Opposing effects of NMDA receptor antagonists on early life stress-induced aggression in mice.

Rates of childhood trauma are high amongst violent offenders who frequently recidivate. Few clinical options are available to treat excessive and recurring violent aggression associated with childhood trauma. Those that do exist are largely ineffective and often replete with side effects. One promising pharmacological target is the glutamate binding N-methyl- d-aspartate receptor (NMDAR). Clinically available NMDAR antagonists have proven successful in mitigating violent and aggressive behavior associated with a host of psychiatric diseases and have both immediate and long-term effects on nervous system function and behavior. This study examined the impact of three NMDAR antagonists on long-lasting aggression brought on by early-life stress: MK-801, memantine, and ketamine. We find that social isolation early in adolescence followed by acute traumatic stress in the form of noncontingent foot shock (FS) late in adolescence works in tandem to promote long-lasting excessive aggression in mice when measured 1 week later. Systemic injections of MK-801 and memantine 30 min before FS suppressed the long-lasting attack behavior induced by our early life stress induction protocol. Systemic injections of ketamine, on the other hand, significantly enhanced the long-lasting attack behavior when injected before FS. These findings indicate that MK-801, memantine, and ketamine have distinct and opposing effects on early life stress-induced aggression, suggesting these drugs may be mechanistically distinct. This study identifies memantine as a promising pharmacological treatment for aggressive behavior associated with early life stress and demonstrates the need for greater care when using glutamate receptor antagonists to treat aggression.

Potentiation of Divergent Medial Amygdala Pathways Drives Experience-Dependent Aggression Escalation.

Heightened aggression can be serious concerns for the individual and society at large and are symptoms of many psychiatric illnesses, such as post-traumatic stress disorder. The circuit and synaptic mechanisms underlying experience-induced aggression increase, however, are poorly understood. Here we find that prior attack experience leading to an increase in aggressive behavior, known as aggression priming, activates neurons within the posterior ventral segment of the medial amygdala (MeApv). Optogenetic stimulation of MeApv using a synaptic depression protocol suppresses aggression priming, whereas high-frequency stimulation enhances aggression, mimicking attack experience. Interrogation of the underlying neural circuitry revealed that the MeApv mediates aggression priming via synaptic connections with the ventromedial hypothalamus (VmH) and bed nucleus of the stria terminalis (BNST). These pathways undergo NMDAR-dependent synaptic potentiation after attack. Furthermore, we find that the MeApv-VmH synapses selectively control attack duration, whereas the MeApv-BNST synapses modulate attack frequency, both with no effect on social behavior. Synaptic potentiation of the MeApv-VmH and MeApv-BNST pathways contributes to increased aggression induced by traumatic stress, and weakening synaptic transmission at these synapses blocks the effect of traumatic stress on aggression. These results reveal a circuit and synaptic basis for aggression modulation by experience that can be potentially leveraged toward clinical interventions.SIGNIFICANCE STATEMENT Heightened aggression can have devastating social consequences and may be associated with psychiatric disorders, such as post-traumatic stress disorder. The circuit and synaptic mechanisms underlying experience-induced aggression escalation, however, are poorly understood. Here we identify two aggression pathways between the posterior ventral segment of the medial amygdala and its downstream synaptic partners, the ventromedial hypothalamus and bed nucleus of the stria terminalis that undergo synaptic potentiation after attack and traumatic stress to enhance aggression. Notably, weakening synaptic transmission in these circuits blocks aggression priming, naturally occurring aggression, and traumatic stress-induced aggression increase. These results illustrate a circuit and synaptic basis of aggression modulation by experience, which can be potentially targeted for clinical interventions.

Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors.

