RGS4 Actions in Mouse Prefrontal Cortex Modulate Behavioral and Transcriptomic Responses to Chronic Stress and Ketamine.

The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.

Microglial Engulfment of Spines in the Ventral Zona Incerta Regulates Anxiety-Like Behaviors in a Mouse Model of Acute Pain.

Although activation of microglial cells is critical in developing brain disorders, their role in anxiety-like behaviors in pain is still vague. This study indicates that alteration of microglia's neuronal spine engulfment capacity in ventral zona incerta (ZI V ) leads to significant pain and anxiety-like behaviors in mice 1-day post-injection of Complete Freud's Adjuvant (CFA1D). Performing whole-cell patch-clamp recordings in GABAergic neurons in the ZI V (ZI V GABA ) in brain slices, we observed decreased activity in ZIv GABA and reduced frequency of the miniature excitatory postsynaptic currents (mEPSCs) in ZI V GABA of CFA1D mice compared with the saline1D mice. Besides, chemogenetic activation of ZI V GABA significantly relieved pain and anxiety-like behaviors in CFA1D mice. Conversely, in naïve mice, chemogenetic inhibition of ZI V GABA induced pain and anxiety-like behaviors. Interestingly, we found changes in the density and morphology of ZI V Microglia and increased microglial engulfment of spines in ZI V of CFA1D mice. Furthermore, pain sensitization and anxiety-like behaviors were reversed when the ZI V Microglia of CFA1D-treated mice were chemically inhibited by intra-ZI V minocycline injection, accompanied by the recovery of decreased ZI V GABA excitability. Conclusively, our results provide novel insights that dysregulation of microglial engulfment capacity encodes maladaptation of ZI V GABA , thus promoting the development of anxiety-like behaviors in acute pain.

Early-life inflammation promotes depressive symptoms in adolescence via microglial engulfment of dendritic spines.

Early-life inflammation increases the risk for depression in later life. Here, we demonstrate how early-life inflammation causes adolescent depressive-like symptoms: by altering the long-term neuronal spine engulfment capacity of microglia. For mice exposed to lipopolysaccharide (LPS)-induced inflammation via the Toll-like receptor 4/NF-κB signaling pathway at postnatal day (P) 14, ongoing longitudinal imaging of the living brain revealed that later stress (delivered during adolescence on P45) increases the extent of microglial engulfment around anterior cingulate cortex (ACC) glutamatergic neuronal (ACCGlu) spines. When the ACC microglia of LPS-treated mice were deleted or chemically inhibited, the mice did not exhibit depressive-like behaviors during adolescence. Moreover, we show that the fractalkine receptor CX3CR1 mediates stress-induced engulfment of ACCGlu neuronal spines. Together, our findings establish that early-life inflammation causes dysregulation of microglial engulfment capacity, which encodes long-lasting maladaptation of ACCGlu neurons to stress, thus promoting development of depression-like symptoms during adolescence.

Distinct thalamocortical circuits underlie allodynia induced by tissue injury and by depression-like states.

In humans, tissue injury and depression can both cause pain hypersensitivity, but whether this involves distinct circuits remains unknown. Here, we identify two discrete glutamatergic neuronal circuits in male mice: a projection from the posterior thalamic nucleus (POGlu) to primary somatosensory cortex glutamatergic neurons (S1Glu) mediates allodynia from tissue injury, whereas a pathway from the parafascicular thalamic nucleus (PFGlu) to anterior cingulate cortex GABA-containing neurons to glutamatergic neurons (ACCGABA→Glu) mediates allodynia associated with a depression-like state. In vivo calcium imaging and multi-tetrode electrophysiological recordings reveal that POGlu and PFGlu populations undergo different adaptations in the two conditions. Artificial manipulation of each circuit affects allodynia resulting from either tissue injury or depression-like states, but not both. Our study demonstrates that the distinct thalamocortical circuits POGlu→S1Glu and PFGlu→ACCGABA→Glu subserve allodynia associated with tissue injury and depression-like states, respectively, thus providing insights into the circuit basis of pathological pain resulting from different etiologies.

