Bilateral Cannulae Research Articles

S-3,4-DCPG, a potent orthosteric agonist for the mGlu8 receptor, facilitates extinction and inhibits the reinstatement of morphine-induced conditioned place preference in male rats

The limbic system, particularly the NAc, shows a high concentration of metabotropic glutamate receptors (mGluRs). Recent evidence suggests the significant involvement of mGluRs in mental disorders, including substance abuse and addiction. The objective of this study was to examine the involvement of mGlu8 receptors in the NAc in the mechanisms underlying the extinction and reinstatement of conditioned place preference (CPP) induced by morphine. Male Wistar rats underwent surgical implantation of bilateral cannulas in the NAc and were assessed in a CPP protocol. In study 1 at the same time as the extinction phase, the rats were given varying doses of S-3,4-DCPG (0.03, 0.3, and 3 μg/0.5 μl). In study 2, rats that had undergone CPP extinction were given S-3,4-DCPG (0.03, 0.3, and 3 μg/0.5 μl) five minutes prior to receiving a subthreshold dose of morphine (1 mg/kg) in order to reactivate the previously extinguished morphine response. The findings demonstrated that administering S-3,4-DCPG directly into the accumbens nucleus resulted in a decrease in the duration of the CPP extinction phase. Moreover, dose-dependent administration of S-3,4-DCPG into the NAc inhibited CPP reinstatement. The observations imply that microinjection of S-3,4-DCPG as a potent orthosteric agonist with high selectivity for the mGlu8 receptor into the NAc promotes the process of extinction while concurrently exerting inhibitory effects on the reinstatement of morphine-induced CPP. This effect may be associated with the modulation of glutamate engagement within the NAc and the plasticity of reward pathways at the synaptic level.

Region-specific targeting of microglia in vivo using direct delivery of tamoxifen metabolites via microfluidic polymer fibers

Region-specific genetic manipulation of glial cells remains challenging due to the lack of anatomically selective transgenic models. Although local transduction is achievable with viral vectors, uniform recombination can be challenging in larger brain regions. We investigated the efficacy of intraparenchymal delivery of the tamoxifen metabolite endoxifen using inducible cre reporter mice. After observing localized reporter induction following stereotaxic injections of endoxifen in CX3CR1creERT2 mice, we carried out chronic delivery via osmotic pumps attached to bilateral cannulas made of stainless steel or microfluidic polymer fibers. Analysis of reporter expression in sections or iDISCO-cleared brains from TMEM119creERT2 mice revealed widespread induction following chronic infusion. Neuronal damage and gliosis were more prevalent around steel cannulas than polymer fibers, and glial reactivity was further attenuated when devices were implanted two months before drug delivery. In summary, region-specific recombination is achievable in glia with minimal tissue damage after endoxifen delivery via microfluidic polymer implants.

Evidence for the existence of facilitatory interactions between the dopamine D2 receptor and the oxytocin receptor in the amygdala of the rat. Relevance for anxiolytic actions.

Introduction: The amygdala is a limbic region of high value for understanding anxiety and its treatment. Dopamine D2 receptors (D2Rs) and oxytocin receptors (OXTRs) have both been shown to participate in modulating anxiety involving effects in the amygdala. The goal is to understand if D2R-OXTR heterocomplexes exist in the central amygdala and if, through enhancing allosteric receptor-receptor interactions, may enhance anxiolytic actions. Methods: The methods used involve the shock-probe burying test, the in situ proximity ligation assay (PLA), image acquisition and analysis, and the BRET2 assay. Bilateral cannulas were introduced into the amygdala, and the effects of the coadministration of oxytocin and the D2R-like agonist quinpirole into the amygdala were studied. Results: The combination treatment enhanced the anxiolytic effects compared to the single treatment. The D2R/D3R antagonist raclopride blocked the effects of the combination treatment of oxytocin and the D2R agonist, although oxytocin is regarded as a distinct modulator of fear-mediating anxiolytic effects. In situ PLA results indicate the existence of D2R-OXTR heteroreceptor complexes and/or the co-location of OXTR and D2R within the same cell membrane nanodomains in the central amygdala. With BRET2, evidence is given for the existence of D2R-OXTR heteromers in HEK293 cells upon co-transfection. Discussion: The enhanced behavioral effects observed upon co-treatment with OXTR and D2R agonists may reflect the existence of improved positive receptor-receptor interactions in the putative D2R-OXTR heterocomplexes in certain neuronal populations of the basolateral and central amygdala. The D2R-OXTR heterocomplex, especially upon agonist co-activation in the central amygdala, may open a new pharmacological venue for the treatment of anxiety.

