Age-Dependent and Pathway-Specific Bimodal Action of Nicotine on Synaptic Plasticity in the Hippocampus of Mice Lacking the miR-132/212 Genes

Nicotine addiction develops predominantly during human adolescence through smoking. Self-administration experiments in rodents verify this biological preponderance to adolescence, suggesting evolutionary-conserved and age-defined mechanisms which influence the susceptibility to nicotine addiction. The hippocampus, a brain region linked to drug-related memory storage, undergoes major morpho-functional restructuring during adolescence and is strongly affected by nicotine stimulation. However, the signaling mechanisms shaping the effects of nicotine in young vs. adult brains remain unclear. MicroRNAs (miRNAs) emerged recently as modulators of brain neuroplasticity, learning and memory, and addiction. Nevertheless, the age-dependent interplay between miRNAs regulation and hippocampal nicotinergic signaling remains poorly explored. We here combined biophysical and pharmacological methods to examine the impact of miRNA-132/212 gene-deletion (miRNA-132/212−/−) and nicotine stimulation on synaptic functions in adolescent and mature adult mice at two hippocampal synaptic circuits: the medial perforant pathway (MPP) to dentate yrus (DG) synapses (MPP-DG) and CA3 Schaffer collaterals to CA1 synapses (CA3–CA1). Basal synaptic transmission and short-term (paired-pulse-induced) synaptic plasticity was unaltered in adolescent and adult miRNA-132/212−/− mice hippocampi, compared with wild-type controls. However, nicotine stimulation promoted CA3–CA1 synaptic potentiation in mature adult (not adolescent) wild-type and suppressed MPP-DG synaptic potentiation in miRNA-132/212−/− mice. Altered levels of CREB, Phospho-CREB, and acetylcholinesterase (AChE) expression were further detected in adult miRNA-132/212−/− mice hippocampi. These observations propose miRNAs as age-sensitive bimodal regulators of hippocampal nicotinergic signaling and, given the relevance of the hippocampus for drug-related memory storage, encourage further research on the influence of miRNAs 132 and 212 in nicotine addiction in the young and the adult brain.


Introduction
Nicotine addiction, as characterized by sustained and uncontrolled tobacco smoking, is behind millions of deaths worldwide and is causally related to conditions, such as coronary

Extracellular Recordings
Slice electrophysiology and recordings at the hippocampal stratum radiatum were conducted as in our previous reports and those from colleagues [30,42,59,60]. fEPSPs recordings from the dentate gyri and from the CA1 regions were conducted as described before by our group [30,42,61,62]. Analysis of basal synaptic transmission (input/output (I/O) curves) and plasticity was conducted as described before [30,42] (see also Figure 1). To induce LTP, we applied 20 pulses of electrical stimulation (200 µs/pulse) at 100 Hz ( Figure 1). Paired-pulse-induced synaptic plasticity (facilitation (PPF) or inhibition (PPI)) was recorded and analyzed as described in [30]. Stimulation protocols were delivered at baseline stimulation intensities. LTP was determined by analyzing changes in the decaying phase of fEPSP slopes after high frequency stimulation, normalized to baseline. Data were averaged within groups and compared between miRNA-132/212 −/− and WT mice, with or without nicotine presence in the recording chamber. In some experiments, slices were simulated twice for 5 min (with a 5 min interval) with bath-applied aCSF solution containing 1 µM nicotine (N5260, Sigma Aldrich) as used in independent experimental settings described before [63][64][65]. In particular, we here decided to use a double nicotinestimulation protocol, as we previously described that this stimulation protocol can acutely induce changes in hippocampal plasticity [30]. Previous reports have shown effects on plasticity upon brief exposures to nicotine preceding the application of stimulation protocols inducing LTP [66], and other groups have also described the use of brief and intermittent stimulations with molecules that activate neuronal cell-surface receptors in in vitro settings in order to induce morphological and functional neuronal responses in studies of synaptic plasticity and memory-related functions [67]. Electrical stimulation was generated from an ISO-STIM 01D (NPI Electronics, Tamm, Germany). An AxoClamp-2B amplifier, a Digidata-1440 interface (Axon Instruments, Molecular Devices, Berkshire, UK), and pClamp-11 software (Molecular Devices, Berkshire, UK) were used for the acquisition and analysis of data. vitro settings in order to induce morphological and functional neuronal responses in studies of synaptic plasticity and memory-related functions [67]. Electrical stimulation was generated from an ISO-STIM 01D (NPI Electronics, Tamm, Germany). An AxoClamp-2B amplifier, a Digidata-1440 interface (Axon Instruments, Molecular Devices, Berkshire, UK), and pClamp-11 software (Molecular Devices, Berkshire, UK) were used for the acquisition and analysis of data.

