Ashlee Van’t Veer

Essential Role for TRPC5 in Amygdala Function and Fear-Related Behavior

The transient receptor potential channel 5 (TRPC5) is predominantly expressed in the brain where it can form heterotetrameric complexes with TRPC1 and TRPC4 channel subunits. These excitatory, nonselective cationic channels are regulated by G protein, phospholipase C-coupled receptors. Here, we show that TRPC5(-/-) mice exhibit diminished innate fear levels in response to innately aversive stimuli. Moreover, mutant mice exhibited significant reductions in responses mediated by synaptic activation of Group I metabotropic glutamate and cholecystokinin 2 receptors in neurons of the amygdala. Synaptic strength at afferent inputs to the amygdala was diminished in P10-P13 null mice. In contrast, baseline synaptic transmission, membrane excitability, and spike timing-dependent long-term potentiation at cortical and thalamic inputs to the amygdala were largely normal in older null mice. These experiments provide genetic evidence that TRPC5, activated via G protein-coupled neuronal receptors, has an essential function in innate fear.

Role of kappa-opioid receptors in stress and anxiety-related behavior

RationaleAccumulating evidence indicates that brain kappa-opioid receptors (KORs) and dynorphin, the endogenous ligand that binds at these receptors, are involved in regulating states of motivation and emotion. These findings have stimulated interest in the development of KOR-targeted ligands as therapeutic agents. As one example, it has been suggested that KOR antagonists might have a wide range of indications, including the treatment of depressive, anxiety, and addictive disorders, as well as conditions characterized by co-morbidity of these disorders (e.g., post-traumatic stress disorder) A general effect of reducing the impact of stress may explain how KOR antagonists can have efficacy in such a variety of animal models that would appear to represent different disease states.ObjectiveHere, we review evidence that disruption of KOR function attenuates prominent effects of stress. We will describe behavioral and molecular endpoints including those from studies that characterize the effects of KOR antagonists and KOR ablation on the effects of stress itself, as well as on the effects of exogenously delivered corticotropin-releasing factor, a brain peptide that mediates key effects of stress.ConclusionCollectively, available data suggest that KOR disruption produces anti-stress effects and under some conditions can prevent the development of stress-induced adaptations. As such, KOR antagonists may have unique potential as therapeutic agents for the treatment and even prevention of stress-related psychiatric illness, a therapeutic niche that is currently unfilled.

Selective κ Opioid Antagonists nor-BNI, GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets. Results In binding assays, the three antagonists showed no detectable affinity (K i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α2C-adrenoceptor (K i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α1A-adrenoceptor (EC50 = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M1 receptor antagonist (K B = 3.7 µM). JDTic bound to the noradrenaline transporter (K i = 54 nM), but only weakly inhibited transport (IC50 = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers. Conclusions Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α1A-adrenoceptors). This may contribute to GNTIs severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.

Activation of CREB in the Nucleus Accumbens Shell Produces Anhedonia and Resistance to Extinction of Fear in Rats

Stress triggers psychiatric conditions including depressive and anxiety disorders. The mechanisms by which stress produces persistent changes in behavior are not fully understood. Here we show in rats that stress (footshock) activates the transcription factor cAMP response element binding protein (CREB) within the nucleus accumbens shell (NAS), a brain area involved in encoding reward and aversion. To examine the behavioral significance of altered CREB function in the NAS, we used viral vectors to elevate or disrupt CREB in this region. Elevated CREB produced increases in intracranial self-stimulation thresholds, a depressive-like sign reflecting anhedonia (decreased sensitivity to reward), whereas disruption of CREB function by expression of a dominant-negative CREB had the opposite effect. To determine whether neuroadaptations that produce anhedonia subsequently affect vulnerability to stress-induced behavioral adaptations, we subjected rats with altered CREB function in the NAS to fear conditioning. Although neither elevation nor disruption of CREB function altered the development of conditioned fear, elevation of CREB impaired extinction of conditioned fear. To mimic downstream effects of CREB activation on expression of the opioid peptide dynorphin, we microinjected the κ-opioid receptor (KOR) agonist U50,488 directly into the NAS. KOR stimulation produced anhedonia but had no effect on expression or extinction of conditioned fear. These findings demonstrate that activation of CREB in the NAS produces multiple behavioral signs (anhedonia, impaired extinction) characteristic of experience-dependent psychiatric conditions such as posttraumatic stress disorder. Although CREB activation is a common trigger, expression of these individual signs appears to involve divergent downstream mechanisms.

