Metabotropic glutamate receptor 3 activation is required for long-term depression in medial prefrontal cortex and fear extinction

Clinical studies have revealed that genetic variations in metabotropic glutamate receptor 3 (mGlu₃) affect performance on cognitive tasks dependent upon the prefrontal cortex (PFC) and may be linked to psychiatric conditions such as schizophrenia, bipolar disorder, and addiction. We have performed a series of studies aimed at understanding how mGlu₃ influences PFC function and cognitive behaviors. In the present study, we found that activation of mGlu₃ can induce long-term depression in the mouse medial PFC (mPFC) in vitro. Furthermore, in vivo administration of a selective mGlu₃ negative allosteric modulator impaired learning in the mPFC-dependent fear extinction task. The results of these studies implicate mGlu₃ as a major regulator of PFC function and cognition. Additionally, potentiators of mGlu₃ may be useful in alleviating prefrontal impairments associated with several CNS disorders.Metabotropic glutamate receptor 3 (mGlu₃) has become of increasing clinical interest due to its genetic association with psychiatric conditions. For example, several studies have identified single-nucleotide polymorphisms (SNPs) in GRM3, the human gene encoding mGlu₃, that are associated with poor performance on cognitive tests that are dependent on function of the prefrontal cortex (PFC) and hippocampus (1, 2). Additionally, these SNPs have also been associated with variations in functional magnetic resonance imaging (fMRI) indexes of prefrontal cortical activity during working memory tasks (1, 3). Moreover, converging lines of evidence indicate that GRM3 represents a major locus associated with schizophrenia (1, 2, 4), bipolar disorder (5, 6), and substance abuse disorders (6–8). Because mGlu₃ is densely expressed in PFC (9), a brain region implicated as a site of pathology in these disorders (10–12), this genetic evidence has led to an increased interest in determining the role of mGlu₃ in regulating PFC function and behavior.Previous studies have revealed that pharmacological activation of group II mGlu receptors (mGlu₂ and mGlu₃) results in long-term depression (LTD) of excitatory transmission in layer V of the rat medial prefrontal cortex (mPFC) (13–16). Although it is not known whether induction of LTD in the mPFC is mediated by mGlu₂ or mGlu₃, previous studies suggest that presynaptically localized mGlu₂ is typically responsible for inhibition of synaptic transmission by group II mGlu receptor agonists at other synapses (17–23). However, evidence suggests that induction of LTD in the mPFC is dependent upon activation of a postsynaptic group II mGlu receptor (15, 16), suggesting that this response is mechanistically distinct from presynaptic effects of group II mGlu receptor agonists on transmission at other synapses. Unfortunately, a lack of pharmacological agents that can selectively antagonize mGlu₃ or mGlu₂ has impaired progress in this area. To allow us to begin studies aimed at understanding the role of mGlu₃ in regulation of mPFC function, we developed a series of negative allosteric modulators (NAMs) that are highly selective for mGlu₃ and are suitable for in vivo use (24). In addition, we now report characterization of a highly selective mGlu₂ NAM. We used these compounds, along with mGlu₂ and mGlu₃ knockout (KO) mice, to evaluate the respective roles of mGlu₂ and mGlu₃ in acute regulation of synaptic transmission and induction of LTD in mPFC. Interestingly, we found that mGlu₂ is involved in acute inhibition of synaptic transmission in the mPFC, but that induction of LTD at this synapse by group II mGlu receptor agonists is mediated exclusively by mGlu₃. Furthermore, we found that mGlu₃ NAMs impair extinction of conditioned fear, a behavioral task that is critically dependent upon the integrity of the mPFC. These data suggest that mGlu₃ plays an essential role in the regulation of a specific form of synaptic plasticity in the mPFC that could be important for forms of cognitive function that require depression of excitatory inputs to mPFC and are thought to be disrupted in patients suffering from a range of CNS disorders.     

Emergence of functional subnetworks in layer 2/3 cortex induced by sequential spikes in vivo

