Extinction of an instrumental response: a cognitive behavioral assay in Fmr1 knockout mice.

Fragile X (FX) is the most common genetic cause of intellectual disability and autism. Previous studies have shown that partial inhibition of metabotropic glutamate receptor signaling is sufficient to correct behavioral phenotypes in a mouse model of FX, including audiogenic seizures, open-field hyperactivity and social behavior. These phenotypes model well the epilepsy (15%), hyperactivity (20%) and autism (30%) that are comorbid with FX in human patients. Identifying reliable and robust mouse phenotypes to model cognitive impairments is critical considering the 90% comorbidity of FX and intellectual disability. Recent work characterized a five-choice visuospatial discrimination assay testing cognitive flexibility, in which FX model mice show impairments associated with decreases in synaptic proteins in prefrontal cortex (PFC). In this study, we sought to determine whether instrumental extinction, another process requiring PFC, is altered in FX model mice, and whether downregulation of metabotropic glutamate receptor signaling pathways is sufficient to correct both visuospatial discrimination and extinction phenotypes. We report that instrumental extinction is consistently exaggerated in FX model mice. However, neither the extinction phenotype nor the visuospatial discrimination phenotype is corrected by approaches targeting metabotropic glutamate receptor signaling. This work describes a novel behavioral extinction assay to model impaired cognition in mouse models of neurodevelopmental disorders, provides evidence that extinction is exaggerated in the FX mouse model and suggests possible limitations of metabotropic glutamate receptor-based pharmacotherapy.

Anatomical origins of ocular dominance in mouse primary visual cortex.

Ocular dominance (OD) plasticity is a classic paradigm for studying the effect of experience and deprivation on cortical development, and is manifested as shifts in the relative strength of binocular inputs to primary visual cortex (V1). The mouse has become an increasingly popular model for mechanistic studies of OD plasticity and, consequently, it is important that we understand how binocularity is constructed in this species. One puzzling feature of the mouse visual system is the gross disparity between the physiological strength of each eye in V1 and their anatomical representation in the projection from retina to the dorsal lateral geniculate nucleus (dLGN). While the contralateral-to-ipsilateral (C/I) ratio of visually evoked responses in binocular V1 is approximately 2:1, the ipsilateral retinal projection is weakly represented in terms of retinal ganglion cell (RGC) density where the C/I ratio is approximately 9:1. The structural basis for this relative amplification of ipsilateral eye responses between retina and V1 is not known. Here we employed neuroanatomical tracing and morphometric techniques to quantify the relative magnitude of each eye's input to and output from the binocular segment of dLGN. Our data are consistent with the previous suggestion that a point in space viewed by both eyes will activate 9 times as many RGCs in the contralateral retina as in the ipsilateral retina. Nonetheless, the volume of the dLGN binocular segment occupied by contralateral retinogeniculate inputs is only 2.4 times larger than the volume occupied by ipsilateral retinogeniculate inputs and recipient relay cells are evenly distributed among the input layers. The results from our morphometric analyses show that this reduction in input volume can be accounted for by a three-to-one convergence of contralateral eye RGC inputs to dLGN neurons. Together, our findings establish that the relative density of feed-forward dLGN inputs determines the C/I response ratio of mouse binocular V1.

Therapeutic implications of the mGluR theory of fragile X mental retardation.

Evidence is reviewed that the consequences of group 1 metabotropic glutamate receptor (Gp1 mGluR) activation are exaggerated in the absence of the fragile X mental retardation protein, likely reflecting altered dendritic protein synthesis. Abnormal mGluR signaling could be responsible for remarkably diverse psychiatric and neurological symptoms in fragile X syndrome, including delayed cognitive development, seizures, anxiety, movement disorders and obesity.

Forebrain-specific calcineurin knockout selectively impairs bidirectional synaptic plasticity and working/episodic-like memory.