Corticosterone injection into the basolateral amygdala before and after memory reactivation impairs the subsequent expression of fear memory in rats: An interaction of glucocorticoids and β-adrenoceptors

AbstractGlucocorticoid administration, before or after fear memory reactivation, impairs subsequent fear memory expression, but the underlying mechanisms are not well understood. The present study examined the role of basolateral amygdala (BLA) β-adrenoceptors in the effects of intra-BLA corticosterone injection on fear memory in rats. Bilateral cannulae were implanted in the BLA of Wistar male rats. The rats were trained and tested using an inhibitory avoidance task (1 mA footshock for 3 s). Forty-eight hours after training, corticosterone (CORT, 5, 10, or 20 ng/0.5 µl/side) and the β2-adrenoceptor agonist clenbuterol (CLEN, 10 or 20 ng/0.5 µl/side) or the β-adrenoceptor antagonist propranolol (PROP, 250 or 500 ng/0.5 µl/side) were injected into the BLA before or right after memory reactivation (retrieval, Test 1). We performed subsequent tests 2 (Test 2), 5 (Test 3), 7 (Test 4), and 9 (Test 5) days after Test 1. The results demonstrated that CORT injection before Test 1 disrupted memory retrieval and reduced fear expression in Tests 2–5, possibly due to enhanced extinction or impaired reconsolidation. CORT injection after Test 1 also impaired reconsolidation and reduced fear expression in Tests 2–5. CLEN prevented, but PROP exacerbated, the effects of CORT on fear expression. The reminder shock did not recover fear memory in CORT-treated animals, suggesting that reconsolidation, not extinction, was affected. These results indicate that glucocorticoids and β-adrenoceptors in the BLA jointly modulate fear memory reconsolidation and expression. Comprehending the neurobiology of stress and the impact of glucocorticoids on fear memory may lead to new treatments for stress and trauma-induced disorders such as PTSD.

Rapid eye movements associated with REM sleep is involved in consolidation of visuospatial learning in rats

Rapid eye movement (REM) sleep plays a significant role in visuospatial learning and memory consolidation; however, its mechanism of action is unknown. Rapid eye movements (REMs), a characteristic active feature of REM sleep, is a potential correlate of neural processing for visual memory consolidation. The superior colliculus (SC) plays a central role in oculomotor control and spatial localization of objects in the visual field. We proposed that local reversible inactivation of the SC during post-learning sessions might interfere with REMs and negatively impact REM sleep associated consolidation of the visuospatial learnt task. Under gaseous anesthesia, bilateral cannulae aiming SC and electrodes for recording electrophysiological signals to classify sleep-waking were implanted. Following standard protocol, all rats were subjected to Morris water maze (MWM) training for 5 consecutive days followed by probe trial. After MWM training, on all except the probe test days, the rat SC were bilaterally infused with either vehicle (control, Group 1), Lidocaine hydrochloride a local anesthetic (Lox 2%, Group 2), or muscimol (Mus, GABA agonist, Group 3) and sleep-wakefulness recorded after day 1, 4, and post-probe learning sessions. Post-learning, compared to vehicle, Mus treated group significantly decreased REMs, phasic REM sleep, percent time spent in REM sleep and REM sleep frequency/hr. Also, during probe test, the escape latency was significantly increased, and the percentage time spent in the platform quadrant were significantly decreased in both, Mus and Lox 2% treated rats, while the number of platform location crossings was decreased in Mus treated group. The results showed that Lox 2% and Mus into SC reduced consolidation of visuospatial learning. The findings support our contention that SC mediated activation of REMs exerts a positive influence in processing and consolidation of visual learning during REM sleep. The findings explain the role of REMs during REM sleep in visual memory consolidation.

