Don't ignore the middle: Distinct mnemonic functions of intermediate hippocampus.

The dorsal region of the hippocampus (dHC) mediates many of the mnemonic functions traditionally associated with the hippocampus proper, such as spatial and episodic memory, whereas ventral hippocampus (vHC) has been extensively implicated in emotional memory and motivational processes. By contrast, the functions of the intermediate hippocampus (iHC) are far less understood. In this study, we aimed to investigate the mnemonic functions of iHC by reversibly inactivating iHC prior to testing memory in behavioral tasks dependent on the integrity of dHC, iHC, or vHC, namely, rapid place water maze, inhibitory avoidance, spontaneous alternation, and temporal ordering of odors. Given our previous findings showing that dHC and vHC are involved in mnemonic control of ingestive behavior, we also assessed the effects of iHC inactivation on sucrose intake. The results showed that pharmacological inhibition of iHC impairs rapid place water maze memory, which has been previously shown to be dependent on iHC but not dHC or vHC. iHC inactivation does not impact memory dependent on dHC (spontaneous alternation), vHC (temporal odor memory), or either dHC or vHC (inhibitory avoidance), and only modestly affects sucrose intake. These findings provide support for the involvement of iHC in mnemonic functions that are distinct from dHC and vHC and highlight the need to further advance our understanding of the functions of this hippocampal region that has been relatively understudied.

Basolateral amygdala corticotropin releasing factor receptor 2 interacts with nonmuscle myosin II to destabilize memory in males.

Preclinical studies show that inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory in male and female adult and adolescent rodents. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g., dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To understand the mechanisms responsible for drug specific selectivity we began by investigating, in male mice, the pharmacokinetic differences in METH and COC brain exposure . Replicating METH's longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, we next assessed transcriptional differences. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotropin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility. Pretreatment with AS2B prevented the ability of Blebb to disrupt an established METH-associated memory. Alternatively, combining CRF2 overexpression and agonist treatment, urocortin 3 (UCN3), in the BLA during conditioning rendered COC-associated memory susceptible to disruption by NMII inhibition, mimicking the Blebb-induced, retrieval-independent memory disruption seen with METH. These results suggest that BLA CRF2 receptor activation during memory formation in male mice can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption by NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Basolateral Amygdala Corticotrophin Releasing Factor Receptor 2 Interacts with Nonmuscle Myosin II to Destabilize Memory.

Inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g. dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To investigate a potential source of this specificity, pharmacokinetic differences in METH and COC brain exposure were examined. Replicating METH's longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, transcriptional differences were next assessed. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotrophin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility after METH conditioning. Pretreatment with AS2B occluded the ability of Blebb to disrupt an established METH-associated memory. Alternatively, the Blebb-induced, retrieval-independent memory disruption seen with METH was mimicked for COC when combined with CRF2 overexpression in the BLA and its ligand, UCN3 during conditioning. These results indicate that BLA CRF2 receptor activation during learning can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption via NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Inhibiting ventral hippocampal NMDA receptors and Arc increases energy intake in male rats.

Research into the neural mechanisms that underlie higher-order cognitive control of eating behavior suggests that ventral hippocampal (vHC) neurons, which are critical for emotional memory, also inhibit energy intake. We showed previously that optogenetically inhibiting vHC glutamatergic neurons during the early postprandial period, when the memory of the meal would be undergoing consolidation, caused rats to eat their next meal sooner and to eat more during that next meal when the neurons were no longer inhibited. The present research determined whether manipulations known to interfere with synaptic plasticity and memory when given pretraining would increase energy intake when given prior to ingestion. Specifically, we tested the effects of blocking vHC glutamatergic N-methyl-D-aspartate receptors (NMDARs) and activity-regulated cytoskeleton-associated protein (Arc) on sucrose ingestion. The results showed that male rats consumed a larger sucrose meal on days when they were given vHC infusions of the NMDAR antagonist APV or Arc antisense oligodeoxynucleotides than on days when they were given control infusions. The rats did not accommodate for that increase by delaying the onset of their next sucrose meal (i.e., decreased satiety ratio) or by eating less during the next meal. These data suggest that vHC NMDARs and Arc limit meal size and inhibit meal initiation.

The role of nonmuscle myosin II in polydrug memories and memory reconsolidation.

