[Show abstract][Hide abstract] ABSTRACT: The tools of molecular biology will bring the field of human genetics into a new era by permitting the analysis of the genetic contribution to disease. Most single gene disorders, inherited in a Mendelian fashion, will be molecularly diagnosed. In addition, the genetic susceptibility of common, complex diseases such a schizophrenia can be clarified, even though the conditions are not inherited as Mendelian characteristics. The mapping of the human genome will increase the rate at which new disease genes are identified and isolated. Finally, the development of genetically engineered animal models will help to dissect the steps involved in physiological and pathophysiological processes and thereby enhance our understanding of complex biological systems.
Journal of Receptor and Signal Transduction Research 06/2009; 17(1-3):1-10. DOI:10.3109/10799899709036591 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GABA(A) receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA(A) receptor subtypes are assembled from alpha5(1-3, 5), beta1-3 and the gamma2 subunit. They exhibit a stoichiometry of two alpha, two beta and one gamma subunit, with two GABA binding sites located at the alpha/beta and one benzodiazepine binding site located at the alpha/gamma subunit interface. Introduction of the H105R point mutation into the alpha5 subunit, to render alpha5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of alpha5 subunit protein in alpha5(H105R) mice. In this study, we show that the alpha5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of alpha5-selective ligand binding. Immunoprecipitation studies suggest that the diminished alpha5-selective binding is presumably due to a repositioning of the alpha5(H105R) subunit in GABA(A) receptor complexes containing two different alpha subunits. These findings imply an important role of histidine 105 in determining the position of the alpha5 subunit within the receptor complex by determining the affinity for assembly with the gamma2 subunit.
Journal of Neurochemistry 05/2009; 110(1):244-54. DOI:10.1111/j.1471-4159.2009.06119.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To identify subunit variants of the GABAA-receptor antisera were developed against specific cDNA-derived peptide sequences of the α1-and α3–subunits of rat brain. The α1-subunit antiserum selectively recognized a protein of 50±1 kDa in rat and bovine GABAA – receptor preparations, while the α3–subunit antiserum interacted with a protein doublet of 59±2 kDa and 61±3 kDa. The α-subunit immunoreactivity resides in a large population of GABAA-receptors as shown by immunoprecipitation of 63+6% of [3H]flumazenil binding sites with the α1-subunit antiserum. In contrast, only 24±3% of receptor binding sites were precipitated with the α3–subunit antiserum. Co-precipitation studies suggest that the α1- and α3–subunit immunoreactivities do not share the same receptor population while the γ2–subunit immunoreactivity is associated with the α1-subunit immunoreactivity.
Journal of Receptor and Signal Transduction Research 09/2008; 11(1-4):407-424. DOI:10.3109/10799899109066418 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The optic tectum of the salmon is a primary visual center with direct input from the retina via the optic tract. The structure is homologous with the superior colliculus of the mammalian brain. We have studied the distribution of immunoreactivity against the GABAA/benzodiazepine receptor beta2/beta3 subunits with a monoclonal antibody (BD-17) in the optic tectum of the salmon brain. A weak immunoreactivity is found in the rostral stratum marginale (SM), strong labelling of the neuropil is shown in a thin band in stratum opticum (SO), two bands in stratum fibrosum et griseum superficiale (SFGS) and two bands in stratum griseum centrale (SGC). Immunoreactive perikarya with neurites that extend radially through the stratum album centrale (SAC) are located in the stratum periventriculare. BD-17 immunoreactivity is to a great extent located in tectal layers that receive direct retinal input, i.e. the SO, SFGS and SGC. These layers are known to receive input also from other visual centers, such as the pretectum (SO, SFGS), the nucleus isthmi (SO, SFGS, SGC), as well as non-visual regions as the telencephalon (SGC). High levels of 2-[125I]-iodomelatonin binding sites have previously been demonstrated in all layers of the salmon optic tectum except the SM and SPV. Thus it appears likely that GABA and/or benzodiazepines and melatonin play a role in visual processing in the optic tectum of teleost fish.
Journal of Receptor and Signal Transduction Research 09/2008; 15(1-4):413-25. DOI:10.3109/10799899509045230 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.
