Nicotine abolishes memory‐related synaptic strengthening and promotes synaptic depression in the neurogenic dentate gyrus of miR‐132/212 knockout mice

Abstract Micro‐RNAs (miRNAs) are highly evolutionarily conserved short‐length/noncoding RNA molecules that modulate a wide range of cellular functions in many cell types by regulating the expression of a variety of targeted genes. miRNAs have also recently emerged as key regulators of neuronal genes mediating the effects of psychostimulant drugs and memory‐related neuroplasticity processes. Smoking is a predominant addictive behaviour associated with millions of deaths worldwide, and nicotine is a potent natural psychoactive agonist of cholinergic receptors, highly abundant in cigarettes. The influence of miRNAs modulation on cholinergic signalling in the nervous system remains however poorly explored. Using miRNA knockout mice and biochemical, electrophysiological and pharmacological approaches, we examined the effects of miR‐132/212 gene disruption on the levels of hippocampal nicotinic acetylcholine receptors, total ERK and phosphorylated ERK (pERK) and MeCP2 protein levels, and studied the impact of nicotine stimulation on hippocampal synaptic transmission and synaptic depression and strengthening. miR‐132/212 deletion significantly altered α7‐nAChR and pERK protein levels, but not total ERK or MeCP2, and resulted in both exacerbated synaptic depression and virtually abolished memory‐related synaptic strengthening upon nicotine stimulation. These observations reveal a functional miRNAs/nicotinergic signalling interplay critical for nicotinic‐receptor expression and neuroplasticity in brain structures relevant for drug addiction and learning and memory functions.

dominant addictive behaviour associated with millions of deaths worldwide, and nicotine is a potent natural psychoactive agonist of cholinergic receptors, highly abundant in cigarettes. The influence of miRNAs modulation on cholinergic signalling in the nervous system remains however poorly explored. Using miRNA knockout mice and biochemical, electrophysiological and pharmacological approaches, we examined the effects of miR-132/212 gene disruption on the levels of hippocampal nicotinic acetylcholine receptors, total ERK and phosphorylated ERK (pERK) and MeCP2 protein levels, and studied the impact of nicotine stimulation on hippocampal synaptic transmission and synaptic depression and strengthening. miR-132/212 deletion significantly altered α7-nAChR and pERK protein levels, but not total ERK or MeCP2, and resulted in both exacerbated synaptic depression and virtually abolished memory-related synaptic strengthening upon nicotine stimulation. These observations reveal a functional miRNAs/nicotinergic signalling interplay critical for nicotinicreceptor expression and neuroplasticity in brain structures relevant for drug addiction and learning and memory functions. tively targeted gene products. [2][3][4][5][6][7] miRNAs are highly evolutionarily conserved in the plant 8 and animal kingdoms. 9,10 In mammals, miRNAs have been involved in the regulation of more than 50% of all genes known to be encoding for the formation of proteins. 11 miRNAs indeed modulate a wide-ranging variety of cellular processes, spanning from early-to-late stages of embryonic development to basic metabolic functions, homeostasis, cellular proliferation and differentiation and secretion. [12][13][14][15][16] miRNAs play also critical roles in the regulation of the neuronal function, a characteristic highly conserved from an evolutionary perspective as demonstrated in organisms such as zebra fish, mice and humans. 14,[17][18][19][20] For example, miRNAs are critical for neural development 21 and adult neurogenesis, 14,18,19 and memory-related synaptic plasticity. 22,23 Dysregulation of miRNAs activity is further associated with a wide variety of severely debilitating human neurodegenerative conditions such as epilepsy, [24][25][26] Alzheimer's [27][28][29][30][31][32][33][34][35] and Parkinson's diseases. 11 The miR-132/212 family of miRNAs has been proposed as a key regulator of spinal cord development, 36 embryonic stem cell differentiation into neurons 37 and memory-related neuroplasticity. 22,38 Dysregulation of miR-132 has been further associated with the genesis and worsening of epilepsy, Alzheimer's and Parkinson's diseases. 39 Little remains known, however, about the involvement of miR-132/212 in the regulation of synaptic functions in neuronal circuits critical for both learning and memory and drug addiction.
Acetylcholine (Ach) 40,41 is a neurotransmitter highly active both in the peripheral and in the central nervous systems, where it is known to act as a key regulator of neuroplasticity and learning and memory processes. Ach interacts with-and regulates-both muscarinic and nicotinic receptors and regulates hippocampal synaptic plasticity and learning and memory processes. [42][43][44][45][46][47][48][49][50][51][52][53][54][55] Whereas both muscarinergic- 56 and nicotinergic-mediated signalling are thus of pivotal importance for the regulation of the brain cognitive function, the predominance of tobacco over mushrooms as the worldwide preferred substance of abuse is disproportionally superior. 57,58 We therefore here focused on nicotinergic-related functions in the mammalian hippocampus. Nicotinic acetylcholine receptors (nAChrs) 59,60 comprise a group of ligandgated ion channels abundant in neurons of the central nervous system 61 that modulate neuroplasticity and memory functions and are importantly implicated in tobacco-derived nicotine addiction. [62][63][64][65] However, although both miRNAs-mediated and nicotinergic signalling have been independently described as critical regulators of neuroplasticity, learning and memory and addiction-related functions, the functional crosstalk between these two pivotal mechanisms of neuronal regulation in brain neuronal circuits have remained virtually unexplored.
Using the mouse as experimental model, this work specifically examined how the knockout (KO) of the gene encoding for miR-132/212 influences the neuroplasticity responses to nicotine stimulation at the hippocampal neurogenic dentate gyrus. Our results provide, to the best of our knowledge, the first biochemical and functional experimental evidence in support of a role of miR-132/212 in the effects of nicotine on memory-related synaptic plasticity in the mammalian hippocampus, a brain region critical for memory storage and importantly implicated in the neurobiology of tobacco-derived nicotine addiction. These results broaden our understanding of the molecular mechanisms influencing the neuronal responses to addictive substances and unveil the miR-132/212 family of miRNAs as putative candidate targets to mediate in the capability of nicotine to alter the function of neuronal circuits underlying memory formation of drug addictions.