Using pharmacologic and genetic approaches targeting actin or the actin-driving molecular motor, nonmuscle myosin II (NMII), we previously discovered an immediate, retrieval-independent, and long-lasting disruption of methamphetamine- (METH-) and amphetamine-associated memories. A single intrabasolateral amygdala complex infusion or systemic administration of the NMII inhibitor Blebbistatin (Blebb) is sufficient to produce this disruption, which is selective, having no retrieval-independent effect on memories for fear, food reward, cocaine, or morphine. However, it was unclear if Blebb treatment would disrupt memories of other stimulants and amphetamine class drugs, such as nicotine (NIC) or mephedrone (MEPH; bath salts). Moreover, many individuals abuse multiple drugs, but it was unknown if Blebb could disrupt polydrug memories, or if the inclusion of another substance would render Blebb no longer able to disrupt METH-associated memories. Therefore, the present study had two primary goals: (1) to determine the ability of Blebb to disrupt NIC- or MEPH-associated memories, and (2) to determine the ability of METH to modify other unconditioned stimulus (US) associations' susceptibility to Blebb. To this end, using the conditional place preference model, mice were conditioned to NIC and MEPH alone or METH in combination with NIC, morphine, or foot shock. We report that, unlike METH, there was no retrieval-independent effect of Blebb on NIC- or MEPH-associated memories. However, similar to cocaine, reconsolidation of the memory for both drugs was disrupted. Further, when combined with METH administration, NIC- and morphine-, but not fear-, associated memories were rendered susceptible to disruption by Blebb. Given the high rate of polydrug use and the resurgence of METH use, these results have important implications for the treatment of substance use disorder.

Memory disrupting effects of nonmuscle myosin II inhibition depend on the class of abused drug and brain region.

Depolymerizing actin in the amygdala through nonmuscle myosin II inhibition (NMIIi) produces a selective, lasting, and retrieval-independent disruption of the storage of methamphetamine-associated memories. Here we report a similar disruption of memories associated with amphetamine, but not cocaine or morphine, by NMIIi. Reconsolidation appeared to be disrupted with cocaine. Unlike in the amygdala, methamphetamine-associated memory storage was not disrupted by NMIIi in the hippocampus, nucleus accumbens, or orbitofrontal cortex. NMIIi in the hippocampus did appear to disrupt reconsolidation. Identification of the unique mechanisms responsible for NMII-mediated, amygdala-dependent disruption of memory storage associated with the amphetamine class may enable induction of retrieval-independent vulnerability to other pathological memories.

Nonmuscle myosin II inhibition disrupts methamphetamine-associated memory in females and adolescents.

Memories associated with drug use can trigger strong motivation for the drug, which increases relapse vulnerability in substance use disorder (SUD). Currently there are no treatments for relapse to abuse of psychostimulants, such as methamphetamine (METH). We previously reported that storage of memories associated with METH, but not those for fear or food reward, and the concomitant spine density increase are disrupted in a retrieval-independent manner by depolymerizing actin in the basolateral amygdala complex (BLC) of adult male rats and mice. Similar results are achieved in males through intra-BLC or systemic inhibition of nonmuscle myosin II (NMII), a molecular motor that directly drives actin polymerization. Given the substantial differences in physiology between genders, we sought to determine if this immediate and selective disruption of METH-associated memory extends to adult females. A single intra-BLC infusion of the NMII inhibitor Blebbistatin (Blebb) produced a long-lasting disruption of context-induced drug seeking for at least 30days in female rats that mirrored our prior results in males. Furthermore, a single systemic injection of Blebb prior to testing disrupted METH-associated memory and the concomitant increase in BLC spine density in females. Importantly, as in males, the same manipulation had no effect on an auditory fear memory or associated BLC spine density. In addition, we established that the NMII-based disruption of METH-associated memory extends to both male and female adolescents. These findings provide further support that small molecular inhibitors of NMII have strong therapeutic potential for the prevention of relapse to METH abuse triggered by associative memories.

Cocaine self-administration induces changes in synaptic transmission and plasticity in ventral hippocampus.

