Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

The Dopaminergic Cells in the Median Raphe Region Regulate Social Behavior in Male Mice.

According to previous studies, the median raphe region (MRR) is known to contribute significantly to social behavior. Besides serotonin, there have also been reports of a small population of dopaminergic neurons in this region. Dopamine is linked to reward and locomotion, but very little is known about its role in the MRR. To address that, we first confirmed the presence of dopaminergic cells in the MRR of mice (immunohistochemistry, RT-PCR), and then also in humans (RT-PCR) using healthy donor samples to prove translational relevance. Next, we used chemogenetic technology in mice containing the Cre enzyme under the promoter of the dopamine transporter. With the help of an adeno-associated virus, designer receptors exclusively activated by designer drugs (DREADDs) were expressed in the dopaminergic cells of the MRR to manipulate their activity. Four weeks later, we performed an extensive behavioral characterization 30 min after the injection of the artificial ligand (Clozapine-N-Oxide). Stimulation of the dopaminergic cells in the MRR decreased social interest without influencing aggression and with an increase in social discrimination. Additionally, inhibition of the same cells increased the friendly social behavior during social interaction test. No behavioral changes were detected in anxiety, memory or locomotion. All in all, dopaminergic cells were present in both the mouse and human samples from the MRR, and the manipulation of the dopaminergic neurons in the MRR elicited a specific social response.

Chemogenetic Manipulation of Amygdala Kappa Opioid Receptor Neurons Modulates Amygdala Neuronal Activity and Neuropathic Pain Behaviors.

Neuroplasticity in the central nucleus of the amygdala (CeA) plays a key role in the modulation of pain and its aversive component. The dynorphin/kappa opioid receptor (KOR) system in the amygdala is critical for averse-affective behaviors in pain conditions, but its mechanisms are not well understood. Here, we used chemogenetic manipulations of amygdala KOR-expressing neurons to analyze the behavioral consequences in a chronic neuropathic pain model. For the chemogenetic inhibition or activation of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (hM4Di) or Gq-DREADD (hM3Dq) was injected stereotaxically into the right CeA of transgenic KOR-Cre mice. The chemogenetic inhibition of KOR neurons expressing hM4Di with a selective DREADD actuator (deschloroclozapine, DCZ) in sham control mice significantly decreased inhibitory transmission, resulting in a shift of inhibition/excitation balance to promote excitation and induced pain behaviors. The chemogenetic activation of KOR neurons expressing hM3Dq with DCZ in neuropathic mice significantly increased inhibitory transmission, decreased excitability, and decreased neuropathic pain behaviors. These data suggest that amygdala KOR neurons modulate pain behaviors by exerting an inhibitory tone on downstream CeA neurons. Therefore, activation of these interneurons or blockade of inhibitory KOR signaling in these neurons could restore control of amygdala output and mitigate pain.

Effect of Coffee and Chocolate Ingestion on Clozapine Dose and on Plasma Clozapine and Norclozapine Concentrations in Clinical Practice.

BACKGROUND: Some reports point to dietary caffeine intake as a cause of increased plasma clozapine concentrations in certain patients. METHODS: We compared clozapine dose and plasma clozapine and N-desmethylclozapine (norclozapine) concentrations in male and female smokers and nonsmokers in relation to reported (i) coffee (caffeine) and (ii) chocolate (caffeine and theobromine) intake in samples submitted for clozapine therapeutic drug monitoring, 1993-2017. RESULTS: There was information on coffee ingestion for 16,558 samples (8833 patients) from males and 5886 samples (3433 patients) from females and on chocolate ingestion for 12,616 samples (7568 patients) from males and 4677 samples (2939 patients) from females. When smoking was considered, there was no discernible effect of either coffee or chocolate ingestion either on the median dose of clozapine or on the median plasma clozapine and norclozapine concentrations in men and in women. However, cigarette smoking was associated with higher coffee and chocolate consumption. Although male nonsmokers who reported drinking 3 or more cups of coffee daily had significantly higher median plasma clozapine and norclozapine concentrations than those who drank less coffee, they were also prescribed a significantly higher clozapine dose. There was no clear effect of coffee ingestion on plasma clozapine and norclozapine in female nonsmokers. IMPLICATIONS: Inhibition of clozapine metabolism by caffeine at the doses of caffeine normally encountered in those treated with clozapine is unlikely even in male nonsmokers. Measurement of plasma caffeine in an appropriate sample should be considered in any future investigation into a presumed clozapine-caffeine interaction.

