The effect of chemogenetic manipulation of DG on alcohol seeking and drinking

To verify the function of DG in regulation of alcohol addiction-related behaviors, we chemogenetically manipulated the activity of DG of C57BL/6J female mice by expression of adeno-associated viral vectors, serotype 1 and 2 (AAV1/2), encoding inhibitory designer receptors exclusively activated by designer drugs (DREADD) (AAV-hSyn-hM4D(Gi)-mCherry), or activatory DREADDs (AAV-hSyn-hM3D(Gq)-mCherry) [43], or the control virus (AAV-CaMKII-mCherry) (Fig. 1a). Post-training analysis of the hippocampal tissue revealed that DREADDs were expressed in ~ 60% of the granule cells of DG (both in cell bodies and dendrites) (Fig. 1a).

Fig. 1 Chemogenetic inhibition of dorsal DG during cue relapse enhances alcohol seeking and drinking. a Expression of AAV1/2 encoding DREADDs. (i) Transfection area in dorsal DG. Expression of the control (AAV-CaMKII-mCherry) (ii), inhibitory (AAV-hSyn-hM4D(Gi)-mCherry) (iii), and activatory DREADDs (AAV-hSyn-hM3D(Gq)-mCherry) (iv) in cell bodies and dendrites of the granule cells (upper blade). (v) Quantification of transfected cells in dorsal DG. b Experimental timeline. c CNO had no effect on alcohol seeking during 2nd cue relapse in any of the experimental groups. d CNO enhanced alcohol seeking during period of free access to alcohol that followed cue relapse in mice expressing Gi (i), but not Gq (ii), or mCherry (iii). e CNO increased alcohol drinking during period of free access to alcohol in mice expressing Gi (i), but not Gq (ii), or mCherry (iii) Full size image

Two weeks after the surgery and viral infection, mice underwent alcohol self-administration training in the IntelliCages [39, 40]. Groups of 14 mice per cage were trained to drink 12% ethyl alcohol (vol/vol). Each cage had two active operant chambers (corners) (Fig. 1b), each visit to a reward corner resulted in presentation of green light (cue) and each nose-poke allowed mice to drink alcohol for 5 s. In a water corner, no specific cue was presented and all mice had unlimited access to water. Free access to alcohol was intermitted by two 7-day withdrawal periods (the reward corner was inactivated) followed by a 24-hour cue relapses induced by the presentation of alcohol-associated cue light in the reward corner without any alcohol available (Fig. 1b). No difference in alcohol seeking between the experimental groups was observed during the first cue relapse and the following period of free access to alcohol (Fig. S1). Twenty minutes before the second cue relapse, mice received i.p. injection of clozapine N-oxide (CNO, 0.5 mg/kg) to activate DREADDs, or saline (Fig. 1b, day 97). We manipulated DG activity at this time point of the training as our data showed that specifically presentation of alcohol-associated cues affects plasticity of DG (Fig. S2). Surprisingly, no effect of CNO injection on alcohol seeking was observed during the cue relapse in any of the experimental groups (repeated measures analysis of variance (RM ANOVA) for Gi: F(1, 12) = 0.3927, p = 0.562; Gq: F(1, 11) = 0.5983, p = 0.455; mCherry: F(1, 11) = 0.162, p = 0.694) (Fig. 1c). We found, however, that inhibition of DG granule cells by CNO in mice expressing inhibitory DREADD receptor hM4D(Gi) had long-lasting effects on alcohol seeking and consumption during the following period of free access to alcohol. The mice performed more nosepokes to the cued corner (RM ANOVA for Gi: F(1, 11) = 10.60, p = 0.007) (Fig. 1d.i) and drank more alcohol (F(1, 11) = 3.402, p = 0.092, CNO × time: F(6, 66) = 3.159, p = 0.008) (Fig. 1e.i), than mice injected with saline. This effect of CNO was not observed, neither in the control mice expressing activatory DREADD hM3D(Gq) (NPs: F(1, 12) = 2.660, p = 0.129; consumption: F(1, 12) = 1.488, p = 0.246) (Fig. 1d–e.ii) nor in the mice expressing mCherry (NPs: F(1, 11) = 0.256, p = 0.622; consumption: F(1, 11) = 0.283, p = 0.615) (Fig. 1d–e.iii).

