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

Voltage-independent GluN2A-type NMDA receptor Ca2+ signaling promotes audiogenic seizures, attentional and cognitive deficits in mice.

The NMDA receptor-mediated Ca2+ signaling during simultaneous pre- and postsynaptic activity is critically involved in synaptic plasticity and thus has a key role in the nervous system. In GRIN2-variant patients alterations of this coincidence detection provoked complex clinical phenotypes, ranging from reduced muscle strength to epileptic seizures and intellectual disability. By using our gene-targeted mouse line (Grin2aN615S), we show that voltage-independent glutamate-gated signaling of GluN2A-containing NMDA receptors is associated with NMDAR-dependent audiogenic seizures due to hyperexcitable midbrain circuits. In contrast, the NMDAR antagonist MK-801-induced c-Fos expression is reduced in the hippocampus. Likewise, the synchronization of theta- and gamma oscillatory activity is lowered during exploration, demonstrating reduced hippocampal activity. This is associated with exploratory hyperactivity and aberrantly increased and dysregulated levels of attention that can interfere with associative learning, in particular when relevant cues and reward outcomes are disconnected in space and time. Together, our findings provide (i) experimental evidence that the inherent voltage-dependent Ca2+ signaling of NMDA receptors is essential for maintaining appropriate responses to sensory stimuli and (ii) a mechanistic explanation for the neurological manifestations seen in the NMDAR-related human disorders with GRIN2 variant-meidiated intellectual disability and focal epilepsy

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

AutonoMouse: High throughput automated operant conditioning shows progressive behavioural impairment with graded olfactory bulb lesions

AbstractOperant 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 seen much less of an improvement. 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 &gt;10^6 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 and resolve the long-standing conundrum about the apparent lack of impact of lesions on olfactory abilities. The modular nature and open-source design of AutonoMouse should allow for similar robust and systematic assessments across neuroscience research areas.</jats:p

AutonoMouse gives high volumes of reliable behavioural data.

(a) Number of trials performed per day by animals housed in AutonoMouse (n = 67, mean +/- std, total trials = 1,351,320). (b) Number of trials that are performed in sessions of continuous trial sequences ( 0.05). (f) Average performance accuracy in each hour of the day, averaged over all animals (mean +/- sem).</p

AutonoMouse schematic.

(a) Basic design of the AutonoMouse system showing the link between the common home cage and the upper behavioural staging area. (i) Main home cage. (ii) Food hopper. (iii) Access door controlled by IR beam detectors. (iv) IR beam detectors, inactive as not blocked by animal. (v) Active IR beam detectors blocked by animal. (vi) Unique RFID readout. (vii) Water reservoir, pump and lickometer. (viii) Odour stimulus production. (ix) Odour exhaust. (x) Access ramp. (b) Time course of a typical olfactory go/no-go stimulus in the system. (c) Response/reward table showing trial outcomes depending on stimulus type and whether animal licks in ≥3 (+ve response) response period quarters or <3 (-ve response) (Q1-Q4 in (b). (d) Performance over trials in the first introduced olfactory discrimination task (n = 29, mean +/- sem; sliding average with 100 trial window).</p

Odour delivery.

(a) Olfactometer schematic. Numerical values shown indicate supplied air flow in cubic centimeters per minute. Black / red lines indicate resting state (between trials) and odour delivery state air pathways respectively. (b) Example PID recorded odour trace. Left: entire recorded pulse, right: at higher temporal resolution. (c) Example recording from the olfactometer switching between final valve only (FV only), empty (non-odourised) input and odourised input (100%, red). (d) Output odour concentration is reliably controlled by airflow dilution. Left: 10 overlaid odour pulses during maximum MFC input (blue), 60% MFC input (green) and 40% MFC input (red). Right: summary of PID recorded odour signal in the three conditions. (e1) Map of trial performance before and after introduction of an extra valve set (as schematised above: red = odour 1, blue = odour 2) into the odour stimulus production, during the first odour pair discrimination learnt by this set of animals (n = 9). Each row corresponds to an animal, with each column in the row corresponding to a trial (pre-switch n = 12, post-switch n = 12). The vertical dashed line indicates the point at which new valves were introduced. Light green: hit, dark green: correct rejection, light red: false alarm, dark red: miss. (e2) Summary of data shown in (e1) showing mean performance before and after for each animal in the group (connecting black line, start and end values jittered for ease of visualisation). Thick black lines indicate the mean of the group pre- and post- new valve introduction. (f1), (f2), (g1), (g2), (h1), (h2) Same analysis as in (e1), (e2) but for novel and familiar odour pair discrimination respectively. (g1), (g2) shows performance on the novel odour pair task before and after a full bottle switch with randomised placement. (i) Performance in a standard odour pair discrimination (EB vs. AA) followed by diversion of the odour stream in the olfactometer final valve (mean +/- sem). Performance analyses in 100 trial bins for each animal.</p