The Journal of Comparative Neurology 02/2008; 506(6):912-29. DOI:10.1002/cne.21573 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment. A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology. Facilitation of spinal gamma-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABA(A) receptors should be able to compensate for this loss. With the use of GABA(A)-receptor point-mutated knock-in mice in which specific GABA(A) receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands, we show here that pronounced analgesia can be achieved by specifically targeting spinal GABA(A) receptors containing the alpha2 and/or alpha3 subunits. We show that their selective activation by the non-sedative ('alpha1-sparing') benzodiazepine-site ligand L-838,417 (ref. 13) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.
[Show abstract][Hide abstract] ABSTRACT: Stressful experiences in early life are known risk factors for anxiety and depressive illnesses, and they inhibit hippocampal neurogenesis and the expression of GABA(A) receptors in adulthood. Conversely, deficits in GABAergic neurotransmission and reduced neurogenesis are implicated in the etiology of pathological anxiety and diverse mood disorders. Mice that are heterozygous for the gamma2 subunit of GABA(A) receptors exhibit a modest functional deficit in mainly postsynaptic GABA(A) receptors that is associated with a behavioral, cognitive, and pharmacological phenotype indicative of heightened trait anxiety. Here we used cell type-specific and developmentally controlled inactivation of the gamma2 subunit gene to further analyze the mechanism and brain substrate underlying this phenotype. Heterozygous deletion of the gamma2 subunit induced selectively in immature neurons of the embryonic and adult forebrain resulted in reduced adult hippocampal neurogenesis associated with heightened behavioral inhibition to naturally aversive situations, including stressful situations known to be sensitive to antidepressant drug treatment. Reduced adult hippocampal neurogenesis was associated with normal cell proliferation, indicating a selective vulnerability of postmitotic immature neurons to modest functional deficits in gamma2 subunit-containing GABA(A) receptors. In contrast, a comparable forebrain-specific GABA(A) receptor deficit induced selectively in mature neurons during adolescence lacked neurogenic and behavioral consequences. These results suggest that modestly reduced GABA(A) receptor function in immature neurons of the developing and adult brain can serve as a common molecular substrate for deficits in adult neurogenesis and behavior indicative of anxious and depressive-like mood states.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 05/2007; 27(14):3845-54. DOI:10.1523/JNEUROSCI.3609-06.2007 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The NMDA receptor is thought to play a central role in some forms of neuronal plasticity, including the induction of long-term potentiation. NMDA receptor hypofunction can result in mnemonic impairment and has been implicated in the cognitive symptoms of schizophrenia. The activity of NMDA receptors is controlled by its endogenous coagonist glycine, and a local elevation of glycine levels is expected to enhance NMDA receptor function. Here, we achieved this by the generation of a novel mouse line (CamKIIalphaCre;Glyt1tm1.2fl/fl) with a neuron and forebrain selective disruption of glycine transporter 1 (GlyT1). The mutation led to a significant reduction of GlyT1 and a corresponding reduction of glycine reuptake in forebrain samples, without affecting NMDA receptor expression. NMDA (but not AMPA) receptor-evoked EPSCs recorded in hippocampal slices of mutant mice were 2.5 times of those recorded in littermate controls, suggesting that neuronal GlyT1 normally assumes a specific role in the regulation of NMDA receptor responses. Concomitantly, the mutants were less responsive to phencyclidine than controls. The mutation enhanced aversive Pavlovian conditioning without affecting spontaneous anxiety-like behavior in the elevated plus maze and augmented a form of attentional learning called latent inhibition in three different experimental paradigms: conditioned freezing, conditioned active avoidance, conditioned taste aversion. The CamKIIalphaCre;Glyt1tm1.2fl/fl mouse model thus suggests that augmentation of forebrain neuronal glycine transmission is promnesic and may also offer an effective therapeutic intervention against the cognitive and attentional impairments characteristic of schizophrenia.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 04/2006; 26(12):3169-81. DOI:10.1523/JNEUROSCI.5120-05.2006 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The metabotropic γ-aminobutyric acid, type B (GABAB) receptors mediate the slow component of GABAergic transmission in the brain. Functional GABAB receptors are heterodimers of the two subunits GABAB1 and GABAB2, of which GABAB1 exists in two main isoforms, GABAB1a and GABAB1b. The significance of the structural heterogeneity of GABAB receptors, the mechanism leading to their differential targeting in neurons as well as the regulation of cell surface numbers
of GABAB receptors, is poorly understood. To gain insights into these processes, we searched for proteins interacting with the C-terminal
domain of GABAB2. Here, we showed that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) directly
interacts with GABAB receptors in a subtype-selective manner to regulate cell surface expression of GABAB1a/GABAB2 receptors upon co-expression in HEK 293 cells. The interaction of CHOP with GABAB1a/GABAB2 receptors resulted in their intracellular accumulation and in a reduced number of cell surface receptors. This regulation