Calcineurin is a calcium-dependent protein phosphatase that has been implicated in various aspects of synaptic plasticity. By using conditional gene-targeting techniques, we created mice in which calcineurin activity is disrupted specifically in the adult forebrain. At hippocampal Schaffer collateral-CA1 synapses, LTD was significantly diminished, and there was a significant shift in the LTD/LTP modification threshold in mutant mice. Strikingly, although performance was normal in hippocampus-dependent reference memory tasks, including contextual fear conditioning and the Morris water maze, the mutant mice were impaired in hippocampus-dependent working and episodic-like memory tasks, including the delayed matching-to-place task and the radial maze task. Our results define a critical role for calcineurin in bidirectional synaptic plasticity and suggest a novel mechanistic distinction between working/episodic-like memory and reference memory.

A role for the cytoplasmic polyadenylation element in NMDA receptor-regulated mRNA translation in neurons.

The ability of neurons to modify synaptic connections based on activity is essential for information processing and storage in the brain. The induction of long-lasting changes in synaptic strength requires new protein synthesis and is often mediated by NMDA-type glutamate receptors (NMDARs). We used a dark-rearing paradigm to examine mRNA translational regulation in the visual cortex after visual experience-induced synaptic plasticity. In this model system, we demonstrate that visual experience induces the translation of mRNA encoding the alpha-subunit of calcium/calmodulin-dependent kinase II in the visual cortex. Furthermore, this increase in translation is NMDAR dependent. One potential source for newly synthesized proteins is the translational activation of dormant cytoplasmic mRNAs. To examine this possibility, we developed a culture-based assay system to study translational regulation in neurons. Cultured hippocampal neurons were transfected with constructs encoding green fluorescent protein (GFP). At 6 hr after transfection, approximately 35% of the transfected neurons (as determined by in situ hybridization) expressed detectable GFP protein. Glutamate stimulation of the cultures at this time induced an increase in the number of neurons expressing GFP protein that was NMDAR dependent. Importantly, the glutamate-induced increase was only detected when the 3'-untranslated region of the GFP constructs contained intact cytoplasmic polyadenylation elements (CPEs). Together, these findings define a molecular mechanism for activity-dependent synaptic plasticity that is mediated by the NMDA receptor and requires the CPE-dependent translation of an identified mRNA.

Long-term potentiation of thalamocortical transmission in the adult visual cortex in vivo.

It has been suggested that NMDA receptor-dependent synaptic strengthening, like that observed after long-term potentiation (LTP), is a mechanism by which experience modifies responses in the neocortex. We report here that patterned (theta burst) stimulation of the dorsal lateral geniculate nucleus reliably induces LTP of field potentials (FPs) evoked in primary visual cortex (Oc1) of adult rats in vivo. The response enhancement is saturable, long-lasting, and dependent on NMDA receptor activation. To determine the laminar locus of these changes, current source density (CSD) analysis was performed on FP profiles obtained before and after LTP induction. LTP was accompanied by an enhancement of synaptic current sinks located in thalamorecipient (layer IV and deep layer III) and supragranular (layers II/III) cell layers. We also examined immunocytochemical labeling for the immediate early gene zif-268 1 hr after induction of LTP. In concert with the laminar changes observed in CSD analyses, we observed a significant increase in the number of zif-268-immunopositive neurons in layers II-IV that occurred over a wide extent of Oc1. Last, we investigated the functional consequences of LTP induction by monitoring changes in visually evoked potentials. After LTP, we observed that the cortical response to a full-field flash was significantly enhanced and that responses to grating stimuli were increased across a range of spatial frequencies. These findings are consistent with growing evidence that primary sensory cortex remains plastic into adulthood, and they show that the mechanisms of LTP can contribute to this plasticity.

Internalization of ionotropic glutamate receptors in response to mGluR activation.