Sequential order spatial memory in male rats: Characteristics and impact of medial prefrontal cortex and hippocampus disruption

Remembering an experience entails linking what happened, where the event transpired, and when it occurred. Most rodent hippocampal studies involve tests of spatial memory, but fewer investigate temporal and sequential order memory. Here we provide a demonstration of rats learning an aversive sequential order task using a radial arm water maze. Male rats learned a fixed sequence of up to seven spatial locations, with each decision session separated by a temporal delay. Rats relied on visuospatial cues and the number of times they had entered the maze for a given day in order to successfully perform the task. Behavioral patterns during asymptotic performance showed similarities to the serial-position effect, especially with regards to faster first choice latency. Rats at asymptotic performance were implanted with bilateral cannula in medial prefrontal cortex, dorsal, and ventral hippocampus. After re-training, we injected muscimol to temporarily disrupt targeted brain regions. While control rats made prospective errors, rats with mPFC muscimol exhibited more retrospective errors. Rats with hippocampal muscimol no longer exhibited a prospective bias and were at chance levels in their error choices. Taken together, our results suggest disruption of mPFC, but not the hippocampus, produced an error choice bias during an aversive sequential order spatial processing task.

GPR55 activation prevents amphetamine-induced conditioned place preference and decrease the amphetamine-stimulated inflammatory response in the ventral hippocampus in male rats

Inflammatory response in the Central Nervous System (CNS) induced by psychostimulants seems to be a crucial factor in the development and maintenance of drug addiction. The ventral hippocampus (vHp) is part of the reward system involved in substance addiction and expresses abundant G protein-coupled receptor 55 (GPR55). This receptor modulates the inflammatory response in vitro and in vivo, but there is no information regarding its anti-inflammatory effects and its impact on psychostimulant consumption. The aim of the present study was to investigate whether vHp GPR55 activation prevents both the inflammatory response induced by amphetamine (AMPH) in the vHp and the AMPH-induced conditioned place preference (A-CPP). Wistar adult male rats with a bilateral cannula into the vHp or intact males were subjected to A-CPP (5 mg/kg). Upon the completion of A-CPP, the vHp was dissected to evaluate IL-1β and IL-6 expression through RT-PCR, Western blot and immunofluorescence. Our results reveal that AMPH induces both A-CPP and an increase of IL-1β and IL-6 in the vHp. The GPR55 agonist lysophosphatidylinositol (LPI, 10 μM) infused into the vHp prevented A-CPP and the AMPH-induced IL-1β increase. CID 16020046 (CID, 10 μM), a selective GPR55 antagonist, abolished LPI effects. To evaluate the effect of the inflammatory response, lipopolysaccharide (LPS, 5 μg/μl) was infused bilaterally into the vHp during A-CPP acquisition. LPS strengthened A-CPP and increased IL-1β/IL-6 mRNA and protein levels in the vHp. LPS also increased CD68, Iba1, GFAP and vimentin expression. All LPS-induced effects were blocked by LPI. Our results suggest that GPR55 activation in the vHp prevents A-CPP while decreasing the local neuro-inflammatory response. These findings indicate that vHp GPR55 is a crucial factor in preventing the rewarding effects of AMPH due to its capacity to interfere with proinflammatory responses in the vHp.

Necessity and recruitment of cue-specific neuronal ensembles within the basolateral amygdala during appetitive reversal learning

Through Pavlovian appetitive conditioning, environmental cues can become predictors of food availability. Over time, however, the food, and thus the value of the associated cues, can change based on environmental variations. This change in outcome necessitates updating of the value of the cue to appropriately alter behavioral responses to these cues. The basolateral amygdala (BLA) is critical in updating the outcomes of learned cues. However, it is unknown if the same BLA neuronal ensembles that are recruited in the initial associative memory are required when the new cue-outcome association is formed during reversal learning. The current study used the Daun02 inactivation method that enables selective targeting and disruption of activated neuronal ensembles in Fos-lacZ transgenic rats. Rats were implanted with bilateral cannulas that target the BLA and underwent appetitive discriminative conditioning in which rats had to discriminate between two auditory stimuli. One stimulus (CS+) co-terminated with food delivery, and the other stimulus was unrewarded (CS−; counterbalanced). Rats were then tested for CS+ or CS− memory retrieval and infused with either Daun02 or a vehicle solution into the BLA to inactivate either CS+ or CS− neuronal ensembles that were activated during that test. To assess if the same neuronal ensembles are necessary to update the value of the new association when the outcomes are changed, rats underwent reversal learning: the CS+ was no longer followed by food (reversal CS−, rCS−), and the CS− was now followed by food (reversal CS+; rCS+). The group that received Daun02 following CS+ session showed a decrease in conditioned responding and increased latency to the rCS− (previously CS+) during the first session of reversal learning, specifically during the first trial. This indicates that the neuronal ensemble that was activated during the recall of the CS+ memory was the same neuronal ensemble needed for learning the new outcome of the same CS, now rCS−. Additionally, the group that received Daun02 following CS− session was slower to respond to the rCS+ (previously CS−) during reversal learning. This indicates that the neuronal ensemble that was activated during the recall of the CS− memory was the same neuronal ensemble needed for learning the new outcome of the same CS. These results demonstrate that different neuronal ensembles within the BLA mediate memory recall of CS+ and CS− cues and reactivation of each cue-specific neuronal ensemble is necessary to update the value of that specific cue to respond appropriately during reversal learning. These results also indicate substantial plasticity within the BLA for behavioral flexibility as both groups eventually showed similar terminal levels of reversal learning.