| Immunoblotting
Brain extraction and whole hippocampi isolation from miRNA-132/212 −/− and WT male mice (n = 6 animals per group) were conducted according to methods established in our laboratory as described before, [66][67][68] with minor modifications. 69 Animals were euthanized by quick cervical dislocation, followed by a swift sharpblade decapitation. Brains were rapidly extracted and submerged in an ice-cold artificial cerebrospinal fluid (aCSF) with pH adjusted to 7.4.
Hippocampi were carefully isolated, and cerebellum was dissected.
Hippocampi and the rest of the brain were individually sampled, frozen in liquid nitrogen and immediately stored at −80 C until further analysis. Brain tissues were further homogenized in lysis buffer (50-mM Tris, 150-mM NaCl, 1% TritonX-100 and 5-mM EDTA) and 1:100 Protease and Phosphatase Inhibitor Cocktail (PIC, Thermo Scientific, Germany) and incubated overnight at 4 C on a tube rotator.
Following overnight incubation, lysates were centrifuged for 5 min at
Following quick cervical dislocation and swift sharp-blade decapitation, brains were extracted by a midline incision in the occipital bone starting at the foramen magnum extending to the sutura frontalis. The parietal bones were folded to the side, and brain was gently removed, using a spatula, and submerged in an ice-cold aCSF solution containing (in mM) 125 NaCl, 2.5 KCl, 20 NaHCO 3 , 2.5 CaCl 2 , 1 MgCl 2 , 25 D-glucose and 1 NaH 2 PO 4 (pH 7.4). Coronal hippocampal slices were cut using a vibrating microtome (7000smz-2, Campden Instruments Ltd., Loughborough, UK) at a frequency of 90 Hz, an amplitude of 0.75 mm and speed of 0.12 mm/s. During cutting, slices remained continually submerged in ice-cold aCSF solution bubbled with a carbogen gas mixture (95% O 2 /5% CO 2 ). After cutting, hemispheres were separated, and slices transferred to a customized recovery chamber containing carbogenated aCSF at 28 C where slices were left to rest for at least 1 h before electrophysiological recording.

