Genetic investigations into the role of ionotropic glutamate receptors in hippocampal learning

AMPA and NMDA receptors are ionotropic glutamate receptors, respectively sensitive to the glutamate analogue alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA), and are essential for hippocampus-dependent learning and memory. As indicated by global and forebrainspecific mutant mouse models of AMPA and NMDA receptors, distinct molecular mechanisms coexist in the dorsal hippocampus, underlying spatial behavior in working and reference memory tasks. The present study is focused on the main ionotropic glutamate receptors (AMPA receptors with GluR-A or GluR-B subunit or NMDA receptors) in principal neurons (DG, CA1, CA2) of the hippocampus in adult mice and the role of these receptors in spatial working and reference memory. Cre recombinase expression in restricted sublayers of the hippocampal formation and the olfactory system was achieved by the use of transgenes of mouse lines TgCN12-itTA and TgLC1 employing the tet-system to prevent widespread recombination in the mouse embryo. Minor recombination, monitored by the use of gene-targeted Rosa26R mice, accumulated in additional forebrain structures but remained sparsely located in one-year-old mice. By employing the TgCN12-itTA / TgLC1 mouse model to deplete GluR-A in Gria1ΔHipOlf mice, GluR-B in Gria2ΔHipOlf mice or all NMDA receptors by NR1 ablation in Grin1ΔHipOlf mice, excitatory neurotransmission was modified in three major ways. Depletion of these receptor subunits was observed with similar spatial and temporal specificity in hippocampal sublayers of adult mice. With these three iGluRΔHipOlf mouse models in hands, behavioral consequences were investigated in spatial working and reference memory tasks in two independent laboratories (Heidelberg, Germany; Oxford, England). Unexpected from our previous observations (Reisel et al. 2002), GluR-A depleted Gria1ΔHipOlf mice performed well in all cognitive tasks of spatial working behavior independent of delay and task composition. However, Gria1ΔHipOlf mice still expressed hyperactivity in a novel environment and little spontaneous alternation. In contrast, GluR-B depletion in Gria2ΔHipOlf mice became manifest in impairment in spatial working memory. Unfortunately, testing of spatial reference memory in Gria2ΔHipOlf mice is still missing. Grin1ΔHipOlf mice exhibited delay- and task-dependent impairment of the IV spatial working memory and in reversal reference learning. Nevertheless, the acquisition of spatial reference memory in Morris watermaze and Y-maze was not affected upon NR1 depletion in dorsal CA1, CA2 and the entire DG subfield of the hippocampal formation. In summary, genetic manipulation of the main ionotropic glutamate receptors in the three mutant mouse models Gria1ΔHipOlf, Gria2ΔHipOlf and Grin1ΔHipOlf demonstrated the essential role of AMPA receptors containing the GluR-B subunit and NMDA receptors in principal DG, CA1 and CA2 neurons of the hippocampal formation in spatial working memory. Spatial reference memory, however, was still intact upon depletion of AMPA receptors containing the GluR-A subunit in Gria1ΔHipOlf mice and NMDA receptors in Grin1ΔHipOlf mice

Different Forms of AMPA Receptor Mediated LTP and Their Correlation to the Spatial Working Memory Formation

Spatial working memory (SWM) and the classical, tetanus-induced long-term potentiation (LTP) at hippocampal CA3/CA1 synapses are dependent on L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) containing GluA1 subunits as demonstrated by knockout mice lacking GluA1. In GluA1 knockout mice LTP and SWM deficits could be partially recovered by transgenic re-installation of full-length GluA1 in principle forebrain neurons. Here we partially restored hippocampal LTP in GluA1-deficient mice by forebrain-specific depletion of the GluA2 gene, by the activation of a hypomorphic GluA2(Q) allele and by transgenic expression of PDZ-site truncated GFP-GluA1(TG). In none of these three mouse lines, the partial LTP recovery improved the SWM performance of GluA1-deficient mice suggesting a specific function of intact GluA1/2 receptors and the GluA1 intracellular carboxyl-terminus in SWM and its associated behavior

Enhanced Odor Discrimination and Impaired Olfactory Memory by Spatially Controlled Switch of AMPA Receptors

Genetic perturbations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) are widely used to dissect molecular mechanisms of sensory coding, learning, and memory. In this study, we investigated the role of Ca(2+)-permeable AMPARs in olfactory behavior. AMPAR modification was obtained by depletion of the GluR-B subunit or expression of unedited GluR-B(Q), both leading to increased Ca(2+) permeability of AMPARs. Mice with this functional AMPAR switch, specifically in forebrain, showed enhanced olfactory discrimination and more rapid learning in a go/no-go operant conditioning task. Olfactory memory, however, was dramatically impaired. GluR-B depletion in forebrain was ectopically variable (“mosaic”) among individuals and strongly correlated with decreased olfactory memory in hippocampus and cortex. Accordingly, memory was rescued by transgenic GluR-B expression restricted to piriform cortex and hippocampus, while enhanced odor discrimination was independent of both GluR-B variability and transgenic GluR-B expression. Thus, correlated differences in behavior and levels of GluR-B expression allowed a mechanistic and spatial dissection of olfactory learning, discrimination, and memory capabilities