Kisspeptin signaling and nNOS neurons in the VMHvl modulate lordosis behavior but not mate preference in female mice

It was recently shown that kisspeptin neurons in the anteroventral periventricular area (AVPV) orchestrate female sexual behavior, including lordosis behavior and mate preference. A potential target of AVPV kisspeptin signaling could be neurons expressing the neuronal form of nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Therefore, in the present study, we further refined the role of the VHMvl in female sexual behavior. Adult female mice received a bilateral cannula aimed at the VMHvl. A single injection with kisspeptin (Kp-10) or SNAP/BAY, a nitric oxide donor, significantly increased lordosis, whereas the nNOS inhibitor l-NAME decreased it. None of these drugs affected mate preference. Interestingly, administration of GnRH into the VMHvl had no effect on lordosis or mate preference. To determine whether the stimulatory effect of Kp-10 on lordosis was specific to the VMHvl, an additional group of females received Kp-10 directly into the paraventricular nucleus (PVN). No effect was found on lordosis and mate preference. These results suggest that kisspeptin most likely modulates lordosis behavior through nNOS neurons in the VMHvl whereas mate preference is modulated by kisspeptin through a separate neuronal circuit not including the VMHvl.

Local administration of bicuculline into the ventrolateral and medial preoptic nuclei modifies sleep and maternal behavior in lactating rats.

The preoptic area (POA) is a brain structure classically involved in a wide variety of animal behavior including sleep and maternal care. In the current study, we evaluate the specific effect of disinhibition of two specific regions of the POA, the medial POA nucleus (mPOA) and the ventrolateral POA area (VLPO) on sleep and maternal behavior in lactating rats. For this purpose, mother rats on postpartum day 1 (PPD1) were implanted for polysomnographic recordings and with bilateral cannulae either in the mPOA or in the VLPO. The rats were tested for sleep and maternal behavior on PPD4–8 after the infusion of the GABA-A antagonist, bicuculline (0, 10 or 30 ng/0.2 µl/side). Infusion of bicuculline into the mPOA augmented retrieving and nest building behaviors and reduced both nursing and milk ejections but had almost no effect on sleep. When bicuculine was microinjected into the VLPO, the rats significantly increase the number of retrievings and mouthings and reduced the nursing time without changes in milk ejections, which was associated with an increase in wakefulness and a reduction in light sleep.Our results show that disinhibition of the mPOA, a key area in the control of maternal behavior, increased active maternal behaviors and reduced nursing without affecting wakefulness or sleep time. In contrast, the enhancement of some active maternal behaviors when the drug was infused into the VLPO, a sleep-promoting area, with a concomitant increase in wakefulness suggests that mother rats devote this additional waking time in the active maternal care of the pups. We hypothesize that maternal behavior changes after bicuculine microinjection into the VLPO are caused by a reduction in the sleep drive, rather than a direct effect on maternal behavior.

Characterizing the Effect of Clodronate in the Locus Coeruleus During Social Stress