Comparison of three DREADD agonists acting on Gq-DREADDs in the ventral tegmental area to alter locomotor activity in tyrosine hydroxylase:Cre male and female rats.

RATIONALE: Despite the increasingly pervasive use of chemogenetic tools in preclinical neuroscience research, the in vivo pharmacology of DREADD agonists remains poorly understood. The pharmacological effects of any ligand acting at receptors, engineered or endogenous, are influenced by numerous factors including potency, time course, and receptor selectivity. Thus, rigorous comparison of the potency and time course of available DREADD ligands may provide an empirical foundation for ligand selection. OBJECTIVES: Compare the behavioral pharmacology of three different DREADD ligands clozapine-N-oxide (CNO), compound 21 (C21), and deschloroclozapine (DCZ) in a locomotor activity assay in tyrosine hydroxylase:cre recombinase (TH:Cre) male and female rats. METHODS: Locomotor activity in nine adult TH:Cre Sprague-Dawley rats (5 female, 4 male) was monitored for two hours following administration of d-amphetamine (vehicle, 0.1-3.2 mg/kg, IP), DCZ (vehicle, 0.32-320 µg/kg, IP), CNO (vehicle, 0.32-10 mg/kg), and C21 (vehicle, 0.1-3.2 mg/kg, IP). Behavioral sessions were conducted twice per week prior to and starting three weeks after bilateral intra-VTA hM3Dq DREADD virus injection. RESULTS: d-Amphetamine significantly increased locomotor activity pre- and post-DREADD virus injection. DCZ, CNO, and C21 did not alter locomotor activity pre-DREADD virus injection. There was no significant effect of DCZ, CNO, and C21 on locomotor activity post-DREADD virus injection; however, large individual differences in both behavioral response and receptor expression were observed. CONCLUSIONS: Large individual variability was observed in both DREADD agonist behavioral effects and receptor expression. These results suggest further basic research would facilitate the utility of these chemogenetic tools for behavioral neuroscience research.

Localized chemogenetic silencing of inhibitory neurons: a novel mouse model of focal cortical epileptic activity.

Focal cortical epilepsies are frequently refractory to available anticonvulsant drug therapies. One key factor contributing to this state is the limited availability of animal models that allow to reliably study focal cortical seizures and how they recruit surrounding brain areas in vivo. In this study, we selectively expressed the inhibitory chemogenetic receptor, hM4D, in GABAergic neurons in focal cortical areas using viral gene transfer. GABAergic silencing using Clozapine-N-Oxide (CNO) demonstrated reliable induction of local epileptiform events in the electroencephalogram signal of awake freely moving mice. Anesthetized mice experiments showed consistent induction of focal epileptiform-events in both the barrel cortex (BC) and the medial prefrontal cortex (mPFC), accompanied by high-frequency oscillations, a known characteristic of human seizures. Epileptiform-events showed propagation indication with favored propagation pathways: from the BC on 1 hemisphere to its counterpart and from the BC to the mPFC, but not vice-versa. Lastly, sensory whisker-pad stimulation evoked BC epileptiform events post-CNO, highlighting the potential use of this model in studying sensory-evoked seizures. Combined, our results show that targeted chemogenetic inhibition of GABAergic neurons using hM4D can serve as a novel, versatile, and reliable model of focal cortical epileptic activity suitable for systematically studying cortical ictogenesis in different cortical areas.

Chemogenetic modulation of the medial prefrontal cortex regulates resistance to acute stress-induced cognitive impairments.