Statistical Analysis
GraphPad Prism-7.0 (San Diego, CA, USA) was used for analysis. Sample data distribution was weighed before statistical analyses by D'Agostino's K 2 normality test. The differences between datasets from two groups were evaluated with a two-sided unpaired Student's t-test. To conduct multiple comparisons, we used repeated-measures (or mixed model) ANOVA with Bonferroni's test. Data in the figures show values of means with standard errors (+/−). A value of 0.05 was used as level of significance (α).

Basal Synaptic Transmission and Paired-Pulse-Induced Plasticity Is Unaltered in Adolescent and Mature Adult miRNA-132/212−/− Mouse Hippocampi
Artificially induced alterations in the frequency of activity at specific hippocampal presynaptic terminals (using electrical stimulation) can result in "plastic" changes characterized by either strengthening (synaptic potentiation; inducible by high-frequency stimulation) or dwindling (synaptic depression; inducible by low-frequency stimulation) the postsynaptic response. The magnitude and persistence in time of the elicited change in the postsynaptic response directly relates to the frequency and intensity of the delivered electrical stimulation [68][69][70][71]. These experimental observations prompted neuroscientists to propose that perhaps the plastic changes associated to memory storage in the mammalian brain could operate following functional principles analogue to those elicited artificially [72][73][74][75][76][77][78]. However, while several modalities of neuronal plasticity are described in ex vivo studies that used brain slices and standardized protocols of electrical stimulation The place where stimulating (S) and recording (R) electrodes were located at the MPP (blue) and SCP (orange) are indicated. Simulation was delivered at the middle inputs coming from the entorhinal cortex molecular layer for recordings at the dentate gyrus and at the CA3 stratum radiatum layer for recordings at the SCP. (C) Graphic detail of an electrode used for recordings positioned at a dendritic zone. (D) Illustrative field potential recording. (E) Drawing for the pattern of pulses of the electrical stimulation used to induce LTP (see also Section 2). PND: postnatal day; MPP: medial perforant pathway; SCP: Schaffer collateral pathway; LTP: long-term potentiation.

Statistical Analysis
GraphPad Prism-7.0 (San Diego, CA, USA) was used for analysis. Sample data distribution was weighed before statistical analyses by D'Agostino's K 2 normality test. The differences between datasets from two groups were evaluated with a two-sided unpaired Student's t-test. To conduct multiple comparisons, we used repeated-measures (or mixed model) ANOVA with Bonferroni's test. Data in the figures show values of means with standard errors (+/−). A value of 0.05 was used as level of significance (α). Artificially induced alterations in the frequency of activity at specific hippocampal presynaptic terminals (using electrical stimulation) can result in "plastic" changes characterized by either strengthening (synaptic potentiation; inducible by high-frequency stimulation) or dwindling (synaptic depression; inducible by low-frequency stimulation) the postsynaptic response. The magnitude and persistence in time of the elicited change in the postsynaptic response directly relates to the frequency and intensity of the delivered electrical stimulation [68][69][70][71]. These experimental observations prompted neuroscientists to propose that perhaps the plastic changes associated to memory storage in the mammalian brain could operate following functional principles analogue to those elicited artificially [72][73][74][75][76][77][78]. However, while several modalities of neuronal plasticity are described in ex vivo studies that used brain slices and standardized protocols of electrical stimulation (see for example [30,79]), a lot is yet to be understood about the molecular elements concomitantly modulating hippocampal synaptic plasticity, memory storage, and nicotine addiction.

