Subunit Knockout Mice Research Articles

Understanding of nicotinic acetylcholine receptors

Understanding of nicotinic acetylcholine receptors

Enhanced long-term and impaired short-term spatial memory in GluA1 AMPA receptor subunit knockout mice: Evidence for a dual-process memory model

The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of GluA1-dependent synaptic plasticity for short-term memory of recently visited places, but not for the ability to form long-term associations between a particular spatial location and an outcome. This hypothesis is in concordance with the theory that short-term and long-term memory depend on dissociable psychological processes. In this study we tested GluA1-/- mice on both short-term and long-term spatial memory using a simple novelty preference task. Mice were given a series of repeated exposures to a particular spatial location (the arm of a Y-maze) before their preference for a novel spatial location (the unvisited arm of the maze) over the familiar spatial location was assessed. GluA1-/- mice were impaired if the interval between the trials was short (1 min), but showed enhanced spatial memory if the interval between the trials was long (24 h). This enhancement was caused by the interval between the exposure trials rather than the interval prior to the test, thus demonstrating enhanced learning and not simply enhanced performance or expression of memory. This seemingly paradoxical enhancement of hippocampus-dependent spatial learning may be caused by GluA1 gene deletion reducing the detrimental effects of short-term memory on subsequent long-term learning. Thus, these results support a dual-process model of memory in which short-term and long-term memory are separate and sometimes competitive processes.

γ-Hydroxybutyrate (GHB) induces GABA B receptor independent intracellular Ca 2+ transients in astrocytes, but has no effect on GHB or GABA B receptors of medium spiny neurons in the nucleus accumbens

We report on cellular actions of the illicit recreational drug γ-hydroxybutyrate (GHB) in the brain reward area nucleus accumbens. First, we compared the effects of GHB and the GABA B receptor agonist baclofen. Neither of them affected the membrane currents of medium spiny neurons in rat nucleus accumbens slices. GABAergic and glutamatergic synaptic potentials of medium spiny neurons, however, were reduced by baclofen but not GHB. These results indicate the lack of GHB as well as postsynaptic GABA B receptors, and the presence of GHB insensitive presynaptic GABA B receptors in medium spiny neurons. In astrocytes GHB induced intracellular Ca 2+ transients, preserved in slices from GABA B receptor type 1 subunit knockout mice. The effects of tetrodotoxin, zero added Ca 2+ with/without intracellular Ca 2+ store depletor cyclopiazonic acid or vacuolar H-ATPase inhibitor bafilomycin A1 indicate that GHB-evoked Ca 2+ transients depend on external Ca 2+ and intracellular Ca 2+ stores, but not on vesicular transmitter release. GHB-induced astrocytic Ca 2+ transients were not affected by the GHB receptor-specific antagonist NCS-382, suggesting the presence of a novel NCS-382-insensitive target for GHB in astrocytes. The activation of astrocytes by GHB implies their involvement in physiological actions of GHB. Our findings disclose a novel profile of GHB action in the nucleus accumbens. Here, unlike in other brain areas, GHB does not act on GABA B receptors, but activates an NCS-382 insensitive GHB-specific target in a subpopulation of astrocytes. The lack of either post- or presynaptic effects on medium spiny neurons in the nucleus accumbens distinguishes GHB from many drugs and natural rewards with addictive properties and might explain why GHB has only a weak reinforcing capacity.

Brain regional distribution of GABA A receptors exhibiting atypical GABA agonism: Roles of receptor subunits

The major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABA A receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[ 35S]thionate ([ 35S]TBPS) binding to GABA A receptors in brain sections and compared the displacing capacities of 10 mM GABA and 1 mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABA A receptor α1, α4, δ, and α4 + δ subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to “GABA-insensitive” (GIS) [ 35S]TBPS binding. THIP displaced more [ 35S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of δ KO and α4 + δ KO mice, being only slightly diminished in that of α1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in α4 KO mice. However, it was fully abolished in α1 KO mice, indicating that the α1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABA A receptors in brain sections showing reduced displacing capacity of [ 35S]TBPS binding by GABA (partial agonism) minimally require the assembly of α1 and β subunits in the forebrain and of α6 and β subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABA A receptors.

