Dlk1 dosage regulates hippocampal neurogenesis and cognition.

Neurogenesis in the adult brain gives rise to functional neurons, which integrate into neuronal circuits and modulate neural plasticity. Sustained neurogenesis throughout life occurs in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and is hypothesized to be involved in behavioral/cognitive processes such as memory and in diseases. Genomic imprinting is of critical importance to brain development and normal behavior, and exemplifies how epigenetic states regulate genome function and gene dosage. While most genes are expressed from both alleles, imprinted genes are usually expressed from either the maternally or the paternally inherited chromosome. Here, we show that in contrast to its canonical imprinting in nonneurogenic regions, Delta-like homolog 1 (Dlk1) is expressed biallelically in the SGZ, and both parental alleles are required for stem cell behavior and normal adult neurogenesis in the hippocampus. To evaluate the effects of maternally, paternally, and biallelically inherited mutations within the Dlk1 gene in specific behavioral domains, we subjected Dlk1-mutant mice to a battery of tests that dissociate and evaluate the effects of Dlk1 dosage on spatial learning ability and on anxiety traits. Importantly, reduction in Dlk1 levels triggers specific cognitive abnormalities that affect aspects of discriminating differences in environmental stimuli, emphasizing the importance of selective absence of imprinting in this neurogenic niche.

Impaired instrumental learning in Spred1-/- mice, a model for a rare RASopathy.

RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modelling these cognitive deficits in a Spred1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if Spred1-/- mice exhibit deficits in other cognitive domains. We show that Spred1-/- mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n = 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signalling pathway could rescue these cognitive impairments, Spred1-/- mice were acutely treated with the clinically relevant mitogen-activated protein kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that Spred1-/- mice can model severe cognitive impairments that cannot be reversed in adulthood.

Learning and reaction times in mouse touchscreen tests are differentially impacted by mutations in genes encoding postsynaptic interacting proteins SYNGAP1, NLGN3, DLGAP1, DLGAP2 and SHANK2.

The postsynaptic terminal of vertebrate excitatory synapses contains a highly conserved multiprotein complex that comprises neurotransmitter receptors, cell-adhesion molecules, scaffold proteins and enzymes, which are essential for brain signalling and plasticity underlying behaviour. Increasingly, mutations in genes that encode postsynaptic proteins belonging to the PSD-95 protein complex, continue to be identified in neurodevelopmental disorders (NDDs) such as autism spectrum disorder, intellectual disability and epilepsy. These disorders are highly heterogeneous, sharing genetic aetiology and comorbid cognitive and behavioural symptoms. Here, by using genetically engineered mice and innovative touchscreen-based cognitive testing, we sought to investigate whether loss-of-function mutations in genes encoding key interactors of the PSD-95 protein complex display shared phenotypes in associative learning, updating of learned associations and reaction times. Our genetic dissection of mice with loss-of-function mutations in Syngap1, Nlgn3, Dlgap1, Dlgap2 and Shank2 showed that distinct components of the PSD-95 protein complex differentially regulate learning, cognitive flexibility and reaction times in cognitive processing. These data provide insights for understanding how human mutations in these genes lead to the manifestation of diverse and complex phenotypes in NDDs.

Enhanced cognition and dysregulated hippocampal synaptic physiology in mice with a heterozygous deletion of PSD-95.

PSD-95 is one of the most abundant proteins of the postsynaptic density of excitatory synapses. It functions as the backbone of protein supercomplexes that mediate signalling between membrane glutamate receptors and intracellular pathways. Homozygous deletion of the Dlg4 gene encoding PSD-95 was previously found to cause a profound impairment in operant and Pavlovian conditioning in Dlg4-/- mice studied in touch screen chambers that precluded evaluation of PSD-95's role in shaping more subtle forms of learning and memory. In this study, using a battery of touch screen tests, we investigated cognitive behaviour of mice with a heterozygous Dlg4 mutation. We found that in contrast to learning deficits of Dlg4-/- mice, Dlg4+/- animals demonstrated enhanced performance in the Visual Discrimination, Visual Discrimination Reversal and Paired-Associates Learning touch screen tasks. The divergent directions of learning phenotypes observed in Dlg4-/- and Dlg4+/- mice also contrasted with qualitatively similar changes in the amplitude and plasticity of field excitatory postsynaptic potentials recorded in the CA1 area of hippocampal slices from both mutants. Our results have important repercussions for the studies of genetic models of human diseases, because they demonstrate that reliance on phenotypes observed solely in homozygous mice may obscure qualitatively different changes in heterozygous animals and potentially weaken the validity of translational comparisons with symptoms seen in heterozygous human carriers.

Haploinsufficiency of EHMT1 improves pattern separation and increases hippocampal cell proliferation.

Heterozygous mutations or deletions of the human Euchromatin Histone Methyltransferase 1 (EHMT1) gene are the main causes of Kleefstra syndrome, a neurodevelopmental disorder that is characterized by impaired memory, autistic features and mostly severe intellectual disability. Previously, Ehmt1+/- heterozygous knockout mice were found to exhibit cranial abnormalities and decreased sociability, phenotypes similar to those observed in Kleefstra syndrome patients. In addition, Ehmt1+/- knockout mice were impaired at fear extinction and novel- and spatial object recognition. In this study, Ehmt1+/- and wild-type mice were tested on several cognitive tests in a touchscreen-equipped operant chamber to further investigate the nature of learning and memory changes. Performance of Ehmt1+/- mice in the Visual Discrimination &Reversal learning, object-location Paired-Associates learning- and Extinction learning tasks was found to be unimpaired. Remarkably, Ehmt1+/- mice showed enhanced performance on the Location Discrimination test of pattern separation. In line with improved Location Discrimination ability, an increase in BrdU-labelled cells in the subgranular zone of the dentate gyrus was observed. In conclusion, reduced levels of EHMT1 protein in Ehmt1+/- mice does not result in general learning deficits in a touchscreen-based battery, but leads to increased adult cell proliferation in the hippocampus and enhanced pattern separation ability.