Basal Synaptic Transmission and
We, therefore, explored the effects of brain maturation and miRNA-132/212 gene deletion on paired-pulse-induced hippocampal synaptic plasticity at MPP-DG (blue in Figure 1B) and CA3-CA1 (orange in Figure 1B) synapses. Field potential recordings were obtained from slices derived from male adolescent and adult miRNA-132/212 gene knock- out (miRNA-132/212 −/− ), as well as on their relative littermate wild-type counterparts used as controls ( Figure 1). The effect of 1 µM nicotine was examined.
Before hand, we used input/output protocols (Section 2) to assess basal synaptic transmission at CA3-CA1 synapses (Figure 2A). Recordings from either miRNA-132/212 −/− or WT groups yielded I/O responses comparable for miRNA-132/212 −/− and either adolescent or adult WT groups ( Figure 2B,D). These results provide experimental evidence indicating that miRNA-132/212 −/− are not required for the proper expression of basal synaptic transmission and voltage stimulation sensitivity, i.e., an observation somewhat different from previous reports [44] and likely attributable to the differences in the age of the used animals and other experimental conditions (e.g., differences can be attributable to types of recording chambers, pattern of stimulation protocol, recovery conditions, etc. [80][81][82]). We then studied paired-pulse-induced synaptic plasticity for both experimental groups and conditions. Slice recordings from nicotine-free (untreated) miRNA-132/212 −/− showed paired-pulse-induced field slopes, in CA3-CA1 synapses comparable to those of WT controls, regardless of their age ( Figure 2C,E). These observations ruled out a requirement of miRNA-132/212 for the impact that nicotine has on proper short-term potentiation in CA3-CA1 synapses in adolescent and adult male mice. We subsequently examined the properties of both synaptic transmission (basal activity) and paired-pulse-induced plasticity at MPP-DG hippocampal synapses of adolescent and mature adult mice (miRNA-132/212 −/− vs. WTs ( Figure 3A)). Recordings for both groups were statistically indistinguishable, irrespective of their age ( Figure 3B-E).
Cells 2022, 11, x FOR PEER REVIEW

Nicotine Bolsters Synaptic Potentiation at CA3-CA1 Synapses of Mature Adult, Not Adolescent Wild-Type Mice
Long-term potentiation (LTP), as experimentally defined by the plastic ability of synapses to undergo stimuli-induced functional strengthening, has been proposed as a potential mechanism for memory formation in vivo [76,[83][84][85][86][87][88][89][90]. In both young and adult mammalian brains, LTP can be experimentally elicited in regions, such as the hippocampal MPP-DG and the CA3-CA1 area, and many authors have also established a link between LTP and addiction [91][92][93][94][95][96][97]. In order to examine the potential age-dependent participation of miRNA-132/212 in the modulation of LTP and the response to nicotine exposure, we obtained hippocampal slices from adolescent and mature adult mice (WT vs. miRNA-132/212 −/− ) and examined the nicotine impact on LTP in developing and fully mature synapses ( Figure 4A). All adolescent mice slices, WT and miRNA-132/212 −/− , showed comparable synaptic potentiation responses. Next, we exposed the adolescent slices from both groups to the double exposure (5-min each time) of 1-µM nicotine. The bath-applied double stimulation with nicotine had no detectable effects on the LTP responses, regardless of genotype ( Figure 4B-D).
= p < 0.05; ANOVA (repeated measures) was followed by the Bonferroni test; error bars presented as SEM). PPF: Paired-Pulse Facilitation; PND: postnatal day. For I/O, ANOVA for adolescent cohort indicated an effect of stimulation voltage (p < 0.0001, F (1.232, 32.03) = 2301) without genotype effect (p = 0.9885, F (1, 26) = 0.0002112), and for adult cohort a predicted main significant effect of input voltage (p > 0.0001, F (1.451, 26.12) = 1286) but no significant effect of genotype (p = 0.9042, F (1, 18) = 0.01490). For paired-pulse-induced plasticity; (ANOVA for adolescent cohort revealed main effect of inter-pulse interval (p < 0.0001, F (2.507, 65    (F) fEPSPs slope changes before and after inducing LTP in slices derived from mature adult (PND 120-180) untreated wild-type (WT untreated, n = 5) and miRNA-132/212 −/− (KO untreated, n = 3) mice (left panel) and for slices from wild-type slices treated with nicotine (WT treated, n = 6) as well as for slices from miRNA-132/212 −/− mice, also treated with nicotine (KO treated, n = 6) in the right panel. Nicotine was applied as for the young animals described above (and as indicated by the shadowed boxes in the figure). Robust but comparable LTP responses were observed in slices from miRNA-132/212 −/− mice, untreated or treated with nicotine. Untreated slices from wild-type mice also showed robust LTP. However, a significant enhancement of LTP was detected in response to nicotine only in slices from wild-type mice (black asterixis represent significant differences in synaptic potentiation between nicotine-treated WT and untreated WT groups, while orange asterixis (set to the right-hand side) between nicotine-treated WT and nicotine-treated miRNA-132/212 −/− groups; ANOVA (repeated measures) was followed by the Bonferroni test; error bars show SEM). Augmentation in LTP upon nicotine treatment of adult wild-type mice and no effect of nicotine treatment on LTP of adolescent wild-type mice (C,F) is observable. We next examined field potential responses in hippocampal slices from mature adult mice (WT and miRNA-132/212 −/− ). Slices obtained from both groups of untreated mature adult mice showed fEPSP responses similar to those observed in their adolescent counterparts, with a rather enhanced LTP response observed only for the adult miRNA-132/212 −/− relative to the other groups, suggesting a tendency to enhanced LTP, which is possibly attributable to aging-related phenomena still remaining to be clarified. Additionally, no significant effects of the treatment with nicotine were apparent in slices from adult miRNA-132/212 −/− mice, which also showed a rather enhanced LTP relative to the responses observed in their young counterparts but not larger than that observed in the adult untreated miRNA-132/212 −/− group, thus indicating that adult miRNA-132/212 −/− have a promoted LTP response in the CA1 region, somewhat analogue to previous observed experiments [44] but without changes in the sensitivity to nicotine stimulation. However, a robust and significant enhancement of synaptic potentiation was observed in mature adult WT mice slices that were treated with nicotine ( Figure 4E-G). Note the circa 25% enhancement in LTP upon nicotine treatment of mature adult WT mice ( Figure 4E-G) versus the lack of effect on LTP upon treatment with nicotine in adolescent WT mice ( Figure 4B-D). Interestingly, in slices obtained from mature adult WT mice, the effects of nicotine on CA3-CA1 synapses became prominent immediately following the induction of LTP (that is, during post-tetanic potentiation (PTP)), with fEPSPs remaining enhanced for the entire time of electrophysiological assessment ( Figure 4F). On the contrary, slices obtained from adolescent WT mice remained unresponsive to the effect of the treatment with the paired 5-min exposure to 1 µM of nicotine throughout the entire electrophysiological assessment ( Figure 4C).