The Hippocampus and Cingulate Cortex Differentially Mediate the Effects of Nicotine on Learning Versus on Ethanol-Induced Learning Deficits Through Different Effects at Nicotinic Receptors

IntroductionThe current study examined the effects of nicotine infusion into the dorsal hippocampus or anterior cingulate on fear conditioning and on ethanol-induced deficits in fear conditioning, and whether these effects involved receptor activation or inactivation.MethodsConditioning consisted of two white noise (30 seconds, 85 dB)–foot shock (2 seconds, 0.57 mA) pairings. Saline or ethanol was administered to C57BL/6 mice 15 minutes before training and saline or nicotine was administered 5 minutes before training or before training and testing. The ability of the high-affinity nicotinic acetylcholinergic receptor (nAChR) antagonist dihydro-beta-erythroidine (DHβE) to modulate the effects of ethanol and nicotine was also tested; saline or DHβE was administered 25 (injection) or 15 (infusion) minutes before training or before training and testing.ResultsInfusion of nicotine into the hippocampus enhanced contextual fear conditioning but had no effect on ethanol-induced learning deficits. Infusion of nicotine into the anterior cingulate ameliorated ethanol-induced deficits in contextual and cued fear conditioning but had no effect on learning in ethanol-naïve mice. DHβE blocked the effects of nicotine on ethanol-induced deficits; interestingly, DHβE alone and co-administration of sub-threshold doses of DHβE and nicotine also ameliorated ethanol-induced deficits but failed to enhance learning. Finally, DHβE failed to ameliorate ethanol-induced deficits in β2 nAChR subunit knockout mice.ConclusionsThese results suggest that nicotine acts in the hippocampus to enhance contextual learning, but acts in the cingulate to ameliorate ethanol-induced learning deficits through inactivation of high-affinity β2 subunit-containing nAChRs.

Presence of α7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective α‐neurotoxins in histochemistry

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.

A novel nicotinic acetylcholine receptor subtype in basal forebrain cholinergic neurons with high sensitivity to amyloid peptides.

Nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunits are thought to assemble as homomers. alpha7-nAChR function has been implicated in learning and memory, and alterations of alpha7-nAChR have been found in patients with Alzheimer's disease (AD). Here we report findings consistent with a novel, naturally occurring nAChR subtype in rodent, basal forebrain cholinergic neurons. In these cells, alpha7 subunits are coexpressed, colocalize, and coassemble with beta2 subunit(s). Compared with homomeric alpha7-nAChRs from ventral tegmental area neurons, functional, presumably heteromeric alpha7beta2-nAChRs on cholinergic neurons freshly dissociated from medial septum/diagonal band (MS/DB) exhibit relatively slow kinetics of whole-cell current responses to nicotinic agonists and are more sensitive to the beta2 subunit-containing nAChR-selective antagonist, dihydro-beta-erythroidine (DHbetaE). Interestingly, presumed, heteromeric alpha7beta2-nAChRs are highly sensitive to functional inhibition by pathologically relevant concentrations of oligomeric, but not monomeric or fibrillar, forms of amyloid beta(1-42) (Abeta(1-42)). Slow whole-cell current kinetics, sensitivity to DHbetaE, and specific antagonism by oligomeric Abeta(1-42) also are characteristics of heteromeric alpha7beta2-nAChRs, but not of homomeric alpha7-nAChRs, heterologously expressed in Xenopus oocytes. Moreover, choline-induced currents have faster kinetics and less sensitivity to Abeta when elicited from MS/DB neurons derived from nAChR beta2 subunit knock-out mice rather than from wild-type mice. The presence of novel, functional, heteromeric alpha7beta2-nAChRs on basal forebrain cholinergic neurons and their high sensitivity to blockade by low concentrations of oligomeric Abeta(1-42) suggests possible mechanisms for deficits in cholinergic signaling that could occur early in the etiopathogenesis of AD and might be targeted by disease therapies.

