Identification of novel biomolecular characteristics and bioinformatic analyses of the anterior cingulate cortex in morphine-dependent mice via proteomic profiling.

Astrocyte neuronal metabolic coupling in the anterior cingulate cortex of mice with inflammatory pain.

The supraspinal pathophysiology of the painful neuropathy induced by paclitaxel, a chemotherapeutic agent, is not well understood. The γ-aminobutyric acid (GABA) neurotransmitter system has been implicated in the pathogenesis of neuropathic pain. Gene expression of GABAergic system molecules was examined in the anterior cingulate cortex (ACC) of mice brains, by real-time PCR, during paclitaxel-induced neuropathic pain, because this area is involved in pain perception and modulation that might contribute to neuropathic pain. Paclitaxel treatment resulted in thermal hyperalgesia and in increased GABA transporter-1 (GAT-1) mRNA expression, but not that of other GABA transporters or GABA(A) ergic enzymes in the ACC compared to vehicle treatment. Among the 18 GABA(A) receptor subunits analyzed, only β2, β3, δ, and γ2 had increased mRNA levels, and for the receptor subunit, only GABA(B2) had increased mRNA levels in the ACC of paclitaxel-treated mice, whereas the rest of the GABA receptor subunits were not altered. The mRNA expression of GABAA receptor subunits α6, θ, π, ρ1, ρ2, and ρ3 were not detected in the ACC. In conclusion, these data show that during paclitaxel-induced neuropathic pain there is significant increase in GAT-1 expression in the ACC. GAT-1 is the main transporter of GABA from the synapse, and thus its increased expression possibly results in less GABA at the synapse and dysregulation of the GABAergic system. GAT-1 is a potential therapeutic target for managing paclitaxel-induced neuropathic pain.

Neuropathic pain (NP) is a severe disease caused by a primary disease or lesion affecting the somatosensory nervous system. It is reported that NP is related to the increased activity of glutamatergic pyramidal cells and changed neural oscillations in the anterior cingulate cortex (ACC). Arginine vasopressin (AVP), a neurohypophyseal hormone, has been shown to cause pain-alleviating effects when applied to the peripheral system. However, the extent to which, and the mechanisms by which, AVP induces analgesic effects in the central nervous system remains unclear. In the present study, we observed that intranasal delivery of AVP inhibited mechanical pain, thermal pain, and spontaneous pain sensitivity in mice with spared nerve injury. Meanwhile, AVP application exclusively reduced the FOS expression in the pyramidal cells but not interneurons in the ACC. In vivo electrophysiological recording of the ACC further showed that AVP application not only inhibited the theta oscillation in local field potential analysis but also reduced the firing rate of spikes of pyramidal cells in the ACC in neuropathic pain mice. In summary, AVP induces analgesic effects by inhibiting neural theta oscillations and the spiking of pyramidal cells of the ACC in mice with neuropathic pain, which should provide new potential noninvasive methods for clinical treatment of chronic pain.NEW & NOTEWORTHY Following intranasal administration of arginine vasopressin (AVP), the pain thresholds for mechanical and thermal nociception significantly increased in the spared nerve injury (SNI) group; exogenous intranasal delivery of AVP improved the physical coordination of SNI mice, resulting in an analgesic effect; AVP treatment significantly reduced the increased firing rate of PYRACC of the SNI group; AVP treatment significantly inhibited the elevated theta oscillation in the anterior cingulate cortex (ACC) in SNI mice.

The anterior cingulate cortex (ACC) is a critical region of the brain for the emotional and affective components of pain in rodents and humans. Hyperactivity in this region has been observed in neuropathic pain states in both patients and animal models and ablation of this region from cingulotomy, or inhibition with genetics or pharmacology can diminish pain and anxiety. Two adenylyl cyclases (AC), AC1 and AC8 play an important role in regulating nociception and anxiety-like behaviors through an action in the ACC, as genetic and pharmacological targeting of these enzymes reduces mechanical hypersensitivity and anxiety-like behavior, respectively. However, the distribution of these ACs in the ACC has not been studied in the context of neuropathic pain. To address this gap in knowledge, we conducted RNAscope in situ hybridization to assess AC1 and AC8 mRNA distribution in mice with spared nerve injury (SNI). Given the key role of AC1 in nociception in neuropathic, inflammatory and visceral pain animal models, we hypothesized that AC1 would be upregulated in the ACC of mice following nerve injury. This hypothesis was also founded on data showing increased AC1 expression in the ACC of mice with zymosan-induced visceral inflammation. We found that AC1 and AC8 are widely expressed in many regions of the mouse brain including the hippocampus, ACC, medial prefrontal cortex and midbrain regions, but AC1 is more highly expressed. Contrary to our hypothesis, SNI causes an increase in AC8 mRNA expression in NMDAR-2B (Nr2b) positive neurons in the contralateral ACC but does not affect AC1 mRNA expression. Our findings show that changes in Adcy1 mRNA expression in the ACC are insufficient to explain the important role of this AC in mechanical hypersensitivity in mice following nerve injury and suggest a potential unappreciated role of AC8 in regulation of ACC synaptic changes after nerve injury.

