Targeting GABAC Receptors Improves Post-Stroke Motor Recovery.

Ischemic stroke remains a leading cause of disability worldwide, with limited treatment options available. This study investigates GABAC receptors as novel pharmacological targets for stroke recovery. The expression of ρ1 and ρ2 mRNA in mice were determined in peri-infarct tissue following photothrombotic motor cortex stroke. (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (R)-4-ACPBPA and (S)-4-ACPBPA were assessed using 2-elecotrode voltage electrophysiology in Xenopus laevis oocytes. Stroke mice were treated for 4 weeks with either vehicle, the α5-selective negative allosteric modulator, L655,708, or the ρ1/2 antagonists, (R)-4-ACPBPA and (S)-4-ACPBPA respectively from 3 days post-stroke. Infarct size and expression levels of GAT3 and reactive astrogliosis were determined using histochemistry and immunohistochemistry respectively, and motor function was assessed using both the grid-walking and cylinder tasks. After stroke, significant increases in ρ1 and ρ2 mRNAs were observed on day 3, with ρ2 showing a further increase on day 7. (R)- and (S)-4-ACPBPA are both potent antagonists at ρ2 and only weak inhibitors of α5ß2γ2 receptors. Treatment with either L655,708, (S)-4-ACPBPA (ρ1/2 antagonist; 5 mM only), or (R)-4-ACPBPA (ρ2 antagonist; 2.5 and 5 mM) from 3 days after stroke resulted in a significant improvement in motor recovery on the grid-walking task, with L655,708 and (R)-4-ACPBPA also showing an improvement in the cylinder task. Infarct size was unaffected, and only (R)-4-ACPBPA significantly increased peri-infarct GAT3 expression and decreased the level of reactive astrogliosis. Importantly, inhibiting GABAC receptors affords significant improvement in motor function after stroke. Targeting the ρ-subunit could provide a novel delayed treatment option for stroke recovery.

The flavonoid, 2'-methoxy-6-methylflavone, affords neuroprotection following focal cerebral ischaemia.

Tonic inhibitory currents, mediated by extrasynaptic GABAA receptors, are elevated at a delay following stroke. Flavonoids minimise the extent of cellular damage following stroke, but little is known about their mode of action. We demonstrate that the flavonoid, 2'-methoxy-6-methylflavone (0.1-10 µM; 2'MeO6MF), increases GABAA receptor tonic currents presumably via δ-containing GABAA receptors. Treatment with 2'MeO6MF 1-6 h post focal ischaemia dose dependently decreases infarct volume and improves functional recovery. The effect of 2'MeO6MF was attenuated in δ-/- mice, indicating that the effects of the flavonoid were mediated via δ-containing GABAA receptors. Further, as flavonoids have been shown to have multiple modes of action, we investigated the anti-inflammatory effects of 2'MeO6MF. Using a macrophage cell line, we show that 2'MeO6MF can dampen an LPS-induced elevation in NFkB activity. Assessment of vehicle-treated stroke animals revealed a significant increase in circulating IL1ß, TNFα and IFγ levels. Treatment with 2'MeO6MF dampened the stroke-induced increase in circulating cytokines, which was blocked in the presence of the pan-AKT inhibitor, GSK690693. These studies support the hypothesis that compounds that potentiate tonic inhibition via δ-containing GABAA receptors soon after stroke can afford neuroprotection.

Antidepressant treatment differentially affects the phenotype of high and low stress reactive mice.

