AMPA receptor subunit 1 (GluR-A) knockout mice model the glutamate hypothesis of depression.

Recent evidence indicates that glutamate homeostasis and neurotransmission are altered in major depressive disorder, but the nature of the disruption and the mechanisms by which it contributes to the syndrome are unclear. Glutamate can act via AMPA, NMDA, or metabotropic receptors. Using targeted mutagenesis, we demonstrate here that mice with deletion of the main AMPA receptor subunit GluR-A represent a depression model with good face and construct validity, showing behavioral and neurochemical features of depression also postulated for human patients. GluR-A(-/-) mice display increased learned helplessness, decreased serotonin and norepinephrine levels, and disturbed glutamate homeostasis with increased glutamate levels and increased NMDA receptor expression. These results correspond well with current concepts regarding the role of AMPA and NMDA receptors in depression, postulating that compounds that augment AMPA receptor signaling or decrease NMDA receptor functions have antidepressant effects. GluR-A(-/-) mice represent a model to investigate the pathophysiology underlying the depressive phenotype and to identify changes in neural plasticity and resilience evoked by the genetic alterations in glutamatergic function. Furthermore, GluR-A(-/-) mice may be a valuable tool to study biological mechanisms of AMPA receptor modulators and the efficacy of NMDA antagonists in reducing behavioral or biochemical changes that correlate with increased helplessness.

Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.

We have recently succeeded in immortalizing rat granulosa cells by co-transfection with SV-40 DNA and the Ha-ras oncogene. These cells lost their response to gonadotropins, but expressed the cytochrome P450scc mitochondrial system enzymes and produced progesterone and 20 alpha-hydroxy-4-pregnan-3-one (20 alpha-OH-P) upon cAMP stimulation (Suh, B. S., and A. Amsterdam. 1990. Endocrinology. 127:2489-2500; Hanukoglu, I., B. S. Suh, S. Himmelhoch, and A. Amsterdam. 1990. J. Cell Biol. 111:1973-1981). In an attempt to restore the steroidogenic response to gonadotropins in immortalized cells, lutropin/choriogonadotropin (LH/CG-R) receptor expression plasmid was prepared by introducing the complete coding region of LH receptor cDNA (McFarland, K. C., R. Sprengel, H. S. Phillips, M. Köhler, N. Rosemblit, K. Nikolics, D. L. Segaloff, and P. H. Seeburg. 1989. Science (Wash. DC). 245:494-499) into a SV-40 early promoter based eucaryotic expression vector. Granulosa cells from preovulatory follicles were transfected with this LH receptor expression plasmid, together with SV-40 DNA and the Ha-ras oncogene. Cell lines obtained after this triple transfection accumulated cAMP in a dose-dependent manner in response to hCG. Moreover, they produced progesterone and 20 alpha-OH-P upon hCG stimulation with an ED50 of 125 pM and 75 pM, respectively, which is within the physiological range. Concomitantly with hCG induced differentiation, inhibition of cell proliferation was evident following stimulation with hormone concentrations as low as 40 pM. The number of hCG receptor sites per cell after numerous passages and several freezing and thawing cycles was 1.9 x 10(4), they showed a Kd of 180 pM. Stimulation with hCG induced pronounced morphological and biochemical changes in these cells including formation of mitochondrial located adrenodoxin, a marker enzyme for enhanced steroidogenesis. These findings make possible the expression in immortalized granulosa cells, of selectively mutated receptor molecules which preserve their steroidogenic potential, thereby opening the way to analysis of structure-function relationships of the receptor molecule.

Mice lacking the AMPA GluR1 receptor exhibit striatal hyperdopaminergia and 'schizophrenia-related' behaviors.

There is growing evidence implicating dysfunctional glutamatergic neurotransmission and abnormal interactions between the glutamate and dopamine (DA) systems in the pathophysiology of various neuropsychiatric disorders including schizophrenia. The present study evaluated knockout (KO) mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) GluR1 receptor subunit for a range of behaviors considered relevant to certain symptoms of schizophrenia. KO showed locomotor hyperactivity during exposure to open field and in response to a novel object, but normal activity in a familiar home cage. Open field locomotor hyperactivity in KO was effectively normalized to WT levels by treatment with the DA antagonist and neuroleptic haloperidol, while locomotor stimulant effects of the NMDA receptor antagonist MK-801 were absent in KO. Social behaviors during a dyadic conspecific encounter were disorganized in KO. KO showed deficits in prepulse inhibition of the acoustic startle response. In vivo chronoamperometric measurement of extracellular DA clearance in striatum demonstrated retarded clearance in KO. These data demonstrate behavioral abnormalities potentially pertinent to schizophrenia in GluR1 KO, together with evidence of dysregulated DA function. Present findings provide novel insight into the potential role of GluR1, AMPA receptors and glutamate x DA interactions in the pathophysiology of schizophrenia and other neuropsychiatric conditions.

Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family.

A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.

Heteromeric NMDA receptors: molecular and functional distinction of subtypes.

The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

Conditional restoration of hippocampal synaptic potentiation in Glur-A-deficient mice.

Plasticity of mature hippocampal CA1 synapses is dependent on l-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors containing the glutamate receptor A (GluR-A) subunit. In GluR-A-deficient mice, plasticity could be restored by controlled expression of green fluorescent protein (GFP)-tagged GluR-A, which contributes to channel formation and displayed the developmental redistribution of AMPA receptors in CA1 pyramidal neurons. Long-term potentiation (LTP) induced by pairing or tetanic stimulation was rescued in adult GluR-A(-/-) mice when (GFP)GluR-A expression was constitutive or induced in already fully developed pyramidal cells. This shows that GluR-A-independent forms of synaptic plasticity can mediate the establishment of mature hippocampal circuits that are prebuilt to express GluR-A-dependent LTP.

Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice.

The arginine residue at position 586 of the GluR-B subunit renders heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele synthesized unedited GluR-B subunits and, in principal neurons and interneurons, expressed AMPA receptors with increased calcium permeability. These mice developed seizures and died by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function.

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning.

Gene-targeted mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit GluR-A exhibited normal development, life expectancy, and fine structure of neuronal dendrites and synapses. In hippocampal CA1 pyramidal neurons, GluR-A-/- mice showed a reduction in functional AMPA receptors, with the remaining receptors preferentially targeted to synapses. Thus, the CA1 soma-patch currents were strongly reduced, but glutamatergic synaptic currents were unaltered; and evoked dendritic and spinous Ca2+ transients, Ca2+-dependent gene activation, and hippocampal field potentials were as in the wild type. In adult GluR-A-/- mice, associative long-term potentiation (LTP) was absent in CA3 to CA1 synapses, but spatial learning in the water maze was not impaired. The results suggest that CA1 hippocampal LTP is controlled by the number or subunit composition of AMPA receptors and show a dichotomy between LTP in CA1 and acquisition of spatial memory.

Restoration of spatial working memory by genetic rescue of GluR-A-deficient mice.

Gene-targeted mice lacking the AMPA receptor subunit GluR-A (also called GluR1 encoded by the gene Gria1,) have deficits in hippocampal CA3-CA1 long-term potentiation (LTP) and have profoundly impaired hippocampus-dependent spatial working memory (SWM) tasks, although their spatial reference memory remains normal. Here we show that forebrain-localized expression of GFP-tagged GluR-A subunits in GluR-A-deficient mice rescues SWM, paralleling its rescue of CA3-CA1 LTP. This provides powerful new evidence linking hippocampal GluR-A-dependent synaptic plasticity to rapid, flexible memory processing.

Two novel GABAA receptor subunits exist in distinct neuronal subpopulations.

Two cDNAs encoding novel GABAA receptor subunits were isolated from a rat brain library. These subunits, gamma 2 and delta, share approximately 35% sequence identity with alpha and beta subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The gamma 2 subunit is the rat homolog of the human gamma 2 subunit recently shown to be important for benzodiazepine pharmacology. Cellular localization of the mRNAs encoding the gamma 2 and delta subunits in rat brain revealed that largely distinct neuronal subpopulations express the two subunits. The delta subunit distribution resembles that of the high affinity GABAA receptor labeled with [3H]muscimol; the gamma 2 subunit distribution resembles that of GABAA/benzodiazepine receptors labeled with [3H]flunitrazepam. These findings have implications for the composition of two different GABAA receptor subtypes and for information processing in networks using GABA for signaling.

The role of the GluR-A (GluR1) AMPA receptor subunit in learning and memory.