Pregnancy-induced analgesia develops in late pregnancy, but its mechanisms are unclear. The anterior cingulate cortex (ACC) plays a key role in the pathogenesis of neuropathic pain. The authors hypothesized that pregnancy-induced analgesia ameliorates neuropathic pain by suppressing activation of microglia and the expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and by upregulating opioid receptors in the ACC in late-pregnant mice. Neuropathic pain was induced in non-pregnant (NP) or pregnant (P) C57BL/6JJmsSlc female mice by partial sciatic nerve ligation (PSNL). The nociceptive response was evaluated by mechanical allodynia and activation of microglia in the ACC was evaluated by immunohistochemistry. The expressions of phosphorylated AMPA receptors and opioid receptors in the ACC were evaluated by immunoblotting. In von Frey reflex tests, NP-PSNL-treated mice showed a lower 50% paw-withdrawal threshold than NP-Naïve mice on experimental day 9. No difference in 50% paw-withdrawal threshold was found among the NP-Naïve, NP-Sham, P-Sham, and P-PSNL-treated mice. The number of microglia in the ACC was significantly increased in NP-PSNL-treated mice compared to NP-Sham mice. Immunoblotting showed significantly increased expression of phosphorylated AMPA receptor subunit GluR1 at Ser831 in NP-PSNL-treated mice compared to NP-Sham mice. Immunoblotting also showed significantly increased δ-opioid receptor in the ACC in P-Sham and P-PSNL-treated mice compared to NP-Sham mice. Pregnancy-induced analgesia ameliorated neuropathic pain by suppressing activation of microglia and the expression of phosphorylated AMPA receptor subunit GluR1 at Ser831, and by upregulation of the δ-opioid receptor in the ACC in late-pregnant mice.

Anterior cingulate cortex (ACC) is a prefrontal area implicated in functions including cognitive control, attention, and prediction. Mouse ACC receives input from the visual system and uses visual information to direct behavior. While extensive work has described experience-dependent plasticity in mouse V1, less is known about how ACC itself adapts to visual experience. Our previous work demonstrated that visual sequences, presented across days, can drive plasticity in the timing of visually evoked responses in mouse ACC. However, it is not known whether this plasticity (“sequence plasticity”) reflects familiarity to the first stimulus in a sequence or expectation of subsequent stimuli—a distinction that is critically important for understanding its functional significance. We recorded visually evoked responses in awake, head-fixed female and male mice trained with visual sequences across days. Visual sequences drove plasticity in ACC, expressed through a change in response timing, that reflects familiarity to the first stimulus. In addition, experience-dependent plasticity could be induced using single-orientation stimuli. Together, these findings suggest that “sequence plasticity” in ACC does not in fact require sequences, but rather reflects a broader phenomenon that we term stimulus-specific response plasticity in timing (SRPT). Our prior work demonstrated that ACC plasticity is impaired in a mouse model of Angelman syndrome (AS). Here, AS model mice showed abnormal responses to familiar visual stimuli in ACC, despite normal plasticity in V1. Together, this work demonstrates how mouse ACC adapts to familiar visual stimuli and describes impaired ACC function in a mouse model of a neurodevelopmental disorder.

Neuropathic pain has a complex pathogenesis. Here, we examined the role of caveolin-1 (Cav-1) in the anterior cingulate cortex (ACC) in a chronic constriction injury (CCI) mouse model for the enhancement of presynaptic glutamate release in chronic neuropathic pain. Cav-1 was localized in glutamatergic neurons and showed higher expression in the ACC of CCI versus sham mice. Moreover, the release of glutamate from the ACC of the CCI mice was greater than that of the sham mice. Inhibition of Cav-1 by siRNAs greatly reduced the release of glutamate of ACC, while its overexpression (induced by injecting Lenti-Cav-1) reversed this process. The chemogenetics method was then used to activate or inhibit glutamatergic neurons in the ACC area. After 21 days of injection of AAV-hM3Dq in the sham mice, the release of glutamate was increased, the paw withdrawal latency was shortened, and expression of Cav-1 in the ACC was upregulated after intraperitoneal injection of 2 mg/kg clozapine N-oxide. Injection of AAV-hM4Di in the ACC of CCI mice led to the opposite effects. Furthermore, decreasing Cav-1 in the ACC in sham mice injected with rAAV-hM3DGq did not increase glutamate release. These findings suggest that Cav-1 in the ACC is essential for enhancing glutamate release in neuropathic pain.

