Michael M. Poe

Regulating anxiety with extrasynaptic inhibition

Aversive experiences can lead to complex behavioral adaptations including increased levels of anxiety and fear generalization. The neuronal mechanisms underlying such maladaptive behavioral changes, however, are poorly understood. Here, using a combination of behavioral, physiological and optogenetic approaches in mouse, we identify a specific subpopulation of central amygdala neurons expressing protein kinase C δ (PKCδ) as key elements of the neuronal circuitry controlling anxiety. Moreover, we show that aversive experiences induce anxiety and fear generalization by regulating the activity of PKCδ+ neurons via extrasynaptic inhibition mediated by α5 subunit-containing GABAA receptors. Our findings reveal that the neuronal circuits that mediate fear and anxiety overlap at the level of defined subpopulations of central amygdala neurons and demonstrate that persistent changes in the excitability of a single cell type can orchestrate complex behavioral changes.

Prior Antipsychotic Drug Treatment Prevents Response to Novel Antipsychotic Agent in the Methylazoxymethanol Acetate Model of Schizophrenia

Trials of novel compounds for the treatment of schizophrenia are typically tested in patients following brief withdrawal of ongoing medication despite known long-term changes in the dopamine (DA) system following chronic antipsychotic drug therapy. The present study explored the impact of withdrawal from repeated haloperidol (HAL) treatment, as well as the response to a novel α5 gamma-aminobutyric acid (GABA(A)) receptor positive allosteric modulator (α5PAM), on the activity of the DA system in the methylazoxymethanol acetate (MAM) neurodevelopmental model of schizophrenia. Electrophysiological recordings were conducted from DA neurons in the ventral tegmental area of MAM and saline (SAL) rats following 7-day withdrawal from repeated HAL (21 d, 0.6 mg/kg, orally). In separate animals, amphetamine-induced locomotion was measured to assess changes in DA behavioral sensitivity. SAL rats withdrawn from HAL demonstrated reduced spontaneous DA neuron activity along with an enhanced locomotor response to amphetamine, indicative of the development of DA supersensitivity. Both α5PAM treatment and ventral hippocampal (vHPC) inactivation reversed the DA neuron depolarization block following HAL withdrawal in SAL rats. In contrast, MAM rats withdrawn from HAL exhibited reduced spontaneous DA activity and enhanced locomotor response to amphetamine compared with untreated SAL rats; however, this condition was unresponsive to α5PAM treatment or vHPC inactivation. Withdrawal from prior HAL treatment interferes with the therapeutic actions of this novel treatment in the MAM model of schizophrenia. Consequently, testing novel compounds on chronically treated schizophrenia patients may be ineffective.

Behavioral Effects of the Benzodiazepine-Positive Allosteric Modulator SH-053-2’F-S-CH3 in an Immune-Mediated Neurodevelopmental Disruption Model

Background: Impaired γ-aminobutyric acid (GABA) signaling may contribute to the emergence of cognitive deficits and subcortical dopaminergic hyperactivity in patients with schizophrenia and related psychotic disorders. Against this background, it has been proposed that pharmacological interventions targeting GABAergic dysfunctions may prove useful in correcting such cognitive impairments and dopaminergic imbalances. Methods: Here, we explored possible beneficial effects of the benzodiazepine-positive allosteric modulator SH-053-2’F-S-CH3, with partial selectivity at the α2, α3, and α5 subunits of the GABAA receptor in an immune-mediated neurodevelopmental disruption model. The model is based on prenatal administration of the viral mimetic polyriboinosinic-polyribocytidilic acid [poly(I:C)] in mice, which is known to capture various GABAergic, dopamine-related, and cognitive abnormalities implicated in schizophrenia and related disorders. Results: Real-time polymerase chain reaction analyses confirmed the expected alterations in GABAA receptor α subunit gene expression in the medial prefrontal cortices and ventral hippocampi of adult poly(I:C) offspring relative to control offspring. Systemic administration of SH-053-2’F-S-CH3 failed to normalize the poly(I:C)-induced deficits in working memory and social interaction, but instead impaired performance in these cognitive and behavioral domains both in control and poly(I:C) offspring. In contrast, SH-053-2’F-S-CH3 was highly effective in mitigating the poly(I:C)-induced amphetamine hypersensitivity phenotype without causing side effects in control offspring. Conclusions: Our preclinical data suggest that benzodiazepine-like positive allosteric modulators with activity at the α2, α3, and α5 subunits of the GABAA receptor may be particularly useful in correcting pathological overactivity of the dopaminergic system, but they may be ineffective in targeting multiple pathological domains that involve the co-existence of psychotic, social, and cognitive dysfunctions.

A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2′F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors.

EEF2K/eEF2 pathway controls the excitation/inhibition balance and susceptibility to epileptic seizures

Abstract Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and &agr;5‐containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal‐dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.