Nicotine Shifts from Promoter to Suppressor of Synaptic Potentiation in the Dentate Gyrus in Absence of miRNA-132/212
The dentate gyrus importantly influences learning and memory, comprises a brain niche for neurogenesis, and is markedly affected by nicotine, as this alkaloid alters the process of synaptic plasticity [23,98,99]. Recently, our group unveiled the existence of a functional interplay between miRNAs and nicotinergic signaling that appears to be involved in the regulation of the expression of cell-surface membrane receptors and that participates in the modulation of neuroplasticity in the mature, fully developed hippocampus [30]. However, the possible role of the miRNAs 132 and 212 as age-dependent regulators of the hippocampal responses to nicotine remain, to the best of our knowledge, unexplored.
We, hence, studied the impact of nicotine exposure on synaptic potentiation in young developing and fully mature MPP-DG synapses using slices from adolescent and mature adult WT and miRNA-132/212 −/− mice ( Figure 5A). To this aim, we used conventional electrophysiological methods previously proven to efficiently induce short-and longterm forms of synaptic potentiation in slices from mice and rat hippocampi [81,[100][101][102]. Field-slope responses in untreated slices from adolescent mice from both groups (WT and miRNA-132/212 −/− ) showed comparable forms of synaptic strengthening in MPP-DG synapses.   In contrast, whereas slices from adolescent WT animals treated with 1 µM of nicotine (two bath applications given with 5-min intervals) showed markedly promoted synaptic strengthening, the slices from adolescent miRNA-132/212 −/− animals showed no potentiation at all and exhibited features resembling those observed after application of pulses of electrical stimulation inducing synaptic depression ( Figure 5B-D).
We subsequently examined the field potential responses in MPP-DG synapses in slices from mature-adult mice, WT, and miRNA-132/212 −/− . Under untreated control conditions, both groups showed synaptic potentiation responses without significant differences ( Figure 5E-G) and comparable to the responses under untreated conditions of adolescent mice as displayed in Figure 5B-D. However, in slices obtained from adult mice the response to 1 µM of nicotine resulted in virtually abolished memory-related synaptic strengthening in miRNA-132/212 −/− mice ( Figure 5E-G), an effect paralleling that observed in adolescent miRNA-132/212 −/− mice, as shown in Figure 5B-D. Nicotine treatment of mature adult WT mice, on the other hand, resulted in slight yet enhanced synaptic potentiation ( Figure 5F), an effect observed in adolescent WT mice (compare black circle symbol in Figure 5C,F).

