ABSTRACT
Strong human genetic evidence points to an essential contribution of the voltage-gated sodium channel Nav1.7 to pain sensation: loss of Nav1.7 function leads to congenital insensitivity to pain, whereas gain-of-function mutations in the SCN9A gene that encodes Nav1.7 cause painful neuropathies, such as inherited erythromelalgia, a syndrome characterized by episodic spontaneous pain. Selective Nav1.7 channel blockers thus hold promise as potential painkillers with improved safety and reduced unwanted side effects compared with existing therapeutics. To determine the maximum effect of a theoretically perfectly selective Nav1.7 inhibitor, we generated a tamoxifen-inducible KO mouse model enabling genetic deletion of Nav1.7 from adult mice. Electrophysiological recordings of sensory neurons from these mice following tamoxifen injection demonstrated the loss of Nav1.7 channel current and the resulting decrease in neuronal excitability of small-diameter neurons. We found that behavioral responses to most, but surprisingly not all, modalities of noxious stimulus are abolished following adult deletion of Nav1.7, pointing toward indications where Nav1.7 blockade should be efficacious. Furthermore, we demonstrate that isoform-selective acylsulfonamide Nav1.7 inhibitors show robust analgesic and antinociceptive activity acutely after a single dose in mouse pain models shown to be Nav1.7-dependent. All experiments were done with both male and female mice. Collectively, these data expand the depth of knowledge surrounding Nav1.7 biology as it relates to pain, and provide preclinical proof of efficacy that lays a clear path toward translation for the therapeutic use of Nav1.7-selective inhibitors in humans.SIGNIFICANCE STATEMENT Loss-of-function mutations in the sodium channel Nav1.7 cause congenital insensitivity to pain in humans, making Nav1.7 a top target for novel pain drugs. Targeting Nav1.7 selectively has been challenging, however, in part due to uncertainties in which rodent pain models are dependent on Nav1.7. We have developed and characterized an adult-onset Nav1.7 KO mouse model that allows us to determine the expected effects of a theoretically perfect Nav1.7 blocker. Importantly, many commonly used pain models, such as mechanical allodynia after nerve injury, appear to not be dependent on Nav1.7 in the adult. By defining which models are Nav1.7 dependent, we demonstrate that selective Nav1.7 inhibitors can approximate the effects of genetic loss of function, which previously has not been directly established.
Subject(s)
NAV1.7 Voltage-Gated Sodium Channel/deficiency , Pain Insensitivity, Congenital/metabolism , Pain Perception/physiology , Pain/metabolism , Sodium Channel Blockers/therapeutic use , Animals , Cells, Cultured , Female , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NAV1.7 Voltage-Gated Sodium Channel/genetics , Pain/drug therapy , Pain/genetics , Pain Insensitivity, Congenital/drug therapy , Pain Insensitivity, Congenital/genetics , Pain Perception/drug effects , Sodium Channel Blockers/pharmacologyABSTRACT
Animals rely on environmental cues to identify potential rewards and select the best reward available. The orbitofrontal cortex (OFC) is proposed to encode sensory-specific representations of expected outcome. However, its contribution to the selection of a preferred outcome among different reward options is still unclear. We investigated the effect of transient OFC inactivation (achieved by presession injection of muscimol and baclofen) in a novel two-reward choice task. In discrete trials, rats could choose between a solution of polycose and an equally caloric, but highly preferred, solution of sucrose by visiting one of two liquid dispensers after the presentation of a specific cue signaling the availability of one or both of the solutions. We found that OFC inactivation did not affect outcome preference: rats maintained high preference for sucrose and adapted their behavioral responding when the cue-outcome contingencies were reversed. However, when rats were tested drug-free 24 h after OFC inactivation and reversal learning, memory for the newly learned contingencies was poor. These results suggest a potential conflict between OFC (encoding pre-reversal contingencies) and other brain circuits (encoding the new contingencies). Remarkably, repeating the OFC inactivation before the reversal memory test restored normal behavior, confirming the hypothesis of a dominant impact of OFC on other decision-making circuits. These results indicate that the representations encoded in the OFC, while not essential to the expression of outcome preference, exert hierarchical control on downstream decision-making circuits.