During cortical circuit development in the mammalian brain, groups of excitatory neurons that receive similar sensory information form microcircuits. However, cellular mechanisms underlying cortical microcircuit development remain poorly understood. Here we implemented combined two-photon imaging and photolysis in vivo to monitor and manipulate neuronal activities to study the processes underlying activity-dependent circuit changes. We found that repeated triggering of spike trains in a randomly chosen group of layer 2/3 pyramidal neurons in the somatosensory cortex triggered long-term plasticity of circuits (LTPc), resulting in the increased probability that the selected neurons would fire when action potentials of individual neurons in the group were evoked. Significant firing pattern changes were observed more frequently in the selected group of neurons than in neighboring control neurons, and the induction was dependent on the time interval between spikes, N-methyl-D-aspartate (NMDA) receptor activation, and Calcium/calmodulin-dependent protein kinase II (CaMKII) activation. In addition, LTPc was associated with an increase of activity from a portion of neighboring neurons with different probabilities. Thus, our results demonstrate that the formation of functional microcircuits requires broad network changes and that its directionality is nonrandom, which may be a general feature of cortical circuit assembly in the mammalian cortex.Layer 2/3 neurons in the barrel cortex play a central role in integrative cortical processing (1–4). Neurons in layer 2/3 are interconnected with each other, and their axons and dendrites traverse adjacent barrel areas (5, 6). Recent calcium (Ca²⁺) imaging studies in awake animals showed that two very closely localized layer 2/3 pyramidal neurons are independently activated by different whiskers (7). In addition, adjacent layer 2/3 neurons have different receptive field properties; signals from different whiskers may emerge on different spines in the same neurons (8, 9). These findings suggest that the organization of functional subnetworks in somatosensory layer 2/3 is heterogeneous at the single-cell level and that microcircuits are assembled at a very fine scale (10). In vivo whole-cell recording experiments have also shown that most, but not all, layer 2/3 pyramidal neurons receive subthreshold depolarization by single-whisker stimulation with much broader receptive fields than neurons in layer 4 (11, 12). These anatomical and functional data suggest that electric signals relayed to the cortex by whisker activation are greatly intermingled within layer 2/3 neurons, and that studying the mechanisms by which these layer 2/3 neurons make connections may be critical for understanding the cortical network organizing principles underlying somatosensation.A previous modeling study suggested that spike timing-dependent plasticity (STDP) can lead to the formation of functional cortical columns and activity-dependent reorganization of neural circuits (13–16). However, how spikes arising in multiple neurons in vivo influence their connectivity is poorly understood. In this study using two-photon glutamate photolysis, which allowed us to control neuronal activity in a spatially and temporally precise manner, we examined activity-dependent cellular mechanisms during network rearrangement generated by repetitive spike trains in a group of neurons. We found that repetitive spikes on a group of neurons induced the probability of the neurons firing together. This circuit plasticity required spiking at short intervals among neurons and is expressed by N-methyl-D-aspartate (NMDA) receptor- and Calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent long-lasting connectivity changes. The probability of firing was differentially affected by the order of the spike sequence but was not dependent on the physical distance between neurons. Thus, our data show that neuronal connectivity within a functional subnetwork is established in not only a preferred but also a directional manner.     

Nanomolar dose of bisphenol A rapidly modulates spinogenesis in adult hippocampal neurons

We demonstrated the rapid effects of 10nM bisphenol A (BPA) on the spinogenesis of adult rat hippocampal slices. The density of spines was analyzed by imaging Lucifer Yellow-injected CA1 neurons in slices. Not only the total spine density but also the head diameter distribution of spine was quantitatively analyzed. Spinogenesis was significantly enhanced by BPA within 2h. In particular, the density of middle-head spine (with head diameter of 0.4-0.5μm) was significantly increased. Hydroxytamoxifen, an antagonist of both estrogen-related receptor gamma (ERRγ) and estrogen receptors (ERα/ERβ), blocked the BPA-induced enhancement of the spine density. However, ICI 182,780, an antagonist of ERα/ERβ, did not suppress the BPA effects. Therefore, ERRγ is deduced to be a high affinity receptor of BPA, responsible for modulation of spinogenesis. The BPA-induced enhancement of spinogenesis was also suppressed by MAP kinase inhibitor, PD98059, and the blocker of NMDA receptors, MK-801. Washout of BPA for additional 2h after 2h BPA treatment abolished the BPA-induced enhancement of spinogenesis, suggesting that the BPA effect was reversible. ERRγ was localized at synapses as well as cell bodies of principal neurons. ERRγ at synapses may contribute to the observed rapid effect. The level of BPA in the hippocampal slices was determined by mass-spectrometric analysis.     

Modulating cognitive deficits and tau accumulation in a mouse model of aging Down syndrome through neonatal implantation of neural progenitor cells

Although Down syndrome (DS) is primarily considered as a pediatric disorder, all DS patients incur Alzheimer's disease (AD)-like pathology and about 60% develop an additional AD-like dementia by 30-40years of age. Cognitive and neuroanatomical changes in DS are least compromised perinatally, indicating there may be an opportunity to modulate their cognitive and neuroanatomical development during aging, preventing or postponing the onset of AD. To this end, neural progenitor cells (NPC) or saline were implanted into the hippocampus of neonatal DS-modeling (trisomic Ts65Dn) mice and non-DS (disomic Ts65Dn) age-matched mice. Twelve months later, implanted and unimplanted mice were assessed for long-term survival of NPC, for cognitive function, hippocampal cell density, and the presence of extracellular tau accumulation. Implantation of NPC in trisomic mice improved learning and memory as assessed by conditioned taste aversion testing, but not on the novel object recognition task. Trisomic mice given saline control injections improved performance on both cognitive tasks compared to unimplanted trisomic mice. In contrast, disomic mice, implanted with either saline or NPC, were impaired in both tasks. Long-term surviving NPC were found in 7 out of 11 disomic brains and 4 out of 5 trisomic brains, with an average survival rate of 3.1% and 5.9% respectively. Extracellular tau aggregations were elevated in trisomic mice, but implantation with NPC was associated with significantly fewer aggregations. This was also seen in disomic mice. Saline injections significantly elevated tau presence in both karyotypes. Based on these results, we conclude that the modest effects of a few surviving NPC cannot be distinguished from those induced by the implant procedure. However, the changes prompted by neonatal treatment were detectable in aged animals. Collectively, our data are consistent with the hypothesis that neonatal therapeutic intervention in DS has the potential to exert positive lasting effects in the later stages of life but that NPC or the implantation approach may not be the most effective strategy and alternative stem cell types or delivery systems merit further investigation.