Allowing rats extended access to cocaine self-administration is thought to recapitulate several key aspects of cocaine addiction in humans. Understanding the mechanisms that underlie drug-induced neuroadaptations that persist in the brain after protracted periods of abstinence is crucial towards the goal of developing therapeutic interventions for this disease state. We have employed both whole-cell voltage clamp and extracellular recording technique to assess changes in neurotransmission and long-term potentiation (LTP) in stratum radiatum of the CA1 region using the rat ventral hippocampal slice preparation. Rats allowed to self-administer cocaine daily, including 'long access' (6 hours) sessions, exhibited an increase in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/N-methyl-d-aspartate current ratio and enhanced excitatory transmission following 3-5 weeks of abstinence. Inhibitory transmission was also significantly decreased in long-access animals, and the AMPA/N-methyl-d-aspartate ratio measured in the absence of GABAergic blockers was greatly enhanced. We also observed a significant reduction of LTP magnitude evoked in the long-access cocaine rats. These findings suggest the presence of synergistic effects of enhanced AMPA and diminished gamma-aminobutyric acid neurotransmission under physiological conditions in the CA1 region of cocaine-taking animals, supporting the conclusion that persisting enhancement of AMPA-mediated transmission and/or inhibition of gamma-aminobutyric acid-mediated transmission promoted a chronic state of potentiation that partially occluded further LTP. This increased output from the ventral hippocampus to other limbic areas would be among the drug-induced neuroadaptations that persist following abstinence from cocaine self-administration and therefore may contribute to the disease state of addiction.

Cyclin-Dependent Kinase Inhibitor 1a (p21) Modulates Response to Cocaine and Motivated Behaviors.

This study investigated the functional role of cyclin-dependent kinase inhibitor 1a (Cdkn1a or p21) in cocaine-induced responses using a knockout mouse model. Acute locomotor activity after cocaine administration (15 mg/kg, i.p.) was decreased in p21(-/-) mice, whereas cocaine-induced place preference was enhanced. Interestingly, κ-opioid-induced place aversion was also significantly enhanced. Concentration-dependent analysis of locomotor activity in response to cocaine demonstrated a rightward shift in the p21(-/-) mice. Pretreatment with a 5-hydroxytryptamine receptor antagonist did not alter the enhancement of cocaine-induced conditioned place preference in p21(-/-) mice, indicating a lack of involvement of serotonergic signaling in this response. Cocaine exposure increased p21 expression exclusively in the ventral sector of the hippocampus of rodents after either contingent or noncontingent drug administration. Increased p21 expression was accompanied by increased histone acetylation of the p21 promoter region in rats. Finally, increased neurogenesis in the dorsal hippocampus of p21(-/-) mice was also observed. These results show that functional loss of p21 altered the acute locomotor response to cocaine and the conditioned responses to either rewarding or aversive stimuli. Collectively, these findings demonstrate a previously unreported involvement of p21 in modulating responses to cocaine and in motivated behaviors.

The Actin Cytoskeleton as a Therapeutic Target for the Prevention of Relapse to Methamphetamine Use.

A high rate of relapse is a defining characteristic of substance use disorder for which few treatments are available. Exposure to environmental cues associated with previous drug use can elicit relapse by causing the involuntary retrieval of deeply engrained associative memories that trigger a strong motivation to seek out drugs. Our lab is focused on identifying and disrupting mechanisms that support these powerful consolidated memories, with the goal of developing therapeutics. A particularly promising mechanism is regulation of synaptic dynamics by actin polymerization within dendritic spines. Emerging evidence indicates that memory is supported by structural and functional plasticity dendritic spines, for which actin polymerization is critical, and that prior drug use increases both spine and actin dynamics. Indeed we have found that inhibiting amygdala (AMY) actin polymerization immediately or twenty-four hours prior to testing disrupted methamphetamine (METH)-associated memories, but not food reward or fear memories. Furthermore, METH training increased AMY spine density which was reversed by actin depolymerization treatment. Actin dynamics were also shifted to a more dynamic state by METH training. While promising, actin polymerization inhibitors are not a viable therapeutic, as a multitude of peripheral process (e.g. cardiac function) rely on dynamic actin. For this reason, we have shifted our focus upstream of actin polymerization to nonmuscle myosin II. We and others have demonstrated that myosin IIb imparts a mechanical force that triggers spine actin polymerization in response to synaptic stimulation. Similar to an actin depolymerizing compound, pre-test inhibition of myosin II ATPase activity in the AMY produced a rapid and lasting disruption of drug-seeking behavior. While many questions remain, these findings indicate that myosin II represents a potential therapeutic avenue to target the actin cytoskeleton and disrupt the powerful, extinction-resistant memories capable of triggering relapse.