A Central Amygdala-Ventrolateral Periaqueductal Gray Matter Pathway for Pain in a Mouse Model of Depression-like Behavior.

BACKGROUND: The mechanisms underlying depression-associated pain remain poorly understood. Using a mouse model of depression, the authors hypothesized that the central amygdala-periaqueductal gray circuitry is involved in pathologic nociception associated with depressive states. METHODS: The authors used chronic restraint stress to create a mouse model of nociception with depressive-like behaviors. They then used retrograde tracing strategies to dissect the pathway from the central nucleus of the amygdala to the ventrolateral periaqueductal gray. The authors performed optogenetic and chemogenetic experiments to manipulate the activity of this pathway to explore its roles for nociception. RESULTS: The authors found that γ-aminobutyric acid-mediated (GABAergic) neurons from the central amygdala project onto GABAergic neurons of the ventrolateral periaqueductal gray, which, in turn, locally innervate their adjacent glutamatergic neurons. After chronic restraint stress, male mice displayed reliable nociception (control, mean ± SD: 0.34 ± 0.11 g, n = 7 mice; chronic restraint stress, 0.18 ± 0.11 g, n = 9 mice, P = 0.011). Comparable nociception phenotypes were observed in female mice. After chronic restraint stress, increased circuit activity was generated by disinhibition of glutamatergic neurons of the ventrolateral periaqueductal gray by local GABAergic interneurons via receiving enhanced central amygdala GABAergic inputs. Inhibition of this circuit increased nociception in chronic restraint stress mice (median [25th, 75th percentiles]: 0.16 [0.16, 0.16] g to 0.07 [0.04, 0.16] g, n = 7 mice per group, P < 0.001). In contrast, activation of this pathway reduced nociception (mean ± SD: 0.16 ± 0.08 g to 0.34 ± 0.13 g, n = 7 mice per group, P < 0.001). CONCLUSIONS: These findings indicate that the central amygdala-ventrolateral periaqueductal gray pathway may mediate some aspects of pain symptoms under depression conditions.

A Central Amygdala Input to the Parafascicular Nucleus Controls Comorbid Pain in Depression.

While comorbid pain in depression (CP) occurs at a high rate worldwide, the neural connections underlying the core symptoms of CP have yet to be elucidated. Here, we define a pathway whereby GABAergic neurons from the central nucleus of the amygdala (GABACeA) project to glutamatergic neurons in the parafascicular nucleus (GluPF). These GluPF neurons relay directly to neurons in the second somatosensory cortex (S2), a well-known area involved in pain signal processing. Enhanced inhibition of the GABACeA→GluPF→S2 pathway is found in mice exhibiting CP symptoms. Reversing this pathway using chemogenetic or optogenetic approaches alleviates CP symptoms. Together, the current study demonstrates the putative importance of the GABACeA→GluPF→S2 pathway in controlling at least some aspects of CP.

Direct auditory cortical input to the lateral periaqueductal gray controls sound-driven defensive behavior.

Threatening sounds can elicit a series of defensive behavioral reactions in animals for survival, but the underlying neural substrates are not fully understood. Here, we demonstrate a previously unexplored neural pathway in mice that projects directly from the auditory cortex (ACx) to the lateral periaqueductal gray (lPAG) and controls noise-evoked defensive behaviors. Electrophysiological recordings showed that the lPAG could be excited by a loud noise that induced an escape-like behavior. Trans-synaptic viral tracing showed that a great number of glutamatergic neurons, rather than GABAergic neurons, in the lPAG were directly innervated by those in layer V of the ACx. Activation of this pathway by optogenetic manipulations produced a behavior in mice that mimicked the noise-evoked escape, whereas inhibition of the pathway reduced this behavior. Therefore, our newly identified descending pathway is a novel neural substrate for noise-evoked escape and is involved in controlling the threat-related behavior.