Expression patterns of promoters for NPY Y1 and Y5 receptors in Y5RitTA and Y1RVenus BAC−transgenic mice

In the rat brain, neuropeptide Y (NPY) Y(1) and Y(5) receptors are coexpressed in various forebrain regions where they mediate several NPY-activated functions, including feeding behaviour, anxiety, neuronal excitability and hormone secretion. We studied the distribution pattern and cellular colocalization of the Y(1) and the Y(5) receptor gene expression in the mouse brain by using transgenic mice with genomically integrated BAC clones, where the coding regions of the Y(1) and Y(5) receptor genes were replaced by Venus and the synthetic transcription factor itTA reporter genes, respectively (Tg(Y5RitTA/Y1RVenus) mice). Analysis of Venus fluorescence and itTA-mediated activation of Cre recombinase revealed copy number-dependent expression levels, between the lines, but similar expression patterns. In three transgenic lines the BAC encoded Y(5) receptor promoter induced strong Cre expression in the olfactory system, cerebral cortex, hippocampus and basal ganglia. Weaker expression was found in most of the hypothalamic nuclei of line 25, the highest-expressing transgenic line. Activation of Cre was itTA-dependent and could be regulated by doxycycline. The Y(1) receptor promoter-induced Venus fluorescence was intense, widely present through the brain and colocalized with Cre immunostaining in neurons of distinct brain regions, including the cerebral cortex, basolateral amygdala, dentate gyrus and paraventricular nucleus. These data provide a detailed and comparative mapping of Y(1) and Y(5) receptor promoter activity within cells of the mouse brain. The Tg(Y5RitTA/Y1RVenus)-transgenic mice generated here also represent a genetic tool for conditional mutagenesis via the Cre lox system, particularly of genes involved in feeding behaviour, neuronal excitability and hormone secretion

AutonoMouse: High throughput operant conditioning reveals progressive impairment with graded olfactory bulb lesions.

Operant conditioning is a crucial tool in neuroscience research for probing brain function. While molecular, anatomical and even physiological techniques have seen radical increases in throughput, efficiency, and reproducibility in recent years, behavioural tools have somewhat lagged behind. Here we present a fully automated, high-throughput system for self-initiated conditioning of up to 25 group-housed, radio-frequency identification (RFID) tagged mice over periods of several months and >106 trials. We validate this "AutonoMouse" system in a series of olfactory behavioural tasks and show that acquired data is comparable to previous semi-manual approaches. Furthermore, we use AutonoMouse to systematically probe the impact of graded olfactory bulb lesions on olfactory behaviour, demonstrating that while odour discrimination in general is robust to even most extensive disruptions, small olfactory bulb lesions already impair odour detection. Discrimination learning of similar mixtures as well as learning speed are in turn reliably impacted by medium lesion sizes. The modular nature and open-source design of AutonoMouse should allow for similar robust and systematic assessments across neuroscience research areas

Different Forms of AMPA Receptor Mediated LTP and Their Correlation to the Spatial Working Memory Formation

Spatial working memory (SWM) and the classical, tetanus-induced long-term potentiation (LTP) at hippocampal CA3/CA1 synapses are dependent on L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) containing GluA1 subunits as demonstrated by knockout mice lacking GluA1. In GluA1 knockout mice LTP and SWM deficits could be partially recovered by transgenic re-installation of full-length GluA1 in principle forebrain neurons. Here we partially restored hippocampal LTP in GluA1-deficient mice by forebrain-specific depletion of the GluA2 gene, by the activation of a hypomorphic GluA2(Q) allele and by transgenic expression of PDZ-site truncated GFP-GluA1(TG). In none of these three mouse lines, the partial LTP recovery improved the SWM performance of GluA1-deficient mice suggesting a specific function of intact GluA1/2 receptors and the GluA1 intracellular carboxyl-terminus in SWM and its associated behavior

Impaired Reproductive Behavior by Lack of GluR-B Containing AMPA Receptors But Not of NMDA Receptors in Hypothalamic and Septal Neurons

