Olga Y. Vekovischeva

From synapse to behavior: Rapid modulation of defined neuronal types with engineered GABAA receptors

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase–induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.

Reduced aggression in AMPA-type glutamate receptor GluR-A subunit-deficient mice

The importance of AMPA‐type glutamate receptors has been demonstrated in neuronal plasticity and in adaptation to drugs of abuse. We studied the involvement of AMPA receptors in social interaction and anxiety and found that in several paradigms of agonistic behavior naïve male mice deficient for the GluR‐A subunit‐ containing AMPA receptors are less aggressive than wild‐type littermates. GluR‐A deficient mice and wild‐type littermates exhibited similar basic behavior and reflexes as monitored by observational Irwins test, but they tended to be less anxious in elevated plus‐maze and light‐dark tests. Maternal aggression or male‐female encounters were not affected which suggests that male hormones are involved in the expression of suppressed aggressiveness. However, testosterone levels and brain monoamines can be excluded and found to be similar between GluR‐A deficient and wild‐type littermates. The reduced AMPA receptor levels caused by the lack of the GluR‐A subunit, and measured by a 30% reduction in hippocampal [3H]‐S‐AMPA binding, seem to be the reason for suppressed male aggressiveness. When we analyzed mice with reduced number of functional AMPA receptors mediated by the genomic introduced GluR‐A(Q582R) channel mutation, we observed again male‐specific suppressed aggression, providing additional evidence for GluR‐A subunit‐containing AMPA receptor involvement in aggression.

TASK-3 Knockout Mice Exhibit Exaggerated Nocturnal Activity, Impairments in Cognitive Functions, and Reduced Sensitivity to Inhalation Anesthetics

The TASK-3 channel is an acid-sensitive two-pore-domain K+ channel, widely expressed in the brain and probably involved in regulating numerous neuronal populations. Here, we characterized the behavioral and pharmacological phenotypes of TASK-3 knockout (KO) mice. Circadian locomotor activity measurements revealed that the nocturnal activity of the TASK-3 KO mice was increased by 38% (P < 0.01) compared with wild-type littermate controls, light phase activity being similar. Although TASK-3 channels are abundant in cerebellar granule cells, the KO mice performed as well as the wild-type mice in walking on a rotating rod or along a 1.2-cm-diameter beam. However, they fell more frequently from a narrower 0.8-cm beam. The KO mice showed impaired working memory in the spontaneous alternation task, with the alternation percentage being 62 ± 3% for the wild-type mice and 48 ± 4% (P < 0.05) for the KO mice. Likewise, during training for the Morris water-maze spatial memory task, the KO mice were slower to find the hidden platform, and in the probe trial, the female KO mice visited fewer times the platform quadrant than the male KO and wild-type mice. In pharmacological tests, the TASK-3 KO mice showed reduced sensitivity to the inhalation anesthetic halothane and the cannabinoid receptor agonist WIN55212-2 mesylate [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] but unaltered responses to the α2 adrenoceptor agonist dexmedetomidine, the i.v. anesthetic propofol, the opioid receptor agonist morphine, and the local anesthetic lidocaine. Overall, our results suggest important contributions of TASK-3 channels in the neuronal circuits regulating circadian rhythms, cognitive functions, and mediating specific pharmacological effects.

The in Vivo Contributions of TASK-1-Containing Channels to the Actions of Inhalation Anesthetics, the alpha~2 Adrenergic Sedative Dexmedetomidine, and Cannabinoid Agonists

Inhalation anesthetics activate and cannabinoid agonists inhibit TWIK-related acid-sensitive K+ channels (TASK)-1 two-pore domain leak K+ channels in vitro. Many neuromodulators, such as noradrenaline, might also manifest some of their actions by modifying TASK channel activity. Here, we have characterized the basal behavioral phenotype of TASK-1 knockout mice and tested their sensitivity to the inhalation anesthetics halothane and isoflurane, the α2 adrenoreceptor agonist dexmedetomidine, and the cannabinoid agonist WIN55212-2 mesylate [R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3,-de]-1,4-benzoxazinyl]-(1-naphtalenyl)methanone mesylate)]. TASK-1 knockout mice had a largely normal behavioral phenotype. Male, but not female, knockout mice displayed an enhanced acoustic startle response. The knockout mice showed increased sensitivity to thermal nociception in a hot-plate test but not in a tail-flick test. The analgesic, sedative, and hypothermic effects of WIN55212-2 (2–6 mg/kg s.c.) were reduced in TASK-1 knockout mice. These results implicate TASK-1-containing channels in supraspinal pain pathways, in particular those modulated by endogenous cannabinoids. TASK-1 knockout mice were less sensitive to the anesthetic effects of halothane and isoflurane than wild-type littermates, requiring higher anesthetic concentrations to induce immobility as reflected by loss of the tail-withdrawal reflex. Our results support the idea that the activation of multiple background K+ channels is crucial for the high potency of inhalation anesthetics. Furthermore, TASK-1 knockout mice were less sensitive to the sedative effects of dexmedetomidine (0.03 mg/kg s.c.), suggesting a role for the TASK-1 channels in the modulation of function of the adrenergic locus coeruleus nuclei and/or other neuronal systems.