Distinguishing between calpain heterodimerization and homodimerization

The two main mammalian calpains, 1 and 2, are heterodimers of a large 80 kDa and a small 28 kDa subunit that together bind multiple calcium ions during enzyme activation. The main contact between the two subunits of these intracellular cysteine proteases is through a pairing of the fifth EF-hand of their C-terminal penta-EF-hand (PEF) domains. From modeling studies and observation of crystal structures, it is not obvious why these calpains form heterodimers with the small subunit rather than homodimers of the large subunit, as suggested for calpain 3 (p94). Therefore, we have used a differential tagging system to determine which of the other PEF domain-containing calpains form heterodimers and which form homodimers. His6-tagged PEF domains of calpains 1, 3, 9 and 13 were coexpressed with the PEF domain of the small subunit that had been tagged with an antifreeze protein. As predicted, the PEF domain of calpain 1 heterodimerized and that of calpain 3 formed a homodimer. The PEF domain of digestive tract-specific calpain 9 heterodimerized with the small subunit, and that of calpain 13, prevalent in lung and testis, was mainly found as a homodimer with a small amount of heterodimer. These results indicate whether recombinant production of a particular calpain requires coexpression of the small subunit, and whether this calpain is likely to be active in a small subunit knockout mouse. Furthermore, as the endogenous inhibitor calpastatin binds to PEF domains on the large and small subunit, it is less likely that the homodimeric calpains 3 and 13 with two active sites will bind or be silenced by calpastatin.

Abnormal adult neurogenesis in the dentate gyrus of GABA(A)R δ subunit knockout mice

Event Abstract Back to Event Abnormal adult neurogenesis in the dentate gyrus of GABA(A)R δ subunit knockout mice Shira Rosenzweig1*, E. Czerwinska1, Beverley A. Orser2 and Martin J. Wojtowicz1 1 University of Toronto, Department of Physiology, Canada 2 University of Toronto, Physiology and Anesthesia, Canada Background: During adult neurogenesis, cells are born in the sub-granular zone (SGZ) of the dentate gyrus (DG) and subsequently undergo differentiation, migration and maturation. The mechanisms that regulate the various stages of adult neurogenesis remain poorly understood. One known regulator is the neurotransmitter GABA, which depolarizes young DG cells before they become responsive to glutamate. This early depolarization initially occurs via activation of extrasynaptic GABAA receptors (GABAARs). As most extrasynaptic GABAARs in the DG contain the delta (δ) subunit, we hypothesized that this receptor subtype is involved in the early depolarization and developmental regulation of young DG neurons. To test this hypothesis we characterized and compared neurogenesis in the DG of mice that are homozygous null for the GABAA receptor δ gene (Garbd-/-) and wild type (WT) C57BL/6J mice. Garbd-/- mice exhibit an increased susceptibility to seizures and attenuated responses to neuroactive steroids which modulate δ-containing GABAARs. Methods: Two months old WT and Garbd-/- mice were injected intra-peritoneally with the mitotic indicator BrdU and sacrificed two weeks later. Hippocampal sections were immunostained for Ki-67, a marker for proliferating cells, doublecortin (DCX), a marker for immature neurons, and BrdU. Labeled cells were counted using a confocal microscope. Results: There were no genotype-related differences in the number of Ki-67+ and two-week old (BrdU+) cells. The total number of immature (DCX+) neurons and the fraction of immature BrdU+ cells were 20% higher in Garbd-/- compared with WT mice (p<0.03 and p<0.01, n=3). Analysis of the position of BrdU+ cells across the granule cell layer (GCL) revealed that in WT mice, 22% of the cells had migrated from the SGZ deeper into the GCL by two weeks. In contrast, only 9.8% of the cells had migrated in Garbd-/- mice (p<0.01, n=3). Conclusions: These results indicate that proliferation and two-week survival are unchanged in Garbd-/- mice. In contrast, a greater number of cells remain immature at two weeks, and their normal migration is disrupted in Garbd-/- mice compared to WT mice. The abnormalities observed in the Garbd-/- mice suggest that δ-containing GABAARs play an important role in the normal development of adult-born DG granule neurons. To fully characterize survival, differentiation and maturation of adult-born neurons in the absence of δ subunits, ongoing experiments will examine more advanced time points of neuronal development. Conference: B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Oral presentations Citation: Rosenzweig S, Czerwinska E, Orser BA and Wojtowicz MJ (2009). Abnormal adult neurogenesis in the dentate gyrus of GABA(A)R δ subunit knockout mice. Front. Neurosci. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.049 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Dec 2009; Published Online: 18 Dec 2009. * Correspondence: Shira Rosenzweig, University of Toronto, Department of Physiology, Toronto, Canada, shirarosen@outlook.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz Google Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz Google Scholar Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz PubMed Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Spontaneous hyperactivity in mutant mice lacking the NMDA receptor GluRε1 subunit is aggravated during exposure to 0.1 MAC sevoflurane and is preserved after emergence from sevoflurane anaesthesia

