotoxin (IBTX, 200 nM) or TEA (5 mM) but was potently inhibited by apamin (1 muM), TEA (30 mM). The IK(ACh) demonstrated a strong sensitivity to alpha-bungarotoxin (alpha-BgTx), bicuculline and strychnine. These results suggested that OHCs display the well-known SK current, which might be gated by the alpha9-containing nAChRs. Two important changes were present after lowering the Ca(2+) concentration in the external conditions from 2 mM to 0.2 mM: one was a flattened N-shape I-V relationship with a maximum shifted toward hyperpolarized potentials from -20 approximately -30 mV approximately -40 to -50 mV, the other was a significant reduction in the agonist maximal response (percentage of maximal response 10.5 +/- 5.4). These results indicated that native nAChRs are both permeable to and modulated by extracellular Ca(2+) ions. Taken together, this work provides direct evidences that SK channels in OHCs of guinea pigs are gated by alpha9-containing nAChRs, which play an important role in the fast cholinergic efferent inhibition. This fast inhibition is both potently dependent on the permeability of Ca(2+) ions through the native nAChRs and modulated by Ca(2+) ions.     

Ventral cochlear nucleus neural discharge characteristics in the absence of outer hair cells

The role of the cochlear outer hair cell (OHC) in auditory processing remains poorly understood. The OHCs possess an independent afferent innervation which constitutes 5-10% of cochlear afferent neurons and which appears to project to the cochlear nucleus (CN). Whether the OHCs contribute to the processing of auditory signals in the CN has not been determined. To address this question, kanamycin ototoxicity was used to produce selective OHC loss while leaving the inner hair cell (IHC) population largely intact, in the basal portion of the cochlea of chinchillas. Single unit responses were then recorded in the ventral cochlear nucleus (VCN), and compared to responses in untreated subjects. Many of the changes observed in VCN neural responses reflected changes which have previously been reported in the VIIIth nerve. However, frequency tuning curve tip segments which were normal in both bandwidth and length were observed in approximately 22% of the units associated with regions of complete OHC loss and preservation of IHCs. This has not been reported in previous OHC lesion studies. Also, first spike latency was found to be significantly lengthened for units associated with the OHC free regions. Those features of VCN neural responses which first arise within the CN, such as non-primary-like post-stimulus-time histogram response patterns, were unaffected by OHC loss. These results suggest that afferent fibers associated with OHCs do not play a major role in signal processing in the VCN.     

Increasing intracellular free calcium induces circumferential contractions in isolated cochlear outer hair cells

The relationship between intracellular free calcium and the motile responses of outer hair cells isolated from the guinea pig cochlea was examined. Calcium levels were modulated by the addition of the calcium ionophores ionomycin or A23187 to the incubation medium and monitored with the fluorescent calcium indicator fluo-3. In the presence of 1.25 mM external calcium, the application of either ionophore (10 microM) led to an increase in intracellular free calcium from 157 +/- 76 nM to 1200 +/- 500 nM within 30-60 sec. Concurrently, cells elongated by 1-2 microns, cell diameter decreased, and cell volume shrank by 269 +/- 220 microns 3 (5.0 +/- 4.1%). The reduction in diameter was most pronounced in the middle portion of the cell (4.4% +/- 4.2%), also evident in the apical region (3.1% +/- 4.8%) but not significant in the basal region near the nucleus. This response was observed in outer hair cells from basal and apical turns of the cochlea and was reversed when the cells were rinsed with calcium-free medium supplemented with 2 mM EGTA. Optical imaging of the cell membrane with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta] [2-(di-n-butylaminol)-6-naphthyl vinyl] pyridinium betaine during the elevation of intracellular calcium demonstrated features of contractility at the lateral cell membrane. A rise in intracellular calcium as well as the motile response was still observed after a 5-min exposure of the cells to a calcium-free solution (supplemented with 2 mM EGTA), indicating that the ionophore was also able to liberate calcium from intracellular sites. However, depletion of calcium stores through prolonged incubation of the cells in calcium-free medium (30-60 min) suppressed both the calcium signal and the cell response. The calmodulin inhibitors trifluoperazine and pimozide (30 microM) blocked the cell motility induced by ionomycin while they left the increase of intracellular calcium unaffected. These observations suggest that calcium-dependent circumferential contractions in outer hair cells are mediated by calmodulin. The application to the extracellular medium of putative neurotransmitters of the cochlear efferent system such as acetylcholine and GABA led to neither an increase in intracellular calcium nor a modification of cell shape. Therefore, these neurotransmitters may not be directly involved in calcium-induced contractions in outer hair cells. The circumferential contractions altered the stiffness of the plasma membrane and the turgor of the cell. Under normal conditions, changes in cell volume were inversely proportional to the osmotic pressure of the extracellular medium following van't Hoff's law.(ABSTRACT TRUNCATED AT 400 WORDS)     

