ABSTRACT Fyn kinase in the dorsomedial striatum (DMS) of rodents plays a central role in mechanisms underlying excessive alcohol intake. The DMS is comprised of medium spiny neurons (MSNs)

that project directly (dMSNs) or indirectly (iMSNs) to the substantia nigra. Here, we examined the cell-type specificity of Fyn’s actions in alcohol use. First, we knocked down Fyn

selectively in DMS dMSNs or iMSNs of mice and measured the level of alcohol consumption. We found that downregulation of Fyn in dMSNs, but not in iMSNs, reduces excessive alcohol but not

saccharin intake. D1Rs are coupled to Gαs/olf, which activate cAMP signaling. To examine whether Fyn’s actions are mediated through cAMP signaling, DMS dMSNs were infected with GαsDREADD,

alcohol intake and observed that CNO-dependent activation of GαsDREADD in DMS dMSNs but not iMSNs increases alcohol but not saccharin intake. Finally, we examined the contribution of Fyn to

GαsDREADD-dependent increase in alcohol intake, and found that systemic administration of the Fyn inhibitor, AZD0503 blocks GαsDREADD-dependent increase in alcohol consumption. Our results

suggest that the cAMP-Fyn axis in the DMS dMSNs is a molecular transducer of mechanisms underlying the development of excessive alcohol consumption. You have full access to this article via

your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS CHEMOGENETIC SELECTIVE MANIPULATION OF NUCLEUS ACCUMBENS MEDIUM SPINY NEURONS BIDIRECTIONALLY CONTROLS ALCOHOL INTAKE IN

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Open access 27 July 2021 INTRODUCTION The dorsomedial striatum (DMS) is critically involved in processes such as locomotion [1], and goal-directed behaviors [2, 3]. The DMS is comprised

primarily of GABAergic medium spiny projection neurons (MSNs) that receive dopaminergic input from the midbrain [4]. MSNs can be divided into two populations of neurons that take part in

opposing activities [5]; MSNs that project directly to the substantia nigra pars reticula (SNr) facilitate actions and are defined as direct MSNs (dMSNs) [5], and MSNs that project

indirectly to the SNr gate actions and are defined as indirect MSNs (iMSNs) [5]. dMSNs selectively express the dopamine D1 receptors (D1Rs) whereas iMSNs express the dopamine D2 receptors

(D2Rs) [5]. In the striatum, D1Rs are coupled to Gαolf, a homolog of Gαs [6]. Stimulation of Gαs/olf-coupled receptors results in the production of the second messenger cyclic adenosine

monophosphate (cAMP) [6, 7], which binds to, and activates, protein kinase A (PKA) [8], a kinase that plays an important role in the adult brain [9,10,11]. In contrast, D2Rs are coupled to

Gαi, which inhibits cAMP signaling [7]. dMSNs and iMSNs exert balanced influence on locomotion and goal-directed behaviors [5], and an imbalance of dMSNs and iMSNs function has been

implicated in neurodegenerative disorders such as Parkinson’s disease [5, 12], as well as psychiatric disorders such as obsessive-compulsive disorder, anxiety, and addiction [13,14,15]. We

previously observed that Fyn kinase is activated in DMS dMSNs upon stimulation of D1Rs [16]. Fyn belongs to the Src family of non-receptor protein tyrosine kinases (PTKs) [17, 18], and is

highly expressed in the developing and adult brain in regions such as cortex, hippocampus, and cerebellum as well as in the striatum [19, 20]. Fyn plays an important role in the CNS [21], as

it modulates excitatory and inhibitory synaptic transmission and participates in learning and memory processes [21,22,23,24,25,26,27,28]. Dysfunction of Fyn signaling has been associated

with Alzheimer’s disease [29] and pain [23]. Accumulating data in humans and rodents also suggest that Fyn plays a central role in cellular neuroadaptations that underlie alcohol use

disorder (AUD) [30, 31]. Specifically, genetic mutations within the Fyn gene have been associated with increased susceptibility for the development and severity of AUD in humans [32,33,34],

and gene network association studies identified a link between Fyn and alcohol dependence [35]. Animal data suggest that Fyn plays a role in the acute tolerance to the hypnotic sedative

effect of alcohol [36, 37], as well as in alcohol drinking behavior [38,39,40]. Molecularly, excessive consumption of alcohol activates Fyn specifically in the DMS of mice and rats [39, 41,

