Saku T. Sinkkonen

Impact of ε and θ subunits on pharmacological properties of α3β1 GABAA receptors expressed in Xenopus oocytes

Backgroundγ-Aminobutyric acid type A (GABAA) receptors provide the main inhibitory control in the brain. Their heterogeneity may make it possible to precisely target drug effects to selected neuronal populations. In situ hybridization using rat brain sections has revealed a unique expression of GABAA receptor ε and θ subunit transcripts in the locus coeruleus, where they are accompanied at least by α3, α2, β1 and β3 subunits. Here, we studied the pharmacology of the human α3β1, α3β1ε, α3β1θ and α3β1εθ receptor subtypes expressed in Xenopus oocytes and compared them with the γ2 subunit-containing receptors.ResultsThe GABA sensitivites and effects of several positive modulators of GABAA receptors were studied in the absence and the presence of EC25 GABA using the two-electrode voltage-clamp method. We found 100-fold differences in GABA sensitivity between the receptors, α3β1ε subtype being the most sensitive and α3β1γ2 the least sensitive. Also gaboxadol dose-response curves followed the same sensitivity rank order, with EC50 values being 72 and 411 μM for α3β1ε and α3β1γ2 subtypes, respectively. In the presence of EC25 GABA, introduction of the ε subunit to the receptor complex resulted in diminished modulatory effects by etomidate, propofol, pregnanolone and flurazepam, but not by pentobarbital. Furthermore, the α3β1ε subtype displayed picrotoxin-sensitive spontaneous activity. The θ subunit-containing receptors were efficiently potentiated by the anesthetic etomidate, suggesting that θ subunit could bring the properties of β2 or β3 subunits to the receptor complex.ConclusionThe ε and θ subunits bring additional features to α3β1 GABAA receptors. These receptor subtypes may constitute as novel drug targets in selected brain regions, e.g., in the brainstem locus coeruleus nuclei.

Mouse models of Angelman syndrome, a neurodevelopmental disorder, display different brain regional GABAA receptor alterations

Angelman syndrome is a severe neurodevelopmental disorder with cognitive impairment and neurological deficits. It results from a maternal deletion of human chromosome 15q11-13 containing two candidate genes E6-P ubiquitin-protein ligase (UBE3A) and GABA(A) receptor beta3 subunit (GABRB3), the latter of which has been also linked to autism. To clarify the potential role of GABA(A) beta3 subunit-containing inhibitory receptors in these disorders, we applied ligand autoradiography on brain sections from mice with inactivated GABRB3 or maternal UBE3A genes. Binding of GABA(A) receptor channel ([(35)S]t-butylbicyclophosphorothionate) and benzodiazepine ([(3)H]Ro 15-4513) site ligands was reduced in selected brain regions of the beta3-deficient mice as compared to controls, while the UBE3A-deficient mice failed to show reduced GABA(A) receptors. The results, suggesting two different pathophysiological mechanisms, are in agreement with positron emission tomography results from Angelman syndrome patients of the corresponding genetic backgrounds.

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.

Autoradiographic imaging of altered synaptic αβγ2 and extrasynaptic αβ GABAA receptors in a genetic mouse model of anxiety

Abstract To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor γ2 subunit (γ2+/−) were studied using ligand autoradiographic assays on brain cryostat sections. The [ 35 S ]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant α1/6β3 than in α1/2β3γ2 or β2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [ 35 S ]TBPS binding in the γ2+/− mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of γ2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [ 3 H ]Ro 15-4513 autoradiography. As the γ2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic αβ3 receptors can be visualized in vitro as atypical GABA-insensitive [ 35 S ]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone γ2+/− mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.

Morphine withdrawal increases expression of GABA(A) receptor epsilon subunit mRNA in locus coeruleus neurons

An increase in the activity of brain stem locus coeruleus noradrenergic neurons has been hypothesised to be a major factor accounting for opiate withdrawal symptoms. These neurons are under GABAergic inhibition. Their GABAA receptors have unique pharmacological properties, most likely due to the enriched expression of GABAA receptor subtypes containing novel &epsis; and &thetas; subunits. Using in situ hybridisation of cryostat sections, we now report a significant increase in the &epsis; subunit mRNA expression after precipitation of opioid withdrawal by naloxone. Similar changes were detected in tyrosine hydroxylase mRNA expression. The results suggest increased formation of unique GABAA receptor subtype(s) in the locus coeruleus neurons during increased neuronal activity.

Human locus coeruleus neurons express the GABAA receptor γ2 subunit gene and produce benzodiazepine binding

Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for gamma-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABA(A) receptors are composed of a variable array of subunits forming heteropentameric chloride channels with different pharmacological properties. The gamma2 subunit is essential for the formation of the binding site for benzodiazepines, allosteric modulators of GABA(A) receptors that are clinically often used as sedatives/hypnotics and anxiolytics. There are contradictory reports in regard to the gamma2 subunits expression and participation in the functional GABA(A) receptors in the mammalian locus coeruleus. We report here that the gamma2 subunit is transcribed and participates in the assembly of functional GABA(A) receptors in the tyrosine hydroxylase-positive neuromelanin-containing neurons within postmortem human locus coeruleus as demonstrated by in situ hybridization with specific gamma2 subunit oligonucleotides and autoradiographic assay for flumazenil-sensitive [(3)H]Ro 15-4513 binding to benzodiazepine sites. These sites were also sensitive to the alpha1 subunit-preferring agonist zolpidem. Our data suggest a species difference in the expression profiles of the alpha1 and gamma2 subunits in the locus coeruleus, with the sedation-related benzodiazepine sites being more important in man than rodents. This may explain the repeated failures in the transition of novel drugs with a promising neuropharmacological profile in rodents to human clinical usage, due to intolerable sedative effects.