Social stress is a leading cause of psychiatric disorders such as depression and anxiety. Studies have linked social stress-evoked neuroinflammation to the development of psychiatric disorders, in which elevated pro-inflammatory cytokines are observed in both plasma and brain tissue of individuals diagnosed with depression. Our goal was to study how microglia, the resident immune cells of the brain, respond to social stress within the stress sensitive locus coeruleus (LC), by measuring the inflammatory-regulating Chemokine Ligand 1 (CX3CL1), High Mobility Group Box 1 (HMGB1), and microglia-specific ionized calcium-binding adapter molecule 1 (IBA1). This study identified a method capable of inducing brain region specific reduction of microglial cells in the LC by administering mannosylated liposomal clodronate (Encapsula NanoSciences). We determined that 25 μg of clodronate (CLD) administered directly into the LC via the mannosylated lipid-based nanoparticle was selectively toxic to microglia of the LC. Pilot studies also determined that 25 ug of the mannosylated liposomal CLD in the central amygdala reduced the population of microglia in that region by approximately 50%. With this knowledge we sought to determine the effects of CLD within the LC at different timepoints during repeated social stress using a witness stress (WS) model in which females were forced daily to witness social defeat between two males. The two experiments conducted in this study included female rats with a bilateral cannula implanted into the LC. In the first study, rats were administered either Veh (empty liposome) or CLD (25ug/side), three days later all rats were exposed to stress on two consecutive days and tissue and blood collected 1hr post stress. In the second experiment, rats were divided into four stress/treatment groups: Con+Veh, Con+CLD, WS+Veh, and WS+CLD. 3 days post treatment, rats were subjected to a handled control condition or WS (5 days, 15 min/day). 5 days after the final stress/control, rats were sacrificed and tissue from the LC was collected and homogenized to conduct immune assessments of protein levels within this region. Western blotting assays were performedto assess levels for CX3CL1, HMGB1, and IBA1. Our findings from study 1 indicate that upon the second exposure to stress, CX3CL1 levels were not different between vehicle and CLD groups, indicating that neuronal silencing of microglia had not been recruited or inhibited. As would be expected, HMGB1 expression was reduced in the CLD treated group compared with vehicle, suggesting that neuroinflammatory priming was delayed by the administration of CLD. Moreover, in study 2, chronic social stress produced an observable increase in CX3CL1 and a decrease in HMGB1 levels in the LC of WS female rats, regardless of treatment. Moreover, by 5 days after the last stress/control there was an observed increase in Iba1 levels, indicating repopulation of the microglia. These data suggest after long-term stress there is a potential delayed repopulation of microglia within the LC, accompanied with a primed inhibitory response from neurons within this region that potentially prevent an exacerbated stress response from these new cells.

Glucagon-like peptide-1 receptors modulate the binge-like feeding induced by µ-opioid receptor stimulation of the nucleus accumbens in the rat.

Neuropeptides and peptide hormones affect food-directed motivation, in part, through actions on brain regions associated with reward processing. For instance, previous reports have shown that stimulating glucagon-like peptide-1 (GLP-1) receptors in the nucleus accumbens (NAc), an area that directs motivational processes towards food and drugs of abuse, has an anorectic effect. In contrast, µ-opioid receptor activation of the NAc increases feeding, particularly on highly palatable diets. While both neurotransmitters act within the NAc to impact food intake, it is not clear if and how they might interact to affect feeding. Therefore, these experiments tested the effects of NAc injections of the GLP-1 receptor agonist Exendin 4 (EX4) or antagonist Exendin 9 (EX9) on the consumption of a sweetened fat diet, with and without simultaneous µ-opioid receptor stimulation. Male Sprague-Dawley rats (n = 8/group, EX4 or EX9) underwent surgery to place bilateral cannula above the NAc core. After recovery, animals were tested following NAc injections of saline or the µ-opioid agonist [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO) (0.025 µg/side), combined with varying doses of EX4 (0, 0.05, or 0.10 µg/side) or EX9 (0, 2.5, 5.0 µg/side), counterbalanced across 6 testing days. Food and water intake, along with locomotor activity, was monitored for 2 h. Mu-opioid receptor stimulation significantly increased feeding, and this effect was reduced by GLP-1 receptor stimulation. In contrast, GLP-1 antagonism with EX9 altered the dynamics of DAMGO-induced binge-like feeding, extending µ-opioid-induced binging, and increasing food consumption. These findings are the first to demonstrate an interaction between NAc µ-opioid and GLP-1 receptors on palatable food intake.

Implantation of Osmotic Pumps and Induction of Stress to Establish a Symptomatic, Pharmacological Mouse Model for DYT/PARK-ATP1A3 Dystonia.

