Hippocampal NMDA receptors are important for behavioural inhibition but not for encoding associative spatial memories.

The idea that an NMDA receptor (NMDAR)-dependent long-term potentiation-like process in the hippocampus is the neural substrate for associative spatial learning and memory has proved to be extremely popular and influential. However, we recently reported that mice lacking NMDARs in dentate gyrus and CA1 hippocampal subfields (GluN1(ΔDGCA1) mice) acquired the open field, spatial reference memory watermaze task as well as controls, a result that directly challenges this view. Here, we show that GluN1(ΔDGCA1) mice were not impaired during acquisition of a spatial discrimination watermaze task, during which mice had to choose between two visually identical beacons, based on extramaze spatial cues, when all trials started at locations equidistant between the two beacons. They were subsequently impaired on test trials starting from close to the decoy beacon, conducted post-acquisition. GluN1(ΔDGCA1) mice were also impaired during reversal of this spatial discrimination. Thus, contrary to the widely held belief, hippocampal NMDARs are not required for encoding associative, long-term spatial memories. Instead, hippocampal NMDARs, particularly in CA1, act as part of a comparator system to detect and resolve conflicts arising when two competing, behavioural response options are evoked concurrently, through activation of a behavioural inhibition system. These results have important implications for current theories of hippocampal function.

Homer 1 - a new player linking the hypothalamic-pituitary-adrenal axis activity to depression and anxiety.

Homer 1 gene products are involved in the regulation of synaptic transmission and plasticity. Beside other deficits, the Homer 1 knockout (KO) mice show distinct behavioural abnormalities, such as anxiety and depression-like behaviours. In addition, we recently reported that the global deletion of the Homer 1 proteins in mice leads to a conspicuous endocrine phenotype linked to hypertrophy of the adrenal cortex, elevated basal and/or adrenocorticotropic hormone-induced corticosterone and aldosterone release in vitro and in vivo, as well as a drastic increase in the adrenocorticotropic hormone receptor mRNA in the adrenocortical cells. Interestingly, the basal secretion of adrenocorticotropic hormone was not changed in these mutants, which is in line with our recent observations, suggesting that the central limb of the hypothalamic-pituitary-adrenal axis (namely hypothalamic corticotropin-releasing hormone levels and the activation of its neurons in response to restraint stress) is not affected in the Homer 1 KO mice. On the contrary, the elevation of both plasma and intra-adrenal corticosterone and aldosterone concentrations in these mutants clearly indicates that the alteration primarily occurred in the adrenal cortex. We propose that excessive steroid release may contribute to depression- and anxiety-like behaviours and that the Homer 1 gene products may be involved in the pathogenesis of these stress-related mood disorders.

Hypertrophy and altered activity of the adrenal cortex in Homer 1 knockout mice.

Homer 1 gene products are involved in synaptic transmission and plasticity, and hence, distinct behavioral abnormalities, including anxiety- and depression-like behaviors, have been observed in Homer 1 knockout (KO) mice. Here we report that Homer 1 KO mice additionally exhibit a pronounced endocrine phenotype, displaying a profoundly increased adrenal gland weight and increased adrenal/body weight ratio. Histological examinations of Homer 1 deficient adrenal glands revealed an increased size of the adrenal cortex, especially the sizes of the zona fasciculata and zona glomerulosa. Moreover, the plasma corticosterone and aldosterone were higher in Homer 1 KO than wild-type (WT) mice while the plasma ACTH levels were not different between the genotypes. The in vivo ACTH test revealed that corticosterone and aldosterone plasma levels were higher in saline injected Homer 1 KO mice than in WT mice (saline injected mice served as controls for the respective groups of ACTH-injected animals), but the magnitude of steroid responses to ACTH was similar in both genotypes. In contrast, an in vitro experiment performed on isolated cells of adrenal cortex clearly showed increased production of both steroids in response to ACTH in Homer 1 KO cells, which is in line with an ~8-fold increase in the expression of ACTH receptor mRNA in the adrenal cortex of these mutants. These results, together with the detection of Homer 1 mRNA and protein in the adrenal cortex of WT mice, indicate that Homer 1 directly affects the steroidogenic function of the adrenal glands.

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).

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.

Impaired spatial working memory but spared spatial reference memory following functional loss of NMDA receptors in the dentate gyrus.