Patients who awake from sevoflurane anaesthesia with symptoms of agitation may have some underlying functional substrate that is sensitive to the low concentrations of anaesthetic encountered during emergence. One candidate for such a substrate could be neurocircuitry implied in the pathophysiology of both agitation and movement disorders with hyperactivity. We postulated that hyperactive animals would show a further increase in activity in the presence of low concentrations of volatile anaesthetics, such as sevoflurane. To confirm our hypothesis, we examined the effects of two subanaesthetic concentrations of sevoflurane, isoflurane and halothane (0.1 and 0.2 MAC (minimum alveolar concentration)) on spontaneous activity in N-methyl-d-aspartate receptor GluRepsilon1 subunit knockout mice exhibiting locomotor hyperactivity in a novel environment and compared these results with those for wild-type controls. We also compared the effects of anaesthetic concentrations of sevoflurane (1.2 MAC) on mice activity during postanaesthesia recovery. Out of the three anaesthetics used, only sevoflurane administered at 0.1 MAC caused a significantly different response between the two experimental groups. Exposure to this subanaesthetic concentration of sevoflurane reduced the activity of wild-type mice, whereas mutant animals showed a further increase in hyperactivity. The effects of 1.2 MAC sevoflurane anaesthesia on mice activity during postanaesthesia recovery also differed significantly between the two genotypes. Exposure to anaesthetic concentrations of sevoflurane had a sedative effect on wild-type mice, whereas mutant mice preserved their high levels of activity upon emergence from the anaesthesia. The presence of an inherent anomaly in mutant mice that becomes more manifest during exposure to 0.1 MAC sevoflurane and is still present after the emergence from sevoflurane anaesthesia suggests the presence of and necessitates a search for some putative substrate that may, by analogy, underlie emergence agitation in the clinical setting.

Varenicline, Appetite, and Weight Reduction

Varenicline, Appetite, and Weight Reduction

Varenicline, Appetite, and Weight Reduction

was normal (2.3 ng/ml), whereas serum prolactin level was suppressed to 1.3 ng/ml. His pathological hypersexuality completely disappeared 2 weeks after discontinuation of bromocriptine. After 4 weeks, pramipexole (0.125 mg b.i.d.), another dopamine receptor agonist, was added instead of bromocriptine since his motor performance deteriorated. After 1 week of pramipexole, all the hypersexual symptoms reemerged. At this point, his serum prolactin level was 2.2 ng/ml. His pathological hypersexuality completely disappeared again just by discontinuing pramipexole. Then we titrated madopar up to 125 mg t.i.d. instead of adding other dopamine receptor agonist. After 3 weeks, his motor performance improved with partial reemergence of hypersexual symptoms (penile erection, facial flushing, sexually suggestive remarks, touching). However, the severity of the reemerged symptoms was much more tolerable than those induced by dopamine receptor agonist. At this point, his serum prolactin level was not suppressed (13.3 ng/ml).

Relationship between AMPK and the transcriptional balance of clock-related genes in skeletal muscle

Circadian clocks coordinate physiological, behavioral, and biochemical events with predictable daily environmental changes by a self-sustained transcriptional feedback loop. CLOCK and ARNTL are transcriptional activators that regulate Per and Cry gene expression. PER and CRY inhibit their own transcription, and their turnover allows this cycle to restart. The transcription factors BHLHB2 and BHLHB3 repress Per activation, whereas orphan nuclear receptors of the NR1D and ROR families control Arntl expression. Here we show the AMP-activated protein kinase (AMPK)gamma(3) subunit is involved in the regulation of peripheral circadian clock function. AMPKgamma3 knockout (Prkag3(-/-)) mice or wild-type littermates were injected with saline or an AMPK activator, 5-amino-4-imidazole-carboxamide riboside (AICAR), and white glycolytic gastrocnemius muscle was removed for gene expression analysis. Genes involved in the regulation of circadian rhythms (Cry2, Nr1d1, and Bhlhb2) were differentially regulated in response to AICAR in wild-type mice but remained unaltered in Prkag3(-/-) mice. Basal expression of Per1 was higher in Prkag3(-/-) mice compared with wild-type mice. Distinct diurnal changes in the respiratory exchange ratio (RER) between the light and dark phase of the day were observed in wild-type mice but not Prkag3(-/-) mice. In summary, the expression profile of clock-related genes in skeletal muscle in response to AICAR, as well as the diurnal shift in energy utilization, is impaired in AMPKgamma(3) subunit knockout mice. Our results indicate AMPK heterotrimeric complexes containing the AMPKgamma(3) subunit may play a specific role in linking circadian oscillators and energy metabolism in skeletal muscle.