| Extracellular recordings
Individual slices were transferred to a low volume submerged recording chamber perfused with a constant flow (2-3 mL/min) of carbogenated aCSF solution maintained at the room temperature.
Evoked field excitatory postsynaptic potentials (fEPSPs) were recorded using borosilicate glass pipettes prepared in a horizontal puller (Sutter Instrument, Novato, CA) and yielding tip resistance of (3 ± 1) MΩ when filled with aCSF. Electrical stimulation was delivered via customized Teflon-coated tungsten wire bipolar stimulating electrodes isolated to the tip (~50-μm diameter tip). Recordings of fEPSPs were obtained from the dentate gyri middle molecular cell layer upon stimulation of the medial perforant pathway (MPP) as previously F I G U R E 1 Schematic representation of the electrophysiological experimental setting. A, Diagram of a hippocampal slice with the positioning of the stimulating and recording electrodes at the neurogenic dentate gyrus. Simulating electrodes are positioned upstream of the middle molecular layer to stimulate the medial perforant path. B, The inset depicts a recording electrode positioned at the dendritic layer of neuronal granule cells, with arrows indicating the direction of the inputs from the perforant path. C, A representation of a recorded extracellular field potential is shown to the right. D, Diagrams of the protocols of electrical stimulation delivered through the stimulation electrodes are described for long-term potentiation (LTP) and for long-term depression (LTD) (see also Section 2) described by our group and others. 68

| Statistical analysis
Statistical analyses were done using GraphPad Prism Software, Version 7.0 (San Diego, CA, USA). The normality test D'Agostino's K 2 test was used prior to any statistical analyses, to determine the sample data distribution. Two-sided unpaired Student's t test was used to determine differences of data sets obtained from two experimental groups. Statistical analysis involving more than two experimental groups was assessed using analysis of variances (ANOVA) as appropriate, specifically repeated-measures ANOVA (or mixed model ANOVA) followed by Bonferroni's test to account for multiple comparisons.    Figure 3C,D). Two-way ANOVA of the 22.5 min F I G U R E 3 Nicotine stimulation abolishes synaptic potentiation in hippocampal dentate gyrus of miRNA-132/212 gene knockout. A, Representative traces of field excitatory postsynaptic potentials (fEPSPs), recorded at baseline conditions and after the application of potentiation-inducing protocols (Section 2), from untreated hippocampal slices derived from untreated wild-type controls and untreated miRNA-132/212 −/− mice. The responses generated after delivery of electrical stimulation were taken at 22.5 min and are indicated as "synaptic potentiation" in the graph. B, Temporal course of the changes in fEPSPs slopes as recorded before and after the application of potentiationinducing protocols in hippocampal slices from untreated wild-type and untreated miRNA-132/212 −/− mice. No statistically significant differences were observed between the groups. C, Representative traces of fEPSPs, recorded at baseline conditions and after the application of potentiationinducing protocols, from untreated hippocampal slices derived from untreated wild-type controls and untreated miRNA-132/212 −/− mice. The responses generated after delivery of electrical stimulation are indicated as "synaptic potentiation" in the graph and were obtained after the dual application of 1-μM nicotine, which was delivered during baseline conditions as described below. D, Temporal course of the changes in fEPSPs slopes as recorded before and after the application of potentiation-inducing protocols in hippocampal slices from untreated wild-type (n = 6) and untreated miRNA-132/212 −/− (n = 5) mice. Two consecutive pulses of 5-min exposure to 1-μM nicotine, spaced by a 5-min washout period, preceded the delivery of the application of potentiation-inducing protocols as illustrated in the figure in the shadowed box insets. A pronounced and statistically significant enhancement of synaptic potentiation was observed in recordings from wild-type animals, whereas a virtual abolishment of the potentiation response was detected in slices from miRNA-132/212 −/− mice. E, Scatter plots showing the field responses after delivering potentiation-inducing protocols in all four investigated groups at 22.5 min. The high-frequency stimulation protocol used to induce potentiation was delivered right after the recording obtained at 20 min as indicated in the figure (HFS + arrow). Data are presented as mean ± SEM. Please see details for the statistics on the main text values showed no significant effect of treatment ( ns P = 0.7234,  Figure 3E).
We therefore next obtained hippocampal slices from miRNA-F I G U R E 4 Nicotine stimulation promotes synaptic depression in hippocampal dentate gyrus of miRNA-132/212 gene knockout. A, Representative traces of field excitatory postsynaptic potentials (fEPSPs), recorded at baseline conditions and after the application of synaptic depression-inducing protocols, from untreated hippocampal slices derived from untreated wild-type controls and untreated miRNA-132/212 −/− mice. The responses generated after delivery depression-inducing electrical stimulation were taken at 22.5 min and are indicated as "synaptic depression" in the graph. B, Temporal course of the changes in fEPSPs slopes as recorded before and after the application of depression-inducing protocols in hippocampal slices from untreated wild-type and untreated miRNA-132/212 −/− mice. No statistically significant differences were observed between the groups. C, Representative traces of fEPSPs, recorded at baseline conditions and after the application of depressioninducing protocols, from untreated hippocampal slices derived from untreated wild-type controls and untreated miRNA-132/212 −/− mice. The responses generated after delivery of electrical stimulation are indicated as "synaptic depression" in the graph and were obtained after the dual application of 1-μM nicotine, which was delivered during baseline conditions as described below. D, Temporal course of the changes in fEPSPs slopes as recorded before and after the application of depression-inducing protocols in hippocampal slices from untreated wild-type and Under untreated control conditions, field potential recordings in slices obtained either from WT or from miRNA-132/212 −/− presented with synaptic depression responses that were statistically indistinguishable between the two groups ( Figure 4A,B). However, while no statistically significant effects of the nicotine treatment was observed in slices obtained from the WT controls after the treatment with the 5-min paired exposure to 1-μM nicotine, a pronounced statistically significant promotion of synaptic depression was apparent in slices obtained from miRNA-132/212 −/− (Figure 4C,E). Mixed-effects ANOVA analysis showed a significant effect of time ( **** P < 0.0001,  Figure 4E).