Activation of group 1 metabotropic glutamate receptors (mGluRs) stimulates dendritic protein synthesis and long-term synaptic depression (LTD), but it remains unclear how these effects are related. Here we provide evidence that a consequence of mGluR activation in the hippocampus is the rapid loss of both AMPA and NMDA receptors from synapses. Like mGluR-LTD, the stable expression of this change requires protein synthesis. These data suggest that expression of mGluR-LTD is at least partly postsynaptic, and that a functional consequence of dendritic protein synthesis is the regulation of glutamate receptor trafficking.

Effect of transgenic overexpression of NR2B on NMDA receptor function and synaptic plasticity in visual cortex.

The NMDA receptor (NMDAR) is a heteromer comprised of NR1 and NR2 subunits. Mice that overexpress the NR2B subunit exhibit enhanced hippocampal LTP, prolonged NMDAR currents, and improved memory ( Tang et al., 1999). In the current study, we explored visual cortex plasticity and NMDAR function in NR2B overexpressing transgenic mice. Unlike the hippocampus, in vitro synaptic plasticity of the visual cortex was unaltered by NR2B overexpression. Consistent with the plasticity findings, NMDAR excitatory postsynaptic current (EPSC) durations from layer 2/3 pyramidal cells were similar in wild-type (wt) and transgenic (tg) mice. Furthermore, temporal summation of NMDAR EPSCs to 10, 20, and 40 Hz stimulation did not differ between cells from wt and tg mice. Finally, although in situ studies clearly demonstrate overexpression of NR2B mRNA in visual cortex, we failed to observe a significant elevation in the synaptic expression of NR2B protein. We conclude that the synaptic ratio of NR2B over NR2A in the NMDA receptor complex in the visual cortex is not significantly influenced by the transgene overexpression. These data suggest that mRNA availability is not a limiting factor for the synthesis of NR2B protein in the visual cortex, and support the hypothesis that levels of NR2A, rather than NR2B, normally determine the subunit composition of NMDARs in visual cortex.

Developmental inhibitory gate controls the relay of activity to the superficial layers of the visual cortex.

A developmental reduction in the radial transmission of synaptic activity has been proposed to underlie the end of the critical period for experience-dependent modification in layers II/III of the visual cortex. Using paired-pulse stimulation, we investigated in visual cortical slices how the propagation of synaptic activity to the superficial layers changes during development and how this process is affected by sensory experience. The results can be summarized as follows. (1) Layers II/III responses to repetitive stimulation of the white matter become increasingly depressed between the third and sixth week of postnatal development, a time course that parallels the end of the critical period. (2) Paired-pulse depression is reduced after dark rearing and also by blocking inhibitory synaptic transmission. (3) Paired-pulse depression and its regulation by age and sensory experience is more pronounced when stimulation is applied to the white matter than when applied to layer IV. Together, these results are consistent with the idea that the maturation of intracortical inhibition reduces the capability of the cortex to relay incoming high-frequency patterns of activity to the supragranular layers.

Heterosynaptic metaplasticity in the hippocampus in vivo: a BCM-like modifiable threshold for LTP.

The homeostatic maintenance of the "modification threshold" for inducing long-term potentiation (LTP) is a fundamental feature of the Bienenstock, Cooper, and Munro (BCM) model of synaptic plasticity. In the present study, two key features of the modification threshold, its heterosynaptic expression and its regulation by postsynaptic neural activity, were tested experimentally in the dentate gyrus of awake, freely moving rats. Conditioning stimulation ranging from 10 to 1,440 brief 400-Hz trains, when applied to medial perforant path afferents, raised the threshold for LTP induction heterosynaptically in the neighboring lateral perforant path synapses. This effect recovered slowly over a 7- to 35-day period. The same conditioning paradigms, however, did not affect the reversal of long-term depression. The inhibition of LTP by medial-path conditioning stimulation was N-methyl-D-aspartate (NMDA) receptor-dependent, but antidromic stimulation of the granule cells could also inhibit lateral path LTP induction, independently of NMDA receptor activation. Increased calcium buffering is a potential mechanism underlying the altered LTP threshold, but the levels of two important calcium-binding proteins did not increase after conditioning stimulation, nor was de novo protein synthesis required for generating the threshold shift. These data confirm, in an in vivo model, two key postulates of the BCM model regarding the LTP threshold. They also provide further evidence for the broad sensitivity of synaptic plasticity mechanisms to the history of prior activity, i.e., metaplasticity.