GABA Site Agonist Gaboxadol Induces Addiction-Predicting Persistent Changes in Ventral Tegmental Area Dopamine Neurons But Is Not Rewarding in Mice or Baboons

Dopamine neurons of the ventral tegmental area (VTA) are involved at early phases of drug addiction. Even the first in vivo dose of various abused drugs induces glutamate receptor plasticity at the excitatory synapses of these neurons. Benzodiazepines that suppress the inhibitory GABAergic interneurons in the VTA via facilitation of synaptic GABAA receptors have induced neuroplasticity in dopamine neurons due to this disinhibitory mechanism. Here, we have tested a non-benzodiazepine direct GABA site agonist 4,5,6,7-tetrahydroisoxazolol[4,5-c]pyridine-3-ol (THIP) (also known as gaboxadol) that acts preferentially via high-affinity extrasynaptic GABAA receptors. A single sedative dose of THIP (6 mg/kg) to mice induced glutamate receptor plasticity for at least 6 d after administration. Increased AMPA/NMDA receptor current ratio and increased frequency, amplitude, and rectification of AMPA receptor responses suggested persistent targeting of GluA2-lacking AMPA receptors in excitatory synapses of VTA dopamine neurons ex vivo after THIP administration. This effect was abolished in GABAA receptor δ−/− mice, which have a loss of extrasynaptic GABAA receptors. In behavioral experiments, we found neither acute reinforcement in intravenous self-administration sessions with THIP at relevant doses using a yoked control paradigm in mice nor in baboons using a standard paradigm for assessing drug abuse liability; nor was any place preference found after conditioning sessions with various doses of THIP but rather a persistent aversion in 6 mg/kg THIP-conditioned mice. In summary, we found that activation of extrasynaptic δ-subunit-containing GABAA receptors leads to glutamate receptor plasticity of VTA dopamine neurons, but is not rewarding, and, instead, induces aversion.

Behavioural correlates of an altered balance between synaptic and extrasynaptic GABAAergic inhibition in a mouse model

GABAA receptors mediate fast phasic inhibitory postsynaptic potentials and participate in slower tonic extrasynaptic inhibition. Thy1α6 mice with ectopic forebrain expression of GABAA receptor α6 subunits exhibit increased extrasynaptic GABAA receptor‐mediated background conductance and reduced synaptic GABAA receptor currents in hippocampal CA1 neurons [W. Wisden et al. (2002) Neuropharmacology 43, 530–549]. Here we demonstrate that isolated CA1 neurons of these mice showed furosemide‐sensitivity of GABA‐evoked currents, confirming the functional expression of α6 subunit. In addition, receptor autoradiography of the CA1 region of Thy1α6 brain sections revealed pharmacological features that are unique for α6βγ2 and α6β receptors. The existence of atypical α6β receptors was confirmed after completely eliminating GABAA receptors containing γ1, γ2, γ3 or δ subunits using serial immunoaffinity chromatography on subunit‐specific GABAA receptor antibodies. Behaviourally, the Thy1α6 mice showed normal features with slightly enhanced startle reflex and struggle‐escape behaviours. However, they were more sensitive to GABAA antagonists DMCM (shorter latency to writhing clonus) and picrotoxinin (shorter latency to generalized convulsions). Tiagabine, an antiepileptic GABA‐uptake inhibitor that increases brain GABA levels, delayed picrotoxinin‐induced convulsions at a low dose of 3.2 mg/kg in Thy1α6 mice, but not in control mice; however, the overall effect of higher tiagabine doses on the convulsion latency remained smaller in the Thy1α6 mice. Altered balance between extrasynaptic and synaptic receptors thus affects seizure sensitivity to GABAergic convulsants. Importantly, the increased extrasynaptic inhibition, even when facilitated in the presence of tiagabine, was not able fully to counteract enhanced seizure induction by GABAA antagonists.