42]. Once activated by alcohol, Fyn phosphorylates its substrate, GluN2B [39, 41, 42]. Alcohol-dependent Fyn-mediated phosphorylation of GluN2B produces a forward trafficking of the channel

and long-lasting enhancement of GluN2B activity in the DMS [39]. Inhibition of Fyn in the DMS of rats attenuates operant self-administration of alcohol [39], and systemic administration of

the Fyn inhibitor, AZD0530, attenuates goal-directed alcohol seeking and facilitates extinction in mice [40]. Together, these data suggest that Fyn in the DMS plays a central role in

neuroadaptations that underlie alcohol use. This study was aimed to explore the cellular specificity of Fyn-dependent molecular and behavioral neuroadaptations that drive AUD. METHODS The

description of purchased reagents, collection of brain samples, western blot analysis, immunoprecipitation, preparation of solutions, and the preparation of FLEX-shRNA-Fyn and FLEX-SCR is

detailed in the Supplementary Information section. ANIMALS C57BL/6 mice were obtained from Jackson Laboratories. Drd1a-Cre (D1-Cre) and AdoraA2-Cre (A2A-Cre) mice both of which are on

C57BL/6 background, were obtained from Mutant Mice Resource and Research Centers (MMRRC) UC Davis (David, CA). Ai14 mice were purchased from Jackson Laboratory (Bar Harbor, Maine). The

generation of D1-Cre/Ai14 mouse line is described in [43]. The same breeding strategy was used to generate the A2A-Cre/Ai14 mouse line. Mice were genotyped by polymerase chain reaction (PCR)

analysis of products derived from tail DNA. Male mice were 8–9 weeks old at the beginning of the experiments and were individually housed in temperature and humidity-controlled rooms under

a reversed 12-h light/dark cycle. Food and water were available ad libitum. All animal procedures were approved by the University of California San Francisco (UCSF) Institutional Animal Care

and Use Committee and were conducted in agreement with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC, UCSF). INFECTION OF THE DMS WITH FLEX-SHFYN AND

FLEX-SCR D1-Cre/Ai14 and A2A-Cre/Ai14 mice were anesthetized using a mixture of ketamine (120 mg/kg) and xylazine (8 mg/kg). Bilateral microinfusions were made using stainless steel

injectors (33 gauge, Small Parts) into the DMS (the stereotaxic coordinates were anterioposterior +1.1 mm from bregma; mediolateral ±1.2 mm from bregma and dorsoventral −2.8 and from bregma

for the first injection site and anterioposterior +1.3 mm from bregma; mediolateral ±1.2 mm from bregma and dorsoventral −3 mm from bregma for the second injection site). Animals were

infused with lentivirus-expressing FLEX-shFyn or its scramble control (FLEX-SCR) (1.2 μl/site with 2 sites of injection per hemisphere) at a concentration of 107 pg/ml at an injection rate

of 0.1 μl/min [16]. After each infusion, the injectors were left in place for an additional 10 min to allow the virus to diffuse. INFECTION OF THE DMS AND THE DORSOLATERAL STRIATUM (DLS)

WITH AAV-DIO-RM3D(GS)-MCHERRY D1-Cre or A2A-Cre mice were anesthetized using a mixture of ketamine (120 mg/kg) and xylazine (8 mg/kg). Bilateral microinfusions were made using stainless

steel injectors (33 gauge, small parts) into the DMS (the stereotaxic coordinates were anterioposterior +1.1 mm from bregma; mediolateral ±1.25 mm from bregma and dorsoventral −2.8 mm from

bregma) or the dorsolateral striatum (DLS) (the stereotaxic coordinates were anterioposterior +1.1, medialateral ±2.3 from bregma, and dorsoventral −2.8 from bregma). Mice were infused with

AAV-DIO-rM3D(Gs)-mCherry (AAV-DIO-Gs-DREADD) (1 μl per hemisphere) at a concentration of 1013 vg/ml and at an injection rate of 0.1 μl/min. After each infusion, the injectors were left in

place for an additional 10 min to allow the virus to diffuse. DRINKING PARADIGM TWO BOTTLE CHOICE—20% ALCOHOL D1-Cre/Ai14 and A2A-Cre/Ai14 mice underwent 1 week of two bottle choice 20%