Apocynin-induced vasodilation involves Rho kinase inhibition but not NADPH oxidase inhibition

The present study was designed to test the hypothesis that NADPH oxidase inhibition with apocynin would lower blood pressure and improve endothelial function in spontaneously hypertensive rats (SHRs). Although apocyin effectively dilated arterial segments in vitro, it failed to lower blood pressure or improve endothelial function. Further experiments were performed in normotensive rats and in NADPH oxidase subunit knock-out mice to test if apocynin-induced vasodilation depends on NADPH oxidase inhibition at all. SHRs were treated with apocynin orally or i.v. Arterial pressure was recorded directly. Rat and mouse arterial function was investigated in vitro by small vessel wire myography. NADPH oxidase activity was measured in human granulocytes and in rat vascular preparations. Rho kinase activity was determined by Western blot analysis. Apocynin did not reduce arterial pressure acutely in SHR when given at 50, 100, or 150 mg kg(-1) day(-1) orally over 1-week intervals or when given i.v. Apocynin potently inhibited granulocyte NADPH oxidase but not vascular NADPH-oxidase-dependent oxygen radical formation unless exogenous peroxidase was added to vascular preparations. Apocynin dilated rat intrarenal and coronary arteries independently of pharmacological interventions that reduce vascular superoxide radical abundance and actions. Aortic rings from p47phox(-/-) mice were more sensitive to apocynin-induced dilation than wild-type aortic rings. Rho kinase inhibition reduced or prevented the inhibitory effect of apocynin on agonist-induced vasoconstriction and apocynin inhibited the phosphorylation of Rho kinase substrates. Apocynin per se does not inhibit vascular NADPH-oxidase-dependent superoxide formation. Its in vitro vasodilator actions are not due to NADPH oxidase inhibition but may be explained at least in part by inhibition of Rho kinase activity. The discrepancy between apocynin-induced vasodilation in vitro and the failure of apocynin to lower arterial pressure in SHR suggests opposing effects on arterial pressure-regulating systems in vivo. Its use as a pharmacological tool to investigate vascular NADPH oxidase should be discontinued.

Cell death and proliferation in NF-κB p50 knockout mouse after cerebral ischemia

The transcription factor NF-κB is a key regulator of inflammation and cell survival. NF-κB activation increases following cerebral ischemia. We previously showed accelerated aging process in NF-κB p50 subunit knockout (p50 −/−) mice under physiological condition. The present investigation concerned the role of NF-κB p50 gene in ischemia-induced neuronal cell death. In an animal model of permanent middle cerebral artery occlusion (MCAO), infarct formation, apoptotic cell death and cell proliferation were examined in adult wild type (WT) and p50 −/− mice. The ischemic infarct volume was significantly larger in p50 −/− mice than that in WT mice. Consistently, the numbers of cells in the penumbra region positive to terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) and caspase-3 staining were significantly more in p50 −/− mice than that in WT mice. To identify proliferation after cerebral ischemia, bromodeoxyurindine (BrdU) was intraperitoneal injected daily after MCAO. Ischemia increased BrdU positive cells in the penumbra, subventricular zone, corpus callosum, and cerebral cortex, while cell proliferation was hampered in p50 −/− mice. These results suggest that NF-κB signaling is a neuroprotective mechanism and may play a role in cell proliferation in the stroke model of permanent MCAO.