Blockade of astrocytic glutamate uptake in the prefrontal cortex induces anhedonia.

Major depression is associated with both dysregulated glutamatergic neurotransmission and fewer astrocytes in limbic areas including the prefrontal cortex (PFC). These deficits may be functionally related. Notably, astrocytes regulate glutamate levels by removing glutamate from the synapse via the glutamate transporter (GLT-1). Previously, we demonstrated that central blockade of GLT-1 induces anhedonia and c-Fos expression in the PFC. Given the role of the PFC in regulating mood, we hypothesized that GLT-1 blockade in the PFC alone would be sufficient to induce anhedonia in rats. We microinjected the GLT-1 inhibitor, dihydrokainic acid (DHK), into the PFC and examined the effects on mood using intracranial self-stimulation (ICSS). At lower doses, intra-PFC DHK produced modest increases in ICSS thresholds, reflecting a depressive-like effect. At higher doses, intra-PFC DHK resulted in cessation of responding. We conducted further tests to clarify whether this total cessation of responding was related to an anhedonic state (tested by sucrose intake), a nonspecific result of motor impairment (measured by the tape test), or seizure activity (measured with electroencephalogram (EEG)). The highest dose of DHK increased latency to begin drinking without altering total sucrose intake. Furthermore, neither motor impairment nor evidence of seizure activity was observed in the tape test or EEG recordings. A decrease in reward value followed by complete cessation of ICSS responding suggests an anhedonic-like effect of intra-PFC DHK; a conclusion that was substantiated by an increased latency to begin sucrose drinking. Overall, these results suggest that blockade of astrocytic glutamate uptake in the PFC is sufficient to produce anhedonia, a core symptom of depression.

Long-acting κ opioid antagonists nor-BNI, GNTI and JDTic: pharmacokinetics in mice and lipophilicity

BackgroundNor-BNI, GNTI and JDTic induce κ opioid antagonism that is delayed by hours and can persist for months. Other effects are transient. It has been proposed that these drugs may be slowly absorbed or distributed, and may dissolve in cell membranes, thus slowing elimination and prolonging their effects. Recent evidence suggests, instead, that they induce prolonged desensitization of the κ opioid receptor.MethodsTo evaluate these hypotheses, we measured relevant physicochemical properties of nor-BNI, GNTI and JDTic, and the timecourse of brain and plasma concentrations in mice after intraperitoneal administration (using LC-MS-MS).ResultsIn each case, plasma levels were maximal within 30 min and declined by >80% within four hours, correlating well with previously reported transient effects. A strong negative correlation was observed between plasma levels and the delayed, prolonged timecourse of κ antagonism. Brain levels of nor-BNI and JDTic peaked within 30 min, but while nor-BNI was largely eliminated within hours, JDTic declined gradually over a week. Brain uptake of GNTI was too low to measure accurately, and higher doses proved lethal. None of the drugs were highly lipophilic, showing high water solubility (> 45 mM) and low distribution into octanol (log D7.4 < 2). Brain homogenate binding was within the range of many shorter-acting drugs (>7% unbound). JDTic showed P-gp-mediated efflux; nor- BNI and GNTI did not, but their low unbound brain uptake suggests efflux by another mechanism.ConclusionsThe negative plasma concentration-effect relationship we observed is difficult to reconcile with simple competitive antagonism, but is consistent with desensitization. The very slow elimination of JDTic from brain is surprising given that it undergoes active efflux, has modest affinity for homogenate, and has a shorter duration of action than nor-BNI under these conditions. We propose that this persistence may result from entrapment in cellular compartments such as lysosomes.