α7 nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic transmission and inflammation. In response to ligands, this receptor channel opens to conduct cations into the cell but desensitizes rapidly. In recent studies we show that α7 nAChRs bind signaling proteins such as heterotrimeric GTP-binding proteins (G proteins). Here, we demonstrate that direct coupling of α7 nAChRs to G proteins enables a downstream calcium signaling response that can persist beyond the expected time course of channel activation. This process depends on a G protein-binding cluster (GPBC) in the M3-M4 loop of the receptor. A mutation of the GPBC in the α7 nAChR (α7345-348A) abolishes interaction with Gαq as well as Gßγ while having no effect on receptor synthesis, cell-surface trafficking, or α-bungarotoxin binding. Expression of α7345-348A, however, did significantly attenuate the α7 nAChR-induced Gαq calcium signaling response as evidenced by a decrease in PLC-ß activation and IP3R-mediated calcium store release in the presence of the α7 selective agonist choline. Taken together, the data provides new evidence for the existence of a GPBC in nAChRs serving to promote intracellular signaling.

Microtubule dynamics at the growth cone are mediated by α7 nicotinic receptor activation of a Gαq and IP3 receptor pathway.

The α7 nicotinic receptor (α7) plays an important role in neuronal growth and structural plasticity in the developing brain. We have recently characterized a G-protein-signaling pathway regulated by α7 that directs the growth of neurites in developing neural cells. Now we show that choline activation of α7 promotes a rise in intracellular calcium from local ER stores via Gαq signaling, leading to IP3 receptor (IP3R) activation at the growth cone of differentiating PC12 cells. A mutant α7 significantly attenuated in calcium conductance (D44A; P<0.001) was found to be unable to promote IP3R signaling and calcium store release. In addition, calcium elevation via α7 correlates with a significant attenuation in the rate of microtubule invasion of the growth cone (P<0.001). This process was also attenuated in the D44A mutant and blocked by an inhibitor of the IP3R, suggesting that calcium flow through the α7 channel and activation of the Gαq pathway are necessary for growth. Taken together, the findings reveal an inhibitory mechanism of α7 on cytoskeletal growth via the intracellular calcium activity of the receptor channel and the Gαq signaling pathway at the growth cone.-Nordman, J. C., Kabbani, N. Microtubule dynamics at the growth cone are mediated by α7 nicotinic receptor activation of a Gαq and IP3 receptor pathway.

Are nicotinic acetylcholine receptors coupled to G proteins?

It was, until recently, accepted that the two classes of acetylcholine (ACh) receptors are distinct in an important sense: muscarinic ACh receptors signal via heterotrimeric GTP binding proteins (G proteins), whereas nicotinic ACh receptors (nAChRs) open to allow flux of Na+, Ca2+, and K+ ions into the cell after activation. Here we present evidence of direct coupling between G proteins and nAChRs in neurons. Based on proteomic, biophysical, and functional evidence, we hypothesize that binding to G proteins modulates the activity and signaling of nAChRs in cells. It is important to note that while this hypothesis is new for the nAChR, it is consistent with known interactions between G proteins and structurally related ligand-gated ion channels. Therefore, it underscores an evolutionarily conserved metabotropic mechanism of G protein signaling via nAChR channels.

The α4 nicotinic receptor promotes CD4+ T-cell proliferation and a helper T-cell immune response.

Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell-based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4(+)CD3(+)CD4(+)) and show that this group of cells is responsive to sustained nicotine exposure. In the circulation, spleen, bone marrow, and thymus, we find that nicotine promotes an increase in CD3(+)CD4(+) cells via its activation of the α4 nAChR and regulation of G protein subunit o, G protein regulated-inducer of neurite outgrowth, and CDC42 signaling within T cells. In particular, nicotine is found to promote a helper T cell 2 adaptive immunologic response within T cells that is absent in α4(-/-) mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T cells, possibly accounting for the effect of smoking on the immune system.

Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth.

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein-regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth-associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

An interaction between α7 nicotinic receptors and a G-protein pathway complex regulates neurite growth in neural cells.

The α7 acetylcholine nicotinic receptor (α7) is an important mediator of cholinergic transmission during brain development. Here we present an intracellular signaling mechanism for the α7 receptor. Proteomic analysis of immunoprecipitated α7 subunits reveals an interaction with a G protein pathway complex (GPC) comprising Gα(i/o), GAP-43 and G protein regulated inducer of neurite outgrowth 1 (Gprin1) in differentiating cells. Morphological studies indicate that α7 receptors regulate neurite length and complexity via a Gprin1-dependent mechanism that directs the expression of α7 to the cell surface. α7-GPC interactions were confirmed in embryonic cortical neurons and were found to modulate the growth of axons. Taken together, these findings reveal a novel intracellular pathway of signaling for α7 within neurons, and suggest a role for its interactions with the GPC in brain development.