Desensitization of nicotinic ACh receptors: shaping cholinergic signaling

Nicotinic ACh receptors (nAChRs) can undergo desensitization, a reversible reduction in response during sustained agonist application. Although the mechanism of desensitization remains incompletely understood, recent investigations have elucidated new properties underlying desensitization, indicating that it might be important to control synaptic efficacy, responses to cholinergic agents, and certain nAChR-related disease states. Thus, studying how different nAChR subunits contribute to desensitization might help to explain variations in responsiveness to drugs, and might thus improve their therapeutic applications. Agonist-specific desensitization, desensitization arising from resting receptors, natural mutations dramatically altering desensitization, and the possibility that recovery from desensitization is an important process for modulating receptor function, together provide a new framework for considering desensitization as a target to shape cholinergic signaling.     

Mice lacking Dfna5 show a diverging number of cochlear fourth row outer hair cells

A complex mutation in DFNA5, resulting in exon 8 skipping, causes autosomal dominant hearing impairment, which starts in the high frequencies between 5 and 15 years of age and progressively affects all frequencies. To study its function in vivo, Dfna5 knockout mice were generated through the deletion of exon 8, simultaneously mimicking the human mutation. To test the hearing impairment, frequency-specific Auditory Brainstem Response (ABR) measurements were performed at different ages in two genetic backgrounds (C57Bl/6J and CBA/Ca), but no differences between Dfna5-/- and Dfna5+/+ mice could be demonstrated. Morphological studies demonstrated significant differences in the number of fourth row outer hair cells between Dfna5-/- mice and their wild-type littermates. These results were obtained in both genetic backgrounds, albeit with opposite effects. In contrast to the results obtained in Dfna5-/- zebrafish, we did not observe different UDP-glucose dehydrogenase and hyaluronic acid levels in Dfna5-/- mice when compared to Dfna5+/+ mice.     

Isolation of outer hair cells from the cochlear sensory epithelium in whole-mount preparation using laser capture microdissection

Outer hair cells (OHCs) play an important role in frequency selectivity and signal amplification in the mammalian cochlea. Because OHCs are relatively few in number and a minority of the cells in the cochlea, separating and isolating them for applications such as cDNA library creation and proteomic studies is a challenging task. Laser capture microdissection (LCM) is designed to capture cells from very thin tissue sections, it can accurately isolate specific cells from large regions of tissue for RNA, DNA, and proteomic studies. Due to the constraints of cochlear anatomy, thin sections of the cochlea contain small numbers of OHCs. Therefore, we adapted the LCM technique to isolate OHCs from organ of Corti whole-mounts, each of which contain hundreds of OHCs that are simultaneously accessible and collectable. For comparison, we also used a more traditional mechanical dissection. The quality of cDNA derived from the OHCs collected with LCM and with the traditional mechanical method are compared and the merits and limitations of the techniques discussed. A similar approach can also be used to isolate large quantities of inner hair cells and selected supporting cells from the whole-mount cochlear preparation.     

High-resolution three-dimensional imaging of the lateral plasma membrane of cochlear outer hair cells by atomic force microscopy

The outer hair cells (OHCs) from the mammalian organ of Corti are assumed to enhance the sensitivity and the selectivity of the cochlea via an electromotile response to sound stimulation. These OHC mechanical changes feed energy back into the cochlea before completion of the transduction process by inner hair cells. OHC electromotility is thought to depend on specific transmembrane motor proteins. Electron microscopy has been used previously to image the OHC lateral plasma membrane, where voltage sensors and motors are located. A very specific and regular organization of membrane particles has been described, together with an equally specific submembraneous meshwork of cytoskeleton anchored to the plasma membrane. To confirm and extend these observations, we have used, for the first time on the OHC lateral wall, atomic force microscopy (AFM). As a result of an improved tapping mode technique as well as the unique ultrastructural organization of the OHC plasma membrane, we have obtained high-resolution three-dimensional (3D) images of a markedly enhanced quality, allowing high-resolution 3D imaging. Tapping-mode AFM confirmed the presence of regularly aligned particles (presumably transmembrane proteins) on both faces of the OHC plasma membrane. It also revealed the presence of markedly different membrane domains, smooth and undulating. The differences between these zones probably are due to local differences in cytoskeleton-membrane interactions. Moreover, 3D reconstructions allowed us to distinguish between globular and pore-like particles, a distinction that may be of great functional significance.     

