Type 2 diabetes and treatment intensification in primary care in Finland.

AIM: To identify how the electronic health record (EHR) systems and national registers can be used for research purposes. We focused on how the primary care physicians adhere to clinical guidelines. METHODS: Study population included incident type 2 diabetes patients from four selected regions. Data were collected in two phases. At the first phase study cohort was identified using the prescription registers of the Social Insurance Institution (SII) and EHR systems used within the study regions. At second phase, data were collected from SII's registers, local EHR systems, the hospital discharge and the primary care registers of National Institute for Health and Welfare. RESULTS: Metformin was the most common choice as first drug. Among all study patients, 8375 (76.0%) started metformin monotherapy or combinations. The treatment was intensified at variable levels of HbA1c depending on the area. DPP4-inhibitors were by far the most common agent for treatment intensification. Sulphonylureas were used less often than basal insulin as the second-line agent. The use of DPP4-inhibitors increased between years 2009-2010, when first DPP4-inhibitor received reimbursement and this class became dominant drug for treatment intensification increasingly thereafter. CONCLUSIONS: The EHR systems and national registers can be used for research purposes in Finland. The realization of diabetes treatment national guidelines are followed in primary care to a large extent. However, the subsequent intensification of therapy was delayed and occurred at elevated Hba1c levels.

Applications of genomic medicine in the treatment of diseases.

Individualized medicine, based on a detailed mapping of the patient's disease mechanisms, is becoming an essential part of treatment for an increasing number of diseases. In the past few years, the possibility to determine the abnormal genome and transcriptome of diseased cells at a reasonable cost has been the major advance. The vast amount of data accumulated from one patient will set requirements for data extraction tools, in order to have the essential information affecting the treatment of the patient information quickly and reliably at the disposal of attending physicians. A computerized decision support system connected to the information systems of the hospital is an integral part of individualized treatment. Although the application of genomic and other profiling information is challenging, individualization of medication provides great promises more effective and safer treatment.

Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABAA Receptor γ2 Subunit in the Cerebellum.

Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABAA receptors. In recombinant receptors and genetic mouse models their actions depend on the α, ß, and δ subunits of the receptor, especially on those that form extrasynaptic GABAA receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunit-containing receptors and even on αß binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5ß-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABAA receptor-mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABAA receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5ß-pregnan-3α-ol-20-one. Motor-impairing effects of 5ß-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABAA responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABAA receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABAA receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic αß or αßδ receptors lead to enhanced changes in the cerebellum-generated behavior.

Removal of GABA(A) receptor γ2 subunits from parvalbumin neurons causes wide-ranging behavioral alterations.

We investigated the behavioral significance of fast synaptic inhibition by αßγ2-type GABA(A) receptors on parvalbumin (Pv) cells. The GABA(A) 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[(35)S]thionate suggested an increased amount of GABA(A) receptors with only α and ß 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.

Actions of two GABAA receptor benzodiazepine-site ligands that are mediated via non-γ2-dependent modulation.

The potent sedative-hypnotic zolpidem and the convulsant methyl-6,7-dimethoxy-4-ethyl-ß-carboline-3-carboxylate (DMCM) act primarily by binding to the benzodiazepine site of the main inhibitory neurotransmitter receptor, the pentameric γ-aminobutyric acid type A receptor (GABA(A)). This binding depends critically on the wild-type F77 residue of the GABA(A) receptor γ2 subunit. Mice with γ2 subunit F77I point mutation (γ2I77 mouse line) lose the high-affinity nanomolar binding of these ligands as well as their most robust behavioral actions at low doses. Interestingly, the γ2I77 mice offer a tool to study the actions of these substances mediated via other possible binding sites of the GABA(A) receptor. In ligand autoradiographic experiments, we discovered in γ2I77 mouse brain sections a significant amount of residual non-γ2 subunit-dependent benzodiazepine site binding enriched to the striatum and septum. Zolpidem only weakly affected this residual binding at micromolar concentrations, and only a high zolpidem dose (≥ 40 mg/kg) caused sedation and deficits in motor coordination in γ2I77 mice. DMCM had an agonistic action through a secondary, low-affinity non-benzodiazepine binding site of the GABA(A) receptor in the forebrain of γ2I77 mice, and this drug also fully displaced the residual benzodiazepine-site labeling. In behavioral tests, a high dose (20mg/kg) of DMCM was sedative and modulated fear learning. DMCM, but not zolpidem, acted as an agonist in recombinant GABA(A) α1/6ß3 receptors studied using ligand binding and electrophysiological assays. Our results highlight the less well-known actions of high doses of DMCM and zolpidem that are not mediated via the γ2 subunit-containing benzodiazepine site of the GABA(A) receptor.