Discussion
The hippocampus of humans and rodents share a large variety of genetic, structural, functional, and pharmacological commonalities and also overlap in key events in maturation during adolescence and adulthood [14,15,20]. While the time scale of the central nervous system developmental phases is considerably different between human and murine species, rodents, however, also exhibit a remarkable vulnerability to nicotine addiction at to human corresponding age stages (e.g., adolescence) and also share numerous features when addiction-related physiological and behavioral changes are examined [103][104][105][106][107] [14,15,[108][109][110]. The signaling elements that become altered and consolidated in the young brain upon nicotine exposure remain, however, poorly understood. Combining molecular biological, pharmacological, and biophysical studies in murine animal models can, therefore, importantly aid in the search for the identification of those still missing molecular elements influencing the brain neuronal vulnerability to nicotine at specific stages of brain development. Using this experimental approach, we here provide experimental evidence proposing an age-dependent and pathway-specific role for miRNA-132/212 in the modulation of the action of nicotine on memory-related hippocampal synaptic functions. Our data further reveal altered acetylcholinesterase and CREB signaling in miRNA132/212 knockout mice, thus encouraging further independent research on these initial, potential molecular elements that could participate in vivo, via miRNA-132/212 regulation, in the brain neuronal response to nicotine.

Discussion
The hippocampus of humans and rodents share a large variety of genetic, structural, functional, and pharmacological commonalities and also overlap in key events in maturation during adolescence and adulthood [14,15,20]. While the time scale of the central nervous system developmental phases is considerably different between human and murine species, rodents, however, also exhibit a remarkable vulnerability to nicotine addiction at to human corresponding age stages (e.g., adolescence) and also share numerous features when addiction-related physiological and behavioral changes are examined [14,15,[103][104][105][106][107][108][109][110]. The signaling elements that become altered and consolidated in the young brain upon nicotine exposure remain, however, poorly understood. Combining molecular biological, pharmacological, and biophysical studies in murine animal models can, therefore, importantly aid in the search for the identification of those still missing molecular elements influencing the brain neuronal vulnerability to nicotine at specific stages of brain development. Using this experimental approach, we here provide experimental evidence proposing an age-dependent and pathway-specific role for miRNA-132/212 in the modulation of the action of nicotine on memory-related hippocampal synaptic functions. Our data further reveal altered acetylcholinesterase and CREB signaling in miRNA132/212 knockout mice, thus encouraging further independent research on these initial, potential molecular elements that could participate in vivo, via miRNA-132/212 regulation, in the brain neuronal response to nicotine.