Acute social defeat during adolescence promotes long-lasting aggression through activation of the medial amygdala.

Traumatic stress, particularly during critical developmental periods such as adolescence, has been strongly linked to an increased propensity and severity of aggression. Existing literature underscores that being a victim of abuse can exacerbate aggressive behaviors, with the amygdala playing a pivotal role in mediating these effects. Historically, animal models have demonstrated that traumatic stressors can increase attack behavior, implicating various amygdala nuclei. Building on this foundation, our previous work has highlighted how traumatic stress invokes long-lasting aggression via an excitatory pathway within the posterior ventral medial amygdala (MeApv). In the current study, we sought to further delineate this mechanism by examining the effects of acute social defeat during adolescence on aggressive behaviors and neural activation in mice. Using a common social defeat paradigm, we first established that acute social defeat during late adolescence indeed promotes long-lasting aggression, measured as attack behavior 7 days after the defeat session. Immunolabeling with c-Fos demonstrated that acute social defeat activates the MeApv and ventrolateral aspect of the ventromedial hypothalamus (VmHvl), consistent with our previous studies that used foot shock as an acute stressor. Finally, chemogenetically inhibiting excitatory MeApv neurons during social defeat significantly mitigated the aggression increase without affecting non-aggressive social behavior. These results strongly suggest that the MeApv plays a critical role in the onset of aggression following traumatic social experience, and offer the MeA as a potential target for therapeutic interventions.

Menthol inhibits 5-HT3 receptor-mediated currents.

The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 µM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 µM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 µM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

Non-angry aggressive arousal and angriffsberietschaft: A narrative review of the phenomenology and physiology of proactive/offensive aggression motivation and escalation in people and other animals.

Human aggression typologies largely correspond with those for other animals. While there may be no non-human equivalent of angry reactive aggression, we propose that human proactive aggression is similar to offense in other animals' dominance contests for territory or social status. Like predation/hunting, but unlike defense, offense and proactive aggression are positively reinforcing, involving dopamine release in accumbens. The drive these motivational states provide must suffice to overcome fear associated with initiating risky fights. We term the neural activity motivating proactive aggression "non-angry aggressive arousal", but use "angriffsberietschaft" for offense motivation in other animals to acknowledge possible differences. Temporal variation in angriffsberietschaft partitions fights into bouts; engendering reduced anti-predator vigilance, redirected aggression and motivational over-ride. Increased aggressive arousal drives threat-to-attack transitions, as in verbal-to-physical escalation and beyond that, into hyper-aggression. Proactive aggression and offense involve related neural activity states. Cingulate, insular and prefrontal cortices energize/modulate aggression through a subcortical core containing subnuclei for each aggression type. These proposals will deepen understanding of aggression across taxa, guiding prevention/intervention for human violence.

A single dose of ketamine enhances early life stress-induced aggression with no effect on fear memory, anxiety-like behavior, or depression-like behavior in mice.

Ketamine is a dissociative anesthetic that has been shown to have antidepressant effects in humans and has been proposed as a potential treatment for mood disorders such as posttraumatic stress disorder and aggression. However, previous studies from our lab and others have demonstrated that ketamine's effects are highly context- and dose-dependent. In a recent study, we found that 10 mg/kg ketamine could exacerbate the effects of early life stress on excessive aggression in mice. To further investigate, the effect of ketamine on moods, such as fear, anxiety, depression, and aggression, we used a mouse model of early life stress, involving chronic social isolation followed by acute traumatic stress in the form of noncontingent, unpredictable foot shock during adolescence. We find this is necessary to induce long-lasting excessive aggression in a novel environment. Seven- to eight-week-old socially isolated mice were given IP injections of 10 mg/kg ketamine 30 min before being subjected to foot shock and then assessed 7 days later for changes in sociability, aggression, mobility, anxiety-like behavior, and depression-like behavior. The results show that ketamine selectively increases long-lasting aggression in mice exposed to foot shock, but does not affect mood-related behaviors or locomotion. These findings suggest that during early life stress, ketamine may exert its effects by specifically targeting aggression brain circuitry that is distinct from brain circuits responsible for nonaggressive social or emotional behaviors. Therefore, while ketamine may be a promising treatment for various mood disorders, caution should be exercised when using ketamine to treat disorders associated with early life stress. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Visualizing traumatic stress-induced structural plasticity in a medial amygdala pathway using mGRASP.