Differential regulation of muscarinic and nicotinic receptors by cholinergic stimulation in cultured avian retina cells

Sustained cholinergic stimulation of retina cells grown in primary aggregate and monolayer cultures regulated the concentration of muscarinic but not nicotinic receptors. Muscarinic receptor sites, quantified by the binding of [³H]quinuclidinyl benzilate to membranes and the binding of [³H]N-methyl-scopolamine to intact cells, decreased up to 84% following long-term incubation of cultures in muscarinic agonists. This decrease was blocked by atropine and was not induced by chronic nicotine treatment. The rate of the muscarinic response was biphasic. A rapid binding decrease of 30% occurred within 15 min. The slower phase was half-maximal by 6 h and was complete by 24 h. Neither the fast nor the slow receptor loss was reversed by the guanine nucleotide GppNp. Three different depolarizing agents (gramicidin D, protoveratrine, and ouabain) blocked the cholinergic-induced receptor loss, but the hyperpolarizing ionophore valinomycin had no effect. In contrast to the muscarinic response, nicotinic receptor binding was not altered by chronic receptor stimulation. Exposure to receptor-saturating doses of carbamylcholine or nicotine for 48 h did not change [¹²⁵I]α-bungarotoxin or [³H]bromoacetylcholine binding. Differential regulation of acetylcholine receptors is discussed in relation to the possible physiological role of receptor regulation by receptor activity.     

Degeneration of sensory outer hair cells following pharmacological blockade of cochlear KCNQ channels in the adult guinea pig

In the inner ear, hair cell function is inextricably linked with intracellular potassium homeostasis. KCNQ potassium channels may play an important role by preventing accumulation of potassium in the hair cells. Linopirdine, a tool useful in targeting native or heterologous KCNQ channels, was used to study the role of KCNQ channels in the guinea pig cochlea. When perfused into intact cochlea, linopirdine transiently increases the summating potential and endocochlear potential, suggesting that it alters K+ homeostasis. The concomitant decrease in cochlear microphonic potential and distortion product otoacoustic emission amplitude indicates that linopirdine has an effect on the outer hair cells (OHCs). To determine the pathological consequences of the inhibition of cochlear KCNQ channels, we developed a hearing loss model based on a chronic intracochlear perfusion of linopirdine via an osmotic minipump. Ultrastructural analysis reveals that KCNQ channel blockade leads to OHC degeneration. Together, these results demonstrate that KCNQ channels, most probably of the KCNQ4 subtype, are crucial for the function and survival of sensory OHCs. Clinically, KCNQ4 channel dysfunction is known to be associated with the DFNA2 form of nonsyndromic dominant deafness. Our study shows that OHC KCNQ4 dysfunction could contribute to the early (40dB) hearing loss, but not for the profound deafness observed at the final stage of this disease.     

Aminoglycoside antibiotics impair calcium entry but not viability and motility in isolated cochlear outer hair cells

Cochlear outer hair cells have been well established as primary targets of the ototoxic actions of aminoglycoside antibiotics. These cells, isolated from the guinea pig cochlea and maintained in short-term culture, were used as a model for evaluating the acute effects of gentamicin on cell viability, depolarization-induced transmembrane calcium flux, and depolarization-induced motile responses. On the basis of morphology and fluorochromasia, the presence of extracellular gentamicin as high as 5 mM did not affect the viability of the cells for up to 6 hr, the longest time tested. Viable cells showed binding of fluorescently tagged gentamicin to their base but excluded the drug from their cytoplasm. In response to [K+]-depolarization, intracellular calcium levels (monitored with the fluorescent calcium-sensitive dye fluo-3) increased from a resting value of 218 +/- 102 nM to 2,018 +/- 1,077 nM concomitant with a cell shortening of 0.7% +/- 1.3%. The depolarization-induced calcium increase was apparently caused by calcium entry into the cell as it was inhibited by the calcium-channel blocker methoxyverapamil and prevented in the absence of extracellular calcium. Both gentamicin and neomycin blocked the [K+]-induced calcium increase at an IC50 of 50 microM. Despite the inhibition of calcium entry the ability of the outer hair cells to shorten under [K+]-depolarization was not impaired; in fact, cell shortening was even more pronounced in the absence of calcium influx (2.6% +/- 1.4%). This argues effectively against the existence of a calcium-dependent actomyosin-mediated component in [K+]-induced shape changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response characteristics of mammalian cochlear hair cells