Acetylcholinergic Signaling and Hippocampal Synaptic Plasticity
We here have studied the effects of nicotine on hippocampal synaptic plasticity in the young and adult wild-type and miRNA-132/212 −/− mice and further examined the expression levels of pCREB, AChE, and M1 in the hippocampi of miRNA-132/212 −/− mice. It must be noted, however, that nicotine is, of course, not degraded by AChE and does not target M1 receptors. We examined the levels of M1 receptors in order to expand our knowledge on the association between miRNAs 132/212 and members of the acethylcolinergic signaling complex since we had previously reported that miRNA-132/212 −/− mice presented with augmented protein levels of the α7-nAChR [30]. The reported unaltered levels of M1 receptors in untreated miRNA-132/212 −/− mice hippocampi, therefore, suggest a potential specificity for the involvement of these microRNAs in the selective regulation of acethylcolinergic pathways. It would be also interesting to explore the effect of nicotine on the levels of AChE in the hippocampus of miR-132/212 knockout mice in future experiments, as nicotine has been proposed as a possible inhibitor of brain AChE [111].
Abundant scientific literature has previously established the impact of nicotinic cholinergic activity on LTP in a variety of hippocampal pathways, including CA3-CA1 synaptic circuits [112][113][114][115][116][117][118] and MPP-DG synapses [30,[119][120][121][122]. At the CA3-CA1 synaptic circuit, nicotine was also shown to promote LTP in the aged brain [114]. In hippocampal slices, 1 µM of nicotine facilitated LTP induction, while having no effect on baseline fEP-SPs [117,123]. On these grounds, we here also applied 1 µM of nicotine and studied its effects on hippocampal LTP in MPP-DG as well as on CA3-CA1 synapses. Our experimental data, derived from the use of a previously characterized knockout mice model [44], for the first time propose a potential role for the microRNAs 132 and 212 in the modulation of the effects that nicotine has on hippocampal synaptic plasticity in both pathway-specific and age-dependent manners. Our observations, thus, suggest the miRNA-132/212 complex as potential in vivo modulator of the selective effects on nicotine on addiction-and memory-related behaviors in the adolescence versus the adulthood through region-specific effects modulated across the life span.
The participation of nicotinic acetylcholine receptor [25,114,[124][125][126][127][128][129] and of the acetylcholinesterase [130,131] in hippocampal synaptic functions has been long established (see also [132][133][134]) [135]. Several groups had also described the relevance of CREB in hippocampal synaptic plasticity and memory-related functions [40,76,136,137] and in nicotinergic signaling [24,[138][139][140]. The miRNA-132/212 family had also been implicated in regulation of synaptic functions [30,[44][45][46], dendritic growth and arborization of hippocampal neurons [141], and higher-order executive functions that are necessary for the cognitive control of behavior [36,47,142]. Previous studies had also shown that miR132 is a target of CREB and that the expression of miRNA-132 is linked to CREB and ERK1/2 activity [37,143]. However, neither the functional crosstalk between the actions of nicotine and of acetylcholinesterase and CREB signaling through miRNA-132/212, or the relevance of age and neural circuitry for these effects, had been previously established. Our group had previously shown that Erk levels are altered in mice lacking the genes encoding for the miRNAs 132 and 212 [30]. Since Erk and CREB are concomitantly associated with both hippocampal synaptic plasticity and memory functions [144][145][146][147][148], we therefore hypothesize that miRNA132/212 might mediate in the modulation of those learning-and memory-related functions that become activated in vivo upon sustained early nicotine exposure through nicotinic acetylcholine receptors, acetylcholinesterase, and Erk/CREB signaling. The relevance of both age and neuronal pathway, apart from the differences introduced through the specific experimental protocols used, can further lie beneath the bimodal actions mediated by miRNA-132/212 and on the diverse alterations in the synaptic function upon deletion of the miRNA-132/212 genes [30,44,149,150]. Given the relevance of the hippocampus in memory functions, miRNA-132/212 advantageous position at the molecular level, thus, emerged through our work as a potential temporal-spatial regulator of nicotine-related gene expression at specific brain developmental stages to influence hip-pocampal neural plasticity and associated behavioral changes during drug-related memory formation and maintenance.

Limitations
It must be noted that, in this work, only male animals were used, and there are important differences reported for several neuronal properties, including synaptic plasticity and memory functions, that are strongly dependent on the sex of the animals used in this type of studies [61,[151][152][153][154]. Therefore, our data here should not be generalized to the female population. For example, some physiological processes and related diseases (e.g., Parkinson's disease) can be differentially modulated in a sex-dependent manner [155][156][157][158]. Similar considerations should be taken in account to avoid a generalization about the scope of our data on the grounds of the possible influence of factors, such as the strain [159][160][161][162]. We, thus, encourage additional independent research to expand our observations on female subjects and with different strains, models, and ages.

Conclusions
Experimental data provided here suggest the existence of an in vivo, fine-tuned ageand pathway-specific mechanism of modulation of the effects of the endogenous neurotransmitter acetylcholine on hippocampal synaptic plasticity. This work expands our current scope of neuronal signaling mechanisms potentially implicated in drug addiction and proposes miR-132/212 as a potential target for pharmacotherapeutic studies searching for ways to ameliorate the deleterious effects of nicotine on the remodeling of neuronal circuits relevant for learning acquisition, memory retention, and drug addiction consolidation. Further research and independent experimental verification of our data might, thus, contribute to enhance our knowledge on the molecular and functional mechanisms determining the age-dependent and pathway specific functional crosstalk between microRNAs and nicotine addiction. Data Availability Statement: All the data generated and/or analyzed in this study has been included in this article.