The amygdala is a chemosensor that detects carbon dioxide and acidosis to elicit fear behavior.

The amygdala processes and directs inputs and outputs that are key to fear behavior. However, whether it directly senses fear-evoking stimuli is unknown. Because the amygdala expresses acid-sensing ion channel-1a (ASIC1a), and ASIC1a is required for normal fear responses, we hypothesized that the amygdala might detect a reduced pH. We found that inhaled CO(2) reduced brain pH and evoked fear behavior in mice. Eliminating or inhibiting ASIC1a markedly impaired this activity, and localized ASIC1a expression in the amygdala rescued the CO(2)-induced fear deficit of ASIC1a null animals. Buffering pH attenuated fear behavior, whereas directly reducing pH with amygdala microinjections reproduced the effect of CO(2). These data identify the amygdala as an important chemosensor that detects hypercarbia and acidosis and initiates behavioral responses. They also give a molecular explanation for how rising CO(2) concentrations elicit intense fear and provide a foundation for dissecting the bases of anxiety and panic disorders.

GluN3-Containing NMDA Receptors in the Rat Nucleus Accumbens Core Contribute to Incubation of Cocaine Craving.

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca2+-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg2+ block and Ca2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain.SIGNIFICANCE STATEMENT "Incubation of craving" is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction.

Chemogenetic inhibition reveals midline thalamic nuclei and thalamo-accumbens projections mediate cocaine-seeking in rats.

Drug addiction is a chronic disease that is shaped by alterations in neuronal function within the cortical-basal ganglia-thalamic circuit. However, our understanding of how this circuit regulates drug-seeking remains incomplete, and relapse rates remain high. The midline thalamic nuclei are an integral component of the cortical-basal ganglia-thalamic circuit and are poised to mediate addiction behaviors, including relapse. It is surprising that little research has examined the contribution of midline thalamic nuclei and their efferent projections in relapse. To address this, we expressed inhibitory, Gi/o -coupled DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) in a subset of the midline thalamic nuclei or in midline thalamic nuclei neurons projecting to either the nucleus accumbens or the amygdala. We examined the effect of transiently decreasing activity of these neuronal populations on cue-induced and cocaine-primed reinstatement of cocaine-seeking. Reducing activity of midline thalamic nuclei neurons attenuated both cue-induced and cocaine-primed reinstatement, but had no effect on cue-induced reinstatement of sucrose-seeking or locomotor activity. Interestingly, attenuating activity of efferent projections from the anterior portion of midline thalamic nuclei to the nucleus accumbens blocked cocaine-primed reinstatement but enhanced cue-induced reinstatement. Decreasing activity of efferent projections from either the posterior midline thalamic nuclei to the nucleus accumbens or the midline thalamic nuclei to amygdala had no effect. These results reveal a novel contribution of subsets of midline thalamic nuclei neurons in drug-seeking behaviors and suggest that modulation of midline thalamic nuclei activity may be a promising therapeutic target for preventing relapse.

Dopamine dependency for acquisition and performance of Pavlovian conditioned response.

During Pavlovian conditioning, pairing of a neutral conditioned stimulus (CS) with a reward leads to conditioned reward-approach responses (CRs) that are elicited by presentation of the CS. CR behaviors can be sign tracking, in which animals engage the CS, or goal tracking, in which animals go to the reward location. We investigated CR behaviors in mice with only ∼5% of normal dopamine in the striatum using a Pavlovian conditioning paradigm. These mice had severely impaired acquisition of the CR, which was ameliorated by pharmacological restoration of dopamine synthesis with l-dopa. Surprisingly, after they had learned the CR, its expression decayed only gradually in following sessions that were conducted without l-dopa treatment. To assess specific contributions of dopamine signaling in the dorsal or ventral striatum, we performed virus-mediated restoration of dopamine synthesis in completely dopamine-deficient (DD) mice. Mice with dopamine signaling only in the dorsal striatum did not acquire a CR, whereas mice with dopamine signaling only in in the ventral striatum acquired a CR. The CR in mice with dopamine signaling only in the dorsal striatum was restored by subjecting the mice to instrumental training in which they had to interact with the CS to obtain rewards. We conclude that dopamine is essential for learning and performance of CR behavior that is predominantly goal tracking. Furthermore, although dopamine signaling in the ventral striatum is sufficient to support a CR, dopamine signaling only in the dorsal striatum can also support a CR under certain circumstances.

Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT(1A) receptor-adenylyl cyclase axis.

Targeting serotonin (5-HT) bioavailability with selective 5-HT reuptake inhibitors (SSRIs) remains the most widely used treatment for mood disorders. However, their limited efficacy, delayed onset of action, and side effects restrict their clinical utility. Endogenous regulator of G-protein signaling (RGS) proteins have been implicated as key inhibitors of 5-HT(1A)Rs, whose activation is believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS proteins involved remains unknown. We identify RGS6 as the critical negative regulator of 5-HT(1A)R-dependent antidepressant actions. RGS6 is enriched in hippocampal and cortical neurons, 5-HT(1A)R-expressing cells implicated in mood disorders. RGS6(-/-) mice exhibit spontaneous anxiolytic and antidepressant behavior rapidly and completely reversibly by 5-HT(1A)R blockade. Effects of the SSRI fluvoxamine and 5-HT(1A)R agonist 8-OH-DPAT were also potentiated in RGS6(+/-) mice. The phenotype of RGS6(-/-) mice was associated with decreased CREB phosphorylation in the hippocampus and cortex, implicating enhanced Gα(i)-dependent adenylyl cyclase inhibition as a possible causative factor in the behavior observed in RGS6(-/-) animals. Our results demonstrate that by inhibiting serotonergic innervation of the cortical-limbic neuronal circuit, RGS6 exerts powerful anxiogenic and prodepressant actions. These findings indicate that RGS6 inhibition may represent a viable means to treat mood disorders or enhance the efficacy of serotonergic agents.

Acid-sensing ion channels 1a (ASIC1a) inhibit neuromuscular transmission in female mice.

Acid-sensing ion channels (ASIC) open in response to extracellular acidosis. ASIC1a, a particular subtype of these channels, has been described to have a postsynaptic distribution in the brain, being involved not only in ischemia and epilepsy, but also in fear and psychiatric pathologies. High-frequency stimulation of skeletal motor nerve terminals (MNTs) can induce presynaptic pH changes in combination with an acidification of the synaptic cleft, known to contribute to muscle fatigue. Here, we studied the role of ASIC1a channels on neuromuscular transmission. We combined a behavioral wire hanging test with electrophysiology, pharmacological, and immunofluorescence techniques to compare wild-type and ASIC1a lacking mice (ASIC1a (-/-) knockout). Our results showed that 1) ASIC1a (-/-) female mice were weaker than wild type, presenting shorter times during the wire hanging test; 2) spontaneous neurotransmitter release was reduced by ASIC1a activation, suggesting a presynaptic location of these channels at individual MNTs; 3) ASIC1a-mediated effects were emulated by extracellular local application of acid saline solutions (pH = 6.0; HEPES/MES-based solution); and 4) immunofluorescence techniques revealed the presence of ASIC1a antigens on MNTs. These results suggest that ASIC1a channels might be involved in controlling neuromuscular transmission, muscle contraction and fatigue in female mice.

Retinoic acid-mediated homeostatic plasticity in the nucleus accumbens core contributes to incubation of cocaine craving.

RATIONALE: Incubation of cocaine craving refers to the progressive intensification of cue-induced craving during abstinence from cocaine self-administration. We showed previously that homomeric GluA1 Ca2+-permeable AMPARs (CP-AMPAR) accumulate in excitatory synapses of nucleus accumbens core (NAcc) medium spiny neurons (MSN) after ∼1 month of abstinence and thereafter their activation is required for expression of incubation. Therefore, it is important to understand mechanisms underlying CP-AMPAR plasticity. OBJECTIVES: We hypothesize that CP-AMPAR upregulation represents a retinoic acid (RA)-dependent form of homeostatic plasticity, previously described in other brain regions, in which a reduction in neuronal activity disinhibits RA synthesis, leading to GluA1 translation and CP-AMPAR synaptic insertion. We tested this using viral vectors to bidirectionally manipulate RA signaling in NAcc during abstinence following extended-access cocaine self-administration. RESULTS: We used shRNA targeted to the RA degradative enzyme Cyp26b1 to increase RA signaling. This treatment accelerated incubation; rats expressed incubation on abstinence day (AD) 15, when it is not yet detected in control rats. It also accelerated CP-AMPAR synaptic insertion measured with slice physiology. CP-AMPARs were detected in Cyp26b1 shRNA-expressing MSN, but not control MSN, on AD15-18. Next, we used shRNA targeted to the major RA synthetic enzyme Aldh1a1 to reduce RA signaling. In MSN expressing Aldh1a1 shRNA, synaptic CP-AMPARs were reduced in late withdrawal (AD42-60) compared to controls. However, we did not detect an effect of this manipulation on incubated cocaine seeking (AD40). CONCLUSIONS: These findings support the hypothesis that increased RA signaling during abstinence contributes to CP-AMPAR accumulation and incubation of cocaine craving.