Electrophysiological characteristics of addicted and non-addicted mice

In the following step we determined whether the function of DG is affected when mice are trained to drink and look for alcohol in the IntelliCages. To this end we applied recently developed model of alcohol addiction, which allows to discern mice controlling their alcohol consumption from those that undergo transition to addiction [39,40,41]. As chemogenetic inhibition of DG enhanced alcohol seeking and consumption, we hypothesized that increased alcohol seeking and drinking in addict mice is linked with weakened DG synapses.

After 68 days of unlimited alcohol self-administration, behaviors that resemble the hallmarks of addiction according to DSM-IV [1] were tested (Fig. 2a): (i) The subject has an extremely high motivation to take the drug, with activities focused on its procurement and consumption. We assessed high motivation for alcohol in progressive schedule of reinforcement in which the number of responses (ratio) to obtain access to alcohol progressively increases within the test. We measured the breakpoint, the last ratio completed, which is considered a reliable index of the motivation for the drug [44] over 7 days of the test. (ii) The subject has difficulty stopping drug use and/or limiting drug intake. These traits were operationalized as persistence in alcohol seeking in persistence test and during alcohol withdrawal as they were previously used to measure addiction-like behaviors in rats and mice [44]. We measured the number of nosepokes performed to the reward corner during periods of alcohol non-availability, as compared with periods when the reward corner was active, in persistence test, and nosepokes performed to the reward corner during 7-day withdrawal. (iii) High propensity to relapse. Again, we measured alcohol seeking during relapse induced by alcohol-associated cue light and excessive alcohol consumption during alcohol relapse after withdrawal. The control, alcohol-naive mice had been tested in the same way as alcohol-exposed mice, however, they had only water in both cage corners during the whole training. Mice performance during all tests was used to indicate addicted and non-addicted individuals. Addicted alcohol drinkers were identified as those, which were positive for at least two addiction-related criteria (top 35% of the population in at least two tests), and non-addict drinkers, which were positive for none of the criteria (for details on how addict and not-addict mice were identified, see Materials and Methods). We also summed up the normalized scores of all tests to obtain addiction score for each animal. Addicted mice had higher total addiction score (t(27) = 6.844, p < 0.0001) (Fig. 2b) and higher performance in all tests as compared with non-addicted mice and alcohol-naive mice (Fig. 2c) (motivation tests: F(2, 45) = 3.235, p = 0.048, persistence: U = 86, p < 0.016; withdrawal: F(1, 43) = 12.59, p = 0.001, cue relapse F(1, 48) = 13.32, p < 0.001; alcohol relapse: F(1, 38) = 5,472, p = 0.024), indicating that the measured behavioral features describe consistent phenotype resembling human disorder. Interestingly, although addict and non-addict mice also differed in general activity and alcohol consumption, the difference developed over time and was not observed during 5-day adaptation to the cage (CA), and initiation of alcohol drinking (4 and 8%) (Fig. 2c.i&ii) (all NPs: t(39) = 3.4303, p = 0.001; alcohol consumption: F(1, 39) = 6.296, p = 0.016). Addicts and non-addicts also did not differ in alcohol preference (Fig. S3).