Modelling key endophenotypes can be a powerful approach to gain insight into mechanisms underlying the aetiology and pathophysiology of neuropsychiatric disorders. Based on evidence of stress hormone system dysregulations in depression, the Stress Reactivity (SR) mouse model has been generated by a selective breeding approach for extremes in HPA axis reactivity, resulting in high (HR), intermediate (IR) and low (LR) reactive mice. The characterisation of their phenotypic alterations has highlighted many similarities of HR and LR mice with the melancholic and atypical depression, respectively. We therefore aimed to examine whether the antidepressant fluoxetine (10 mg/kg/day i.p., 4-5 weeks) can ameliorate the phenotypic characteristics of HR and LR mice in neuroendocrine functions (HPA axis basal activity, stress reactivity, negative feedback), emotional reactivity/coping-strategy (open field, forced swim tests), spatial learning/memory (Morris water-maze) and hippocampal neurogenesis. Line differences in HPA axis reactivity were maintained under fluoxetine treatment. However, we observed fluoxetine effects on glucocorticoid-induced negative feedback, stress-coping behaviours, cognitive functions and neurogenesis. Specifically, our results revealed line-dependent consequences of fluoxetine treatment: (1) an amelioration of the 'melancholic-like' features of HR mice (reversing the negative feedback resistance, the hyperactive coping style and the memory deficits; increasing hippocampal neurogenesis); (2) an exacerbation of the phenotypic deviations of LR mice (increasing their pronounced negative feedback and passive coping style). Thus, these findings support the predictive validity of antidepressant treatment in the HR mouse line and emphasize the translational value of the SR mouse model for the development of therapeutic strategies based on endophenotype-driven classifications.

Zolpidem is a potent stoichiometry-selective modulator of α1ß3 GABAA receptors: evidence of a novel benzodiazepine site in the α1-α1 interface.

Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1ß3 GABAA receptors in either the 3α1:2ß3 or 2α1:3ß3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a ß3-ß3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn(2+). At the 3α1:2ß3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3ß3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1ß3 (3α1:2ß3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target.

Hippocampal protein expression is differentially affected by chronic paroxetine treatment in adolescent and adult rats: a possible mechanism of "paradoxical" antidepressant responses in young persons.

Selective serotonin reuptake inhibitors (SSRIs) are commonly recognized as the pharmacological treatment of choice for patients with depressive disorders, yet their use in adolescent populations has come under scrutiny following reports of minimal efficacy and an increased risk of suicidal ideation and behavior in this age group. The biological mechanisms underlying these effects are largely unknown. Accordingly, the current study examined changes in hippocampal protein expression following chronic administration of paroxetine in drinking water (target dose = 10 mg/kg for 22 days) to adult and adolescent rats. Results indicated age-specific changes in protein expression, with paroxetine significantly altering expression of 8 proteins in adolescents only and 10 proteins solely in adults. A further 12 proteins were significantly altered in both adolescents and adults. In adults, protein changes were generally suggestive of a neurotrophic and neuroprotective effect of paroxetine, with significant downregulation of apoptotic proteins Galectin 7 and Cathepsin B, and upregulation of the neurotrophic factor Neurogenin 1 and the antioxidant proteins Aldose reductase and Carbonyl reductase 3. Phosphodiesterase 10A, a signaling protein associated with major depressive disorder, was also downregulated (-6.5-fold) in adult rats. Adolescent rats failed to show the neurotrophic and neuroprotective effects observed in adults, instead displaying upregulation of the proapoptotic protein BH3-interacting domain death agonist (4.3-fold). Adolescent protein expression profiles also suggested impaired phosphoinositide signaling (Protein kinase C: -3.1-fold) and altered neurotransmitter transport and release (Syntaxin 7: 5.7-fold; Dynamin 1: -6.9-fold). The results of the present study provide clues as to possible mechanisms underlying the atypical response of human adolescents to paroxetine treatment.

'When an old rat smells a cat': A decline in defense-related, but not accessory olfactory, Fos expression in aged rats.