Traumatic stress has been shown to contribute to persistent behavioral changes, yet the underlying neural pathways are not fully explored. Structural plasticity, a form of long-lasting neural adaptability, offers a plausible mechanism. To scrutinize this, we used the mGRASP imaging technique to visualize synaptic modifications in a pathway formed between neurons of the posterior ventral segment of the medial amygdala and ventrolateral segment of the ventromedial hypothalamus (MeApv-VmHvl), areas we previously showed to be involved in stress-induced excessive aggression. We subjected mice (7-8 weeks of age) to acute stress through foot shocks, a reliable and reproducible form of traumatic stress, and compared synaptic changes to control animals. Our data revealed an increase in synapse formation within the MeApv-VmHvl pathway post-stress as evidenced by an increase in mGRASP puncta and area. Chemogenetic inhibition of CaMKIIα-expressing neurons in the MeApv during the stressor led to reduced synapse formation, suggesting that the structural changes were driven by excitatory activity. To elucidate the molecular mechanisms, we administered the NMDAR antagonist MK-801, which effectively blocked the stress-induced synaptic changes. These findings suggest a strong link between traumatic stress and enduring structural changes in an MeApv-VmHvl neural pathway. Furthermore, our data point to NMDAR-dependent mechanisms as key contributors to these synaptic changes. This structural plasticity could offer insights into persistent behavioral consequences of traumatic stress, such as symptoms of PTSD and social deficits.

Anger management: Mechanisms of glutamate receptor-mediated synaptic plasticity underlying animal aggression.

Excessive and recurring violent aggression is a serious concern for society and a symptom of many psychiatric diseases. Substance abuse, attack experience, and social and traumatic stress increase vulnerability to developing this type of aggression. Glutamate receptors are an intriguing target for long-term treatment. This review will assess the importance of glutamate receptors and glutamatergic pathways in aggression, focusing on the role of glutamate receptor-mediated synaptic plasticity in experience-dependent long-lasting aggression. By synthesizing what is known about glutamatergic systems in aggression, it is hoped more effective treatments can be developed.

Promises and Pitfalls of NMDA Receptor Antagonists in Treating Violent Aggression.

Treatment options for chronically aggressive individuals remain limited despite recent medical advances. Traditional pharmacological agents used to treat aggression, such as atypical antipsychotics, have limited efficacy and are often replete with dangerous side effects. The non-competitive NMDAR antagonists ketamine and memantine are promising alternatives, but their effects appear to be highly dependent on dosage, context, and personal experience. Importantly, these drugs can increase aggression when combined with substances of abuse or during periods of heightened stress. This is likely due to mechanistic differences operating at specific synapses under different contexts. Previous findings from our lab and others have shown that early life stress, substance abuse, and attack experience promote aggression through NMDAR-dependent synaptic plasticity within aggression-related brain circuits. Ketamine and memantine affect these types of aggression in opposite ways. This has led us to propose that ketamine and memantine oppositely affect aggression brought on by early life stress, substance abuse, or attack experience through opposite effects on NMDAR-dependent synaptic plasticity. This would account for the persistent effects of these drugs on aggression and suggest they could be leveraged as a more long-lasting treatment option. However, a more thorough examination of the effects of ketamine and memantine on cellular and synaptic function will be necessary for responsible administration. Additionally, because the effects of ketamine and memantine are highly dependent on prior drug use, traumatic stress, or a history of aggressive behavior, we propose a more thorough medical evaluation and psychiatric assessment will be necessary to avoid possible adverse interactions with these drugs.