(v/v) alcohol drinking paradigm (IA20%2BC) as described in [44, 45]. Specifically, 1 month after stereotaxic surgery and infection of the DMS with FLEX-shFyn or FLEX-SCR, mice had 24 h

access to one bottle of 20% alcohol and one bottle of water on Monday, Wednesday, and Friday with alcohol drinking sessions starting 2 h into the dark cycle. During the 24 or 48 h (weekend)

of alcohol deprivation periods, mice had access to two bottles of water. TWO BOTTLE CHOICE—10% ALCOHOL One month after stereotaxic surgery and the infection of the DMS or the DLS of D1-Cre

or A2A-Cre with AAV-DIO-Gs-DREADD, mice were habituated by intraperitoneal (IP) injection of saline for three days. On test day, mice received a systemic administration of vehicle (0.5%

DMSO) or Clozapine N-Oxide (CNO, 3 mg/kg). Fifteen minutes later, mice had access to one bottle of 10% (v/v) alcohol and one bottle of water. Alcohol and water intake were measured 4 h

later. Mice were then given water only for one week and were tested again using a counterbalanced, within subject design. A separate cohort of D1-Cre mice were infected with

AAV-DIO-Gs-DREADD in the DMS. One month later, mice were systemically administered with vehicle (20% HPBCD) or AZD0530 (10 mg/kg) 3 h prior to the beginning of the drinking session.

Subsequently, animals received a systemic administration of vehicle (0.5% DMSO) or CNO, (3 mg/kg) 15 min before the beginning of the drinking session, and alcohol and water intake were

measured 4 h later. Mice were tested in a counterbalanced, within-subjects design. TWO BOTTLE CHOICE—0.03% SACCHARIN D1-Cre/Ai14 mice underwent an intermittent saccharin intake procedure

[46]. Specifically, 2 weeks after the end of the alcohol drinking paradigm during which mice consumed only water, mice had access to one bottle of water and one bottle of saccharin (0.03%)

for 1 week (2 session) and saccharin and water intake were evaluated. The DMS of D1-Cre mice were infected with AAV-DIO-Gs-DREADD. On test day, mice received a systemic administration of

vehicle (0.5% DMSO) or CNO (3 mg/kg). Fifteen minutes later, mice had access to a bottle of saccharin (0.03%) and one bottle of water. Saccharin and water intake were measured 4 h later.

Mice were then given water only for one week and were tested again using a counterbalanced, within subject design. To measure the level of Fyn signaling activation by saccharin, C57BL/6 were

subjected to one week of home cage 0.03% intermittent saccharin intake and the DMS was dissected at the last 4 h drinking session. FLUID CONSUMPTION MEASUREMENTS Alcohol, saccharin, and

water bottles were presented in 50 ml graduated polypropylene cylinders with stainless steel drinking spouts inserted through 2 grommets in front of the cage. Bottles were weighted before

and 4 h after the drinking session in order to determine the volume of the consumed fluid. The weight of each mouse was measured the day before the drinking session to calculate the grams of

alcohol intake per kilogram of body weight. The placement (right or left) of the bottles was alternated in each session to control for side preference. Two bottles containing water and

alcohol in a cage without mice were used to evaluate the spillage due to the experimental manipulations during the test sessions. The spillage was always ≤0.2 ml. Alcohol (g/kg), saccharin

(ml/kg), and water (ml/kg) intake were recorded at the end of each 4-h drinking session. STATISTICAL ANALYSIS GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used to plot

and analyze the data. D’Agostino–Pearson normality test and _F_-test/Levene tests were used to verify the normal distribution of variables and the homogeneity of variance, respectively.