Novel spatially restricted genetic manipulations can be used to assess contributions made by synaptic plasticity to learning and memory, not just selectively within the hippocampus, but even within specific hippocampal subfields. Here we generated genetically modified mice (NR1(deltaDG) mice) exhibiting complete loss of the NR1 subunit of the N-methyl-D-aspartate receptor specifically in the granule cells of the dentate gyrus. There was no evidence of any reduction in NR1 subunit levels in any of the other hippocampal subfields, or elsewhere in the brain. NR1(deltaDG) mice displayed severely impaired long-term potentiation (LTP) in both medial and lateral perforant path inputs to the dentate gyrus, whereas LTP was unchanged in CA3-to-CA1 cell synapses in hippocampal slices. Behavioural assessment of NR1(deltaDG) mice revealed a spatial working memory impairment on a three-from-six radial arm maze task despite normal hippocampus-dependent spatial reference memory acquisition and performance of the same task. This behavioural phenotype resembles that of NR1(deltaCA3) mice but differs from that of NR1(deltaCA1) mice which do show a spatial reference memory deficit, consistent with the idea of subfield-specific contributions to hippocampal information processing. Furthermore, this pattern of selective functional loss and sparing is the same as previously observed with the global GluR-A L-alpha-amino-3-hydroxy-5-methyl-4-isoxazelopropionate receptor subunit knockout, a mutation which blocks the expression of hippocampal LTP. The present results show that dissociations between spatial working memory and spatial reference memory can be induced by disrupting synaptic plasticity specifically and exclusively within the dentate gyrus subfield of the hippocampal formation.

Complex, multimodal behavioral profile of the Homer1 knockout mouse.

Proteins of the Homer1 immediate early gene family have been associated with synaptogenesis and synaptic plasticity suggesting broad behavioral consequences of loss of function. This study examined the behavior of male Homer1 knockout (KO) mice compared with wild-type (WT) and heterozygous mice using a battery of 10 behavioral tests probing sensory, motor, social, emotional and learning/memory functions. KO mice showed mild somatic growth retardation, poor motor coordination, enhanced sensory reactivity and learning deficits. Heterozygous mice showed increased aggression in social interactions with conspecifics. The distribution of mGluR5 and N-methyl-D-aspartate receptors (NMDA) receptors appeared to be unaltered in the hippocampus (HIP) of Homer1 KO mice. The results indicate an extensive range of disrupted behaviors that should contribute to the understanding of the Homer1 gene in brain development and behavior.

GluR-A-dependent synaptic plasticity is required for the temporal encoding of nonspatial information.

Four related experiments studied operant performance of mice on differential reinforcement of low rates of responding (DRL) paradigms. Experiment 1 showed that excitotoxic hippocampal lesions impaired performance of a 10-s DRL schedule (DRL-10). Experiments 2 and 3 showed that GluR-A AMPA receptor subunit knockout mice, which are deficient in CA3-CA1 long-term potentiation (LTP), were markedly impaired at 15 s (DRL-15), but less impaired at DRL-10. Experiment 4 compared DRL-15 performance in mice from the 2 strains from which the GluR-A colony was derived and showed that they did not differ. The results show that GluR-A-containing AMPA receptors are required for normal performance on hippocampus-dependent, nonspatial working memory tasks, consistent with a role for GluR-A in the temporal encoding (what happened when) of nonspatial information.

SVC: structured visualization of evolutionary sequence conservation.

We have developed a web application for the detailed analysis and visualization of evolutionary sequence conservation in complex vertebrate genes. Given a pair of orthologous genes, the protein-coding sequences are aligned. When these sequences are mapped back onto their encoding exons in the genomes, a scaffold of the conserved gene structure naturally emerges. Sequence similarity between exons and introns is analysed and embedded into the gene structure scaffold. The visualization on the SVC server provides detailed information about evolutionarily conserved features of these genes. It further allows concise representation of complex splice patterns in the context of evolutionary conservation. A particular application of our tool arises from the fact that around mRNA editing sites both exonic and intronic sequences are highly conserved. This aids in delineation of these sites. SVC is available at http://svc.molgen.mpg.de.

Behavioral and neurochemical phenotyping of Homer1 mutant mice: possible relevance to schizophrenia.

Homer proteins are involved in the functional assembly of postsynaptic density proteins at glutamatergic synapses and are implicated in learning, memory and drug addiction. Here, we report that Homer1-knockout (Homer1-KO) mice exhibit behavioral and neurochemical abnormalities that are consistent with the animal models of schizophrenia. Relative to wild-type mice, Homer1-KO mice exhibited deficits in radial arm maze performance, impaired prepulse inhibition, enhanced 'behavioral despair', increased anxiety in a novel objects test, enhanced reactivity to novel environments, decreased instrumental responding for sucrose and enhanced MK-801- and methamphetamine-stimulated motor behavior. No-net-flux in vivo microdialysis revealed a decrease in extracellular glutamate content in the nucleus accumbens and an increase in the prefrontal cortex. Moreover, in Homer1-KO mice, cocaine did not stimulate a rise in frontal cortex extracellular glutamate levels, suggesting hypofrontality. These behavioral and neurochemical data derived from Homer1 mutant mice are consistent with the recent association of schizophrenia with a single-nucleotide polymorphism in the Homer1 gene and suggest that the regulation of extracellular levels of glutamate within limbo-corticostriatal structures by Homer1 gene products may be involved in the pathogenesis of this neuropsychiatric disorder.