The medial prefrontal cortex (mPFC) has been implicated in regulating resistance to the effects of acute uncontrollable stress. We previously showed that mPFC-lesioned animals exhibit impaired object recognition memory after acute exposure to a brief stress that had no effect in normal animals. Here, we used designer receptors exclusively activated by designer drugs to determine how modulating mPFC activity affects recognition-memory performance under stressful conditions. Specifically, animals with chemogenetic excitation or inhibition of the mPFC underwent either a brief ineffective stress (20-min restraint + 20 tail shocks) or a prolonged effective stress (60-min restraint + 60 tail shocks). Subsequent recognition memory tests showed that animals with chemogenetic mPFC inhibition exposed to brief stress showed impairment in an object recognition memory task, whereas those with chemogenetic mPFC excitation exposed to prolonged stress did not. Thus, the present findings the decreased mPFC activity exacerbates acute stress effects on memory function whereas increased mPFC activity counters these stress effects provide evidence that the mPFC bidirectionally modulates stress resistance.

Metabolite Profiling of Clozapine in Patients Switching Versus Maintaining Treatment: A Retrospective Pilot Study.

PURPOSE/BACKGROUND: Pharmacokinetics may be of relevance for the risk of clozapine discontinuation. We compared metabolite profiles, accounting for smoking habits, in patients switching versus maintaining clozapine treatment at therapeutic concentrations. METHODS/PROCEDURES: Adult patients with clozapine serum levels above 1070 nmol/L (350 ng/mL) were retrospectively included from a Norwegian therapeutic drug monitoring service during 2018-2020. Inclusion criteria were (1) known smoking habits, (2) blood sample drawn within 10 to 30 hours after last clozapine intake, and (3) detectable levels of N -desmethylclozapine, clozapine -N -oxide, clozapine-5 N -glucuronide, or clozapine- N + - glucuronide. Patients comedicated with cytochrome P450 enzyme inducers, inhibitors, or valproic acid were excluded. The high-resolution mass spectrometry assay enabled detection of 21 clozapine metabolites. Metabolite profiles were compared between patients switching treatment (switchers), measured as clozapine being replaced by another antipsychotic drug in blood samples, versus maintaining clozapine treatment (nonswitchers) during the study period. FINDINGS/RESULTS: Of the 84 patients fulfilling the study criteria, 7 patients (8.3%) were identified as clozapine switchers. After correcting for smoking habits, the clozapine-5 N -glucuronide/clozapine ratio was 69% lower ( P < 0.001), while the clozapine- N + -glucuronide/clozapine-5 N -glucuronide ratio was 143% higher ( P = 0.026), respectively, in switchers versus nonswitchers. The other metabolite ratios did not significantly differ between switchers and nonswitchers. IMPLICATIONS/CONCLUSIONS: The present study found a significantly reduced 5 N -glucuronidation phenotype in patients switching from clozapine at therapeutic serum concentrations (>1070 nmol/L) to other antipsychotic drugs. This may indicate that glucuronidation, as a potential detoxification mechanism, is related to clozapine tolerability. However, the causality of this observation needs to be investigated in future studies with larger patient populations.

Plasma Clozapine and Norclozapine After Use of Either Crushed Tablets or Suspension Compared With Tablets.

BACKGROUND: With clozapine, either crushed tablets suspended in an aqueous medium or proprietary suspension is sometimes prescribed as an alternative to tablets, but bioequivalence data are scant. METHODS: We compared clozapine dose, and plasma clozapine and N -desmethylclozapine (norclozapine) concentrations after use of either tablets or crushed tablets/suspension in samples submitted for clozapine therapeutic drug monitoring, 1993 to 2017. RESULTS: There were 846 patients (1646 samples) given crushed tablets/suspension and 6065 patients (10,779 samples) given tablets. The median dose (mg d -1 ) was significantly higher in men (500 vs 450) and women (500 vs 400) given crushed tablets/suspension, but the median plasma clozapine and norclozapine concentrations (mg L -1 ) were significantly lower (men: 0.29 and 0.22 vs 0.39 and 0.28; women: 0.35 and 0.26 vs 0.50 and 0.32, respectively). A subgroup of 480 patients was prescribed either crushed tablets/suspension (1016 samples) or tablets (1259 samples) at different times. The median dose was again significantly higher in men (500 vs 500) and women (500 vs 450), but the median plasma clozapine and norclozapine concentrations were significantly lower (men: 0.29 and 0.22 vs 0.32 and 0.24; women: 0.30 and 0.24 vs 0.38 and 0.29, respectively). IMPLICATIONS: Poor adherence, sedimentation of suspension before use, and incomplete dosage are potential contributors to the lower median plasma clozapine and norclozapine concentrations observed after use of either crushed clozapine tablets or suspension as compared with tablets. Those administering crushed tablets/suspension should be aware of these factors.