Involvement of orexinergic receptors in the nucleus accumbens, in the effect of forced swim stress on the reinstatement of morphine seeking behaviors.

Orexinergic system is involved in primary rewards; the neural circuit of the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex and amygdala represents overlapping elements mediating the rewarding effects of drugs and stressful experiences. The NAc integrates reward-related information from the VTA. Also, it has been indicated that orexinergic system activates the mesolimbic dopamine projecting neurons to the NAc and promotes the development of reward in rodents. Therefore, in the present study, the conditioned place preference (CPP) paradigm was used to determine the role of the two types of orexin receptors (OXR) in the NAc in forced swim stress (FSS), as physical stress, and/or priming-induced reinstatement of morphine. The CPP was induced by injecting morphine (5 mg/kg, SC for 3 days) and lasted for eight free-morphine days; the reinstatement was induced by administration of effective priming dose of morphine (1 mg/kg; sc). The extinguished rats received intra-NAc injection of SB334867 as OX1R antagonist or TCSOX229 as OX2R antagonist before effective priming dose injection of morphine (1 mg/kg; sc). In others, the extinguished rats were given intra-NAc injection of SB334867 or TCSOX229 and then, they underwent FSS before injection of ineffective priming dose of morphine (0.5 mg/kg; sc). Our results showed that intra-accumbal administration of SB334867 or TCSOX229 could inhibit morphine priming- and FSS-induced reinstatement of extinguished morphine-seeking in the rats. It seems that OXR in the NAc may be involved in reward and could play an important role in the effect of stress on reinstatement of morphine-seeking behaviors in this area.

Role of Orexin-1 Receptor Within the Ventral Tegmental Area in Mediating Stress- and Morphine Priming-induced Reinstatement of Conditioned Place Preference in Rats.

INTRODUCTION: Orexin-containing neurons exist in the lateral hypothalamic region, sending their projections toward mesolimbic regions such as the Ventral Tegmental Area (VTA). METHODS: In the current study, a Reinstatement model is used to examine the effects of intra-VTA administration of SB334867 as an Orexin-1 Receptor (OX1R) antagonist on drug priming- and Forced Swim Stress (FSS)-induced reinstatement of morphine. Eighty-eight male adult albino Wistar rats, weighing 200-280 g, were bilaterally implanted by cannulas into the VTA. We induced the Conditioned Place Preference (CPP) by Subcutaneous (SC) injection of morphine (5 mg/kg) daily in three days. Then, the CPP score was calculated. After a 24-h "off" period following achievement of extinction criterion, the rats were tested for drug priming-induced reinstatement by a priming dose of morphine (1 mg/kg, SC) and for FSS-induced reinstatement 10 min after FSS. In the next experiments, the animals received different doses of intra-VTA administration of SB334867 (0.3, 3, and 1 nM/0.3 µL 12% DMSO per side) and bilaterally were subsequently tested for FSS- and morphine priming-induced reinstatement. RESULTS: Our findings indicated that the FSS could induce the reinstatement of seeking behaviors. Furthermore, intra-VTA administration of OX1R antagonists suppressed FSS- and drug priming-induced reinstatement dose-dependently. CONCLUSION: It is concluded that FSS and drug priming-induced reinstatement might be mediated, at least in part, by stimulation of orexin receptors in the VTA.

Antagonism of the D1- and D2-like dopamine receptors in the nucleus accumbens attenuates forced swim stress- and morphine priming-induced reinstatement of extinguished rats.