| miRNA-132/212 deletion alters protein levels of α7-nAChR and pERK in mouse hippocampus
Abundant scientific literature has recently started to emerge describing miR-132 as a strong regulator of members of the cholinergic F I G U R E 5 The miRNA-132/212 deletion alters protein levels of α7-nAChR and pERK in mouse hippocampus. Total cell lysates from male mouse bilateral hippocampi were analysed by Western blot to determine the protein expression levels of α7-nAChR, ERK and pERK. A, Representative immunoblots and relative levels of α7-nAChR protein together with corresponding GAPDH levels, indicating significant increase in protein levels of α7-nAChR in the hippocampus of miRNA-132/212 −/− mice, compared with wild-type mice. B, Representative immunoblots and relative levels of ERK1 (p44) and ERK2 (p41) protein together with corresponding GAPDH levels in the hippocampus of miRNA-132/212 −/− mice. Analysis showed no changes in protein levels of ERK compared with wild-type mice. C, Representative immunoblots and relative levels of pERK and GAPDH levels indicating significant decrease in protein levels of pERK in the hippocampus of miRNA-132/212 −/− mice compared with wild-type mice. No significant differences were observed in protein levels of pERK. D, Representative immunoblots and relative levels of MeCP2 protein together with corresponding GAPDH levels, indicating no significant differences in protein levels of MeCP2 in the hippocampus of miRNA-132/212 −/− mice, compared with wild-type mice. Results are shown relative to GAPDH. Data are expressed as mean ± SD. n = 5 per group. WT, wild type; kDa, kilodalton. Please see details for the statistics on the main text signalling pathway in several different tissues. [122][123][124][125][126][127][128][129][130]
We postulate that miR-132/212 gene disruption results, either directly or indirectly, in altered levels of α7-nAChR, which results in a bimodal response to nicotine that affects both synaptic depression and strengthening through mechanism that still remain to be characterized.