It is widely believed that synaptic plasticity may provide the neural mechanism that underlies certain kinds of learning and memory in the mammalian brain. The expression of long-term potentiation (LTP) in the hippocampus, an experimental model of synaptic plasticity, requires the GluR-A subunit of the AMPA subtype of glutamate receptor. Genetically modified mice lacking the GluR-A subunit show normal acquisition of the standard, fixed-location, hidden-platform watermaze task, a spatial reference memory task that requires the hippocampus. In contrast, these mice are dramatically impaired on hippocampus-dependent, spatial working memory tasks, in which the spatial response of the animal is dependent on information in short-term memory. Taken together, these results argue for two distinct and independent spatial information processing mechanisms: (i) a GluR-A-independent associative learning mechanism through which a particular spatial response is gradually or incrementally strengthened, and which presumably underlies the acquisition of the classic watermaze paradigm and (ii) a GluR-A-dependent, non-associative, short-term memory trace which determines performance on spatial working memory tasks. These results are discussed in terms of Wagner's SOP model (1981).

Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B.

We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.

Deletion of glutamate receptor-A (GluR-A) AMPA receptor subunits impairs one-trial spatial memory.

Genetically modified mice lacking the glutamate receptor A (GluR-A) subunit of the AMPA receptor (GluR-A-/- mice) display normal spatial reference memory but impaired spatial working memory (SWM). This study tested whether the SWM impairment in these mice could be explained by a greater sensitivity to within-session proactive interference. The SWM performance of GluR-A-/- and wild-type mice was assessed during nonmatching-to-place testing under conditions in which potential proactive interference from previous trials was reduced or eliminated. SWM was impaired in GluR-A-/- mice, both during testing with pseudotrial-unique arm presentations on the radial maze and when conducting each trial on a different 3-arm maze, each in a novel testing room. Experimentally naive GluR-A-/- mice also exhibited chance performance during a single trial of spontaneous alternation. This 1-trial spatial memory deficit was present irrespective of the delay between the sample information and the response choice (0 or 45 min) and the length of the sample phase (0.5 or 5 min). These results imply that the SWM deficit in GluR-A-/- mice is not due to increased susceptibility to proactive interference.

Tetracycline-controlled genetic switches.

Unlike recombinase-mediated gene manipulations, tetracycline (Tet)-controlled genetic switches permit reversible control of gene expression in the mouse. Trancriptional activation can be induced by activators termed tTA (Tet-Off) or rtTA (Tet-On) in the absence and presence of Tet, respectively. The Tet-Off and Tet-On systems are complementary, and the decision to choose one over the other depends on the particular experimental strategy. Both systems were optimized over the years and can now be used to develop mouse models.

A GFP-equipped bidirectional expression module well suited for monitoring tetracycline-regulated gene expression in mouse.

Doxycycline (Dox)-sensitive co-regulation of two transcriptionally coupled transgenes was investigated in the mouse. For this, we generated four independent mouse lines carrying coding regions for green fluorescent protein (GFP) and beta-galactosidase in a bicistronic, bidirectional module. In all four lines the expression module was silent but was activated when transcription factor tTA was provided by the alpha-CaMKII-tTA transgene. In vivo analysis of GFP fluorescence, beta-galactosidase and immunochemical stainings revealed differences in GFP and beta-galactosidase levels between the lines, but comparable patterns of expression. Strong signals were found in neurons of the olfactory system, neocortical, limbic lobe and basal ganglia structures. Weaker expression was limited to thalamic, pontine and medullary structures, the spinal cord, the eye and to some Purkinje cells in the cerebellum. Strong GFP signals were always accompanied by intense beta-galactosidase activity, both of which could be co-regulated by Dox. We conclude that the tTA-sensitive bidirectional expression module is well suited to express genes of interest in a regulated manner and that GFP can be used to track transcriptional activity of the module in the living mouse.

Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies.

This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons. Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins beta-galactosidase, GFP, luciferase, and beta-lactamase, respectively. Targeted transgenic expression of these reporter proteins is obtained by fusing the corresponding reporter gene, with or without a subcellular localization signal, to a cell type- or brain region-specific gene promoter. Mice carrying GnRH promoter-driven reporter genes have proven useful for revealing the promoter elements required for cell type-specific expression of GnRH, the full anatomical profile of the GnRH neuronal network, and its electrophysiological activity, suggesting that similar approaches will assist in elucidating the properties of other neuronal populations as well.

Molecular biology of glutamate receptors.