Genetically modified mouse models face limitations, especially when studying movement disorders, where most of the available transgenic rodent models do not present a motor phenotype resembling the clinical aspects of the human disease. Pharmacological mouse models allow for a more direct study of the pathomechanisms and their effect on the behavioral phenotype. Osmotic pumps connected to brain cannulas open up the possibility of creating pharmacological mouse models via local and chronic drug delivery. For the hereditary movement disorder of rapid-onset dystonia-parkinsonism, the loss-of-function mutation in the α3-subunit of the Na+/K+-ATPase can be simulated by a highly specific blockade via the glycoside ouabain. In order to locally block the α3-subunit in the basal ganglia and the cerebellum, which are the two brain structures believed to be heavily involved in the pathogenesis of rapid-onset dystonia-parkinsonism, a bilateral cannula is stereotaxically implanted into the striatum and an additional single cannula is introduced into the cerebellum. The cannulas are connected via vinyl tubing to two osmotic pumps, which are subcutaneously implanted on the back of the animals and allow for the chronic and precise delivery of ouabain. The pharmacological mouse model for rapid-onset dystonia-parkinsonism carries the additional advantage of recapitulating the clinical and pathological features of asymptomatic and symptomatic mutation carriers. Just like mutation carriers of rapid-onset dystonia parkinsonism, the ouabain-perfused mice develop dystonia-like movements only after additional exposure to stress. We demonstrate a mild stress paradigm and introduce two modified scoring systems for the assessment of a motor phenotype.

Differential effects of GABAA receptor activation in the prelimbic and orbitofrontal cortices on anxiety.

The development of effective anxiety treatments has been hindered by limited understanding of the neurobiological mechanisms involved in anxiety regulation. Whilst gamma-aminobutyric acid (GABA) neurotransmission in the prefrontal cortex (PFC) is one mechanism consistently implicated in anxiety regulation, PFC subregions may contribute uniquely. The present study examined the effects of inactivating the PFC subregions of the prelimbic cortex (PrL) or orbitofrontal cortex (OFC) through GABAA receptor (GABAAR) activation, on anxiety behaviours in male Wistar rats. Sixty-six male Wistar rats were surgically implanted with bilateral cannulae into the PrL (n = 33) or the OFC (n = 33). Rats then received a microinjection of either the GABAA receptor agonist muscimol or vehicle prior to each experiment, conducted 1week apart. Measures of anxiety were examined using the elevated plus maze (EPM) and the emergence test (ET). The effect on locomotor activity (baseline or methamphetamine-induced) was also tested. Differential effects of brain region inactivation on anxiety-like behaviour were shown by measures in the EPM and ET; muscimol infused into the PrL-reduced anxiety-like behaviour, yet had no significant effect when infused into the OFC, compared with control treated rats. No effects on locomotor activity at baseline or following methamphetamine treatment were found. This study highlights that activation of GABAARs specifically within the PrL, but not OFC, reduces anxiety behaviours in male rats. This suggests that activity of the PrL plays a more important role than the OFC in the neurobiological mechanisms of unconditioned anxiety and should be targeted for future therapies.

The nucleus accumbens core and shell sub‐regions differentially regulate conditioned fear expression and extinction

The nucleus accumbens (NAcc), consisting of core (NAcC) and shell (NAcS) sub‐regions, has primarily been studied as a locus mediating the effects of drug reward and addiction. This is due to the region’s role of ascribing valence and salience to internal and external cues. However, there is emerging evidence that this region is also involved in regulating aversive responses, but its exact role is not yet clear. Further, the two sub‐regions differ in their connectivity, neurochemical and physiological properties, and each sub‐regions specific involvement in fear processing has not been determined. In our studies, we sought to elucidate the differential role of the two sub‐regions on cued fear expression and extinction. Using adult male C57BL/6 mice, we stereotaxically implanted bilateral cannulae directed at either the NAcC or NAcS. Twenty‐four hours following fear training to an auditory tone, we infused lidocaine, a sodium channel blocker, into the targeted subregion to temporarily inactivate the subregion 5 mins prior to a fear expression test which also served as extinction training. Within the NAcS, fear expression was not influenced by lidocaine infusions, however, when the mice were tested 24 h later for extinction retention, mice that had their shell subregion inactivated exhibited enhanced fear, indicating that this subregion is important for fear extinction consolidation. Mice that had their NAcC inactivated showed an attenuated fear response during expression testing, suggesting that the core sub‐region is necessary for the expression of fear. This effect was not due to alterations in the ability to produce freezing behavior. In addition, there was no effect on extinction consolidation. We next examined whether activation of ERK signaling, downstream of mGluR1, in the nucleus accumbens shell is necessary for extinction consolidation. Thus, we infused the MEK inhibitor, U0126, prior to extinction training and observed a facilitation of extinction consolidation during the retention test. However, with pre‐extinction infusions we also observed within‐session changes in fear behavior during extinction training. To control for this we conducted post‐extinction infusions. Here, mice that were infused with the MEK inhibitor exhibited enhanced freezing during the retention test suggesting poor extinction consolidation and that this pathway is necessary for appropriate extinction consolidation. These findings suggest that the NAc may be considered a critical region involved in regulating both fear expression and its extinction. It’s dual role in appetitive and aversive regulation likely underlies the high co‐morbidity between anxiety disorders and substance use disorders.Support or Funding InformationThis work was supported by funds from grant NIMH1R15MH118705‐01 to Dr. Aaron M Jasnow.