Removal of GABAA Receptor γ2 Subunits from Parvalbumin Neurons Causes Wide-Ranging Behavioral Alterations

We investigated the behavioral significance of fast synaptic inhibition by αβγ2-type GABAA receptors on parvalbumin (Pv) cells. The GABAA receptor γ2 subunit gene was selectively inactivated in Pv-positive neurons by Cre/loxP recombination. The resulting Pv-Δγ2 mice were relatively healthy in the first postnatal weeks; but then as Cre started to be expressed, the mice progressively developed wide-ranging phenotypic alterations including low body weight, motor deficits and tremor, decreased anxiety levels, decreased pain sensitivity and deficient prepulse inhibition of the acoustic startle reflex and impaired spatial learning. Nevertheless, the deletion was not lethal, and mice did not show increased mortality even after one year. Autoradiography with t-butylbicyclophosphoro[35S]thionate suggested an increased amount of GABAA receptors with only α and β subunits in central nervous system regions that contained high levels of parvalbumin neurons. Using BAC-transgenesis, we reduced some of the Pv-Δγ2 phenotype by selectively re-expressing the wild-type γ2 subunit back into some Pv cells (reticular thalamic neurons and cerebellar Pv-positive neurons). This produced less severe impairments of motor skills and spatial learning compared with Pv-Δγ2 mice, but all other deficits remained. Our results reveal the widespread significance of fast GABAergic inhibition onto Pv-positive neurons for diverse behavioral modalities, such as motor coordination, sensorimotor integration, emotional behavior and nociception.

DECOMPRESSIVE CRANIECTOMY FOR INTRACEREBRAL HEMORRHAGE

OBJECTIVEIntracerebral hemorrhage (ICH) has a high mortality rate and leaves most survivors disabled. The dismal outcome is mostly due to the mass effect of hematoma plus edema. Major clinical trials show no benefit from surgical or medical treatment. Decompressive craniectomy has, however, proven beneficial for large ischemic brain infarction with massive swelling. We hypothesized that craniectomy can improve ICH outcome as well. METHODSWe used the model of autologous blood injection into the basal ganglia in rats. After induction of ICH and then magnetic resonance imaging, animals were randomly allocated to groups representing no craniectomy (n = 10) or to craniectomy at 1, 6, or 24 hours. A fifth group without ICH underwent craniectomy only. Neurological and behavioral outcomes were assessed on days 1, 3, and 7 after ICH induction. Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells were counted. RESULTSAfter 7 days, compared with the ICH + no craniectomy group, all craniectomy groups had strikingly lower mortality (P < 0.01), much better neurological outcome (P < 0.001), and more favorable behavioral outcome. A trend occurred in the ICH + no craniectomy group toward more robust apoptosis. CONCLUSIONDecompressive craniectomy performed up to 24 hours improved outcome after experimental ICH, with earlier intervention of greater benefit.

Neurosteroid Agonist at GABAA Receptor Induces Persistent Neuroplasticity in VTA Dopamine Neurons

The main fast-acting inhibitory receptors in the mammalian brain are γ-aminobutyric acid type-A (GABAA) receptors for which neurosteroids, a subclass of steroids synthesized de novo in the brain, constitute a group of endogenous ligands with the most potent positive modulatory actions known. Neurosteroids can act on all subtypes of GABAA receptors, with a preference for δ-subunit-containing receptors that mediate extrasynaptic tonic inhibition. Pathological conditions characterized by emotional and motivational disturbances are often associated with perturbation in the levels of endogenous neurosteroids. We studied the effects of ganaxolone (GAN)—a synthetic analog of endogenous allopregnanolone that lacks activity on nuclear steroid receptors—on the mesolimbic dopamine (DA) system involved in emotions and motivation. A single dose of GAN in young mice induced a dose-dependent, long-lasting neuroplasticity of glutamate synapses of DA neurons ex vivo in the ventral tegmental area (VTA). Increased α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/N-methyl-D-aspartate ratio and rectification of AMPA receptor responses even at 6 days after GAN administration suggested persistent synaptic targeting of GluA2-lacking AMPA receptors. This glutamate neuroplasticity was not observed in GABAA receptor δ-subunit-knockout (δ-KO) mice. GAN (500 nM) applied locally to VTA selectively increased tonic inhibition of GABA interneurons and triggered potentiation of DA neurons within 4 h in vitro. Place-conditioning experiments in adult wild-type C57BL/6J and δ-KO mice revealed aversive properties of repeated GAN administration that were dependent on the δ-subunits. Prolonged neuroadaptation to neurosteroids in the VTA might contribute to both the physiology and pathophysiology underlying processes and changes in motivation, mood, cognition, and drug addiction.

Importance of GluA1 Subunit-Containing AMPA Glutamate Receptors for Morphine State-Dependency

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.