The roles of ionotropic glutamate receptors in mammalian reproduction are unknown. We therefore generated mice lacking a major subtype of (S)−−amino−3−hydroxy−5−methyl−isoxazolepropionic acid (AMPA) receptors or all N−methyl−D−aspartate (NMDA) receptors in GnRH neurons and other mainly limbic system neurons, primarily in hypothalamic and septal areas. Male mice without NMDA receptors in these neurons were not impaired in breeding and exhibited similar GnRH secretion as control littermates. However, male mice lacking GluR−B containing AMPA receptors in these neurons were poor breeders and severely impaired in reproductive behaviors such as aggression and mounting. Testis and sperm morphology, testis weight, and serum testosterone levels, as well as GnRH secretion, were unchanged. Contact with female cage bedding failed to elicit male sexual behavior in these mice, unlike in control male littermates. Their female counterparts had unchanged ovarian morphology, had bred successfully, and had normal litter sizes but exhibited pronounced impairments in maternal behaviors such as pup retrieval and maternal aggression. Our results suggest that NMDA receptors and GluR−B containing AMPA receptors are not essential for fertility, but that GluR−B containing AMPA receptors are essential for male and female reproduction−related behaviors, perhaps by mediating responses to pheromones or odorants

Deciphering the Contributions of CRH Receptors in the Brain and Pituitary to Stress-Induced Inhibition of the Reproductive Axis

Based on pharmacological studies, corticotropin-releasing hormone (CRH) and its receptors play a leading role in the inhibition of the hypothalamic-pituitary-gonadal (HPG) axis during acute stress. To further study the effects of CRH receptor signaling on the HPG axis, we generated and/or employed male mice lacking CRH receptor type 1 (CRHR1) or type 2 (CRHR2) in gonadotropin-releasing hormone neurons, GABAergic neurons, or in all central neurons and glia. The deletion of CRHRs revealed a preserved decrease of plasma luteinizing hormone (LH) in response to either psychophysical or immunological stress. However, under basal conditions, central infusion of CRH into mice lacking CRHR1 in all central neurons and glia, or application of CRH to pituitary cultures from mice lacking CRHR2, failed to suppress LH release, unlike in controls. Our results, taken together with those of the earlier pharmacological studies, suggest that inhibition of the male HPG axis during acute stress is mediated by other factors along with CRH, and that CRH suppresses the HPG axis at the central and pituitary levels via CRHR1 and CRHR2, respectively

Somatic Accumulation of GluA1-AMPA Receptors Leads to Selective Cognitive Impairments in Mice

The GluA1 subunit of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) plays a crucial, but highly selective, role in cognitive function. Here we analyzed AMPAR expression, AMPAR distribution and spatial learning in mice (Gria1R/R), expressing the “trafficking compromised” GluA1(Q600R) point mutation. Our analysis revealed somatic accumulation and reduction of GluA1(Q600R) and GluA2, but only slightly reduced CA1 synaptic localization in hippocampi of adult Gria1R/R mice. These immunohistological changes were accompanied by a strong reduction of somatic AMPAR currents in CA1, and a reduction of plasticity (short-term and long-term potentiation, STP and LTP, respectively) in the CA1 subfield following tetanic and theta-burst stimulation. Nevertheless, spatial reference memory acquisition in the Morris water-maze and on an appetitive Y-maze task was unaffected in Gria1R/R mice. In contrast, spatial working/short-term memory during both spontaneous and rewarded alternation tasks was dramatically impaired. These findings identify the GluA1(Q600R) mutation as a loss of function mutation that provides independent evidence for the selective role of GluA1 in the expression of short-term memory

Specific Hippocampus and Piriform Cortex Expression of Transgenic ^GFPGluR-B

<div><p>(A) Schematic diagrams depicting forebrain-specific GluR-B deletion as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-g002" target="_blank">Figure 2</a>A and itTA-dependent expression of ^GFPGluR-B and nuclear-localized β-galactosidase (nLacZ) in <i>GluR-B^ΔFB</i> mice (termed <i>GluR-B^Rescue</i>). ^GFPGluR-B and nLacZ are both encoded by <i>Tg^OCN1,</i> and itTA is controlled by a fusion of the NR2C silencer element [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-b91" target="_blank">91</a>] and the αCaMKII promoter (termed <i>Tg^CN12-itTA</i> [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030354#pbio-0030354-b92" target="_blank">92</a>]).</p> <p>(B) In coronal brain sections of mice positive for both transgenes <i>(Tg^CN12-itTA</i> and <i>Tg^OCN1)</i> β-galactosidase activity (blue, X-gal, counterstain by eosin) is restricted to hippocampal neurons in CA1, DG, and neurons in the piriform cortex. The same neurons show ^GFPGluR-B expression when analyzed in immunohistochemical sections with an antibody against GFP. Scale bars: 500 μm.</p> <p>(C) Immunoblot detecting endogenous GluR-B and transgenic ^GFPGluR-B in the hippocampus of three different mice (#1, #2, #3).</p> <p>(D) Relative quantification from (C) of transgenic ^GFPGluR-B compared with endogenous GluR-B in the hippocampus.</p></div