The touchscreen operant platform for testing learning and memory in rats and mice.

An increasingly popular method of assessing cognitive functions in rodents is the automated touchscreen platform, on which a number of different cognitive tests can be run in a manner very similar to touchscreen methods currently used to test human subjects. This methodology is low stress (using appetitive rather than aversive reinforcement), has high translational potential and lends itself to a high degree of standardization and throughput. Applications include the study of cognition in rodent models of psychiatric and neurodegenerative diseases (e.g., Alzheimer's disease, schizophrenia, Huntington's disease, frontotemporal dementia), as well as the characterization of the role of select brain regions, neurotransmitter systems and genes in rodents. This protocol describes how to perform four touchscreen assays of learning and memory: visual discrimination, object-location paired-associates learning, visuomotor conditional learning and autoshaping. It is accompanied by two further protocols (also published in this issue) that use the touchscreen platform to assess executive function, working memory and pattern separation.

The touchscreen operant platform for testing working memory and pattern separation in rats and mice.

The automated touchscreen operant chamber for rats and mice allows for the assessment of multiple cognitive domains within the same testing environment. This protocol presents the location discrimination (LD) task and the trial-unique delayed nonmatching-to-location (TUNL) task, which both assess memory for location. During these tasks, animals are trained to a predefined criterion during ∼20-40 daily sessions. In LD sessions, touching the same location on the screen is rewarded on consecutive trials, followed by a reversal of location-reward contingencies. TUNL, a working memory task, requires animals to 'nonmatch' to a sample location after a delay. In both the LD and TUNL tasks, spatial similarity can be varied, allowing assessment of pattern separation ability, a function that is thought to be performed by the dentate gyrus (DG). These tasks are therefore particularly useful in animal models of hippocampal, and specifically DG, function, but they additionally permit discernment of changes in pattern separation from those in working memory.

The touchscreen operant platform for assessing executive function in rats and mice.

This protocol details a subset of assays developed within the touchscreen platform to measure various aspects of executive function in rodents. Three main procedures are included: extinction, measuring the rate and extent of curtailing a response that was previously, but is no longer, associated with reward; reversal learning, measuring the rate and extent of switching a response toward a visual stimulus that was previously not, but has become, associated with reward (and away from a visual stimulus that was previously, but is no longer, rewarded); and the 5-choice serial reaction time (5-CSRT) task, gauging the ability to selectively detect and appropriately respond to briefly presented, spatially unpredictable visual stimuli. These protocols were designed to assess both complementary and overlapping constructs including selective and divided visual attention, inhibitory control, flexibility, impulsivity and compulsivity. The procedures comprise part of a wider touchscreen test battery assessing cognition in rodents with high potential for translation to human studies.

Depletion of perineuronal nets enhances recognition memory and long-term depression in the perirhinal cortex.

Perineuronal nets (PNNs) are extracellular matrix structures surrounding cortical neuronal cell bodies and proximal dendrites and are involved in the control of brain plasticity and the closure of critical periods. Expression of the link protein Crtl1/Hapln1 in neurons has recently been identified as the key event triggering the formation of PNNs. Here we show that the genetic attenuation of PNNs in adult brain Crtl1 knock-out mice enhances long-term object recognition memory and facilitates long-term depression in the perirhinal cortex, a neural correlate of object recognition memory. Identical prolongation of memory follows localized digestion of PNNs with chondroitinase ABC, an enzyme that degrades the chondroitin sulfate proteoglycan components of PNNs. The memory-enhancing effect of chondroitinase ABC treatment attenuated over time, suggesting that the regeneration of PNNs gradually restored control plasticity levels. Our findings indicate that PNNs regulate both memory and experience-driven synaptic plasticity in adulthood.

A touch screen-automated cognitive test battery reveals impaired attention, memory abnormalities, and increased response inhibition in the TgCRND8 mouse model of Alzheimer's disease.

Transgenic mouse models of Alzheimer's disease (AD) with abundant ß-amyloid develop memory impairments. However, multiple nonmnemonic cognitive domains such as attention and executive control are also compromised early in AD individuals, but have not been routinely assessed in animal models. Here, we assessed the cognitive abilities of TgCRND8 mice-a widely used model of ß-amyloid pathology-with a touch screen-based automated test battery. The test battery comprises highly translatable tests of multiple cognitive constructs impaired in human AD, such as memory, attention, and response control, as well as appropriate control tasks. We found that familial AD mutations affect not only memory, but also cause significant alterations of sustained attention and behavioral flexibility. Because changes in attention and response inhibition may affect performance on tests of other cognitive abilities including memory, our findings have important consequences for the assessment of disease mechanisms and therapeutics in animal models of AD. A more comprehensive phenotyping with specialized, multicomponent cognitive test batteries for mice might significantly advance translation from preclinical mouse studies to the clinic.