Dopamine transmission at D1 and D2 receptors in the nucleus accumbens contributes to the expression of incubation of cocaine craving.

Relapse represents a consistent clinical problem for individuals with substance use disorder. In the incubation of craving model of persistent craving and relapse, cue-induced drug seeking progressively intensifies or 'incubates' during the first weeks of abstinence from drug self-administration and then remains high for months. Previously, we and others have demonstrated that expression of incubated cocaine craving requires strengthening of excitatory synaptic transmission in the nucleus accumbens core (NAcc). However, despite the importance of dopaminergic signaling in the NAcc for motivated behavior, little is known about the role that dopamine (DA) plays in the incubation of cocaine craving. Here we used fiber photometry to measure DA transients in the NAcc of male and female rats during cue-induced seeking tests conducted in early abstinence from cocaine self-administration, prior to incubation, and late abstinence, after incubation of craving has plateaued. We observed DA transients time-locked to cue-induced responding but their magnitude did not differ significantly when measured during early versus late abstinence seeking tests. Next, we tested for a functional role of these DA transients by injecting DA receptor antagonists into the NAcc just before the cue-induced seeking test. Blockade of either D1 or D2 DA receptors reduced cue-induced cocaine seeking after but not before incubation. We found no main effect of sex in our experiments. These results suggest that DA contributes to incubated cocaine seeking but the emergence of this role reflects changes in postsynaptic responsiveness to DA rather than presynaptic alterations.

Persistent Neuroadaptations in the Nucleus Accumbens Core Accompany Incubation of Methamphetamine Craving in Male and Female Rats.

Relapse is a major problem in treating methamphetamine use disorder. "Incubation of craving" during abstinence is a rat model for persistence of vulnerability to craving and relapse. While methamphetamine incubation has previously been demonstrated in male and female rats, it has not been demonstrated after withdrawal periods greater than 51 d and most mechanistic work used males. Here, we address both gaps. First, although methamphetamine intake was higher in males during self-administration training (6 h/d × 10 d), incubation was similar in males and females, with "incubated" craving persisting through withdrawal day (WD)100. Second, using whole-cell patch-clamp recordings in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) core, we assessed synaptic levels of calcium-permeable AMPA receptors (CP-AMPARs), as their elevation is required for expression of incubation in males. In both sexes, compared with saline-self-administering controls, CP-AMPAR levels were significantly higher in methamphetamine rats across withdrawal, although this was less pronounced in WD100-135 rats than WD15-35 or WD40-75 methamphetamine rats. We also examined membrane properties and NMDA receptor (NMDAR) transmission. In saline controls, MSNs from males exhibited lower excitability than females. This difference was eliminated after incubation because of increased excitability of MSNs from males. NMDAR transmission did not differ between sexes and was not altered after incubation. In conclusion, incubation persists for longer than previously described and equally persistent CP-AMPAR plasticity in NAc core occurs in both sexes. Thus, abstinence-related synaptic plasticity in NAc is similar in males and females although other methamphetamine-related behaviors and neuroadaptations show differences.

Post-acquisition CO2 Inhalation Enhances Fear Memory and Depends on ASIC1A.