Subject(s)
Behavior, Animal/physiology , Choice Behavior/physiology , Frontal Lobe/physiology , Nerve Net/physiology , Reversal Learning/physiology , Animals , Baclofen/pharmacology , Behavior, Animal/drug effects , Choice Behavior/drug effects , Frontal Lobe/drug effects , GABA-A Receptor Agonists/pharmacology , GABA-B Receptor Agonists/pharmacology , Male , Muscimol/pharmacology , Nerve Net/drug effects , Rats , Rats, Long-Evans , Reversal Learning/drug effects , RewardABSTRACT
The development of alcoholism may involve a shift from goal-directed to habitual drinking. These action control systems are distinct in the dorsal striatum, with the dorsomedial striatum (DMS) important for goal-directed behavior and the dorsolateral striatum (DLS) required for habit formation. Goal-directed behavior can be modeled in rats with a fixed ratio (FR) reinforcement schedule, while a variable interval (VI) schedule promotes habitual behavior (e.g. insensitivity to contingency degradation). Using extracellular recordings from chronically implanted electrodes, we investigated how DMS and DLS neurons encoded lever-press responses and conditioned cues during operant alcohol self-administration in these two models. In rats self-administering 10% alcohol on an FR schedule, the DMS neuronal population showed increased firing at the onset of start-of-session stimuli. During self-administration, the most prominent phasic firing patterns in the DMS occurred at the time of reinforcement and reinforcement-associated cues, while the most prominent phasic activity in the DLS surrounded the lever response. Neural recordings from an additional cohort of rats trained on a VI schedule revealed a similar pattern of results; however, phasic changes in firing were smaller and differences between the medial and lateral dorsal striatum were less marked. In summary, the DMS and DLS exhibited overlapping but specialized phasic firing patterns: DMS excitations were typically time-locked to reinforcement, while DLS excitations were generally associated with lever responses. Furthermore, the regional specificities and magnitudes of phasic firing differed between reinforcement schedules, which may reflect differences in behavioral flexibility, reward expectancy and the action sequences required to procure reinforcement.
Subject(s)
Alcohol Drinking/physiopathology , Corpus Striatum/physiopathology , Neurons/physiology , Reinforcement Schedule , Animals , Ethanol/administration & dosage , Male , Rats , Rats, Long-Evans , Self AdministrationABSTRACT
Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.
Subject(s)
Analgesics, Opioid , Nociceptors , Mice , Humans , Animals , Analgesics, Opioid/pharmacology , Action Potentials , NAV1.7 Voltage-Gated Sodium Channel/genetics , Pain/genetics , Sensory Receptor Cells , Opioid Peptides , Enkephalins , Ganglia, SpinalABSTRACT
Context: Positron emission tomography imaging with 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) is used clinically for initial staging, restaging, and assessing therapy response in breast cancer. Tumor FDG uptake in steroid hormone receptor-positive breast cancer and physiologic FDG uptake in normal breast tissue can be affected by hormonal factors such as menstrual cycle phase, menopausal status, and hormone replacement therapy. Objective: The purpose of this study was to determine the role of the progesterone receptor (PR) in regulating glucose and FDG uptake in breast cancer cells. Methods and Results: PR-positive T47D breast cancer cells treated with PR agonists had increased FDG uptake compared with ethanol control. There was no significant change in FDG uptake in response to PR agonists in PR-negative MDA-MB-231 cells, MDA-MB-468 cells, or T47D PR knockout cells. Treatment of T47D cells with PR antagonists inhibited the effect of R5020 on FDG uptake. Using T47D cell lines that only express either the PR-A or the PR-B isoform, PR agonists increased FDG uptake in both cell types. Experiments using actinomycin D and cycloheximide demonstrated the requirement for both transcription and translation in PR regulation of FDG uptake. GLUT1 and PFKFB3 mRNA expression and the enzymatic activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were increased after progestin treatment of T47D cells. Conclusion: Thus, progesterone and progestins increase FDG uptake in T47D breast cancer cells through the classical action of PR as a ligand-activated transcription factor. Ligand-activated PR ultimately increases expression and activity of proteins involved in glucose uptake, glycolysis, and the pentose phosphate pathway.