Insomnia, depression, and anxiety disorder are common problems for people with neuropathic pain. In this study, mild noxious heat stimuli increased the duration and number of spontaneous pain-like behaviors in sciatic nerve-ligated mice. We used functional magnetic resonance imaging to visualize the increased blood oxygenation level-dependent signal intensity in the anterior cingulate cortex (ACC) of mice with sciatic nerve ligation under mild noxious stimuli. Such stimuli significantly increased the release of glutamate in the ACC of nerve-ligated mice. In addition, sciatic nerve ligation and mild noxious stimuli changed the morphology of astrocytes in the ACC. Treatment of cortical astrocytes with glutamate caused astrocytic activation, as detected by a stellate morphology. Furthermore, glutamate induced the translocation of GAT-3 to astrocyte cell membranes using primary cultured glial cells from the mouse cortex. Moreover, the GABA level at the synaptic cleft in the ACC of nerve-ligated mice was significantly decreased exposure to mild noxious stimuli. Finally, we investigated whether astrocytic activation in the ACC could directly mediate sleep disorder. With the optogenetic tool channel rhodopsin-2 (ChR2), we demonstrated that selective photostimulation of these astrocytes in vivo triggered sleep disturbance. Taken together, these results suggest that neuropathic pain-like stimuli activated astrocytes in the ACC and decreased the extracellular concentration of GABA via an increase in the release of glutamate. Furthermore, these findings provide novel evidence that astrocytic activation in the ACC can mimic sleep disturbance in mice.

The anterior cingulate cortex (ACC) is a key cortical region that plays an important role in pain perception and emotional functions. Previous studies of the ACC projections have been collected primarily from monkeys, rabbits and rats. Due to technological advances, such as gene manipulation, recent progress has been made in our understanding of the molecular and cellular mechanisms of the ACC-related chronic pain and emotion is mainly obtained from adult mice. Few anatomic studies have examined the whole-brain projections of the ACC in adult mice. In the present study, we examined the continuous axonal outputs of the ACC in the whole brain of adult male mice. We used the virus anterograde tracing technique and an ultrahigh-speed imaging method of Volumetric Imaging with Synchronized on-the-fly-scan and Readout (VISoR). We created a three-dimensional (3D) reconstruction of mouse brains. We found that the ACC projected ipsilaterally primarily to the caudate putamen (CPu), ventral thalamic nucleus, zona incerta (ZI), periaqueductal gray (PAG), superior colliculus (SC), interpolar spinal trigeminal nucleus (Sp5I), and dorsal medullary reticular nucleus (MdD). The ACC also projected to contralateral brain regions, including the ACC, reuniens thalamic nucleus (Re), PAG, Sp5I, and MdD. Our results provide a whole-brain mapping of efferent projections from the ACC in adult male mice, and these findings are critical for future studies of the molecular and synaptic mechanisms of the ACC and its related network in mouse models of brain diseases.

Anterior cingulate cortex (ACC) is known to play important roles in key brain functions such as pain perception, cognition, and emotion. Different forms of homosynaptic plasticity such as long-term potentiation (LTP) and long-term depression have been studied in ACC synapses. However, heterosynaptic plasticity such as synaptic tagging has not been reported. Here, we demonstrate synaptic tagging in the ACC of adult male mice by using a 64-channel multielectrode array recording system. Weak theta burst stimulation (TBS), normally inducing early-phase LTP or No-LTP in most of the activated channels, produced late phase-LTP (L-LTP) in a majority of channels when a strong TBS was applied earlier to a separate input within a certain time window. Similar to hippocampus, synaptic tagging in the ACC depends on the synthesis of new proteins. Tail amputation-induced peripheral injury caused a loss of this heterosynaptic L-LTP and occluded strong TBS-evoked L-LTP as well. Together, we provide the first report of the synaptic tagging-like phenomenon in the ACC of adult mice, and the loss of synaptic tagging to amputation may contribute to injury-related cognitive changes and phantom limb sensation and pain.SIGNIFICANCE STATEMENT ACC is an important cortical region involved in many brain functions. Previous studies have dissected the molecular mechanism of multiple types of homosynaptic plasticity of ACC synapses. Here, we report a novel form of heterosynaptic plasticity occurring in the ACC. This newly identified, protein synthesis-dependent neocortical synaptic tagging is sensitive to peripheral tail amputation injury and may provide basic mechanisms for synaptic pathophysiology of phantom pain and related cognitive changes.