A growing body of evidence suggests that memories of fearful events may be altered after initial acquisition or learning. Although much of this work has been done in rodents using Pavlovian fear conditioning, it may have important implications for fear memories in humans such as in post-traumatic stress disorder (PTSD). A recent study suggested that cued fear memories, made labile by memory retrieval, were made additionally labile and thus more vulnerable to subsequent modification when mice inhaled 10% carbon dioxide (CO2) during retrieval. In light of this finding, we hypothesized that 10% CO2 inhalation soon after fear acquisition might affect memory recall 24 h later. We found that both cue and context fear memory were increased by CO2 exposure after fear acquisition. The effect of CO2 was time-dependent, as CO2 inhalation administered 1 or 4 h after cued fear acquisition increased fear memory, whereas CO2 inhalation 4 h before or 24 h after cued fear acquisition did not increase fear memory. The ability of CO2 exposure following acquisition to enhance fear memory was not a general consequence of stress, as restraining mice after acquisition did not alter cued fear memory. The memory-enhancing action of CO2 may be relatively specific to fear conditioning as novel object recognition was impaired by post-training CO2 inhalation. To explore the molecular underpinnings of these effects, we tested if they depended on the acid-sensing ion channel-1a (ASIC1A), a proton-gated cation channel that mediates other effects of CO2, likely via its ability to sense acidosis induced during CO2 inhalation. We found that CO2 inhalation did not alter cued or context fear memory in Asic1a-/- mice, suggesting that this phenomenon critically depends on ASIC1A. These results suggest that brain acidosis around the time of a traumatic event may enhance memory of the trauma, and may thus constitute an important risk factor for developing PTSD. Moreover, preventing peritraumatic acidosis might reduce risk of PTSD.

Acid-sensing ion channel-1a in the amygdala, a novel therapeutic target in depression-related behavior.

No animal models replicate the complexity of human depression. However, a number of behavioral tests in rodents are sensitive to antidepressants and may thus tap important underlying biological factors. Such models may also offer the best opportunity to discover novel treatments. Here, we used several of these models to test the hypothesis that the acid-sensing ion channel-1a (ASIC1a) might be targeted to reduce depression. Genetically disrupting ASIC1a in mice produced antidepressant-like effects in the forced swim test, the tail suspension test, and following unpredictable mild stress. Pharmacologically inhibiting ASIC1a also had antidepressant-like effects in the forced swim test. The effects of ASIC1a disruption in the forced swim test were independent of and additive to those of several commonly used antidepressants. Furthermore, ASIC1a disruption interfered with an important biochemical marker of depression, the ability of stress to reduce BDNF in the hippocampus. Restoring ASIC1a to the amygdala of ASIC1a(-/-) mice with a viral vector reversed the forced swim test effects, suggesting that the amygdala is a key site of ASIC1a action in depression-related behavior. These data are consistent with clinical studies emphasizing the importance of the amygdala in mood regulation, and suggest that ASIC1a antagonists may effectively combat depression.

Restoring Acid-sensing ion channel-1a in the amygdala of knock-out mice rescues fear memory but not unconditioned fear responses.

Acid-sensing ion channel-1a (ASIC1a) contributes to multiple fear behaviors, however the site of ASIC1a action in behavior is not known. To explore a specific location of ASIC1a action, we expressed ASIC1a in the basolateral amygdala of ASIC1a-/- mice using viral vector-mediated gene transfer. This rescued context-dependent fear memory, but not the freezing deficit during training or the unconditioned fear response to predator odor. These data pinpoint the basolateral amygdala as the site where ASIC1a contributes to fear memory. They also discriminate fear memory from fear expressed during training and from unconditioned fear. Furthermore, this work illustrates a strategy for identifying discrete brain regions where specific genes contribute to complex behaviors.

Histologic examination of the eye of acid-sensing ion channel 1a knockout mice.

Various subtypes of the acid sensing ion channel have been detected in the retina of rodents and other mammalian species, but the functional importance of this finding is not clearly understood. The purpose of the study was to determine if retinal degeneration was present in ASIC1a-/- mice. The eyes of ASIC1a-/- mice, heterozygote ASIC1a+/- mice, and wild type ASIC1a+/+ mice that were 5 or 22-27 weeks old were processed by routine histotech-nological methods and examined for histologic changes in the retina and other portions of the eye. Additional sections of eyes from ASIC1a-/- and ASIC1a+/+ mice were labeled with peanut agglutinin (PNA) to evaluate cone pho-toreceptors. The retinas of ASIC1a-/-, ASIC1a+/-, and ASIC1a+/+ mice at 5 or 22-27 weeks of age were unremarkable and no morphologic changes in cone photo receptors were detected. Additional findings detected in the eye of ASIC1a+/+ mice included swelling of lens fibers or cataract that were also detected in some of the ASIC1a-/- or ASIC1a+/- mice. Lenticular findings were not considered to be associated with an absence of ASIC1a.