Complication rates of radiofrequency surgery in the upper airways: a single institution experience

Conclusion: Radiofrequency (RF) surgery of the upper airways appears to be a safe procedure with an acceptable incidence of minor and moderate complications. Objectives: RF surgery is increasingly used in the treatment of patients with sleep disordered breathing and inferior turbinate hypertrophy. Our aim was to investigate the incidence and the severity of the complications of RF surgery in the upper airways. Patients and methods: This was a retrospective, observational study at a tertiary care centre, academic teaching hospital during 1 year. Data from medical records were collected on 753 consecutive patients treated with RF surgery of the inferior turbinate, soft palate and base of the tongue. Patients with synchronous surgical treatment were excluded. Results: In all, 413 patients (66.3% males) with a mean age of 44.7 years (range 8–83 years) were treated with 2926 RF surgery ablations in 524 treatment sessions. There were no severe complications. The overall incidence of minor and moderate complications was low, i.e. 2.7% (11/524) and 0.6% (3/524) of the treatment sessions, and 0.5% (11/2926) and 0.1% (3/2926) of the ablations, respectively.

Compensation by reduced L‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor responses in a mouse model with reduced γ‐aminobutyric acid type A receptor‐mediated synaptic inhibition

L‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor antagonists increase the threshold for electroshock‐induced convulsions. Here, we show that a transgenic mouse line overexpressing cerebellum‐restricted γ‐aminobutyric acid type A (GABAA) receptor α6 subunit in the hippocampal CA1 pyramidal cells (Thy1α6 mouse line) exhibits about a 20% increase in the electroshock current intensity inducing tonic hindlimb extension convulsion in 50% of the mice compared with that of their wild‐type controls. AMPA receptor‐mediated miniature excitatory postsynaptic currents (mEPSCs) in patch clamp recordings of CA1 pyramidal neurons in hippocampal slices had decreased amplitudes (8.4 ± 2.2 pA) in the transgenics compared with the wild types (10.3 ± 2.5 pA) but showed no change in current decay or frequency. Our results suggest that decreased AMPA‐mediated neurotransmission might explain the increased threshold for electroconvulsions and warrant further studies on the regulation between various components of inhibition and excitation in neurons.

Radiofrequency ablation for treatment of auricular keloids: Our experience in eleven patients

Keloids are caused by abnormal wound healing when scar tissue invades surrounding normal skin. The growth of keloids is typically unregulated and may continue indefinitely. Auricular keloids typically occur after trauma, piercing or surgery. Clinical diagnosis is the golden standard. There is racial variation in the incidence of keloids and genetic predisposition is significant. The treatment of auricular keloids is challenging due to their tendency to reoccur. Numerous different options and protocols have been suggested. Surgical treatment options include conventional surgery with or without skin grafts and cryosurgery. Medical treatment such as steroid injections or more experimental options such as verapamil, 5-Fluorouracil, interferon, mitomycin C injections or imiquimod cream are often combined to surgical treatment. Radiation therapy with doses ranging from 3 to 10 Gy has also been used as a treatment modality. Pressure therapy in a variety of modifications is widely used as an accompanying option. The reported success rates for the treatment of auricular keloids varies greatly ranging from 0 to 60%. The true recurrence rate is unknown due to study designs and limited patient numbers. There are no randomised studies, and majority of reports are based on small case series with no control groups. It is safe to say that a significant part of keloids do recur and the treatment of auricular keloids is based on experiences and institutional or individual preferences rather than scientific basis. Fruth and co-workers described the usage of radiofrequency ablation for the treatment of auricular keloids. Radiofrequency ablation is widely used for tissue volume reduction for disorders such as habitual snoring (soft palate) and chronic nasal obstruction (nasal turbinates). Radiofrequency energy is used to create controlled protein denaturation or necrosis in soft-tissue structures leading to scar formation and volume reduction. Radiofrequency ablation can be considered as minimally invasive treatment modality with no major disadvantages. Fruth and co-workers reported promising results in their limited patient material, and good cosmetic results were achieved in 10 of 14 patients. In this correspondence, we confirm the findings by Fruth and co-workers and present our results for the treatment of auricular keloids with radiofrequency ablation. This is the second paper on this promising novel treatment option.

Balloon Eustachian tuboplasty under local anesthesia: Is it feasible?: Feasibility of BET Under Local Anesthesia

To study whether balloon Eustachian tuboplasty (BET) is a feasible and safe procedure under local anesthesia.