required the interaction of CHOP via two distinct domains with the heterodimeric receptor; its C-terminal leucine zipper associates
with the leucine zipper present in the C-terminal domain of GABAB2, and its N-terminal domain associates with an as yet unidentified site on GABAB1a. In conclusion, the data indicated a subtype-selective regulation of cell surface receptors by interaction with the transcription
[Show abstract][Hide abstract] ABSTRACT: The occupation of the glycine binding-site is a prerequisite for NMDA receptor activation by glutamate. To analyze the regulation of NMDA receptor function by the glycine transporter 1 (GlyT1), we generated heterozygous constitutive GlyT1 knockout mice (GlyT1tm1.1(+/-)). These animals were fully viable. Using a newly generated antibody, the pattern of GlyT1 expression in brain was found to be unaltered in the mutants while the level of expression was strongly reduced in all brain regions, as shown immunohistochemically. In hippocampal slices the ratio of the peak amplitude of NMDA and AMPA receptor evoked excitatory postsynaptic currents (EPSCs), recorded in CA1 pyramidal cells, was significantly enhanced by 36% in Glyt1tm1.1(+/-) compared to wild-type slices. The frequency and amplitude of AMPA miniature events in Glyt1tm1.1(+/-) mice were indistinguishable from those recorded in wild type. These results provide proof that the NMDA receptor function is enhanced by a reduction of GlyT1 expression. Thus, GlyT1 function is a controlling factor for an enhancement of the NMDA receptor response. These findings are of relevance for the development of GlyT1 inhibitory drugs.
[Show abstract][Hide abstract] ABSTRACT: Gamma-aminobutyric acid, type A (GABAA) receptors are pentameric proteins of which the majority is composed of two alpha subunits, two beta subunits and one gamma subunit. It is well documented that two different types of alpha subunits can exist in a singles GABAA receptor complex. However, information on the abundance of such GABAA receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the alpha1, alpha2, or alpha3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABAA receptors totally insensitive to diazepam binding that contain two mutated alpha subunits in the receptor complex, whereas receptors containing one mutated and one heterologous alpha subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABAA receptors containing heterologous alpha subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated alpha subunit with those present in mouse lines containing two different mutated alpha subunits. The data indicate that the alpha1alpha1-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the alpha1alpha2 (13%), alpha1alpha3 (15%), alpha2alpha2 (12%), alpha2alpha3 (2%), and alpha3alpha3 combinations (4%) are considerably less expressed. Only within the alpha1-containing receptor population does the combination of equal alpha subunits (84% alpha1alpha1, 7% alpha1alpha2, and 8% alpha1alpha3) prevail, whereas in the alpha2-containing receptor population (46% alpha2alpha2, 36% alpha2alpha1, and 19% alpha2alpha3) and particularly in the alpha3-containing receptor population (27% alpha3alpha3, 56% alpha3alpha1, and 19% alpha3alpha2), the receptors with two different types of alpha subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABAA receptors containing heterologous alpha subunits on a brain regional level.
[Show abstract][Hide abstract] ABSTRACT: Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially unknown. The acute sedative action of diazepam is mediated via alpha1-GABA(A) receptors. Therefore, we tested whether chronic activation of these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which thealpha1-,alpha2-,alpha3-, oralpha(5)-GABA(A) receptors had been rendered insensitive to diazepam by histidine-arginine point mutation, were chronically treated with diazepam (8 d; 15 mg x kg(-1) x d(-1)) and tested for motor activity. Wild-type, alpha2(H101R), and alpha3(H126R) mice showed a robust diminution of the motor-depressant drug action. In contrast, alpha5(H105R) mice failed to display any sedative tolerance. alpha1(H101R) mice showed no alteration of motor activity with chronic diazepam treatment. Autoradiography with [3H]flumazenil revealed no change in benzodiazepine binding sites. However, a decrease in alpha5-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of diazepam is associated with a downregulation of alpha5-GABA(A) receptors in the dentate gyrus. Thus, the chronic activation of alpha(5)-GABA(A) receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with alpha1-GABA(A) receptors.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 08/2004; 24(30):6785-90. DOI:10.1523/JNEUROSCI.1067-04.2004 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gamma2 subunit of GABA(A) receptor chloride channels is required for normal channel function and for postsynaptic clustering of these receptors during synaptogenesis. In addition, GABA(A) receptor function is thought to contribute to normal postnatal maturation of neurons. Loss of postsynaptic GABA(A) receptors in gamma2-deficient neurons might therefore reflect a deficit in maturation of neurons due to the reduced channel function. Here, we have used the Cre-loxP strategy to examine the clustering function of the gamma2 subunit at mature synapses. Deletion of the gamma2 subunit in the third postnatal week resulted in loss of benzodiazepine-binding sites and parallel loss of punctate immunoreactivity for postsynaptic GABA(A) receptors and gephyrin. Thus, the gamma2 subunit contributes to postsynaptic localization of GABA(A) receptors and gephyrin by a mechanism that is operant in mature neurons and not limited to immature neurons, most likely through interaction with proteins involved in trafficking of synaptic GABA(A) receptors.