WO03/062224 is an in vivo selective agonist at nicotinic β4 receptors

Pharmacological agents that increase cholinergic transmission have considerable use in cognitive disorders and evidence from both human and animal studies suggests that nicotinic acetylcholine receptors (nAChRs) represent an attractive target for treating certain neurological disorders. This investigation aimed to provide an in vivo verification of the in vitro data on WO03/062224, an agonist selective at β4 subunit-containing nicotinic receptors. The effects of WO03/062224 were tested on wildtype and β4 nAChR null mice on two behavioural paradigms; locomotor behaviour and instrumental responding for food on a second order schedule. Separate groups of wildtype and β4 nAChR subunit knockout mice were tested in each paradigm with instrumental responding and forward locomotion being measured. WO03/062224 had a greater effect in the wildtype mice than the β4 knockout mice in both locomotor activity (unconditioned behaviour) and instrumental responding (conditioned behaviour). In wildtype mice WO03/062224 caused a significant initial depression in locomotor activity followed by a significant increase in activity. The β4 knockout mice displayed no significant drug-induced alterations in locomotor activity at any time point. In wildtype mice WO03/062224 caused a significant depression in instrumental responding throughout the session at both 3 mg/kg and 10 mg/kg. The β4 knockout mice only displayed a reduction in initial responding at 10 mg/kg. The present study demonstrated that the effects of WO03/062224 at 3 mg/kg on locomotor activity and instrumental responding are likely occurring through a β4 nicotinic mechanism. This investigation has shown that at an appropriate dose WO03/062224 is a suitable in vivo probe for the contribution of β4-containing nAChRs to behaviour and suggests that their involvement is greater than previously recognised.

Ethanol Modulates Synaptic and Extrasynaptic GABAA Receptors in the Thalamus

Drinking alcohol is associated with the disturbance of normal sleep rhythms, and insomnia is a major factor in alcoholic relapse. The thalamus is a brain structure that plays a pivotal role in sleep regulation and rhythmicity. A number of studies have implicated GABA(A) receptors (GABA(A)-Rs) in the anxiolytic, amnestic, sedative, and anesthetic effects of ethanol. In the present study, we examined the effects of ethanol on both synaptic and extrasynaptic GABA(A)-Rs of relay neurons in the thalamus. We found that ethanol (> or =50 mM) elicits a sustained current in thalamocortical relay neurons from the mouse ventrobasal thalamus, and this current is associated with a decrease in neuronal excitability and firing rate in response to depolarization. The steady current induced by ethanol was totally abolished by gabazine and was absent in relay neurons from GABA(A)-R alpha(4) subunit knockout mice, indicating that the effect of ethanol is to enhance tonic GABA-mediated inhibition. Ethanol (50 mM) enhanced the amplitude of tonic inhibition by nearly 50%. On the other hand, ethanol had no effect on spontaneous or evoked inhibitory postsynaptic currents (IPSCs) at 50 mM but did prolong IPSCs at 100 mM. Ethanol had no effect on the paired-pulse depression ratio, suggesting that the release of GABA from presynaptic terminals is insensitive to ethanol. We conclude that ethanol, at moderate (50 mM) but not low (10 mM) concentrations, can inhibit thalamocortical relay neurons and that this occurs mainly via the actions of ethanol at extrasynaptic GABA(A)-Rs containing GABA(A)-R alpha(4) subunits.

Association of the GABRB3 Gene with Nonsyndromic Oral Clefts

Nonsyndromic oral clefts are common craniofacial anomalies classified into two subgroups: cleft lip with or without cleft palate and isolated cleft palate. Nonsyndromic oral clefts are multifactorial diseases, with both genetic and environmental factors involved in their pathogenesis. The inhibitory neurotransmitter, gamma-aminobutyric acid plays a role in normal embryonic, and particularly facial, development and gamma-aminobutyric acid receptor type A beta-3 subunit (GABRB3) knockout mice have been shown to have cleft palate. The GABRB3 gene is therefore a strong candidate gene for nonsyndromic oral clefts. We investigated here whether genetic variations of the GABRB3 gene affect the risk for nonsyndromic oral clefts. In this case-control study, a total of 178 Japanese patients with cleft lip with or without cleft palate and 374 unrelated controls were recruited and were genotyped for six single nucleotide polymorphisms and a dinucleotide repeat marker of the GABRB3 gene. None of the single nucleotide polymorphisms showed complete linkage disequilibrium with other single nucleotide polymorphisms. In a case-control association study with the six-locus haplotype of the gene, TGTGCT haplotype frequency in patients with cleft lip with or without cleft palate was significantly higher than in the controls (corrected p value = .029). None of the alleles of the dinucleotide repeat marker showed significant association with cleft lip with or without cleft palate. Our data suggest that the GABRB3 gene is involved in the pathogenesis of cleft lip with or without cleft palate in the Japanese population.