Data were analyzed using the appropriate statistical test, including two-tailed unpaired or paired _t_-test, two-way analysis of variance (ANOVA) followed by post hoc tests as detailed in

the figure legends. Experiments designed to evaluate the consequence of Fyn knockdown using FLEX-shFyn were analyzed with unpaired _t_-test. Experiments designed to test the contribution of

cAMP signaling on alcohol intake using AAV-DIO-Gs-DREADD were analyzed with paired _t_-test since within subject design was used. All data are expressed as mean ± SEM, and statistical

significance was set at _p_ < 0.05. RESULTS To determine whether Fyn’s actions are localized to DMS dMSNs and/or iMSNs, we used transgenic mice that express Cre recombinase and tdTomato

specifically in dMSNs (D1-Cre/Ai14), or that express Cre recombinase and tdTomato specifically in iMSNs (A2A-Cre/Ai14), in combination with a Cre-dependent Flip Excision (FLEX) approach to

downregulate _Fyn_ mRNA in dMSNs or iMSNs, respectively, as described in [16]. First, we examined the consequence of Fyn knockdown in DMS dMSNs on alcohol drinking. To do so, the DMS of

D1-Cre/Ai14 was infected bilaterally with a lentivirus expressing a short hairpin mRNA sequence targeting Fyn inserted in a FLEX cassette (FLEX-shFyn) (107 pg/ml, 1.2 μl per site, two sites

per hemisphere) or with a FLEX virus expressing a scramble sequence, which was used as a control (FLEX-SCR, 107 pg/ml, 1.2 μl per site, two sites per hemisphere) (Fig. 1a). After 4 weeks,

which enabled maximal viral infection and knockdown of the gene [16], mice were subjected to week of IA20%2BC and alcohol intake was measured at the last 4 h binge drinking session, a period

in which mice drink the majority of alcohol, and in which mice reach a blood alcohol concentration (BAC) of over 80 mg% [44, 47] (Timeline, Fig. 1b). Alcohol intake was significantly

reduced in D1-Cre/Ai14 mice infected with FLEX-shFyn in dMSNs as compared with the FLEX-SCR infected mice (Fig. 1c, Supplementary Table 1). We then assessed whether Fyn’s action in DMS dMSNs

is specific for alcohol or is shared with other rewarding substances. To examine this question, we tested the consequences of Fyn knockdown in DMS dMSNs on the consumption of 0.03%

saccharin (Timeline Fig. 1b). Knockdown of Fyn in DMS dMSNs did not alter saccharin intake (Fig. 1d, Supplementary Table 1). As detailed in the introduction alcohol activates Fyn in the DMS

of rodents [39, 41, 42] and that the activation of Fyn results in GluN2B phosphorylation [39, 41, 42]. To solidify the conclusion that Fyn in the DMS does not contribute to saccharin intake,

we measured Fyn activation and GluN2B phosphorylation in the DMS of C57BL/6 mice consuming saccharin (Timeline, Supplementary Fig. 1a). As shown in Supplementary Fig. 1b, c, saccharin

consumption (Supplementary Table 1), does not alter Fyn/GluN2B signaling in the DMS. Together, these results suggest that Fyn’s regulation of consummatory behavior in DMS dMSNs is specific

for alcohol and is not generalized to other reinforcing agents. Next, to determine whether downregulation of Fyn in iMSNs also affects alcohol intake, the DMS of A2A-Cre/Ai14 mice was

infected with FLEX-shFyn or FLEX-SCR (Timeline, Fig. 1b). After 4 weeks of recovery allowing maximal virus infection, mice were subjected to 1 week of IA20%2BC period, and alcohol intake was

measured at the last 4 h binge drinking session. We found that knockdown of Fyn in iMSNs does not alter alcohol intake (Fig. 1e, Supplementary Table 1) suggesting that Fyn participates in

mechanisms underlying alcohol consumption through its actions in dMSNs but not in iMSNs. As stated above, D1Rs are selectively expressed in dMSNs [5]. D1Rs are coupled to Gαs/olf, and

stimulation of Gαs/olf-coupled receptors activates cAMP/PKA signaling [6, 7]. Ex vivo data suggest that Fyn is activated in the hippocampal neurons through the cAMP/PKA pathway [24,

48,49,50]. We, therefore, postulated that alcohol activates Fyn through the stimulation of cAMP/PKA signaling in DMS dMSNs. To test this possibility, we utilized the Designer Receptor

Exclusively Activated by Designer Drug (DREADD) system to remotely activate Gαs in DMS dMSNs or iMSNs [51]. First, the DMS of D1-Cre mice was infected bilaterally with