ABSTRACT
Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant posttranslational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group revealed enrichment of the GRHL2 motif near pS118-ER binding sites. In this study, we used cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300, compared to sites where each factor binds independently. Transcriptomic analysis yielded four subsets of ER/GRHL2-coregulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis indicated that coregulated genes are involved in cell migration. Accordingly, knockdown of GRHL2, combined with estrogen treatment, resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to selected enhancers and facilitates pS118-ER recruitment to chromatin, which then results in differential activation and repression of genes that control estrogen-regulated ER-positive breast cancer cell migration.
Subject(s)
Estrogen Receptor alpha , Receptors, Estrogen , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Transcriptional Activation/genetics , Chromatin , Ligands , Estrogens/metabolism , Serine/metabolism , Cell Line, TumorABSTRACT
Transient receptor potential ankyrin 1 (TRPA1) antagonists have generated broad interest in the pharmaceutical industry for the treatment of both pain and asthma. Over the past decade, multiple antagonist classes have been reported in the literature with a wide range of structural diversity. Our own work has focused on the development of proline sulfonamide and hypoxanthine-based antagonists, two antagonist classes with distinct physicochemical properties and pharmacokinetic (PK) trends. Late in our discovery program, cryogenic electron microscopy (cryoEM) studies revealed two different antagonist binding sites: a membrane-exposed proline sulfonamide transmembrane site and an intracellular hypoxanthine site near the membrane interface. A retrospective look at the discovery program reveals how the different binding sites, and their location relative to the cell membrane, influenced the optimization trajectories and overall drug profiles of each antagonist class.
ABSTRACT
Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.
Subject(s)
Asthma/drug therapy , Furans/therapeutic use , Purines/therapeutic use , TRPA1 Cation Channel/antagonists & inhibitors , Animals , Asthma/chemically induced , Asthma/complications , CHO Cells , Cricetulus , Furans/chemical synthesis , Furans/metabolism , Guinea Pigs , Humans , Inflammation/drug therapy , Inflammation/etiology , Ligands , Male , Molecular Structure , Ovalbumin , Oxadiazoles/chemical synthesis , Oxadiazoles/metabolism , Oxadiazoles/therapeutic use , Protein Binding , Purines/chemical synthesis , Purines/metabolism , Rats, Sprague-Dawley , Structure-Activity Relationship , TRPA1 Cation Channel/metabolismABSTRACT
Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.
Subject(s)
Asthma/drug therapy , Neurogenic Inflammation/drug therapy , Pain/drug therapy , Pruritus/drug therapy , Pyridines/pharmacology , Pyridines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , TRPA1 Cation Channel/antagonists & inhibitors , Adolescent , Adult , Animals , Cohort Studies , Disease Models, Animal , Dogs , Double-Blind Method , Female , Guinea Pigs , Healthy Volunteers , Humans , Isothiocyanates/administration & dosage , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Pain/chemically induced , Pruritus/chemically induced , Rats , Rats, Sprague-Dawley , TRPA1 Cation Channel/deficiency , Treatment Outcome , Young AdultABSTRACT
Transcriptional regulation of ESR1, the gene that encodes for estrogen receptor α (ER), is critical for regulating the downstream effects of the estrogen signaling pathway in breast cancer such as cell growth. ESR1 is a large and complex gene that is regulated by multiple regulatory elements, which has complicated our understanding of how ESR1 expression is controlled in the context of breast cancer. Early studies characterized the genomic structure of ESR1 with subsequent studies focused on identifying intrinsic (chromatin environment, transcription factors, signaling pathways) and extrinsic (tumor microenvironment, secreted factors) mechanisms that impact ESR1 gene expression. Currently, the introduction of genomic sequencing platforms and additional genome-wide technologies has provided additional insight on how chromatin structures may coordinate with these intrinsic and extrinsic mechanisms to regulate ESR1 expression. Understanding these interactions will allow us to have a clearer understanding of how ESR1 expression is regulated and eventually provide clues on how to influence its regulation with potential treatments. In this review, we highlight key studies concerning the genomic structure of ESR1, mechanisms that affect the dynamics of ESR1 expression, and considerations towards affecting ESR1 expression and hormone responsiveness in breast cancer.