| Putative direct/indirect molecular elements linking miR-132/212 down-regulation and nicotinergic signalling
Nicotine facilitates the release of Ach, which plays an important role in cognitive functions 170,171 and facilitates LTP induction by interacting with the nAChRs subtypes α7 in the hippocampus. 172 In line with these previously described observations, we detected enhanced synaptic transmission (potentiation) upon high-frequency electrical stimulation (known to induce LTP) in WT mice that were previously exposed to nicotine, effects that were abolished in miR-132/212 KO mice. Furthermore, our protein analysis for the first time describes that miR-132/212 gene disruption results in a marked enhancement in the protein expression levels of the α7-nAChR paralleled by a pronounced decrease of pERK, with no changes in total ERK or MeCP2.
Interestingly, while MeCP2 has been proposed as a target of miR-132 in neurological disorders like Autism spectrum disorder (ASD) and Huntington and Parkinson's diseases, 173 181 Previous reports have described that AChE is capable of acting as a modulator of the activity of α7-nAChRs. 182 More importantly, AChE is indeed a target of miRNAs from the miR-132/212 family. 122,123,[126][127][128] Additionally, disruption in the proper targeting and inhibition of AChE expression by miR-132 (due to the down-regulation it this miRNA) have been further proposed to be causally linked with the progression of dementia after ischaemic stroke. 130 Recent experiments have also described that inhibitors of the AChEs can act to promote neuroprotection or neurogenesis via the activation of α7-nAChR thus resulting in an increase in the levels of growth factors in the mouse hippocampus. 183 Indeed, compounds acting as inhibitors of AChEs have been clinically used for the treatment of Alzheimer's disease. 184 The targeting of AChE by miR-132 suggest thus that AChE could indirectly mediate in the here described effects of miR-132/212 gene disruption on the neuroplasticity responses to nicotine in the dentate gyrus, a hypothesis requiring future research for clarification.

| Conclusions
The specific molecular mechanisms linking the action of drugs of abuse with brain neuroplasticity and memory storage remain elusive, and consequently, clinical interventions continue to fail to alter drugdependence-related behaviours, particularly in the case of nicotine addiction. ACh affects the hippocampus due to its selective effect on episodic and semantic memory formation. 185 Therefore, the expression and correct regulation of ACh synthesis, its release, and the properties of its effect on specific receptors are likely key factors in preserving and enhancing memory-related neural functions during drug addiction in the mammalian nervous systems. Interestingly, the overexpression of specific miRNAs in the CA1 hippocampal region (an area also of central importance for spatial memory) has been shown to alter some long-term forms of synaptic plasticity. 38,186 Moreover, in the hippocampus, the prevalent subunit subtypes are the α7 and α4β2 subtypes, with α7-nAChRs having the highest density in the pyramidal neurons, localized presynaptically and postsynaptically. 187 Dysfunction or deactivation of those receptors has shown to lead to various serious neuropathologies including Alzheimer's disease, 181,188 perhaps the most debilitating and widespread form of memory dysfunction. It is noteworthy to highlight that whereas data presented here indicate that miRNA-132/212 −/− mice show an increase in the levels of alpha-7 nicotinic Ach receptors, they however have a decreased response to nicotine administration (to LTP) compared with controls, thus suggesting that inhibitory mechanism has been promoted, including either enhancing of reversal of LTP (depotentiation; see also Fujii and Sumikawa 52 ) or promotion of LTD as indicated here by nicotine-enhanced LTD in miRNA-132/212 −/− KO animals). The molecular mechanisms responsible for these rather paradoxical effects remain unclear, and the capability of nicotine to induce a large variety of different actions is surely determined by its capability to interact with diverse nicotinic Ach receptor subtypes. For example, whereas some authors have described that nicotine can enhance LTP, 189,190 other authors have described that nicotine can act as both synaptic enhancer and depressor depending on the levels of A2 nicotinic Ach receptors and in a pathway-specific manner. 191 Nicotine (3 mg/kg, i.p.) delivered 1 h prior to the LTP induction has been also shown not to enhance LTP recorded in vivo but rather to exacerbate the impairments of LTP induced by Aβ 1-40 treatment. 192 In slices, nicotine can also fail to induce synaptic potentiation in CA1 pyramidal neurons of AD11 antinerve growth factor transgenic mice in a manner related to the Aβ levels. 193 Interestingly, a recent manuscript has described that the deficiency in miRNA-132/212 results in enhanced levels of Aβ production in mouse models of Alzheimer's disease. 194 These observations thus also suggest that the deficiency of nicotine to positively influence LTP in our experiments can be related to the effects of miRNA-132/212 gene deletion on the levels of Aβ, a hypothesis currently under investigation in our laboratory. Nicotine appears thus to have the capability to exert bimodal effects, inducing either inhibition or strengthening of synaptic responses on the same circuits.
Interestingly, it has been demonstrated that stimulation with the parasympathomimetic choline ester carbachol (CCh), which can simultaneously act as a nicotinic and muscarinic Ach receptor agonist, can result in a bimodal effect either suppressing or potentiating the synaptic responses depending upon the centration and duration of CCh exposure, 43

DISCLOSURE/CONFLICT OF INTEREST
The authors report no conflicts of interest.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.