Ablation of Kappa-Opioid Receptors from Brain Dopamine Neurons has Anxiolytic-Like Effects and Enhances Cocaine-Induced Plasticity

Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR−/−) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KORlox/lox). Autoradiography demonstrated complete ablation of KOR binding in the KOR−/− mutants, and reduced binding in the DAT-KORlox/lox mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR−/− mice appeared normal in the open field and light/dark box tests, DAT-KORlox/lox mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR−/− mutants, but was exaggerated in DAT-KORlox/lox mutants. Increased sensitivity to cocaine in the DAT-KORlox/lox mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR−/− mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.

Corticotropin-releasing factor (CRF)-induced disruption of attention in rats is blocked by the κ-opioid receptor antagonist JDTic.

Stress often disrupts behavior and can lead to psychiatric illness. Considerable evidence suggests that corticotropin-releasing factor (CRF) plays an important role in regulating the effects of stress. CRF administration produces stress-like effects in humans and laboratory animals, and CRF levels are elevated in individuals with stress-related illness. Recent work indicates that κ-opioid receptor (KOR) antagonists can block CRF effects, raising the possibility that at least some of the effects of stress are mediated via KORs. Here we examined the effects of CRF on performance in the 5-choice serial reaction time task (5CSRTT), a test used to quantify attention in rodents, as well as functional interactions between CRF and KORs. Male Sprague-Dawley rats were trained in the 5CSRTT and then each was implanted with an intracerebroventricular (ICV) cannula. After recovery and restabilization of performance, they received a single intraperitoneal (IP) injection of vehicle or JDTic (10 mg/kg), a KOR antagonist with long-lasting (>14 days) effects. In subsequent sessions, rats received ICV infusions of CRF (0.25–1.0 μg) or vehicle and were tested 60 min later. CRF dose-dependently disrupted performance as reflected by decreases in correct responding, increases in omission errors, increases in latencies to respond correctly, and increases in time to complete the session. JDTic attenuated each of these CRF-induced deficits while having no effects on its own. The persistent ability of JDTic to disrupt KOR function was confirmed using the tail immersion assay. These findings indicate that KOR antagonists can prevent acute stress-related effects that degrade performance in tasks requiring attention.

Knowledge gaps and research recommendations for essential tremor

Essential tremor (ET) is a common cause of significant disability, but its etiologies and pathogenesis are poorly understood. Research has been hampered by the variable definition of ET and by non-standardized research approaches. The National Institute of Neurological Disorders and Stroke (USA) invited experts in ET and related fields to discuss current knowledge, controversies, and gaps in our understanding of ET and to develop recommendations for future research. Discussion focused on phenomenology and phenotypes, therapies and clinical trials, pathophysiology, pathology, and genetics. Across all areas, the need for collaborative and coordinated research on a multinational level was expressed. Standardized data collection using common data elements for genetic, clinical, neurophysiological, and pathological studies was recommended. Large cohorts of patients should be studied prospectively to collect bio-samples, characterize the natural history of the clinical syndrome including patient-oriented outcomes, investigate potential etiologies of various phenotypes, and identify pathophysiological mechanisms. In particular, cellular and system-level mechanisms of tremor oscillations should be elucidated because they may yield effective therapeutic targets and biomarkers. A neuropathology consortium was recommended to standardize postmortem analysis and further characterize neuropathological observations in the cerebellum and elsewhere. Furthermore, genome-wide association studies on large patient cohorts (>10,000 patients) may allow the identification of common genes contributing to risk, and whole exome or genome sequencing may enable the identification of genetic risk and causal mutations in cohorts and well-characterized families.

Kappa-opioid receptors differentially regulate low and high levels of ethanol intake in female mice.

Studies in laboratory animals and humans indicate that endogenous opioids play an important role in regulating the rewarding value of various drugs, including ethanol (EtOH). Indeed, opioid antagonists are currently a front‐line treatment for alcoholism in humans. Although roles for mu‐ and delta‐opioid receptors have been characterized, the contribution of kappa‐opioid receptors (KORs) is less clear. There is evidence that changes in KOR system function can decrease or increase EtOH drinking, depending on test conditions. For example, female mice lacking preprodynorphin – the precursor to the endogenous KOR ligand dynorphin – have reduced EtOH intake. Considering that KORs can regulate dopamine (DA) transmission, we hypothesized that KORs expressed on DA neurons would play a prominent role in EtOH intake in females.