Fig. 2 Addicted mice have more silent synapses in DG during cue relapse than non-addict mice. a Experimental timeline and IntelliCage setups for alcohol drinking and alcohol-naive mice. Mice went through tests measuring addiction-related behaviors: motivation, persistence, withdrawal, cue relapse, and alcohol relapse spaced by periods of free access to alcohol, to identify addict and non-addict animals. b Addicts had significantly higher addiction score than non-addicts (t(27) = 6.844, p < 0.0001). c Mice performance during the training. (i) Addict and non-addict mice did not differ in nose-poke activity during the cage adaptation (CA) and initial 30 days of free access to alcohol (30d). (ii) They also did not differ in alcohol consumption during initiation of alcohol consumption (4 and 8%). Later (90d) addicts, as compared with non-addict mice, (i) performed more nosepokes to cage corners, and (ii) consumed more alcohol. They also (iii) reached higher breakpoint during Motivation tests, (iv) showed higher increase of reward nosepokes during non-rewarded (nR) phases as compared to rewarded (R) phases during persistence tests; (v) performed more nosepokes to the reward corner during withdrawal; (vi) performed more nosepokes to the reward corner during presentation of alcohol predicting cue; (vii) drank more alcohol during 12 h of relapse (test) as compared with non-addicts, and to the last 12 h of the last day of free alcohol access period (“0”). d Electrophysiological analysis of the granule cells in dorsal DG. (i) Experimental timelines and cage setups for alcohol drinking and alcohol-naive mice. Mice were killed during period of free access to alcohol (day 115), withdrawal (day 122) or cue relapse (+90’). (ii) Recording electrode in dorsal DG. Stimulating electrode was in perforant path. (iii) Example EPSCs (successes and failures) elicited by minimal stimulations at +45 mV (top) and −60 mV (bottom). Frequency of successes and failures was used to calculated % of silent synapses (see Materials and Methods for details). e Electrophysiological analysis. Trial plots of EPSCs elicited by minimal stimulations at +45 and −60 mV from alcohol-naive, non-addict and addict mice killed (i) during free alcohol drinking, (ii) withdrawal, and (iii) cue relapse, and mice drinking water. (iv) Frequency of silent synapses was increased in alcohol-drinking mice (both addict and non-addict) as compared to alcohol-naive animals during free access to alcohol, and addict mice as compared to non-addict and alcohol-naive mice during cue relapse *p < 0.05, **p < 0.01 by Tukey’s multiple comparisons test Full size image

Next, the mice continued the training (Fig. 2d) and we looked at the electrophysiological differences between alcohol-naive, addicted, and non-addicted mice. We investigated how the extended alcohol drinking affects synaptic plasticity in DG by performing whole-cell voltage clamp recordings from the granule cells of the upper blade of DG, whereas stimulating the perforant path (Fig. 2d, c.ii). Following the observation that chemogenetic inhibition of DG activity enhanced subsequent alcohol consumption and seeking, we measured the content of AMPAR-silent synapses [45, 46], as their appearance is a potential mechanism to weaken basal synaptic transmission. Silent synapses were previously linked with drug craving and addiction in multiple experimental paradigms [28,29,30, 34]. Moreover, our previous findings show a robust increase of silent synapse number after alcohol consumption in the IntelliCages [39]. Using the minimal stimulation protocol [42] we recorded the frequency of instances when minimal stimulation failed to elicit changes in EPSCs higher than five pA-baseline noise (failures) (Fig. 2d.iii). This was used to calculate % of silent synapses (see Supplementary Materials and Methods for details of the protocol). When alcohol-naive, non-addict, and addict mice were killed during period of free access to alcohol (Fig. 2d.i, day 115) we observed increased frequency of silent synapses both in non-addict and addict drinkers, as compared with the alcohol-naive mice (Fig. 2e) (addiction score: F(2, 37) = 12.92, p < 0.001; time: F(2, 37) = 12.47, p < 0.001), suggesting that generation of silent synapses during alcohol consumption was not specific to the severity of alcohol addiction. After 7-day withdrawal (Fig. 2d.i, day 122), the levels of silent synapses were very low (<5%) for all experimental groups and again no significant differences between the experimental groups were found (Fig. 2e). Finally, after 90-min presentation of alcohol-associated cue light (Fig. 2d, day 122 + 90’), we observed a significant increase in the frequency of silent synapses in addict drinkers, as compared with non-addict mice and alcohol-naive controls (Fig. 2e).