AAV8-hSyn-DIO-rM3D(Gs)-mCherry (1013 vg/ml, 1 μl per hemisphere) (Fig. 2a). Four weeks after surgery, vehicle or CNO (3 mg/kg) was administered systemically, and the DMS was harvested 30 min

later (Timeline, Fig. 2b). As shown in Fig. 2c, d, CNO administration produced a robust increase in the phosphorylation and thus activation of Fyn in the DMS. To measure Fyn activation, we

utilized anti-phosphoTyrosine418/420Src/Fyn antibodies, which recognize the autophosphorylated active form of Fyn and Src [17, 18]. To confirm that the kinase that was activated in response

to CNO administration was indeed Fyn and not Src, the DMS of D1-Cre mice of another cohort of animals was infected bilaterally with AAV8-hSyn-DIO-rM3D(Gs)-mCherry and treated with vehicle or

CNO (3 mg/kg). Src was then immunoprecipitated using specific anti-Src antibodies (Supplementary Fig. 2a, b), and the level of Src activation in response to CNO treatment was measured using

the anti-phosphoTyrosine418/420Src/Fyn antibodies. As shown in Supplementary Fig. 2c, Src was not activated upon stimulation of GαsDREADD in DMS dMSNs. We also measured the phosphorylation

level of the Fyn substrate, GluN2B [24], and found that Fyn activation in dMSNs was accompanied by the phosphorylation of GluN2B (Fig. 2c, e). In contrast, CNO administration did not alter

Fyn’s activity or GluN2B phosphorylation in the DLS, a neighboring striatal region that was not infected with AAV8-hSyn-DIO-rM3D(Gs)-mCherry (Supplementary Fig. 3). Together, these data

suggest that Fyn/GluN2B signaling is enhanced in response to remote activation of GαsDREADD in DMS dMSNs. Next, we examined the level of Fyn signaling activation in the DLS dMSNs upon remote

simulation of GαsDREADD. To do so, the DLS of Drd1-Cre mice was infected bilaterally with AAV8-hSyn-DIO-rM3D(Gs)-mCherry (1013 vg/ml, 1 μl per hemisphere) (Fig. 3a). Four weeks after

surgery, vehicle or CNO (3 mg/kg) was administered systemically, and the DLS was harvested 30 min later (Timeline, Fig. 3b). Strikingly, as shown in Fig. 3c–e, remote activation of GαsDREADD

in DLS dMSNs did not alter Fyn’s activity or GluN2B phosphorylation. These data suggest that GαsDREADD-dependent activation of Fyn signaling in dMSNs is centered in the DMS. We then

determined whether the cAMP-dependent activation of Fyn in dMSNs alters alcohol drinking. As Drd1-Cre mice consume large quantities of 20% alcohol ([43], Fig. 1c), we used a lower alcohol

concentration (10% v/v) in order to avoid a confounding ceiling effect of alcohol intake due to Gα_s_DREADD activation. The DMS of Drd1-Cre mice was infected bilaterally with

AAV-hSyn-DIO-rM3D(Gs)-mCherry, 4 weeks later, vehicle or CNO (3 mg/kg) was administered systemically 15 min before the beginning of a 10% alcohol drinking session, and alcohol intake was

measured after 4 h (Timeline, Fig. 4a). As shown in Fig. 4b, Supplementary Table 1, remote activation of GαsDREADD in DMS dMSNs significantly increased alcohol intake. Next, we examined

whether remote activation of GαsDREADD in DMS dMSNs alters the consumption of saccharin. Two weeks after the end of the alcohol drinking experiment, vehicle, or CNO (3 mg/kg) was

administered systemically 15 min prior to the initiation of the saccharin (0.03%) drinking session, and saccharin intake was measured after 4 h (Timeline, Fig. 4a). Activation of GαsDREADD

in dMSNs did not alter saccharin intake (Fig. 4c, Supplementary Table 1) suggesting that the increase in consumption upon activation of GαsDREADD in DMS dMSNs is specific for alcohol. We

also examined whether remote activation of cAMP signaling in DMS iMSNs also alters alcohol intake. Interestingly, we found that CNO-dependent activation of GαsDREADD in DMS iMSNs does not

affect alcohol intake (Fig. 4d, Supplementary Table 1) suggesting that cAMP signaling in dMSNs but not iMSNs contributes to the development of excessive alcohol consumption. Gomez et al.