Chemical induction of mGluR5- and protein synthesis--dependent long-term depression in hippocampal area CA1.

Recent work has demonstrated that specific patterns of synaptic stimulation can induce long-term depression (LTD) in area CA1 that depends on activation of metabotropic glutamate receptors (mGluRs) and rapid protein synthesis. Here we show that the same form of synaptic modification can be induced by brief application of the selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG). DHPG-LTD 1) is a saturable form of synaptic plasticity, 2) requires mGluR5, 3) is mechanistically distinct from N-methyl-D-aspartate receptor (NMDAR)--dependent LTD, and 4) shares a common expression mechanism with protein synthesis-dependent LTD evoked using synaptic stimulation. DHPG-LTD should be useful for biochemical analysis of mGluR5- and protein synthesis-dependent synaptic modification.

Visual experience and deprivation bidirectionally modify the composition and function of NMDA receptors in visual cortex.

The receptive fields of visual cortical neurons are bidirectionally modified by sensory deprivation and experience, but the synaptic basis for these changes is unknown. Here we demonstrate bidirectional, experience-dependent regulation of the composition and function of synaptic NMDA receptors (NMDARs) in visual cortex layer 2/3 pyramidal cells of young rats. Visual experience decreases the proportion of NR2B-only receptors, shortens the duration of NMDAR-mediated synaptic currents, and reduces summation of synaptic NMDAR currents during bursts of high-frequency stimulation. Visual deprivation exerts an opposite effect. Although the effects of experience and deprivation are reversible, the rates of synaptic modification vary. Experience can induce a detectable change in synaptic transmission within hours, while deprivation-induced changes take days. We suggest that experience-dependent changes in NMDAR composition and function regulate the development of receptive field organization in visual cortex.

Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity.

Bidirectional changes in the efficacy of neuronal synaptic transmission, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD), are thought to be mechanisms for information storage in the brain. LTP and LTD may be mediated by the modulation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazloe proprionic acid) receptor phosphorylation. Here we show that LTP and LTD reversibly modify the phosphorylation of the AMPA receptor GluR1 subunit. However, contrary to the hypothesis that LTP and LTD are the functional inverse of each other, we find that they are associated with phosphorylation and dephosphorylation, respectively, of distinct GluR1 phosphorylation sites. Moreover, the site modulated depends on the stimulation history of the synapse. LTD induction in naive synapses dephosphorylates the major cyclic-AMP-dependent protein kinase (PKA) site, whereas in potentiated synapses the major calcium/calmodulin-dependent protein kinase II (CaMKII) site is dephosphorylated. Conversely, LTP induction in naive synapses and depressed synapses increases phosphorylation of the CaMKII site and the PKA site, respectively. LTP is differentially sensitive to CaMKII and PKA inhibitors depending on the history of the synapse. These results indicate that AMPA receptor phosphorylation is critical for synaptic plasticity, and that identical stimulation conditions recruit different signal-transduction pathways depending on synaptic history.

Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression.

A hippocampal pyramidal neuron receives more than 10(4) excitatory glutamatergic synapses. Many of these synapses contain the molecular machinery for messenger RNA translation, suggesting that the protein complement (and thus function) of each synapse can be regulated on the basis of activity. Here, local postsynaptic protein synthesis, triggered by synaptic activation of metabotropic glutamate receptors, was found to modify synaptic transmission within minutes.

Bidirectional, activity-dependent regulation of glutamate receptors in the adult hippocampus in vivo.

