Ayako M. Watabe

Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome

Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22,2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine‐phosphorylated in the brain, with Tyr‐1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr‐1472 site to phenylalanine (Y1472F) and show that Tyr‐1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear‐related learning and reduced amygdaloid long‐term potentiation. NMDA receptor‐mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr‐1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

Involvement of NMDAR2A tyrosine phosphorylation in depression‐related behaviour

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression‐related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression‐related behaviour. The NR2A subunit of the NMDA receptor is tyrosine‐phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr‐1325‐Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock‐in mice show antidepressant‐like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock‐in mice, DARPP‐32 phosphorylation at Thr 34, which is important for the regulation of depression‐related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src‐induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor‐mediated downstream signalling to modulate depression‐related behaviour.

Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS.

The NMDA‐type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long‐term potentiation (LTP) and long‐term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit‐containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit‐containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala.

Dysfunction of the RAR/RXR signaling pathway in the forebrain impairs hippocampal memory and synaptic plasticity

BackgroundRetinoid signaling pathways mediated by retinoic acid receptor (RAR)/retinoid × receptor (RXR)-mediated transcription play critical roles in hippocampal synaptic plasticity. Furthermore, recent studies have shown that treatment with retinoic acid alleviates age-related deficits in hippocampal long-term potentiation (LTP) and memory performance and, furthermore, memory deficits in a transgenic mouse model of Alzheimers disease. However, the roles of the RAR/RXR signaling pathway in learning and memory at the behavioral level have still not been well characterized in the adult brain. We here show essential roles for RAR/RXR in hippocampus-dependent learning and memory. In the current study, we generated transgenic mice in which the expression of dominant-negative RAR (dnRAR) could be induced in the mature brain using a tetracycline-dependent transcription factor and examined the effects of RAR/RXR loss.ResultsThe expression of dnRAR in the forebrain down-regulated the expression of RARβ, a target gene of RAR/RXR, indicating that dnRAR mice exhibit dysfunction of the RAR/RXR signaling pathway. Similar with previous findings, dnRAR mice displayed impaired LTP and AMPA-mediated synaptic transmission in the hippocampus. More importantly, these mutant mice displayed impaired hippocampus-dependent social recognition and spatial memory. However, these deficits of LTP and memory performance were rescued by stronger conditioning stimulation and spaced training, respectively. Finally, we found that pharmacological blockade of RARα in the hippocampus impairs social recognition memory.ConclusionsFrom these observations, we concluded that the RAR/RXR signaling pathway greatly contributes to learning and memory, and LTP in the hippocampus in the adult brain.

The mechanisms of the strong inhibitory modulation of long-term potentiation in the rat dentate gyrus

The hippocampus is essential for the formation of certain types of memory, and synaptic plasticity such as long‐term potentiation (LTP) is widely accepted as a cellular basis of hippocampus‐dependent memory. Although LTP in both perforant path–dentate gyrus (DG) granule cell and CA3–CA1 pyramidal cell synapses is similarly dependent on activation of postsynaptic N‐methyl‐D‐aspartate receptors, several reports suggest that modulation of LTP by γ‐aminobutyric acid (GABA) receptor‐mediated inhibitory inputs is stronger in perforant path–DG granule cell synapses. However, little is known about how different the mechanism and physiological relevance of the GABAergic modulation of LTP induction are among different brain regions. We confirmed that the action of GABAA receptor antagonists on LTP was more prominent in the DG, and explored the mechanism introducing such difference by examining two types of GABAA receptor‐mediated inhibition, i.e. synaptic and tonic inhibition. As synaptic inhibition, we compared inhibitory vs. excitatory monosynaptic responses and their summation during an LTP‐inducing stimulus, and found that the balance of the summated postsynaptic currents was biased toward inhibition in the DG. As tonic inhibition, or sustained activation of extrasynaptic GABAA receptors by ambient GABA, we measured the change in holding currents of the postsynaptic cells induced by GABAA receptor antagonists, and found that the tonic inhibition was significantly stronger in the DG. Furthermore, we found that tonic inhibition was associated with LTP modulation. Our results suggest that both the larger tonic inhibition and the larger inhibitory/excitatory summation balance during conditioning are involved in the stronger inhibitory modulation of LTP in the DG.

Functional coupling of the metabotropic glutamate receptor, InsP3 receptor and L-type Ca2+ channel in mouse CA1 pyramidal cells

•  While the metabotropic glutamate receptor (mGluR) is supposed to modulate L‐type voltage‐dependent calcium channels (L‐VDCCs), its reported actions include both facilitation and suppression, and thus the modulation of L‐VDCCs by synaptic activity has still been under debate. •  In this study, using acute hippocampal slices of subtype‐specific knockout mice, we have shown that mGluR5 induces facilitation of the depolarization‐evoked calcium current. •  This facilitation was not accompanied by the change in single‐channel properties of the L‐VDCC itself, but required the activation of calcium‐induced calcium release that was triggered by L‐VDCC opening. •  L‐VDCCs and mGluR5 were shown to form a complex by coimmunoprecipitation, suggesting that the specific functional coupling between mGluR5, InsP3 receptors and L‐VDCCs played a pivotal role in the calcium‐current facilitation. •  Our study has identified a novel mechanism of the interaction between the mGluR and calcium signalling, and suggested a contribution of mGluR5 to synaptic plasticity.