reported that CNO is converted to clozapine prior to binding and activating DREADDs [52], and furthermore, CNO was reported to produce some behavioral effects on its own [53, 54]. Therefore,

to ensure that the increase in alcohol intake was not due to off-target effects of the drug, we measured the level of alcohol consumption upon vehicle or CNO (3 mg/kg) treatment in D1-Cre

mice that were not infected with AAV-hSyn-DIO-rM3D(Gs)-mCherry (Timeline, Supplementary Fig. 4a). CNO administration did not alter alcohol intake in uninfected D1-Cre mice (Supplementary

Fig. 4b, Supplementary Table 1) suggesting that the increase in alcohol intake by CNO-dependent activation of Gα_s_DREADD in DMS dMSNs is not due to off-target effects of the drug itself or

its metabolite, clozapine. Finally, we set out to test whether Fyn is required for GαsDREADD-dependent increase in alcohol consumption. To test this possibility, the DMS of D1-Cre mice was

infected bilaterally with AAV-hSyn-DIO-rM3D(Gs)-mCherry. Four weeks later, vehicle or the Src/Fyn inhibitor AZD0530 (10 mg/kg) [29, 40, 55] was administered 3 h before the beginning of a 10%

alcohol drinking session followed by the administration of vehicle or CNO (3 mg/kg) 15 min prior to the start of the session. Alcohol intake was measured at the end of a 4 h session

(Timeline, Fig. 5a). In accordance with Fig. 4b, the amount of alcohol consumed by mice was elevated after the remote activation of GαsDREADD in DMS dMSNs, but the increase in alcohol intake

was blocked when AZD0530 was administered prior to CNO (Fig. 5b, Supplementary Table 1). In order to ensure that the differences observed above are not due to a change in alcohol

metabolism, we measured BAC after AZD0530 administration. To do so, mice received a systemic administration of vehicle or AZD0530 (10 mg/kg), 3 h later, mice received a systemic

administration of alcohol (2 g/kg), and BAC was measured 30 min later (Timeline, Supplementary Fig. 5a). As shown in Supplementary Fig. 5b, BAC was similar in vehicle and AZD0530 treated

mice suggesting that the drug does not affect alcohol metabolism. Together, these results suggest alcohol intake is driven through the cAMP/PKA/Fyn signaling in DMS dMSNs. DISCUSSION Here,

we present data to suggest that cAMP/PKA-dependent activation of Fyn kinase in the DMS dMSNs participates in mechanisms underlying the development of excessive alcohol intake. Specifically,

we show that downregulation of Fyn levels in DMS dMSNs prior to the initiation of the IA20%2BC regimen attenuates alcohol intake. We further report that the stimulation of Gαs signaling in

DMS dMSNs activates Fyn/GluN2B signaling and enhances alcohol intake in a Fyn-dependent manner. In contrast, knockdown of Fyn or activation of cAMP signaling in iMSNs does not alter alcohol

intake and preference. Finally, we show that the cAMP/Fyn signaling in DMS dMSNs is specific for alcohol and does not contribute to mechanisms underlying consummatory behavior per se. Based

on previous data showing that Fyn is activated in the DMS through the stimulation of D1R in DMS dMSNs [16], and since dopamine levels in the dorsal striatum are elevated by drugs of abuse

including alcohol [56, 57], we propose a model in which dopamine, released in the DMS in response to alcohol exposure, stimulates D1R/cAMP/PKA signaling in dMSNs which in turn activates Fyn

to initiate neuroadaptations such as GluN2B phosphorylation that promote the development of excessive alcohol use (Supplementary Fig. 6). Our data suggest that Fyn in DMS dMSNs contributes

to the development of excessive alcohol consumption. The DMS is essential for goal-directed behaviors [2, 3], and dMSNs contribute to reward learning and reinforcement [58, 59]. Thus, it is

plausible that Fyn in dMSNs promotes reward learning, which in turn initiates and maintains goal-directed alcohol seeking. This possibility is in line with the finding that systemic

administration of the Src/Fyn inhibitor, AZD0530, attenuates goal-directed alcohol self-administration in mice [40]. Xie et al. previously showed that administration of the Src/Fyn inhibitor

PP2 into the dorsal hippocampus attenuates context-dependent cocaine seeking [60], and more recently, Belin–Rauscent reported that oral administration of the Src/Fyn s inhibitor, Masitinib,

attenuates self-administration of cocaine [61]. Thus, it would be of interest to determine whether Fyn in DMS dMSNs contributes to goal-directed seeking of other drugs of abuse.