Dopaminergic pathways from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) play a critical role in reward-related phenomena as well as in the reinstatement of drug-seeking behavior. Stress is a major trigger for inducing reinstatement, however, the interaction between stress and the dopaminergic system is not well known. The present study was undertaken to investigate the effect of D1- and D2-like dopamine receptors within the NAc in forced swim stress (FSS)- and priming-induced reinstatement of morphine-seeking behaviors. The conditioned place preference (CPP) was induced by injecting morphine (5 mg/kg, SC for 3 days) and lasted for eight days after cessation of the morphine treatment. The FSS (6 min) and effective priming dose of morphine (1 mg/kg, sc) reinstated the extinguished morphine-induced CPP. In order to investigate the effect of intra-accumbal injection of SCH23390 as a D1-like receptor antagonist, or Sulpiride as a D2-like receptor antagonist on the FSS-induced reinstatement of morphine extinguished rats, animals received bilaterally intra-NAc injection of SCH23390 or Sulpiride (0.25, 1 and 4 µg/side) before application of FSS, and then, they were tested in the reinstatement day. Our results showed that the intra-accumbal administration of D1- and D2-like receptors antagonists dose-dependently blocked the effect of FSS on the reinstatement and significantly modulated morphine priming-induced reinstatement as well. These findings suggested that the D1- and D2-like dopamine receptors in the NAc involve in morphine-seeking behaviors and antagonism of these receptors can reduce the effect of stress on rewarding properties of morphine.

Reversible inactivation of the lateral hypothalamus reversed high reward choices in cost-benefit decision-making in rats.

The Lateral hypothalamus (LH) is an important component of the networks underlying the control of feeding and other motivated behaviors. Cost-benefit decision-making is mediated largely by the prefrontal cortex (PFC) which strongly innervates the LH. Therefore, in the current study, we conducted a series of experiments to elucidate the role of the perifornical area of the lateral hypothalamus (PeF-LH) in effort and/or delay-based decision-making. We trained different groups of rats in a delay-based and/or an effort-based form of cost-benefit T-maze decision- making task in which they could either choose to pay the cost to obtain a high reward in one arm or could obtain a low reward in the other arm with no cost. During test days, the rats received local injections of either vehicle or lidocaine4% (0.5 µl/side), in the PeF-LH. In an effort-based decision task, PeF-LH inactivation led to decrease in high reward choice. Similarly, in a delay-based decision task animals' preference changed to a low but immediately available reward. This was not caused by a spatial memory or motor deficit. PeF-LH inactivation modified decision behavior. The results imply that PeF-LH is important for allowing the animal to pay a cost to acquire greater rewards.

Role of intra-accumbal orexin receptors in the acquisition of morphine-induced conditioned place preference in the rats.

Orexin receptor shave essential role in the induction of reward-related behaviors to several drugs of abuse. In the present study, we investigated the effects of bilateral administration of SB334867, as an orexin-1 receptor antagonist, and TCS OX2 29, as an orexin-2 receptor antagonist, into the nucleus accumbens (NAc) on the acquisition of morphine-induced conditioned place preference (CPP) in the rats. Adult male Wistar rats (n=80; 220-250g) were entered in a CPP paradigm. Bilateral microinjections of different doses of SB334867 (1, 3, 10 and 30nM) or TCS OX2 29 (3, 10, 30 and 100nM) into the NAc (0.5µl/side) were done 5min before subcutaneous injection of morphine (5mg/kg) during 3-dayconditioning (acquisition) phase. The CPP scores and locomotor activity of animals were recorded by video tracking system and Ethovision software. The results demonstrated that intra-NAc microinjection of 3, 10 and 30nM solutions of SB334867 markedly decreased the acquisition of morphine-induced CPP in a dose-dependent manner. Intra-accumbal injection of 10, 30 and 100nM solutions of TCS OX2 29 significantly attenuated the acquisition of morphine CPP as well. In addition, contribution of orexin-1 receptors to development of morphine reward-related behaviors was more than orexin-2 receptors. Our results suggest that both orexin-1 and -2 receptors in the NAc are involved in the development of morphine-induced CPP. It seems that orexin-1 receptors in this region are more effective in development of drug seeking behaviors in the rats.