Intracellular recordings were made from the low frequency region (third turn) of the guinea pig cochlea. Response characteristics are compared to gross potentials obtained from the organ of Corti fluid space. Inner hair cells (IHCs) possess relatively low (median, -32 mV) initial membrane potentials, whereas that of outer hair cells (OHCs) is higher (median, -53.5 mV). In response to tone burst stimuli, both cell types produce a combination of AC and DC responses. The latter are depolarizing for IHCs but may be of either polarity for OHCs. In terms of their AC responses, IHCs are about 12 dB more sensitive than OHCs. At low sound levels these cells are more linear than high frequency hair cells (Russell, I. J., and P. M. Sellick (1978) J. Physiol. (Lond.) 284: 261-290), judging from the relation between AC and DC response components. At high sound levels pronounced response saturation is seen. The overall tuning properties of the two hair cell types are rather similar, even though IHCs exhibit low frequency velocity dependence, whereas OHCs are displacement sensitive and the cell membrane time constant is larger for IHCs. In order to fit IHC experimental data it is necessary to assume the presence of an underdamped complex pole above the best frequency. The electrical behavior of the OHC does not disqualify it as a conveyor of auditory information to the central nervous system, even though its primary function may be that of a mechanical effector (evidence summarized by Dallos, P. (1985) in Contemporary Sensory Neurobiology, Alan R. Liss, Inc., New York, pp. 207-230).     

Properties and regional distribution of nicotinic cholinergic receptors in the rat hypothalamus

The rat hypothalamus has the capacity to bind α-bungarotoxin with high affinity to a saturable number of non-interacting receptors with a pharmacologic profile consistent with a nicotinic receptor. Studies of the hypothalamic nuclear distribution of cholinergic receptors showed no specific pattern of enrichment of muscarinic receptors. In contrast, there was a distinct distribution of nicotinic receptors with high concentrations in the suprachiasmatic, dorsomedial and preoptic suprachiasmatic nuclei. Thus, the quantitative distribution of nicotinic receptors in hypothalamic nuclei is in general agreement with the observed autoradiographic distribution of radioactive alpha bungarotoxin. Further, these results confirm the existence of high concentrations of nicotinic receptors in hypothalamic regions of the rat implicated in neuroendocrine function.     

Effects of protein kinase and phosphatase inhibitors on slow shortening of guinea pig cochlear outer hair cells

The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation, and changes in the cell length, volume and intracellular Cl⁻ concentration were microscopically monitored using a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor, W-7, and a calcium calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K⁺ with valinomycin. Among the protein phosphatase inhibitors tested, a type 1 and 2A protein phosphatase inhibitor, calyculin A, inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased Cl⁻ permeability, but neither by the increased K⁺ conductance with valinomycin nor by the increased Ca²⁺ conductance with A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine, inhibited the shortening, which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor, okadaic acid, but not by calyculin A. These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells; the K⁺ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the Cl⁻ channel opening depends on calyculin A-sensitive protein phosphatase activity.     

The voltage-sensitive motor protein and the Ca2+-sensitive cytoskeleton in developing rat cochlear outer hair cells

Cochlear outer hair cells (OHCs) possess a unique fast voltage-driven motility associated with a voltage-sensitive motor protein embedded in the basolateral membrane. This mechanism is believed to underlie the cochlear amplification in mammals. OHCs also have a Ca2+/calmodulin-dependent mechanical pathway which involves a submembranous circumferential cytoskeleton. The purpose of this study was to compare the functional appearance of the voltage-sensitive motor proteins with that involving the Ca2+-sensitive cytoskeleton during postnatal development of rat OHCs. We demonstrate that whole-cell electromotility and Ca2+-voked mechanical responses, by ionomycin, develop concomitantly after postnatal day 5 (P5). These two mechanical properties also develop simultaneously in OHCs isolated from two-week-old cultures of P0-P1 organs of Corti. This excludes the participation of neural innervation in the postnatal maturation of the OHCs' motile properties. In addition, we show that the expression of the membranous voltage-sensitive motor protein precedes, by several days, the appearance of whole-cell electromotility. The concomitant development of whole-cell electromotility and Ca2+-sensitive motility, both in vivo and in vitro, underlines the cytoskeleton as an important factor in the functional organization of the voltage-sensitive motor proteins within the plasma membrane.     