Normal sleep homeostasis and lack of epilepsy phenotype in GABAA receptor α3 subunit-knockout mice

Thalamo-cortical networks generate specific patterns of oscillations during distinct vigilance states and epilepsy, well characterized by electroencephalography (EEG). Oscillations depend on recurrent synaptic loops, which are controlled by GABAergic transmission. In particular, GABA A receptors containing the alpha3 subunit are expressed predominantly in cortical layer VI and thalamic reticular nucleus (nRT) and regulate the activity and firing pattern of neurons in relay nuclei. Therefore, ablation of these receptors by gene targeting might profoundly affect thalamo-cortical oscillations. Here, we investigated the role of alpha3-GABA A receptors in regulating vigilance states and seizure activity by analyzing chronic EEG recordings in alpha3 subunit-knockout (alpha3-KO) mice. The presence of postsynaptic alpha3-GABA A receptors/gephyrin clusters in the nRT and GABA A-mediated synaptic currents in acute thalamic slices was also examined. EEG spectral analysis showed no difference between genotypes during non rapid-eye movement (NREM) sleep or at waking-NREM sleep transitions. EEG power in the spindle frequency range (10-15 Hz) was significantly lower at NREM-REM sleep transitions in mutant compared with wild-type mice. Enhancement of sleep pressure by 6 h sleep deprivation did not reveal any differences in the regulation of EEG activities between genotypes. Finally, the waking EEG showed a slightly larger power in the 11-13-Hz band in alpha3-KO mice. However, neither behavior nor the waking EEG showed alterations suggestive of absence seizures. Furthermore, alpha3-KO mice did not differ in seizure susceptibility in a model of temporal lobe epilepsy. Strikingly, despite the disruption of postsynaptic gephyrin clusters, whole-cell patch clamp recordings revealed intact inhibitory synaptic transmission in the nRT of alpha3-KO mice. These findings show that the lack of alpha3-GABA(A) receptors is extensively compensated for to preserve the integrity of thalamo-cortical function in physiological and pathophysiological situations.

Facilitation of Cortico–Amygdala Synapses by Nicotine: Activity-Dependent Modulation of Glutamatergic Transmission

The basolateral nucleus of the amygdala (BLA) receives cholinergic innervation from the basal forebrain and nicotine, via activation of neuronal nicotinic acetylcholine receptors (nAChRs), can improve performance in amygdala-based learning tasks. We tested the hypothesis that acute and prenatal nicotine exposure modulates cortico-amygdala synaptic transmission. We found that low-dose, single-trial exposures to nicotine can elicit lasting facilitation, the extent of which is dependent on the level of stimulation of the cortical inputs to the BLA. In addition, sustained facilitation is ablated by prenatal exposure to nicotine. This study examined synaptic transmission in 238 patch-clamp recordings from BLA neurons in acute slice from mouse brain. Pharmacological studies in wild-type and nAChR subunit knock-out mice reveal that activation of presynaptic alpha 7, containing (alpha 7*) and non-alpha 7* nAChRs, facilitates glutamatergic transmission in an activity-dependent manner. Without prior stimulation, application of nicotine elicits modest and transient facilitation of glutamatergic postsynaptic currents (PSCs) in about 40% of BLA neurons. With low-frequency stimulation of cortical inputs nicotine elicits robust facilitation of transmission at about 60% of cortico-BLA synapses and synaptic strength remains elevated at about 40% of these connections for >15 min after nicotine washout. Following paired-pulse stimulation nicotine elicits long-lasting facilitation of glutamatergic transmission at about 70% of cortico-BLA connections. Nicotine reduces the threshold for activation of long-term potentiation of cortico-BLA synapses evoked by patterned stimulation. Prenatal exposure to nicotine reduced subsequent modulatory responses to acute nicotine application.