[Show abstract][Hide abstract] ABSTRACT: Recombinant GABAA receptors, expressed from α-, β-, and γ2-subunits, are diazepam-insensitive when the α-subunit is either α4 or α6. In situ, diazepam-insensitive receptors containing the α6-subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam-insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam-insensitive GABAA receptors in various brain areas containing the α4-subunit. GABAA receptors immunoprecipitated with a newly developed α4-subunit-specific antiserum displayed a drug binding profile that was indistinguishable from those of α4β2γ2-recombinant receptors and diazepam-insensitive [3H]Ro 15-4513 binding sites in rat brain membranes. In addition, α4-subunit containing receptors and forebrain diazepam-insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of α4-subunit containing receptors revealed that the α4-subunit contributes to several receptor subtypes. Depending on the brain region, the α4-subunit can be coassembled with a second type of α4-subunit variant being α1, α2, or α3. The data demonstrate that native receptors containing the α4-subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam-insensitive [3H]Ro 15-4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15-4513 and bretazenil.
[Show abstract][Hide abstract] ABSTRACT: Neonatal hepatic steatosis (OMIM 228100) is a fatal condition of unknown etiology characterized by a pale and yellow liver and early postnatal mortality. In the present study, a deficit in adenosine-dependent metabolism is proposed as a causative factor. Physiologically, adenosine is efficiently metabolized to AMP by adenosine kinase (ADK), an enzyme highly expressed in liver. ADK not only ensures normal adenine nucleotide levels but also is essential for maintaining S-adenosylmethionine-dependent transmethylation processes, where adenosine, an obligatory product, has to be constantly removed. Homozygous Adk(-/-) mutants developed normally during embryogenesis. However, within 4 days after birth they displayed microvesicular hepatic steatosis and died within 14 days with fatty liver. Adenine nucleotides were decreased and S-adenosylhomocysteine, a potent inhibitor of transmethylation reactions, was increased in the mutant liver. Thus, a deficiency in adenosine metabolism is identified as a powerful contributor to the development of neonatal hepatic steatosis, providing a model for the rapid development of postnatally lethal fatty liver.
Proceedings of the National Academy of Sciences 06/2002; 99(10):6985-90. DOI:10.1073/pnas.092642899 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the search for yet unknown subtypes of GABAB receptors, the subunit architecture of GABAB receptors in the retina was analyzed using selective antisera. Immunopurification of the splice variants GABAB1a and GABAB1b demonstrated that both were associated with GABAB2. Quantitative immunoprecipitation experiments indicated that practical the entire GABAB receptor population in the retina consists of the receptor subtypes GABAB1a/GABAB2 and GABAB1b/GABAB2, although low levels of GABAB1c/GABAB2 cannot be excluded. The data rule out the existence of GABAB receptors containing the splice variants GABAB1d and GABAB1e. Moreover, no evidence for homomeric GABAB1 receptors was found. Among the splice variants, GABAB1a is by far the predominant one in neonatal and adult retina, whereas GABAB1b is expressed only late in postnatal development and in the adult retina. Since GABAB1a is expressed at high levels before functional synapses are formed, this specific receptor subtype might be involved in the maturation of the retina. Finally, subcellular fractionation demonstrated that GABAB1a, but not GABAB1b, is present in postsynaptic densities, suggesting a differential pre- and postsynaptic localisation of both splice variants.
Journal of Receptor and Signal Transduction Research 01/2002; 22(1-4):253-66. DOI:10.1081/RRS-120014600 · 2.28 Impact Factor