Sex-Dependent Anti-Stress Effect of an α5 Subunit Containing GABAA Receptor Positive Allosteric Modulator

Rationale: Current first-line treatments for stress-related disorders such as major depressive disorder (MDD) act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2’F-R-CH3 (denoted “α5-PAM”), a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites, has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS) and treated with α5-PAM acutely (30 min prior to assessing behavior) or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as “behavioral emotionality”) across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in the hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusion: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

Neonatal bladder inflammation induces long-term visceral pain and altered responses of spinal neurons in adult rats

Painful events early in life have been shown to increase the incidence of interstitial cystitis/painful bladder syndrome in adulthood. However, the intrinsic mechanism is not well studied. We previously reported that neonatal bladder inflammation causes chronic visceral hypersensitivity along with molecular disruption of spinal GABAergic system in rats. The present study investigates whether these molecular changes affect the integrative function and responses of bladder-sensitive primary afferent and spinal neurons. Neonatal bladder inflammation was induced by intravesicular injection of zymosan during postnatal (P) days 14-16. In adulthood (P60), the viscero-motor response (VMR) to visceral stimuli was significantly inhibited by intrathecal (i.t) HZ166 (GABAAα-2 agonist) only in neonatally saline-treated, but not in neonatally zymosan-treated rats. HZ166 significantly inhibited the responses of bladder-responsive lumbosacral (LS) spinal neurons to urinary bladder distension (UBD) and slow infusion (SI) in neonatally saline-treated rats. Similar results were also observed in naïve adult rats where HZ166 produced significant inhibition of bladder-responsive spinal neurons. However, HZ166 did not inhibit responses of UBD-responsive spinal neurons from neonatally zymosan-treated rats. The drug did not attenuate the responses of UBD-sensitive pelvic nerve afferent (PNA) fibers to UBD and SI in either group of rats tested. Immunohistochemical studies showed a significantly lower level of GABAAα-2 receptor expression in the LS spinal cord of neonatally zymosan-treated rats compared to saline-treated rats. These findings indicate that neonatal bladder inflammation leads to functional and molecular alteration of spinal GABAAα-2 receptor subtypes, which may result in chronic visceral hyperalgesia in adulthood.

Pharmacological and antihyperalgesic properties of the novel α2/3 preferring GABAA receptor ligand MP-III-024

γ-Aminobutyric acid type A (GABAA) receptors are located in spinal nociceptive circuits where they modulate the transmission of pain sensory signals from the periphery to higher centers. Benzodiazepine-type drugs bind to GABAA receptors containing α1, α2, α3, and α5 subunits (α1GABAA, α2GABAA, α3GABAA and α5GABAA receptors, respectively) through which they inhibit the transmission of these signals. In the present study we describe the novel benzodiazepine site positive allosteric modulator modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024). MP-III-024 displayed preference for α2GABAA and α3GABAA receptors relative to α1GABAA and α5GABAA receptors as well as an improved metabolic profile relative to subtype-selective positive modulators that are available currently. Administration of MP-III-024 resulted in a dose- and time-dependent reversal of mechanical hyperalgesia. On locomotor activity and schedule-controlled responding, MP-III-024 was ineffective across the doses tested. These data provide further evidence that α2GABAA and α3GABAA receptors play an important role in the antihyperalgesic effects and may not be involved in some of the undesired effects of benzodiazepine-like drugs. Further, these findings suggest that MP-III-024 is a suitable research tool for investigating the role of α2GABAA and α3GABAA receptors in the behavioral properties of benzodiazepine-like drugs in mice.

Antinociceptive Effects of a Novel α2/α3-Subtype Selective GABAA Receptor Positive Allosteric Modulator

Pain remains a challenging clinical condition and spinal GABAA receptors are crucial modulators of pain processing. α2/α3-subtype GABAA receptors mediate the analgesic actions of benzodiazepines. Positive allosteric modulators (PAMs) at α2/α3-subtype GABAA receptors may have analgesic potential. Here we report a new selective α2/α3-subtype GABAA receptor PAM in in vitro and in vivo pain assays. KRM-II-81 demonstrated similar efficacy at α1/α2/α3 GABAA receptors and negligible efficacy at α4/α5/α6 GABAA receptors, with α2 and α3-subtypes being 17- and 28-fold more potent than α1 subtypes in HEK-293T cells expressing GABAA receptors with different α subunits. In contrast, KRM-II-18B showed significant efficacy at α1/α2/α3/ α5 subtypes, with similar potency at α1/α2/α3 subtypes. Both PAMs and morphine dose-dependently decreased 0.6% acetic acid- and 0.32% lactic acid-induced writhing. The effects of both PAMs were reversed by the benzodiazepine receptor antagonist flumazenil, confirming their action at the benzodiazepine binding site of GABAA receptors. Both PAMS and morphine all dose-dependently reversed 0.32% lactic acid (but not 0.6% acetic acid) induced suppression of nesting behavior. Acetaminophen, but not the PAMs, reversed acid-depressed locomotor activity. Combined, these findings suggest that KRM-II-81 is a selective α2/α3 subtype GABAA PAM with significant antinociceptive effects in chemical stimulation-induced pain in mice.

Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments

Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.