Comparisons were made between young (3-6 months) and aged (20-30 months) Wistar rats on locomotor activity, emergence, social interaction and cat odor avoidance. Aged rats were less active and spent less time in the open field during the emergence test than younger rats. Older rats also showed fewer contacts with a novel conspecific in the social interaction test, although total duration of interaction did not differ. There were very few behavioral differences between male and female rats. Older rats were less reactive than younger rats in a test of cat odor avoidance. However, they expressed similar amounts of cat odor-induced Fos in the posterior accessory olfactory bulb, a critical region for processing the predator odor stimulus. Older rats had reduced Fos expression in several defense-related brain regions that are normally activated by predator odors such as the medial amygdala and dorsal premammillary nucleus. These results indicate that aged rats are less reactive than younger rats to predator odors due to decreased responsiveness in defense-related but not necessarily olfactory circuits.

Residual social, memory and oxytocin-related changes in rats following repeated exposure to γ-hydroxybutyrate (GHB), 3,4-methylenedioxymethamphetamine (MDMA) or their combination.

RATIONALE: There has been little investigation of the possible lasting adverse effects of γ-hydroxybutyrate (GHB). OBJECTIVES: This study aims to study whether GHB produces residual adverse effects on memory and social behaviour in rats and lasting changes in brain monoamines and oxytocin-related gene expression. METHODS: Rats received daily intraperitoneal injections of GHB (500 mg/kg), methylenedioxymethamphetamine (MDMA; 5 mg/kg) or their combination (GHB/MDMA) over ten consecutive days. Locomotor activity and body weight were assessed during the dosing period and withdrawal-related anxiety was assessed 24 h after drug cessation. After a washout of 4 weeks, rats were tested on the emergence, social interaction, and object recognition tasks over a 2-week period. Monoamine levels in cortex and striatum, and hypothalamic oxytocin and oxytocin receptor mRNA, were then assessed. RESULTS: MDMA and GHB/MDMA caused modest sensitization of locomotor activity over time, while sedative effects of GHB diminished with repeated exposure. GHB-treated rats showed reduced social interaction 24 h after the final dose, indicating GHB withdrawal-induced anxiety. All drug-treated groups displayed residual deficits in social interaction and object recognition. No changes in monoamine levels were detected 8 weeks post-drug. However, MDMA pre-exposure increased hypothalamic oxytocin mRNA while GHB pre-exposure upregulated oxytocin receptor mRNA. GHB/MDMA pre-exposure caused intermediate changes in both of these measures. CONCLUSIONS: GHB treatment caused residual impairments in memory and social behaviour and increases in anxiety, paralleling the lasting adverse effects of MDMA. Both drugs caused lasting neuroadaptations in brain oxytocin systems and this may be related to the long-term social interaction deficiencies caused by both drugs.

A long hangover from party drugs: residual proteomic changes in the hippocampus of rats 8 weeks after γ-hydroxybutyrate (GHB), 3,4-methylenedioxymethamphetamine (MDMA) or their combination.

3,4-Methylenedioxymethamphetamine (MDMA) and gamma-hydroxybutyrate (GHB) are popular party drugs that are used for their euphoric and prosocial effects, and sometimes in combination. Both drugs increase markers of oxidative stress in the hippocampus and can cause lasting impairments in hippocampal-dependent forms of memory. To gain further information on the biochemical mechanisms underlying these effects, the current study examined residual changes in hippocampal protein expression measured 8 weeks after chronic administration of GHB (500mg/kg), MDMA (5mg/kg) or their combination (GHB/MDMA). The drugs were administered once a day for 10 days in an environment with an elevated ambient temperature of 28 degrees C. Results showed significant changes in protein expression, relative to controls, in all three groups: MDMA and GHB given alone caused residual changes in 8 and 5 proteins respectively, while the GHB/MDMA combination significantly changed 6 proteins. The altered proteins had roles in neuroplasticity, neuroprotection, intracellular signalling and cytoskeletal function. The largest change (-4.3-fold) was seen in the MDMA group with the protein C-crk: a protein implicated in learning-related neuroplasticity. The second largest change (3.0-fold) was seen in the GHB group in Glutathione-S-transferase (GST), a protein that protects against oxidative stress. Two cytoskeletal proteins (Tubulin Folding Cofactor B and Tropomyosin-alpha-3 chain) and one plasticity related protein (Neuronal Pentraxin-1 NP1) were similarly changed in both the MDMA and the GHB groups, while two intracellular signalling proteins (alpha-soluble NSF-attachment protein and subunits of the V-type proton ATPase) were changed in both the MDMA/GHB and the MDMA groups. These results provide some insight into the molecular pathways possibly underlying the lasting cognitive deficits arising from GHB and/or MDMA use.