In the following experiment we tested whether silent synapses in DG are specific for alcohol-associated cues or are generated during presentation of other salient cues as well. To this end, mice were trained to drink 5% sucrose or 12% alcohol for 90 days (Fig. 3a). During that time, mice drinking sucrose were more active than mice drinking alcohol, measured as the number of nosepokes performed to the active corners (Fig. 3b.ii) (t(26) = 5.09, p < 0.0001), as they drank much more liquids. They performed, however, the same number of nosepokes to the cued corner during withdrawal (Fig. 3b.iii) and cue relapse (Fig. 3b.iv) (RM ANOVA withdrawal: F(1, 9) = 2.805, p > 0.05; cue relapse: F(1, 9) = 0.237, p > 0.01), indicating that sucrose-, as much as alcohol-associated corner and cue light, gained rewarding properties. To measure the levels of silent synapses, alcohol and sucrose drinking mice were killed after 90 min of the cue relapse (Fig. 3c.i, day 122 + 90’). The level of silent synapses in the sucrose mice was significantly lower than in the alcohol drinking mice (~ 4% vs 24%) (Fig. 3c.ii) (t(13) = 2.68; p < 0.05). This finding indicates that generation of silent synapses in DG during cue relapse is specific for alcohol-associated cues and does not result only from intensive reward seeking.

Fig. 3 Sucrose-associated cues do not generate AMPAR-silent synapses in DG. a Experimental timeline and IntelliCage setups for alcohol drinking (i) and sucrose drinking mice (ii). b Mice performance during the training. (i) Alcohol consumption in the group of alcohol drinking mice. (ii) Sucrose mice performed more nosepokes to cage corners during the free access period, as compared with alcohol drinking mice. They were, however, as active as alcohol drinking mice during withdrawal (iii), and cue relapse (iv). c Electrophysiological analysis of the granule cells in dorsal DG. (i) Experimental timeline. Mice were killed after cue relapse (+90’). (ii) Trial plots of EPSCs elicited by minimal stimulations at +45 and −60 mV from alcohol and sucrose drinking mice killed during cue relapse. (iii) Frequency of silent synapses was increased in alcohol drinking mice as compared to sucrose drinking animals Full size image

Addiction score affects density of dendritic spines in DG

Formation of silent synapses has been linked with generation of new dendritic spines (after cocaine and nicotine) or rejuvenation of existing synaptic contacts (after morphine) to support addiction-related remodeling of the brain circuits [34, 47]. Alcohol is known to affect morphology and density of dendritic spines [48], it is however unknown how this is linked with electrophysiological properties of the spines and whether addicted individuals differ in this regard from non-addicted ones. We imaged dendritic spines of the granule cells of DG using DiI staining [34], focusing on the dendrites in the medial part of the molecular layer (upper blade) (Fig. 4a, left) that are innervated by the perforant path. Spine density and size, as well as density of spines in three categories (mushroom, stubby, and thin) were automatically assessed using NeuronStudio software (Fig. 4a, right). During the period of free access to alcohol (Fig. 2d.i, day 115) the addict mice had significantly fewer spines than non-addict mice (Fig. 4c) (two-way ANOVA, addiction score: F(1, 25) = 0.620, p = 0.438; time: F(2, 25) = 3.529, p = 0.044, interaction: F(2, 25) = 5.071, p = 0.014). This impairment was reversed during withdrawal (day 122) and no further change in total spine density was detected after 90 min of cue relapse (day 122 + 90’). Detailed analysis of the spines in three shape categories showed that the density of thin spines was decreased during free access to alcohol in addict mice, as compared with non-addicts (Fig. 4d) (addiction score: F(1, 25) = 0.041, p = 0.839; time: F(2, 25) = 1.969, p = 0.160, interaction: F(2, 25) = 6.115, p = 0.007). This was reversed during withdrawal and not affected by cue relapse. No statistically significant change in density of mushroom or stubby spines was observed (Fig. 4e, f) (mushroom, addiction score: F(1, 26) = 0.095, p = 0.760; time: F(2, 26) = 1.531, p = 0.235, interaction: F(2, 26) = 2.499, p = 0.101); Stubby, addiction score: F(1, 26) = 3.938, p = 0.057; time: F(2, 26) = 2.443, p = 0.106, interaction: F(2, 26) = 1.681, p = 0.205). Furthermore, addict mice had bigger spines than the controls during free access to alcohol. Their size decreased during withdrawal and increased during cue relapse (Fig. 4g) (addiction score: F(1, 25) = 0.007, p = 0.929; time: F(2, 25) = 0.633, p = 0.539, interaction: F(2, 25) = 4.555, p = 0.020), suggesting that these changes resulted from disappearance and reappearance of small, thin dendritic spines. Notably, no change in density and size of dendritic spines was observed in non-addict mice during the training. In conclusions, our data show that changes in the density of thin spines, next to the changes of silent synapses, are the hallmark of addict mice.