Furthermore, Goto et al. previously reported that mating behavior increases PKA activity in dorsal striatal dMSNs [62]. As PKA in DMS dMSNs is upstream of Fyn, it is plausible that the Fyn

in these neurons plays a role in other goal-directed behaviors such as mating. Finally, Bocarsly et al. previously showed that enhanced D1R signaling in the dorsal striatum of mice is

required for the consumption of alcohol despite negative consequences, and for the enhancement of alcohol-dependent hyperlocomotion [13]. Therefore, it plausible that Fyn in DMS dMSNs also

contributes to other alcohol-dependent behaviors. We found that stimulation of cAMP/PKA signaling activates Fyn but not Src in DMS dMSNs. This observation is in line with previous data

showing that Fyn but not Src is activated in the dorsal striatum by alcohol exposure [38] or D1R stimulation [16]. However, it is plausible that the cAMP/PKA-dependent activation of Src in

other brain regions contribute to excessive alcohol use. For instance, Zhang et al. reported that opiate withdrawal activates Src in the locus coeruleus [63]. The data herein and previous

findings [31] suggest that Fyn exerts its action on alcohol drinking in dMSNs through the phosphorylation and activation of GluN2B. However, we cannot exclude the possibility that other

molecular transducers of Fyn contribute to the development of _excessive_ alcohol use. For example, protein translation plays a critical role in mechanisms underlying AUD [64], and Fyn was

shown to enhance protein translation in oligodendrocytes [65] and in neurons [66]. Fyn was also shown to promote ERK1/2 phosphorylation in oligodendrocytes [67], and NfkB signaling in

microglia [68]; both signaling cascades have been linked to alcohol use [30, 69]. Finally, Fyn was reported to phosphorylate the metabotropic glutamate receptor 1 (mGluR1) [70] and the

Collapsin Response Mediator Protein 2 (CRMP2) [71], which have also been implicated in alcohol’s actions in the brain [72, 73]. Exploring the contribution of these substrates and others in

DMS dMSNs to the neuroadaptations underlying AUD merits further investigation. It is highly likely that the activation of PKA in DMS dMSNs produces additional cellular consequences. For

example, PKA phosphorylates the Striatal-Enriched Protein Tyrosine Phosphatase (STEP) [50] resulting in the inhibition of the activity of the phosphatase [50]. STEP is an endogenous Fyn

inhibitor and is responsible for the termination of Fyn activation [50]. We previously reported that alcohol increases PKA phosphorylation of STEP in the DMS, and that knockdown of STEP in

the DMS [41], or global knockout of the phosphatase increases alcohol intake [74]. It is therefore plausible that one of the consequences of PKA activation in DMS dMSNs is the

phosphorylation of STEP, enabling Fyn in these neurons to stay active for a prolonged period of time. Using the DREADD/CNO methodology, we report that the activation of GαsDREADD in dMSNs

but not in iMSNs initiates the consumption of alcohol. We further show that GαsDREADD-dependent increase in alcohol intake depends, at least in part, on Fyn. To our knowledge this is the

first study that provides a link between GαsDREADD activation and alcohol consummatory behavior. Cheng et al. previously showed that GαiDREADD-mediated inhibition of iMSNs enhances alcohol

intake [75]. Stimulation of Gαs-coupled receptors activates adenylate cyclase that increases cAMP production, whereas the stimulation of Gαi-coupled receptors inhibits adenylate cyclase

activity and cAMP production [7]. Thus, it is plausible that the development of excessive alcohol consumption depends on the inhibition of cAMP signaling in iMSNs and on the activation of

cAMP signaling in dMSNs. Our data suggest that alcohol-dependent molecular adaptations are highly specific and are segregated to a subpopulation of neurons. Future studies are necessary to

determine whether this cell-type specificity is unique for the cAMP/PKA/Fyn signaling or that this is a common feature shared by some or all of the molecular targets of alcohol [30].