Sedative and hypothermic effects of gamma-hydroxybutyrate (GHB) in rats alone and in combination with other drugs: assessment using biotelemetry.

The recreational drug gamma-hydroxybutyrate (GHB) has euphoric effects and can induce sedation and body temperature changes. GHB is frequently combined with other recreational drugs although these interactions are not well characterised. The present study used biotelemetry to provide a fine-grained analysis of the effects of GHB on body temperature and locomotor activity in freely moving rats, and investigated interactions between GHB and 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine (METH) and various antagonist drugs. GHB (1000mg/kg) caused profound sedation for more than 2h and a complex triphasic effect on body temperature: an initial hypothermia (5-40min), followed by hyperthermia (40-140min), followed again by hypothermia (140-360min). A lower GHB dose (500mg/kg) also caused sedation but only a hypothermic effect that lasted up to 6h. The dopamine D(1) receptor antagonist SCH 23390 (1mg/kg), the opioid antagonist naltrexone (1mg/kg), the benzodiazepine antagonist flumazenil (10mg/kg), and the 5-HT(2A/2C) receptor antagonist ritanserin (1mg/kg) did not prevent the overall sedative or body temperature effects of GHB (1000mg/kg). However the GABA(B) antagonist SCH 50911 (50mg/kg) prevented the hyperthermia induced by GHB (1000mg/kg). Repeated daily administration of GHB (1000mg/kg) produced tolerance to the sedative and hyperthermic effects of the drug and cross-tolerance to the sedative effects of the GABA(B) receptor agonist baclofen (10mg/kg). A high ambient temperature of 28 degrees C prevented the hypothermia obtained with GHB (500mg/kg) at 20 degrees C, while GHB (500mg/kg) reduced the hyperthermia and hyperactivity produced by co-administered doses of MDMA (5mg/kg) or METH (1mg/kg) at 28 degrees C. These results further confirm a role for GABA(B) receptors in the hypothermic and sedative effects of GHB and show an interaction between GHB and MDMA, and GHB and METH, that may be relevant to the experience of recreational users who mix these drugs.

Rats discriminate individual cats by their odor: possible involvement of the accessory olfactory system.

Social behavior in mammals often relies upon the discrimination of same-species individuals via olfactory processing of unique chemosensory signatures. The ability to identify individuals from a different species by their odor (heterospecific discrimination) is less well documented. Here we used a habituation-dishabituation paradigm to demonstrate that rats can discriminate individual cats by their odor. Rats were repeatedly exposed to a collar previously worn by a domestic cat. Strong initial defensive responses (hiding in a small box and vigilant "head out" behavior from the box entrance) habituated with repeated exposure to the same collar. Brain activation following repeated presentation of the same odor - as indexed by c-Fos expression - also habituated in accessory olfactory regions (mitral and granular layers of the posterior accessory olfactory bulb and posteroventral medial amygdala), as well as regions involved in defensive behavior, including the ventromedial and dorsal premammillary hypothalamic nuclei, basolateral amygdala and periaqueductal grey. When a collar taken from a different cat was presented to habituated rats, defensive responses (hiding, vigilance, suppression of grooming) were dishabituated, and c-Fos expression was reinstated in the accessory olfactory system and in defense-related hypothalamic, amygdaloid and brainstem nuclei. Results indicate that rats may process and store details of the chemosensory signatures of individual predators using the accessory olfactory system.