Relationship between plasma clozapine/N-desmethylclozapine and changes in basal forebrain-dorsolateral prefrontal cortex coupling in treatment-resistant schizophrenia.

Clozapine (CLZ) demonstrates a unique clinical efficacy relative to other antipsychotic drugs. Previous work has linked the plasma ratio of CLZ and its major metabolite, N-desmethylclozapine (NDMC), to an inverse relationship with cognition via putative action on the cholinergic system. However, neuroimaging correlates of CLZ/NDMC remain unknown. Here, we examined changes in basal forebrain functional connectivity with the dorsolateral prefrontal cortex, and secondly, cognition in relation to the CLZ/NDMC ratio. A cohort of nineteen chronically ill participants with treatment-resistant schizophrenia (TRS) undergoing 12 weeks of CLZ treatment were included. Measures of cognition and plasma CLZ/NDMC ratios were obtained in addition to resting-state functional neuroimaging scans, captured at baseline and after 12 weeks of CLZ treatment. We observed a significant correlation between basal forebrain-DLPFC connectivity and CLZ/NDMC ratios across CLZ treatment (p = 0.02). Consistent with previous findings, we also demonstrate a positive relationship between CLZ/NDMC ratio and working memory (p = 0.03). These findings may reflect the action of CLZ and NDMC on the muscarinic cholinergic system, highlighting a possible neural correlate of cognition across treatment.

CNO Administration Increases Dopamine and Glutamate in the Medial Prefrontal Cortex of Wistar Rats: Further Concerns for the Validity of the CNO-activated DREADD Procedure.

The chemogenetic procedure DREADD (designer receptor exclusively activated by designer drugs) is an inventive way to selectively affect g-coupled protein receptors. In theory, DREADD receptors are only activated by administering inert compounds, primarily clozapine N-oxide (CNO). Research has shown that CNO does not cross the blood-brain barrier, and CNO is converted back to clozapine and N-desmethylclozapine (N-Des) in the brain. Clozapine and N-Des have many neurological effects including alterations in glutamate and dopamine (DA) levels in multiple brain regions. The current study examined the effects of peripheral administration of CNO on glutamate and DA levels in the medial prefrontal cortex (mPFC). Wistar rats were administered CNO, and microdialysis samples were collected from the mPFC. Administration of CNO significantly increased glutamate (31-87%) and DA (65-126%), CNO-induced increases in DA occurred for a longer duration than glutamate, and that for the two highest doses of CNO there was a significant correlation between the increase in glutamate and DA in the mPFC. In the mPFC, CNO-induced increases in DA occurred at 0.5 mg/kg, while increases in glutamate were observed at doses greater than 1.0 mg/kg. The source of the DA and glutamate could be caused by activation of projection neurons or local effects. The data replicate findings that CNO is not an inert compound and that interpretation of CNO-activated DREADD findings should be done with caution. The data indicate that low ('safe') doses of CNO still have neurochemical effects and that controlling for the actions of clozapine/N-Des in CNO-DREADD studies has many concerns.

Determination of clozapine and norclozapine in dried plasma spot and dried blood spot by liquid chromatography-tandem mass spectrometry.