Possible interaction between the ventral hippocampal cannabinoid CB2 and muscarinic acetylcholine receptors on the modulation of memory consolidation in mice.

To clarify possible interaction between the ventral hippocampal cannabinoid CB2 receptors and the cholinergic system in control of the memory process, the effects of cannabinoid and acetylcholine receptor agents on memory consolidation have been investigated in mice. Animals implanted with bilateral cannulas at the CA3 region of the ventral hippocampus and microinjected with scopolamine and cannabinergic agents. These animals were tested using a one-trial step-down inhibitory avoidance task. The results indicated impairment of memory consolidation by posttraining intra-CA3 microinjection of scopolamine (1 and 2 µg/mouse). Nevertheless, coinjection of various doses of scopolamine (0.01, 1 and 2 µg/mouse) with an ineffective dose of AM630 (1 µg/mouse) or GP1a (1 µg/mouse) did not show any significant effect on deficiency of memory consolidation produced by scopolamine. Posttraining application of cannabinoid CB2 receptor antagonist, AM630 (1, 10 and 100 µg/mouse; intra-CA3) alone had no significant influence on memory performance, but its coinjection with significant dose of scopolamine (1 µg/mouse) decreased memory consolidation. Moreover, posttraining injection of GP1a, cannabinoid CB2 receptor agonist, (10 and 100 µg/mouse; intra-CA3) decreased memory consolidation. Posttraining coadministration of diverse doses of GP1a (1, 10 and 100 µg/mouse; intra-CA3) with an effective dose of scopolamine (1 µg/mouse) meaningfully increased deficiency of memory consolidation produced by GP1a (100 µg/mouse). In addition, all drugs had no significant effect on locomotion. Consequently, these results propose that a probable interaction between the CA3 cannabinoid CB2 receptors and muscarinic acetylcholine receptors (mAChR) modulates memory consolidation process in mice.

Cross state-dependent memory retrieval between morphine and norharmane in the mouse dorsal hippocampus

State-dependent memory (SDM) describes a phenomenon that memory is efficiently restored only when the brain state during restoration matches the state during encoding. Some psychoactive drugs such as morphine, ethanol, and cocaine evoke SDM. The scope of this study was to investigate the cross SDM between morphine and norharmane injected into the dorsal hippocampus of male NMRI mice, and the involvement of μ-opioid receptors (MORs) in the SDM of the drugs. Bilateral cannulae were implanted into the CA1 regions (intra-CA1), and memory retrieval was measured by the step-down apparatus. Results showed that pre-test microinjection of morphine (1 μg/mouse, intra-CA1) reversed amnesia induced by pre-training administration of the same dose of morphine, indicating morphine SDM. Moreover, norharmane (10 μg/mouse) also exerted a SDM. Pre-test microinjection of naloxone (0.5 μg/mouse) abolished amnesia induced by morphine or norharmane, and impaired SDM produced by each drug. The results demonstrated the contribution of MORs in the SDM induced by morphine as well as norharmane. Pre-test administration of morphine (1 μg/mouse, intra-CA1) also inhibited amnesia induced by pre-training intra-CA1 microinjection of norharmane (10 μg/mouse) and vice versa, suggesting a cross SDM between the drugs. In conclusion, it seems that there may be a cross SDM between morphine and norharmane, and MORs have a critical role in this phenomenon.