Strategies for replacing lost cochlear hair cells

The auditory sensory epithelium is a mosaic composed of sensory (hair) cells and several types of non-sensory (supporting) cells. All these cells are highly differentiated in their structure and function. Mosaic epithelia (and other complex tissues) are generally formed by differentiation of distinct and specialized cell types from common progenitors. Most types of epithelial tissues maintain a population of undifferentiated (basal) cells which facilitate turnover (renewal) and repair, but this is not the case for the organ of Corti in the cochlea. Therefore, when cochlear hair cells are lost they cannot be replaced. Consequently, sensorineural hearing loss is permanent. In designing therapy for sensorineural deafness, the most important task is to find a way to generate new cochlear hair cells to replace lost cells.     

Selective inhibition of nicotinic cholinergic receptors by proadrenomedullin N-terminal 12 peptide in bovine adrenal chromaffin cells

We studied whether a novel proadrenomedullin derived peptide was present and what was its physiological function in cultured bovine adrenal chromaffin cells. We found a high level of proadrenomedullin N-terminal 12 peptide (PAMP-12) which consists of a peptide from 9th amino acid to 20th amino acid of proadrenomedullin N-terminal 20 peptide (PAMP-20). PAMP-12 was released from the cells along with catecholamine upon stimulation of nicotinic cholinergic receptors. When PAMP-12 was added in the incubation medium, this peptide inhibited nicotinic receptor-mediated catecholamine release and influx of Na(+) and Ca(2+) into the cells. PAMP-12 did not affect catecholamine release evoked by histamine or by depolarization by high concentration of potassium. PAMP-12 also inhibited synthesis of catecholamines as well as the activation of tyrosine hydroxylase by nicotinic stimulation. Thus, PAMP-12 is an endogenous peptide that regulates release and synthesis of catecholamines by acting on nicotinic cholinergic receptors in an autocrine manner in adrenal chromaffin cells.     

Two distinct Ca(2+)-dependent signaling pathways regulate the motor output of cochlear outer hair cells.

The outer hair cells (OHCs) of the cochlea have an electromotility mechanism, based on conformational changes of voltage-sensitive "motor" proteins in the lateral plasma membrane. The translocation of electrical charges across the membrane that accompanies electromotility imparts a voltage dependency to the membrane capacitance. We used capacitance measurements to investigate whether electromotility may be influenced by different manipulations known to affect intracellular Ca(2+) or Ca(2+)-dependent protein phosphorylation. Application of acetylcholine (ACh) to the synaptic pole of isolated OHCs evoked a Ca(2+)-activated apamin-sensitive outward K(+) current. It also enhanced electromotility, probably because of a phosphorylation-dependent decrease of the cell's axial stiffness. However, ACh did not change the voltage-dependent capacitance either in conventional whole-cell experiments or under perforated-patch conditions. The effects produced by the Ca(2+) ionophore ionomycin mimicked those produced by ACh. Hyperpolarizing shifts of the voltage dependence of capacitance and electromotility were induced by okadaic acid, a promoter of protein phosphorylation, whereas trifluoperazine and W-7, antagonists of calmodulin, caused opposite depolarizing shifts. Components of the protein phosphorylation cascade-IP(3) receptors and calmodulin-dependent protein kinase type IV-were immunolocalized to the lateral wall of the OHC. Our results suggest that two different Ca(2+)-dependent pathways may control the OHC motor output. The first pathway modulates cytoskeletal stiffness and can be activated by ACh. The second pathway shifts the voltage sensitivity of the OHC electromotile mechanism and may be activated by the release of Ca(2+) from intracellular stores located in the proximity of the lateral plasma membrane.     

Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission

Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys‐loop ligand‐gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss‐of‐function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss‐of‐function insertions or deletions (indels) by these ‘all‐in‐one’ HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type‐specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre‐dependent manner were validated using viral microinjections and genetic crosses to common Cre‐driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno‐associated virus (AAV) delivery of sgRNAs. Finally, the mouse β2 nAChR gene was successfully targeted in dopamine transporter (DAT)‐positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre‐dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.