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.

A comparison of GluR-A-deficient and wild-type mice on a test battery assessing sensorimotor, affective, and cognitive behaviors.

Previous studies have demonstrated a spatial working memory deficit in glutamate receptor (GluR)-A (GluR1) AMPA receptor subunit knockout mice. The present study evaluated male and female wild-type and GluR-A-/- mice on a test battery that assessed sensorimotor, affective, and cognitive behaviors. Results revealed a behavioral phenotype more extensive than previously described. GluR-A-/- mice were hyperactive, displayed a subtle lack of motor coordination, and were generally more anxious than wild-type controls. In addition, they showed a deficit in spontaneous alternation, consistent with previous reports of a role for GluR-A-dependent plasticity in hippocampus-dependent, spatial working memory. Although changes in motor coordination or anxiety cannot explain the dissociations already reported within the spatial memory domain, it is clear that they could significantly affect interpretation of results obtained in other kinds of behavioral tasks.

Spatial reference memory in GluR-A-deficient mice using a novel hippocampal-dependent paddling pool escape task.

Genetically modified mice lacking the L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit, GluR-A (GluR1), and deficient in hippocampal CA3-CA1 long-term potentiation (LTP), were assessed on a novel, hippocampal-dependent spatial reference memory, paddling pool escape task. The mice were required to use the extramaze cues around the laboratory to find a hidden escape tube that was in a constant location at one of 12 possible positions around the perimeter of the paddling pool, in order to escape from shallow water. The knockout mice performed well on this task. They displayed a small initial impairment (in terms of both escape latencies and choice errors), but they were soon as efficient as the wild-type mice in escaping from the water. This was further demonstrated by performance during a 20-s probe trial in which the exit tube was blocked. Both groups of mice spent most of the time searching in the quadrant of the pool in which the exit tube had previously been located. In a subsequent experiment, entirely normal spatial acquisition was observed in the knockout mice when the paddling pool was moved to a novel spatial environment. The GluR-A -/- mice were also unimpaired in a further reversal phase in which the correct exit location was moved by 180 degrees around the perimeter wall. These results are consistent with previous watermaze studies, providing further demonstration of intact hippocampus-dependent spatial reference memory in GluR-A knockout mice. They contrast strikingly with the profound deficits in hippocampus-dependent, short-term, flexible spatial working memory observed in these knockout mice. This study also demonstrates a novel behavioral task for assessing spatial memory in genetically modified mice. This task shares the behavioral profile of the well-established watermaze paradigm, but may have advantages for the study of genetically modified mice.

GluR-A-Deficient mice display normal acquisition of a hippocampus-dependent spatial reference memory task but are impaired during spatial reversal.

Acquisition and reversal of a spatial discrimination were assessed in an appetitive, elevated plus-maze task in 4 groups of mice: knockout mice lacking the AMPA receptor subunit GluR-A (GluR1), wild-type controls, mice with cytotoxic hippocampal lesions, and controls that had undergone sham surgery. In agreement with previous studies using tasks such as the water maze, GluR-A(-/-) mice were unimpaired during acquisition of the spatial discrimination task, whereas performance in the hippocampalgroup remained at chance levels. In contrast to their performance during acquisition, the GluR-A(-/-) mice displayed a mild deficit during reversal of the spatial discrimination and were profoundly impaired during discrete trial, rewarded-alternation testing on the elevated T maze. The latter result suggests a short-term, flexible spatial working memory impairment in GluR-A(-/-) mice, which might also underlie their mild deficit during spatial reversal.

Spatial memory dissociations in mice lacking GluR1.

Gene-targeted mice lacking the AMPA receptor subunit GluR1 (GluR-A) have deficits in hippocampal CA3-CA1 long-term potentiation. We now report that they showed normal spatial reference learning and memory, both on the hidden platform watermaze task and on an appetitively motivated Y-maze task. In contrast, they showed a specific spatial working memory impairment during tests of non-matching to place on both the Y-maze and an elevated T-maze. In addition, successful watermaze and Y-maze reference memory performance depended on hippocampal function in both wild-type and mutant mice; bilateral hippocampal lesions profoundly impaired performance on both tasks, to a similar extent in both groups. These results suggest that different forms of hippocampus-dependent spatial memory involve different aspects of neural processing within the hippocampus.