Subject(s)
Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Neoplastic/genetics , Cell Proliferation , Female , Humans , MaleABSTRACT
The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1 KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.
Subject(s)
Asthma/genetics , Behavior, Animal/physiology , Pain/genetics , Pruritus/genetics , TRPA1 Cation Channel/genetics , Animals , Asthma/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Pain/metabolism , Phenotype , Pruritus/metabolism , Rats , Rats, Transgenic , TRPA1 Cation Channel/metabolismABSTRACT
In mammals, the grainyhead-like transcription factor (GRHL) family is composed of three nuclear proteins that are responsible for driving epithelial cell fate: GRHL1, GRHL2, and GRHL3. GRHL2 is important in maintaining proper tubulogenesis during development and in suppressing the epithelial-to-mesenchymal transition. Within the last decade, evidence indicates both tumor-suppressive and oncogenic roles for GRHL2 in various types of cancers. Recent studies suggest that GRHL2 may be especially important in hormone-dependent cancers, as correlative relationships exist between GRHL2 and various steroid receptors, such as the androgen and estrogen receptors. Acting as a pioneer factor and coactivator, GRHL2 may directly affect steroid receptor transcriptional activity. This review will highlight recent discoveries of GRHL2 activity in cancer and in maintaining the epithelial state, while also exploring recent literature on the role of GRHL2 in hormone-dependent cancers and epigenetics.
Subject(s)
DNA-Binding Proteins/genetics , Epigenesis, Genetic/genetics , Gene Expression Regulation, Neoplastic , Neoplasms/genetics , Receptors, Steroid/genetics , Transcription Factors/genetics , Animals , Cell Line, Tumor , Cell Proliferation/genetics , DNA-Binding Proteins/metabolism , Epithelial-Mesenchymal Transition/genetics , Humans , Neoplasms/metabolism , Neoplasms/pathology , Receptors, Steroid/metabolism , Transcription Factors/metabolismABSTRACT
Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.
Subject(s)
DNA, Neoplasm/metabolism , DNA-Binding Proteins/metabolism , Estrogen Receptor alpha/metabolism , Transcription Factors/metabolism , Binding Sites , Breast Neoplasms/metabolism , Cell Line, Tumor , Chromatin/metabolism , Enhancer Elements, Genetic , Estrogens/metabolism , Female , Humans , MCF-7 Cells , Phosphorylation , Protein Binding , Signal TransductionABSTRACT
Mesolimbic dopamine perturbations modulate performance of reward-seeking behavior, with tasks requiring high effort being especially vulnerable to disruption of dopamine signaling. Previous work primarily investigated long-term perturbations such as receptor antagonism and dopamine depletion, which constrain the ability to assess dopamine contributions to effort expenditure in isolation from other behavior events, such as reward consumption. Also unclear is if dopamine is required for both initiation and maintenance when a sequence of multiple instrumental responses is required. Here we used optogenetic inhibition of midbrain TH+ â¯neurons to probe the role of dopamine neuron activity during instrumental responding for reward by varying the time epoch of neural inhibition relative to the time of response initiation. Within a fixed-ratio procedure, requiring eight nosepoke responses per reinforcer delivery, or a progressive ratio (PR) procedure, in which within-session response requirements increased exponentially, inhibiting dopamine neurons while mice were engaged in response bouts decreased the probability of continued responding. If inhibition occurred during each attempted bout, the effect was to decrease total responses, and thus amount of rewards earned, over a session. In contrast, if inhibition was applied only during some bouts, mice increased the number of bouts initiated to earn control levels of reward. Inhibiting dopamine neurons while mice were not responding decreased the probability of initiating an instrumental response but had no effect on the amount of effort exerted over the entire session. We conclude that midbrain dopamine signaling promotes initiation of instrumental responding and maintains motivation to continue ongoing bouts of effortful responses.
Subject(s)
Conditioning, Operant/physiology , Dopaminergic Neurons/physiology , Mesencephalon/metabolism , Animals , Inhibition, Psychological , Male , Mice, Transgenic , Motor Activity/physiology , Optogenetics , Reinforcement Schedule , Reward , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.