Effects of Menthol on Nicotine Pharmacokinetic, Pharmacology and Dependence in Mice.

Although menthol, a common flavoring additive to cigarettes, has been found to impact the addictive properties of nicotine cigarettes in smokers little is known about its pharmacological and molecular actions in the brain. Studies were undertaken to examine whether the systemic administration of menthol would modulate nicotine pharmacokinetics, acute pharmacological effects (antinociception and hypothermia) and withdrawal in male ICR mice. In addition, we examined changes in the brain levels of nicotinic receptors of rodents exposed to nicotine and menthol. Administration of i.p. menthol significantly decreased nicotine's clearance (2-fold decrease) and increased its AUC compared to i.p. vehicle treatment. In addition, menthol pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg, s.c.) for periods up to 180 min post-nicotine administration. Repeated administration of menthol with nicotine increased the intensity of mecamylamine-precipitated withdrawal signs in mice exposed chronically to nicotine. The potentiation of withdrawal intensity by menthol was accompanied by a significant increase in nicotine plasma levels in these mice. Western blot analyses of α4 and ß2 nAChR subunit expression suggests that chronic menthol impacts the levels and distribution of these nicotinic subunits in various brain regions. In particular, co-administration of menthol and nicotine appears to promote significant increase in ß2 and α4 nAChR subunit expression in the hippocampus, prefrontal cortex and striatum of mice. Surprisingly, chronic injections of menthol alone to mice caused an upregulation of ß2 and α4 nAChR subunit levels in these brain regions. Because the addition of menthol to tobacco products has been suggested to augment their addictive potential, the current findings reveal several new pharmacological molecular adaptations that may contribute to its unique addictive profile.

Chemical crosslinkers enhance detection of receptor interactomes.

Receptor function is dependent on interaction with various intracellular proteins that ensure the localization and signaling of the receptor. While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor-protein interaction networks (interactomes) in cells, the capture of receptor interactomes and their dynamic properties remains a challenge. In particular, the study of interactome components that bind to the receptor with low affinity or can rapidly dissociate from the macromolecular complex is difficult. Here we describe how chemical crosslinking (CC) can aid in the isolation and proteomic analysis of receptor-protein interactions. The addition of CC to standard affinity purification and mass spectrometry protocols boosts the power of protein capture within the proteomic assay and enables the identification of specific binding partners under various cellular and receptor states. The utility of CC in receptor interactome studies is highlighted for the nicotinic acetylcholine receptor as well as several other receptor types. A better understanding of receptors and their interactions with proteins spearheads molecular biology, informs an integral part of bench medicine which helps in drug development, drug action, and understanding the pathophysiology of disease.

Capture of D2 dopamine receptor signaling complexes in striatal cells for mass spectrometry proteomic analysis.

In recent years advancements in proteomic techniques have contributed to the understanding of protein interaction networks (Interactomes) in various cell types. Today, high throughput proteomics promises to define virtually all of the components of a signaling and a regulatory network within cells for various molecules including membrane-spanning receptors. The D2 dopamine receptor (D2R) is a primary mediator of dopamine transmission in the brain. Signaling through D2Rs has been linked to dopamine-mediated effects on motivation, reward, locomotion and addiction to drugs of abuse. In the striatum, the D2R is a key mediatory of dopamine transmission. Actions on this receptor are an important pharmacological property of various drugs including typical antipsychotics and drugs of abuse. Here we provide an approach for the identification protein interaction networks of the D2R within striatal cells. We discuss key assays and techniques, such as cellular membrane protein fractionation, western blot analysis, magnetic bead coimmunoprecipitation, and liquid chromatography electrospray ionization (LC-ESI) mass spectrometry, that can be used for the isolation and characterization of D2R protein interaction networks. This approach presents a reliable method for the identification and characterization of D2R signaling within cells.

Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+)-dependent Cl(-) channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing Ba(2+). Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125)I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+) transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.