The use of alternative blood sampling strategies in clozapine (CLZ) therapeutic drug monitoring (TDM) aims to facilitate collection and improve drug therapy and adherence. This study aimed to develop and validate two methods for the determination CLZ and norclozapine (NOR) in dried blood spots (DBS) and dried plasma spots (DPS) by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The analytes were extracted from one 10 microliter volumetric DBS disc punch and from one 6 mm DPS disc punch with methyl tert-butyl ether: methanol (1:9, v/v) and injected into the HPLC-MS/MS with Atmospheric pressure chemical ionization (APCI) source. Separation was performed in a phenyl column, with mobile phase ammonium formate 1 mM pH 4.0 with methanol in gradient mode. The method was linear from 50 to 1500 ng/ml (r > 0.99), with accuracy between 98% and 105% in DBS and 91-101% in DPS, and intra- and inter-assay CV% from 5.23% to 9.35% in DBS and 2.22-11.36% in DPS for both analytes. The matrix effect was compensated by the internal standard, between - 5.1-6.89% in DBS and - 2.45-5.74% in DPS. The average extraction efficiency was 63-67% for CLZ and 58-69% for NOR with no significant impact of hematocrit (HCT). The analytes were stable in the dried matrices stored up to 42 °C for 26 days. The method was applied in the evaluation of clozapine therapy in 13 schizophrenic patients with mean serum levels of 401 ng/ml (43-914 ng/ml). Only 38% were within the therapeutic range, 46% below and 23% above. CLZ and NOR concentrations in dried samples were highly correlated to serum levels, with greater accuracy for DPS compared to DBS (97 versus 89%, and 99 versus 131%, for CLZ and NOR, respectively). Our data support the use of DBS and DPS as alternative sampling strategies in CLZ therapeutic drug monitoring, with satisfactory performance and logistics advantages.

Body weight gain in clozapine-treated patients: Is norclozapine the culprit?

The antipsychotic drug clozapine is associated with weight gain. The proposed mechanisms include blocking of serotonin (5-HT2a/2c ), dopamine (D2 ) and histamine (H1 ) receptors. Clozapine is metabolized by cytochrome P450 1A2 (CYP1A2) to norclozapine, a metabolite with more 5-HT2c -receptor and less H1 blocking capacity. We hypothesized that norclozapine serum levels correlate with body mass index (BMI), waist circumference and other parameters of the metabolic syndrome. We performed a retrospective cross-sectional study in 39 patients (female n = 8 (20.5%), smokers n = 18 (46.2%), average age 45.8 ± 9.9 years) of a clozapine outpatient clinic in the Netherlands between 1 January 2017 and 1 July 2020. Norclozapine concentrations correlated with waist circumference (r = 0.354, P = .03) and hemoglobin A1c (HbA1c) (r = 0.34, P = .03). In smokers (smoking induces CYP1A2), norclozapine concentrations correlated with waist circumference (r = 0.723, P = .001), HbA1c (r = 0.49, P = .04) and BMI (r = 0.63, P = .004). Elucidating the relationship between norclozapine and adverse effects of clozapine use offers perspectives for interventions and treatment options.

Chemogenetic stimulation of the Gi pathway in astrocytes suppresses neuroinflammation.

Engineered G protein-coupled receptors (GPCRs) are commonly used in chemogenetics as designer receptors exclusively activated by designer drugs (DREADDs). Although several GPCRs have been studied in astrocytes using a chemogenetic approach, the functional role of the astrocytic Gi pathway is not clear, as the literature is conflicting depending on the brain regions or behaviors investigated. In this study, we evaluated the role of the astrocytic Gi pathway in neuroinflammation using a Gi -coupled DREADD (hM4Di). Gi -DREADD was expressed in hippocampal astrocytes of a lipopolysaccharide (LPS)-induced neuroinflammation mouse model using adeno-associated viruses. We found that astrocyte Gi -DREADD stimulation using clozapine N-oxide (CNO) inhibits neuroinflammation, as characterized by decreased levels of proinflammatory cytokines, glial activation, and cognitive impairment in mice. Subsequent experiments using primary astrocyte cultures revealed that Gi -DREADD stimulation significantly downregulated LPS-induced expression of Nos2 mRNA and nitric oxide production. Similarly, in vitro calcium imaging showed that activation of the astrocytic Gi pathway attenuated intracellular calcium transients triggered by LPS treatment, suggesting a positive correlation between enhanced calcium transients and the inflammatory phenotype of astrocytes observed in the inflamed brain. Taken together, our results indicate that the astrocytic Gi pathway plays an inhibitory role in neuroinflammation, providing an opportunity to identify potential cellular and molecular targets to control neuroinflammation.