Subject(s)
Prodrugs/pharmacology , Proline/analogs & derivatives , Proline/pharmacology , Sulfonamides/pharmacology , TRPA1 Cation Channel/antagonists & inhibitors , Animals , Dogs , Drug Discovery , Drug Stability , Humans , Ligands , Madin Darby Canine Kidney Cells , Microsomes, Liver/metabolism , Models, Molecular , Molecular Conformation , Prodrugs/chemical synthesis , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Proline/chemical synthesis , Proline/pharmacokinetics , Rats , Solubility , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , TRPA1 Cation Channel/chemistryABSTRACT
The environmental context in which a discrete Pavlovian conditioned stimulus (CS) is experienced can profoundly impact conditioned responding elicited by the CS. We hypothesized that alcohol-seeking behavior elicited by a discrete CS that predicted alcohol would be influenced by context and require glutamate signaling in the basolateral amygdala (BLA). Male, Long-Evans rats were allowed to drink 15% ethanol (v/v) until consumption stabilized. Next, rats received Pavlovian conditioning sessions in which a 10 s CS (15 trials/session) was paired with ethanol (0.2 ml/CS). Entries into a port where ethanol was delivered were measured. Pavlovian conditioning occurred in a specific context (alcohol context) and was alternated with sessions in a different context (non-alcohol context) where neither the CS nor ethanol was presented. At test, the CS was presented without ethanol in the alcohol context or the non-alcohol context, following a bilateral microinfusion (0.3 µl/hemisphere) of saline or the AMPA glutamate receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt) in the BLA (0, 0.3, or 1.0 µg/0.3 µl). The effect of NBQX (0, 0.3 µg/0.3 µl) in the caudate putamen (CPu) on CS responding in the non-alcohol context was also tested. The discrete alcohol CS triggered more alcohol-seeking behavior in the alcohol context than the non-alcohol context. NBQX in the BLA reduced CS responding in both contexts but had no effect in the CPu. These data indicate that AMPA glutamate receptors in the BLA are critical for alcohol-seeking elicited by a discrete CS and that behavior triggered by the CS is strongly invigorated by an alcohol context.
Subject(s)
Alcohol Drinking/physiopathology , Amygdala/metabolism , Conditioning, Classical/physiology , Cues , Drug-Seeking Behavior/physiology , Glutamates/metabolism , Signal Transduction/physiology , Alcohol Drinking/psychology , Analysis of Variance , Animals , Central Nervous System Depressants/administration & dosage , Conditioning, Classical/drug effects , Dose-Response Relationship, Drug , Drug-Seeking Behavior/drug effects , Ethanol/administration & dosage , Excitatory Amino Acid Antagonists/pharmacology , Male , Quinoxalines/pharmacology , Rats , Rats, Long-Evans , Signal Transduction/drug effectsABSTRACT
Neurobiological mechanisms that influence behavior in the presence of alcohol-associated stimuli involve processes that organize behavior during the presence of these cues, and separately, regulation of behavior in their absence. However, little is known about anatomical structures that might mediate this regulation. Here we examined nucleus accumbens shell (AcbSh) as a possible neural substrate mediating behavior modulation triggered by the presence and absence of alcohol-associated environmental cues and contexts. We also examined subregions of basal amygdala nuclei- rostral basolateral (BLA) and basal posterior (BAP)- as they provide a major source of glutamatergic input to the AcbSh. Animals were trained to associate an auditory conditioning stimulus with alcohol in a discriminative context and then subsequently tested for conditioned port-entries across contexts either previously associated or not associated with alcohol. We found that, on test to the alcohol cue alone, AcbSh inactivation prevented conditioned port-entries in contexts that either were associated with alcohol or were novel, while also increasing unconditioned port-entries during the intertrial intervals. When tested to alcohol-reinforced cues, AcbSh inactivation produced more cue-trial omissions and elevated unconditioned port-entries. Interestingly, BLA and BAP inactivation produced dissociable effects. BAP but not BLA increased unconditioned port-entries, while both manipulations prevented conditioned port-entries during the alcohol-cue. We conclude that AcbSh is necessary for modulating control over behavior otherwise guided by the presence of alcohol-predictive environmental stimuli and contexts. Moreover, this role may involve integration of functionally segregated inputs from rostral and posterior portions of basal amygdala nuclei.