Experience-dependent regulation of synaptic strength has been suggested as a physiological mechanism by which memory storage occurs in the brain. Although modifications in postsynaptic glutamate receptor levels have long been hypothesized to be a molecular basis for long-lasting regulation of synaptic strength, direct evidence obtained in the intact brain has been lacking. Here we show that in the adult brain in vivo, synaptic glutamate receptor trafficking is bidirectionally, and reversibly, modified by NMDA receptor-dependent synaptic plasticity and that changes in glutamate receptor protein levels accurately predict changes in synaptic strength. These findings support the idea that memories can be encoded by the precise experience-dependent assignment of glutamate receptors to synapses in the brain.

Induction of NMDA receptor-dependent long-term depression in visual cortex does not require metabotropic glutamate receptors.

We tested the role of group I mGluRs in the induction of long-term depression (LTD) in the visual cortex, using the novel mGluR antagonist LY341495 and mice lacking mGluR5, the predominant phosphoinositide (PI)-linked mGluR in the visual cortex. We find that LY341495 is a potent blocker of glutamate-stimulated PI hydrolysis in visual cortical synaptoneurosomes, and that it effectively antagonizes the actions of the mGluR agonist 1S, 3R-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on synaptic transmission in visual cortical slices. However, LY341495 has no effect on the induction of LTD by low-frequency stimulation. Furthermore, mice lacking mGluR5 show normal NMDA receptor-dependent LTD. These results indicate that group I mGluR activation is not required for the induction of NMDA receptor-dependent LTD in the visual cortex.

Bidirectional, experience-dependent regulation of N-methyl-D-aspartate receptor subunit composition in the rat visual cortex during postnatal development.

In the visual cortex, as elsewhere, N-methyl-D-aspartate receptors (NMDARs) play a critical role in triggering long-term, experience-dependent synaptic plasticity. Modifications of NMDAR subunit composition alter receptor function, and could have a large impact on the properties of synaptic plasticity. We have used immunoblot analysis to investigate the effects of age and visual experience on the expression of different NMDAR subunits in synaptoneurosomes prepared from rat visual cortices. NMDARs at birth are comprised of NR2B and NR1 subunits, and, over the first 5 postnatal weeks, there is a progressive inclusion of the NR2A subunit. Dark rearing from birth attenuates the developmental increase in NR2A. Levels of NR2A increase rapidly (in <2 hr) when dark-reared animals are exposed to light, and decrease gradually over the course of 3 to 4 days when animals are deprived of light. These data reveal that NMDAR subunit composition in the visual cortex is remarkably dynamic and bidirectionally regulated by sensory experience. We propose that NMDAR subunit regulation is a mechanism for experience-dependent modulation of synaptic plasticity in the visual cortex, and serves to maintain synaptic strength within an optimal dynamic range.

Molecular basis for induction of ocular dominance plasticity.

The most dramatic example of experience-dependent cortical plasticity is the shift in ocular dominance that occurs in visual cortex as a consequence of monocular deprivation during early postnatal life. Many of the basic properties of this type of synaptic plasticity have been described in detail. The important challenge that remains is to understand the molecular basis for these properties. By combining theoretical analysis with experiments in vivo and in vitro, some of the elementary molecular mechanisms for visual cortical plasticity have now been uncovered.

BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex.

Maturation of the visual cortex is influenced by visual experience during an early postnatal period. The factors that regulate such a critical period remain unclear. We examined the maturation and plasticity of the visual cortex in transgenic mice in which the postnatal rise of brain-derived neurotrophic factor (BDNF) was accelerated. In these mice, the maturation of GABAergic innervation and inhibition was accelerated. Furthermore, the age-dependent decline of cortical long-term potentiation induced by white matter stimulation, a form of synaptic plasticity sensitive to cortical inhibition, occurred earlier. Finally, transgenic mice showed a precocious development of visual acuity and an earlier termination of the critical period for ocular dominance plasticity. We propose that BDNF promotes the maturation of cortical inhibition during early postnatal life, thereby regulating the critical period for visual cortical plasticity.