Fig. 4 Density of dendritic spines in DG in addict mice is regulated during free access to alcohol and withdrawal, but not cue relapse. a DiI staining. (i) The analyzed region of DG. The gray line delineates the outer boundary of the granular layer of DG. Inset: magnification of the stained granule cell. Scale bar, 200 μm. (ii) Exemplary microphotograph of dendritic spines traced with NeuronStudio. Scale bar, 2 μm. b Exemplary microphotographs of dendrites from the medial part of the molecular layer of dorsal DG (upper blade) stained with DiI in alcohol-naive, non-addict, and addict animals killed during free access to alcohol, after 7-day withdrawal or 90-min cue relapse. Scale bars, 1 μm. Summary of data showing changes in density of all spines c, thin spines d, mushroom spines e, stubby spines f. g Summary of data showing changes in size of spines. For all graphs *p < 0.05, **p < 0.01 by Fisher’s test Full size image

The effect of acamprosate on alcohol drinking and seeking, and silent synapses in DG

Some of the behavioral manifestations of alcohol addiction, such as alcohol craving and consumption, can be suppressed by acamprosate treatment [49]. Therefore, we determined whether acamprosate can also prevent cue relapse-induced generation of silent synapses in alcohol addict mice. To this end, mice underwent alcohol self-administration training followed by the tests to select addict and non-addict mice (Fig. 5a). After the tests half of the addict and non-addict mice (as well as the alcohol-naive mice) had been given acamprosate in drinking water (250 mg/kg/day, ACA) (Fig. 5a). The rest of alcohol-drinking mice drank only alcohol and water. We observed that acamprosate decreased alcohol consumption during the period of free access to alcohol (Fig. 5c) (alcohol-naive F(1, 9) = 0.077, p = 0.786; non-addict: F(1, 6) = 0.227, p = 0.650, addict: F(1, 7) = 18.50, p = 0.003) and alcohol seeking during withdrawal of addict mice (Fig. 5d) (alcohol-naive: F(1, 7) = 0.561, p = 0.478; non-addict: F(1, 7) = 3.271, p = 0.113, addict: F(1, 6) = 8.686, p = 0.025), but not in non-addict mice. Neither did we observe any effect of acamprosate on the behavior of alcohol-naive mice. Next, the mice were killed after 90 min of the cue presentation to measure the number of silent synapses (Fig. 5a, day 122 + 90’). We did not observe the differences in the behavior of the mice from the experimental groups during this short period (Fig. S4). Consumption of acamprosate prevented, however, formation of silent synapses during presentation of cue light associated with alcohol in addicted mice (Fig. 5e) (addiction: F(2, 19) = 20.65, p < 0.0001, acamprosate: F(1, 19) = 15.98, p = 0.0008, interaction: F(2, 19) = 8.094, p = 0.0029). Acamprosate did not alter silent synapse number neither in alcohol-naive nor non-addict mice.