On-Site Energy Supply at Synapses through Monocarboxylate Transporters Maintains Excitatory Synaptic Transmission

ATP production through oxidative phosphorylation in the mitochondria is the most efficient way to provide energy to various energy-consuming activities of the neurons. These processes require a large amount of ATP molecules to be maintained. Of these, synaptic transmission is most energy consuming. Here we report that lactate transported through monocarboxylate transporters (MCTs) at excitatory synapses constitutively supports synaptic transmission, even under conditions in which a sufficient supply of glucose and intracellular ATP are present. We analyzed the effects of MCT inhibition on neuronal activities using whole-cell recordings in brain slices of rats in the nucleus of the solitary tract. MCT inhibitors (α-cyano-4-hydroxycinnamic acid (4-CIN), phloretin, and d-lactate) significantly decreased the amplitude of EPSCs without reducing release probability. Although 4-CIN significantly reduced currents mediated by heterologously expressed AMPA-Rs in oocytes (a novel finding in this study), the IC50 of the inhibitory effect on EPSC in brain slices was ∼3.8 times smaller than that on AMPA-R currents in oocytes. Removal of intracellular ATP significantly potentiated the inhibition of EPSC with 4-CIN in a manner that was counteracted by intracellular lactate addition. In addition, extracellular lactate rescued aglycemic suppression of EPSC, in a manner that was prevented by 4-CIN. Inhibition of MCTs also reduced NMDA-R-mediated EPSCs and, to a lesser extent, the IPSC. The reduction in EPSC amplitude by γ-d-glutamylglycine was enhanced by 4-CIN, suggesting also a decreased quantal content. We conclude that “on-site” astrocyte-neuron lactate transport to presynaptic and postsynaptic elements is necessary for the integrity of excitatory synaptic transmission.

The lateral parabrachial nucleus is actively involved in the acquisition of fear memory in mice

BackgroundPavlovian fear conditioning is a form of learning accomplished by associating a conditioned stimulus (CS) and an unconditioned stimulus (US). While CS–US associations are generally thought to occur in the amygdala, the pathway mediating US signal processing has only been partially identified. The external part of the pontine lateral parabrachial nucleus (elPB) is well situated for providing US nociceptive information to the central amygdala (CeA), which was recently revealed to play a primary role in fear acquisition. Therefore, we manipulated the elPB activity to examine its role in the regulation of fear learning.ResultsFirst, we transiently inactivate the elPB during the acquisition of fear memory. Mice received bilateral elPB injections of the GABAA agonist muscimol (MUS) or phosphate-buffered saline (drug control), with bilateral misplacement of MUS defined as a placement control group. After the injection, mice were conditioned with a pure tone and foot-shock. On a memory retrieval test on day 2, the freezing ratio was significantly lower in the MUS group compared with that in the drug control or placement control groups. A second retrieval test using a pip tone on day 4 following de novo training on day 3, resulted in significant freezing with no group differences, indicating integrity of fear learning and a temporary limited effect of MUS. Next, we examined whether selectively activating the elPB-CeC pathway is sufficient to induce fear learning when paired with CS. Mice with channelrhodopsin2 (ChR2) expressed in the elPB received a pure tone (CS) in association with optical stimulation in the CeA (CS-LED paired group). On the retrieval test, CS-LED paired mice exhibited significantly higher freezing ratios evoked by CS presentation compared with both control mice receiving optical stimulation immediately after being placed in the shock chamber and exposed to the CS much later (immediate shock group) and those expressing only GFP (GFP control group). These results suggest that selective stimulation of the elPB-CeC pathway substitutes for the US to induce fear learning.ConclusionsThe elPB activity is necessary and sufficient to trigger fear learning, likely as a part of the pathway transmitting aversive signals to the CeA.

Non-Hebbian synaptic plasticity induced by repetitive postsynaptic action potentials.

Modern theories on memory storage have mainly focused on Hebbian long-term potentiation (LTP), which requires coincident activation of presynaptic and postsynaptic neurons for its induction. In addition to Hebbian LTP, the roles of non-Hebbian plasticity have also been predicted by some neuronal network models. However, still only a few pieces of evidence have been presented for the presence of such plasticity. In this study, we show in mouse hippocampal slices that LTP can be induced by postsynaptic repetitive depolarization alone in the absence of presynaptic inputs. The induction was dependent on voltage-dependent calcium channels instead of NMDA receptors (NMDARs), whereas the expression mechanism was shared with conventional NMDAR-dependent LTP. During the potentiation, the amplitude of spontaneous EPSCs was increased, suggesting a novel neuron-wide nature of this form of LTP. Furthermore, we also successfully induced LTP with trains of action potentials, which supported the possible existence of depolarizing pulse-induced LTP in vivo. Based on these findings, we suggest a model in which neuron-wide LTP works in concert with synapse-specific Hebbian plasticity to help information processing in memory formation.