Longitudinal functional imaging of VIP interneurons reveals sup-population specific effects of stroke that are rescued with chemogenetic therapy.

Stroke profoundly disrupts cortical excitability which impedes recovery, but how it affects the function of specific inhibitory interneurons, or subpopulations therein, is poorly understood. Interneurons expressing vasoactive intestinal peptide (VIP) represent an intriguing stroke target because they can regulate cortical excitability through disinhibition. Here we chemogenetically augmented VIP interneuron excitability in a murine model of photothrombotic stroke and show that it enhances somatosensory responses and improves recovery of paw function. Using longitudinal calcium imaging, we discovered that stroke primarily disrupts the fidelity (fraction of responsive trials) and predictability of sensory responses within a subset of highly active VIP neurons. Partial recovery of responses occurred largely within these active neurons and was not accompanied by the recruitment of minimally active neurons. Importantly, chemogenetic stimulation preserved sensory response fidelity and predictability in highly active neurons. These findings provide a new depth of understanding into how stroke and prospective therapies (chemogenetics), can influence subpopulations of inhibitory interneurons.

Pharmacogenetic excitation of the median raphe region affects social and depressive-like behavior and core body temperature in male mice.

AIMS: Median raphe region (MRR) is an important bottom-up regulatory center for various behaviors as well as vegetative functions, but detailed descriptions and links between the two are still largely unexplored. METHODS: Pharmacogenetics was used to study the role of MRR in social (sociability, social interaction, resident intruder test) and emotional behavior (forced swim test) parallel with some vegetative changes (biotelemetry: core body temperature). Additionally, to validate pharmacogenetics, the effect of clozapine-N-oxide (CNO), the ligand of the artificial receptor, was studied by measuring (i) serum and brainstem concentrations of CNO and clozapine; (ii) MRR stimulation induced neurotransmitter release in hippocampus; (iii) CNO induced changes in body temperature and locomotor activity. KEY FINDINGS: MRR stimulation decreased locomotion, increased friendly social behavior in the resident intruder test and enhanced depressive-like behavior. The latter was accompanied by diminished decrease in core body temperature. Thirty minutes after CNO injection clozapine was predominant in the brainstem. Nonetheless, peripheral CNO injection was able to induce glutamate release in the hippocampus. CNO had no immediate (<30 min) or chronic (repeated injections) effect on the body temperature or locomotion. SIGNIFICANCE: We confirmed the role of MRR in locomotion, social and depressive-like behavior. Most interestingly, only depressive-like behavior was accompanied by changed body temperature regulation, which was also observed in human depressive disorders previously. This indicates clinical relevance of our findings. Despite low penetration, CNO acts centrally, but does not influence the examined basic parameters, being suitable for repeated behavioral testing.

Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition.

Acetylcholine (ACh), released in the hippocampus from fibers originating in the medial septum/diagonal band of Broca (MSDB) complex, is crucial for learning and memory. The CA2 region of the hippocampus has received increasing attention in the context of social memory. However, the contribution of ACh to this process remains unclear. Here, we show that in mice, ACh controls social memory. Specifically, MSDB cholinergic neurons inhibition impairs social novelty discrimination, meaning the propensity of a mouse to interact with a novel rather than a familiar conspecific. This effect is mimicked by a selective antagonist of nicotinic AChRs delivered in CA2. Ex vivo recordings from hippocampal slices provide insight into the underlying mechanism, as activation of nAChRs by nicotine increases the excitatory drive to CA2 principal cells via disinhibition. In line with this observation, optogenetic activation of cholinergic neurons in MSDB increases the firing of CA2 principal cells in vivo. These results point to nAChRs as essential players in social novelty discrimination by controlling inhibition in the CA2 region.