Codon-improved Cre recombinase (iCre) expression in the mouse.

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

Impaired NMDA receptor function in mouse olfactory bulb neurons by tetracycline-sensitive NR1 (N598R) expression.

High Ca(2+) permeability and its control by voltage-dependent Mg(2+) block are defining features of NMDA receptors. These features are lost if the principal NR1 subunit carries an asparagine (N) to arginine (R) substitution in a critical channel site at NR1 position 598. NR1(R) expression from a single allele in gene-targeted NR1(+/R) mice is lethal soon after birth, precluding analysis of altered synaptic functions later in life. We therefore employed the forebrain specific alphaCaMKII promoter to drive tTA-mediated tetracycline sensitive transcription of transgenes for NR1(R) and for lacZ as reporter. Transgene expression was observed in cortex, striatum, hippocampus, amygdala and olfactory bulb and was mosaic in all these forebrain regions. It was highest in olfactory bulb granule cells, in most of which Ca(2+) permeability and voltage-dependent Mg(2+) block of NMDA receptors were reduced to different extents. This indicates significant impairment of NMDA receptor function by NR1(R) in presence of the wild-type NR1 complement. Indeed, even though NR1(R) mRNA constituted only 18% of the entire NR1 mRNA population in forebrain, the transgenic mice died during adolescence unless transgene expression was suppressed by doxycycline. Thus, glutamate receptor function can be altered in the mouse by regulated NR1(R) transgene expression.

Dominance of the lurcher mutation in heteromeric kainate and AMPA receptor channels.

Homomeric glutamate receptor (GluR) channels become spontaneously active when the last alanine residue within the invariant SYTANLAAF-motif in the third membrane segment is substituted by threonine. The same mutation in the orphan GluRdelta2 channel is responsible for neurodegeneration in "Lurcher" (Lc) mice. Since most native GluRs are composed of different subunits, we investigated the effect of an Lc-mutated subunit in heteromeric kainate and AMPA receptors expressed in HEK293 cells. Kainate receptor KA2 subunits, either wild type or carrying the Lc mutation (KA2(Lc)), are retained inside the cell but are surface-expressed when assembled with GluR6 subunits. Importantly, KA2(Lc) dominates the gating of KA2(Lc)/GluR6(WT) channels, as revealed by spontaneous activation and by slowed desensitization and deactivation kinetics of ligand-activated whole-cell currents. Moreover, the AMPA receptor subunit GluR-B(Lc)(Q) which forms spontaneously active homomeric channels with rectifying current-voltage relationships, dominates the gating of heteromeric GluR-B(Lc)(Q)/GluR-A(R) channels. The spontaneous currents of these heteromeric AMPAR channels show linear current-voltage relationships, and the ligand-activated whole-cell currents display slower deactivation and desensitization kinetics than the respective wild-type channels. For heteromeric Lc-mutated kainate and AMPA receptors, the effects on kinetics were reduced relative to the homomeric Lc-mutated forms. Thus, an Lc-mutated subunit can potentially influence heteromeric channel function in vivo, and the severity of the phenotype will critically depend on the levels of homomeric GluR(Lc) and heteromeric GluR(Lc)/GluR(WT) channels.

Channel-lining residues of the AMPA receptor M2 segment: structural environment of the Q/R site and identification of the selectivity filter.

In AMPA receptor channels, a single amino acid residue (Q/R site) of the M2 segment controls permeation of calcium ions, single-channel conductance, blockade by intracellular polyamines, and permeation of anions. The structural environment of the Q/R site and its positioning with regard to a narrow constriction were probed with the accessibility of substituted cysteines to positively and negatively charged methanethiosulfonate reagents, applied from the extracellular and cytoplasmic sides of the channel. The accessibility patterns confirm that the M2 segment forms a pore loop with the Q/R site positioned at the tip of the loop (position 0) facing the extracellular vestibule. Cytoplasmically accessible residues on the N- and C-terminal sides of position 0 form the ascending alpha-helical (-8 to -1) and descending random coil (+1 to +6) components of the loop, respectively. Substitution of a glycine residue at position +2 with alanine strongly decreased the permeability of organic cations, indicating that position +2 contributes to the narrow constriction. The anionic 2-sulfonatoethyl-methanethiosufonate reacted with a cysteine at position 0 only from the external side and with cysteines at positions +1 to +4 only from the cytoplasmic side. These results suggest that charge selectivity occurs external to the constriction (+2) and possibly involves interactions of ions with the negative electrostatic potential created by the dipole of the alpha-helix formed by the ascending limb of the loop.

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.