The ligand-gated receptors for L-glutamate play a central role in acute neuronal degeneration. Recently cDNAs have been isolated for subunits of several glutamate receptor subtypes. By sequence homology all these subunits clearly belong to one large gene family. Several subfamilies exist and match roughly previously pharmacologically and electrophysiologically defined subtypes of glutamate receptors. Currently four genes (GluR A, B, C and D) are known that code for the AMPA subtypes of glutamate receptors. Recombinant expression of wild type and mutated sequences identified a critical residue in the putative TM2 channel-lining segment that controls Ca2+ ion permeability. The arginine (R) found in GluR B subunits at that position renders AMPA channels impermeable for Ca2+ ions, whereas glutamine (Q) containing GluR A, C and D subunits give rise to Ca2+ permeable channels. RNA editing converts the genomically encoded glutamine codon into the arginine codon found in GluR B cDNAs for the Q/R site. NMDA subtypes of glutamate receptors are formed after coexpression of the NR1 cDNA with a cDNA of the NR2 family. Depending on the member of the NR2 family used, NMDA receptors with different kinetical and pharmacological properties are generated. Common to all channels of these NMDA receptors is a high permeability for Ca2+ ions and a voltage dependent block by Mg2+ ions. All currently known NMDA receptor subunits have an asparagine at the Q/R homologous position. We found that this residue is critical for Mg2+ block and Ca2+ permeability of NMDA receptor channels.

Knockout of NMDA-receptors from parvalbumin interneurons sensitizes to schizophrenia-related deficits induced by MK-801.

It has been suggested that a functional deficit in NMDA-receptors (NMDARs) on parvalbumin (PV)-positive interneurons (PV-NMDARs) is central to the pathophysiology of schizophrenia. Supportive evidence come from examination of genetically modified mice where the obligatory NMDAR-subunit GluN1 (also known as NR1) has been deleted from PV interneurons by Cre-mediated knockout of the corresponding gene Grin1 (Grin1(ΔPV) mice). Notably, such PV-specific GluN1 ablation has been reported to blunt the induction of hyperlocomotion (a surrogate for psychosis) by pharmacological NMDAR blockade with the non-competitive antagonist MK-801. This suggests PV-NMDARs as the site of the psychosis-inducing action of MK-801. In contrast to this hypothesis, we show here that Grin1(ΔPV) mice are not protected against the effects of MK-801, but are in fact sensitized to many of them. Compared with control animals, Grin1(ΔPV)mice injected with MK-801 show increased stereotypy and pronounced catalepsy, which confound the locomotor readout. Furthermore, in Grin1(ΔPV)mice, MK-801 induced medial-prefrontal delta (4 Hz) oscillations, and impaired performance on tests of motor coordination, working memory and sucrose preference, even at lower doses than in wild-type controls. We also found that untreated Grin1(ΔPV)mice are largely normal across a wide range of cognitive functions, including attention, cognitive flexibility and various forms of short-term memory. Taken together these results argue against PV-specific NMDAR hypofunction as a key starting point of schizophrenia pathophysiology, but support a model where NMDAR hypofunction in multiple cell types contribute to the disease.

Morphine-induced dependence and sensitization are altered in mice deficient in AMPA-type glutamate receptor-A subunits.

AMPA-type glutamate receptors have been suggested to be involved in the neurobiological mechanisms of drug addiction. We have made use of two mouse lines, which both have modulated AMPA receptor responses. The first line is entirely deficient in glutamate receptor-A (GluR-A) subunits (A-/- knock-out line) and, in the second one, the Q582 residue of GluR-A subunits is replaced by an arginine residue (R/R mutants), which reduces the calcium permeability and channel conductance of the receptors containing this mutated subunit. Mice of both lines are healthy, but they show slightly increased locomotor activity. Acute morphine administration enhanced locomotor activity of the GluR-A-/- and GluR-A(R/R) mice, at least as much as that of their wild-type littermates. Only in the GluR-A-/- mice did we observe reduced tolerance development in tail-flick antinociception and less severe naloxone-precipitated withdrawal symptoms after treatment with increasing morphine doses, without differences in plasma and brain morphine levels when compared with wild type. Repeated daily morphine administration sensitized the locomotor activity responses in the GluR-A-/- and GluR-A(R/R) mice only when given in the measuring cages, whereas the wild-type mice showed slightly increased responses also when the repeated treatment was given in their home cages. Normal or even enhanced context-dependent sensitization was observed also with repeated amphetamine administration in the GluR-A subunit-deficient mice. The results indicate that AMPA receptors are involved in the acute and chronic effects of morphine, including context-independent sensitization, and that the GluR-A subunit itself is important for morphine tolerance and dependence.