Ro 15-4513 Antagonizes Alcohol-Induced Sedation in Mice Through αßγ2-type GABA(A) Receptors.

Ethyl alcohol (ethanol) has many molecular targets in the nervous system, its potency at these sites being low compared to those of sedative drugs. This has made it difficult to discover ethanol's binding site(s). There are two putative binding sites at γ-aminobutyric acid (GABA) type A receptor subtypes for the proposed ethanol antagonist Ro 15-4513, the established γ2 subunit-dependent benzodiazepine site and the recently reported δ subunit-dependent Ro 15-4513/ethanol binding site. Here, we aimed at clarifying the in vivo role of Ro 15-4513 at these two sites. We found that the antagonism of ethanol actions by Ro 15-4513 in wildtype mice was dependent on the test: an open field test showed that light sedation induced by 1.5-1.8 g/kg ethanol was sensitive to Ro 15-4513, whereas several tests for ethanol-induced anxiolytic effects showed that the ethanol-induced effects were insensitive to Ro 15-4513. Antagonism of ethanol-induced sedation by Ro 15-4513 was unaffected in GABA(A) receptor δ subunit knockout mice. By contrast, when testing the GABA(A) receptor γ2 subunit F77I knock-in mouse line (γ2I77 mice) with its strongly reduced affinity of the benzodiazepine sites for Ro 15-4513, we found that the ethanol-induced sedation was no longer antagonized by Ro 15-4513. Indeed, γ2I77 mice had only a small proportion of high-affinity binding of [(3)H]Ro 15-4513 left as compared to wildtype mice, especially in the caudate-putamen and septal areas, but these residual sites are apparently not involved in ethanol antagonism. In conclusion, we found that Ro 15-4513 abolished the sedative effect of ethanol by an action on γ2 subunit-dependent benzodiazepine sites.

K+ channel TASK-1 knockout mice show enhanced sensitivities to ataxic and hypnotic effects of GABA(A) receptor ligands.

TASK two-pore-domain leak K(+) channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA(A) receptors in GABA(A) alpha6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA(A) receptor subtypes: benzodiazepines (diazepam and flurazepam, active at alpha1betagamma2, alpha2betagamma2, alpha3betagamma2, and alpha5betagamma2 subtypes), zolpidem (alpha1betagamma2 subtype), propofol (beta2-3-containing receptors), gaboxadol (alpha4betadelta and alpha6betadelta subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA(A) receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of alpha2beta2/3gamma2 and alpha3beta2/3gamma2 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of alpha4betadelta or alpha6betadelta subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA(A) receptors, the up-regulation of GABA(A) receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.

Does ethanol act preferentially via selected brain GABAA receptor subtypes? the current evidence is ambiguous.

In rodent models, gamma-aminobutyric acid A (GABAA) receptors with the alpha6 and delta subunits, expressed in the cerebellar and cochlear nucleus granule cells, have been linked to ethanol sensitivity and voluntary ethanol drinking. Here, we review the findings. When considering both in vivo contributions and data on cloned receptors, the evidence for direct participation of the alpha6-containing receptors to increased ethanol sensitivity is poor. The alpha6 subunit-knockout mouse lines do not have any changed sensitivity to ethanol, although these mice do display increased benzodiazepine sensitivity. However, in general the compensations occurring in knockout mice (regardless of which particular gene is knocked out) tend to fog interpretations of drug actions at the systems level. For example, the alpha6 knockout mice have increased TASK-1 channel expression in their cerebellar granule cells, which could influence sensitivity to ethanol in the opposite direction to that obtained with the alpha6 knockouts. Indeed, TASK-1 knockout mice are more impaired than wild types in motor skills when given ethanol; this might explain why GABAA receptor alpha6 knockout mice have unchanged ethanol sensitivities. As an alternative to studying knockout mice, we examined the claimed delta subunit-dependent/gamma2 subunit-independent ethanol/[3H]Ro 15-4513 binding sites on GABAA receptors. We looked at [3H]Ro 15-4513 binding in HEK 293 cell membrane homogenates containing rat recombinant alpha6/4beta3delta receptors and in mouse brain sections. Specific high-affinity [3H]Ro 15-4513 binding could not be detected under any conditions to the recombinant receptors or to the cerebellar sections of gamma2(F77I) knockin mice, nor was this binding to brain sections of wild-type C57BL/6 inhibited by 1-100 mM ethanol. Since ethanol may act on many receptor and channel protein targets in neuronal membranes, we consider the alpha6 (and alpha4) subunit-containing GABAA receptors unlikely to be directly responsible for any major part of ethanol's actions. Therefore, we finish the review by discussing more generally alcohol and GABAA receptors and by suggesting potential future directions for this research.

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 gamma2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase-induced swapping of the gamma2 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.

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 alpha(2) 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.