Effects of inactivation of the ventral tegmental area on prefronto-accumbens activity and sexual motivation in male rats

Sexual motivation requires the processing of sexual stimuli. The prefrontal cortex (PFC) and nucleus accumbens (NAcc) receive dopaminergic innervation from the ventral tegmental area (VTA). Both structures participate in processing stimuli, and their adequate functioning is modulated by dopamine and other neurotransmitters. This study was designed to determine the effect of inactivation of the VTA on sexual motivation, relative power (RP) and electroencephalographic (EEG) correlation of the PFC and NAcc in male rats. A total of 20 rats implanted with electrodes in the left medial prefrontal cortex (mPFC) and NAcc, and with bilateral cannulae in the VTA, were divided into two groups of 10 rats each, one injected with tetrodotoxin (TTX), the other with a vehicle solution (VEH). EEGs from the mPFC and NAcc were recorded during the awake-quiet state in the presence of either a receptive or non-receptive female. The TTX group showed a lower preference for the receptive female accompanied by a lower RP of the 8–13 and 14–30 Hz bands in the mPFC. Also, in the presence of the receptive female, the TTX group had a lower RP of the 8–13 Hz band in the NAcc, but a higher prefronto-accumbens correlation in the same band. These results provide evidence that VTA activity is necessary for the adequate functioning of the mPFC and NAcc and, therefore, also for the adequate processing of sexually-relevant stimuli that allows the induction and maintenance of sexual motivation in male rats.

Voluntary wheel running effects on intra-accumbens opioid driven diet preferences in male and female rats

Palatability driven feeding and voluntary physical activity are mediated by and influence similar neural mechanisms, notably through the actions of opioids within the nucleus accumbens. Recent studies suggest that access to a voluntary running wheel results in sex dependent behavioral and physiological adaptations related to opioid mediated palatability-driven feeding. To explore this relationship, male and female Wistar rats were given either access to a voluntary running wheel (RUN group) or no access (SED group) for one week prior to being stereotaxically implanted with bilateral cannulae targeting the nucleus accumbens. Following 7 days of recovery, with RUN or SED conditions continuing the duration of the experiment, all rats were assessed daily (2 h/day) for feeding behavior of concurrently accessible high-carbohydrate and high-fat diet for one week. Following this week, all rats were administered the μ-opioid receptor agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO) (0.0025 μg, 0.025 μg, or 0.25 μg/0.5 μl/side) or the opioid antagonist naloxone (20 μg/0.5 μl/side) into the nucleus accumbens and given concurrent access (2 h) to both diets. All groups expressed a significant baseline preference for the high-carbohydrate diet. DAMGO administration, compared to saline treatment, led to significant increased consumption of the high-carbohydrate diet in all treatment groups. While high-fat diet consumption also increased following DAMGO administration, the influence of DAMGO was much more robust for the preferred high-carbohydrate diet in all groups. Compared to males, females consumed significantly more of both diets at baseline and following DAMGO treatment. Both male and female rats in the RUN condition consumed more high-carbohydrate diet compared to rats in the SED condition. While males exhibited similar increased consumption of both diets regardless of RUN or SED condition, females in the RUN condition displayed a greater sensitivity to DAMGO-driven consumption of the preferred high-carbohydrate, compared to SED females.

Voluntary wheel running effects on intra-accumbens opioid high-fat feeding and locomotor behavior in Sprague-Dawley and Wistar rat strains

The present study examined the influence of physical activity vs. sedentary home cage conditions on baseline and opioid-driven high-fat feeding behaviors in two common strains of laboratory rats. Sprague-Dawley and Wistar rats were singly housed with either access to a voluntary running wheel (RUN) or locked-wheel (SED) for 5 weeks, before being stereotaxically implanted with bilateral cannulae targeting the nucleus accumbens. Following recovery, with RUN or SED conditions continuing the duration of the experiment, all rats were given 2 h daily access to a high-fat diet for 6 consecutive days to establish a stable baseline intake. Over the next 2 weeks, all subjects were administered the μ-opioid agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO) (multiple dose range) or saline into the nucleus accumbens, immediately followed by 2 h access to a high-fat diet. Drug treatments were separated by at least 1 day and treatment order was counterbalanced. Baseline consumption of the high-fat diet during the 1-week baseline acclimation period did not differ between RUN and SED groups in either rat strain. Higher doses of DAMGO produced increased fat consumption in both strains of rats, yet no differences were observed between RUN vs. SED treated groups. However, SED treatment produced a greater locomotor response following intra-accumbens DAMGO administration, compared to the RUN condition, during the 2 h feeding session. The data suggest that the animals housed in sedentary versus voluntary wheel running conditions may differ in behavioral tolerance to the locomotor but not the orexigenic activating properties of intra-accumbens DAMGO treatment.