Although we provide a strong evidence linking PKA signaling in dMSNs to alcohol drinking behaviors, we cannot exclude the possibility that other cAMP effectors such as the guanine nucleotide

exchange factor EPAC (exchange protein directly activated by cAMP) [76], and/or cyclic nucleotide-gated ion channels (CNGC) [77] also contribute to the development of excessive alcohol use.

Finally, further research is required to monitor cAMP production and PKA activation in behaving animals. Recent advances in the development of reporters for cAMP [78], and PKA [79] will

enable a spatial and temporal analysis of cAMP/PKA signaling in animals consuming alcohol. Finally, we previously showed that treatment of mice with AZD530 attenuates alcohol-dependent Fyn

activation and GluN2B phosphorylation in the DMS [40], and reduces goal-directed alcohol seeking. We show herein that the enhancement of alcohol intake upon activation of GαsDREADD in DMS

dMSNs is inhibited upon the administration of AZD530. AZD530 is well-tolerated in humans, as phase I and II clinical trials indicate that the drug does not produce significant side effects

[80, 81], and our preclinical mouse studies show that systemic administration of the drug does not alter basal levels of locomotion [40], and does not change BAC. Together, these data give

rise to the potential use of AZD530 in AUD. FUNDING AND DISCLOSURE This research was supported by the National Institute of Alcohol Abuse and Alcoholism, UO1 AA023489 (DR and VAA). The

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  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank AstraZeneca for providing us with AZD5030. The authors thank Ellanor Whiteley for her contribution. AUTHOR INFORMATION

Author notes * Nadege Morisot Present address: Nkarta Therapeutics, San Francisco, CA, USA * Samuel A. Sakhai Present address: Sage Therapeutics, San Francisco, CA, USA * Martin F. Adrover

Present address: INGEBI, CONICET, Buenos Aires, Argentina AUTHORS AND AFFILIATIONS * Department of Neurology, University of California San Francisco (UCSF), San Francisco, CA, 94143, USA

Yann Ehinger, Nadege Morisot, Khanhky Phamluong, Samuel A. Sakhai, Drishti Soneja & Dorit Ron * National Institutes of Alcohol Abuse and Alcoholism, National Institute of Health,

Bethesda, MD, 20892, USA Martin F. Adrover & Veronica A. Alvarez * Center on Compulsive Behaviors, Intramural Research Program, National Institute of Health, Bethesda, MD, 20892, USA

Veronica A. Alvarez Authors * Yann Ehinger View author publications You can also search for this author inPubMed Google Scholar * Nadege Morisot View author publications You can also search

for this author inPubMed Google Scholar * Khanhky Phamluong View author publications You can also search for this author inPubMed Google Scholar * Samuel A. Sakhai View author publications

You can also search for this author inPubMed Google Scholar * Drishti Soneja View author publications You can also search for this author inPubMed Google Scholar * Martin F. Adrover View

author publications You can also search for this author inPubMed Google Scholar * Veronica A. Alvarez View author publications You can also search for this author inPubMed Google Scholar *

Dorit Ron View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS YE contributed to the design of the experiments, the acquisition of data, data

analysis, and to the preparation and revision of the manuscript. NM, SAS, and KP contributed to the design of the experiments, the acquisition of the data and data analysis. MFA contributed

to the acquisition of data. VAA contributed to the conception of the study. DR contributed to the conception of the study, the design of the experiments, and wrote the manuscript.

CORRESPONDING AUTHOR Correspondence to Dorit Ron. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

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cAMP-Fyn signaling in the dorsomedial striatum direct pathway drives excessive alcohol use. _Neuropsychopharmacol._ 46, 334–342 (2021). https://doi.org/10.1038/s41386-020-0712-1 Download

citation * Received: 07 January 2020 * Accepted: 11 May 2020 * Published: 17 May 2020 * Issue Date: January 2021 * DOI: https://doi.org/10.1038/s41386-020-0712-1 SHARE THIS ARTICLE Anyone

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