Long-term potentiation (LTP) is an important molecular mechanism for chronic pain in the anterior cingulate cortex (ACC), a key cortical region for pain perception and emotional regulation. Inhibiting ACC LTP via various manipulations or pharmacological treatments blocks chronic pain. Long-term depression (LTD) is another form of synaptic plasticity in the ACC, which is also proved to be involved in the mechanisms of chronic pain. However, less is known about the interactive relationship between LTP and LTD in the ACC. Whether the synaptic depression could be induced after synaptic LTP in the ACC is not clear. In the present study, we used multi-channel field potential recording systems to study synaptic depression after LTP in the ACC of adult mice. We found that low frequency stimulus (LFS: 1 Hz, 15 min) inhibited theta burst stimulation (TBS)-induced LTP at 30 min after the induction of LTP. However, LFS failed to induce depression at 90 min after the induction of LTP. Furthermore, NMDA receptor antagonist AP-5 blocked the induction of synaptic depression after potentiation. The GluN2B-selective antagonist Ro25-6981 also inhibited the phenomenon in the ACC, while the GluN2A-selective antagonist NVP-AAM077 and the GluN2C/D-selective antagonist PPDA and UBP145 had no any significant effect. These results suggest that synaptic LTP can be depressed by LTD in a time dependent manner, and GluN2B-containing NMDA receptors play important roles in this form of synaptic depression.

Role of microglia in mechanical allodynia in the anterior cingulate cortex

Gentiopicroside (Gent) is one of the secoiridoid compound isolated from Gentiana lutea. This compound exhibits analgesic activities and inhibits the expression of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors in the anterior cingulate cortex in mice. Nucleus accumbens (NAc) is a forebrain structure known for its role in drug addiction. However, little is known about the role of Gent on morphine dependence and synaptic transmission changes in the NAc. Conditioned place preference (CPP) test and behavioral sensitization of locomotor activity were used to investigate drug-seeking related behaviors. Brain slices containing NAc were prepared, and whole-cell patch-clamp recordings were performed to record the excitatory postsynaptic currents (EPSCs). Expression of proteins was detected by Western blot analysis. Systemic administration of Gent attenuated the CPP effect induced by morphine, but had no effect on morphine-induced behavioral sensitization. Gent significantly reversed overexpression of GluN2B-containing NMDA receptors and dopamine D2 receptors in NAc during the first week of morphine withdrawal. However, the compound did not affect the overexpression of GluN2A-containing NMDA receptors, GluA1, and dopamine D1 receptors. Lastly, Gent significantly reduced NMDA receptors-mediated EPSCs in the NAc. Our study provides strong evidence that Gent inhibits morphine dependence through downregulation of GluN2B-containing NMDA receptors in the NAc.

Observational fear (OF) is the ability to vicariously experience and learn from another’s fearful situation, enabling adaptive responses crucial for survival. It has been shown that the anterior cingulate cortex (ACC) and basolateral amygdala (BLA) are crucial for OF. A subset of neurons in the ACC is activated when observing aversive events in the demonstrator, which elicits OF. However, the neural circuit mechanisms underlying the expression of OF-related activity in the ACC remain unexplored. Previous studies have shown that the mediodorsal thalamus (MD) is crucial for OF, and MD neurons project to the ACC. Therefore, we hypothesize that the projection from MD to ACC may facilitate the OF-related activity in the ACC. By utilizing in vivo calcium imaging combined with the optogenetic terminal inhibition of MD-ACC pathway, we found that a subset of ACC neurons was activated when observing demonstrator’s fearful situation in male mice. Furthermore, the optogenetic inhibition of the MD-ACC projection during the demonstrator’s aversive moments significantly suppressed the OF-related activity in the ACC. Our data suggests that the MD-ACC projection plays a role in OF-related activity in ACC neurons.

Lower urinary tract (LUT) function is controlled by the central nervous system, including higher-order cognitive brain regions. The anterior cingulate cortex (ACC) is one of these regions, but the role of its activity in LUT function remains poorly understood. In the present study, we conducted optogenetic experiments to manipulate neural activity in mouse ACC while monitoring bladder pressure to elucidate how the activity of ACC regulates LUT function. Selective optogenetic stimulation of excitatory neurons in ACC induced a sharp increase in bladder pressure, whereas activation of inhibitory neurons in ACC prolonged the interval between bladder contractions. Pharmacological manipulation of ACC also altered bladder contractions, consistent with those observed in optogenetic experiments. Optogenetic mapping of the cortical area responsible for eliciting the increase in bladder pressure revealed that stimulation to ACC showed more potent effects than the neighboring motor cortical areas. These results suggest that ACC plays a crucial role in initiating the bladder pressure change and the micturition reflex. Thus, the balance between excitation and inhibition in ACC may regulate the reflex bidirectionally.