Vanderbilt Institute of Chemical Biology



Discovery at the VICB







New Insights into the Neuronal Response to Stress


By: Carol A. Rouzer, VICB Communications
Published:  January 11, 2018



Stress interferes with the ability of the mGlu3 receptor to modulate excitatory synaptic transmission in the prefrontal cortex.  


Stress alters synaptic transmission in the prefrontal cortex (PFC) of the brain, leading to changes in motivation and cognition. These changes may play a significant role in the pathogenesis or exacerbation of psychiatric disorders. Glutamate is the primary excitatory neurotransmitter in the brain, and stress results in increased activity at glutamatergic synapses. Increasing evidence suggests that aberrant glutamate-dependent signaling in the PFC may contribute to the motivational and cognitive effects of stress. This led Vanderbilt Institute of Chemical Biology members Jeff Conn and Craig Lindsley to explore the role of metabotropic glutamate receptor-3 (mGlu3) in the modulation of glutamate-dependent neurotransmission during stress. They now show that mGlu3 suppresses glutamatergic signaling by inducing internalization of a key glutamate receptor in the post-synaptic neuron, and that stress interferes with this regulation. Furthermore, failure of mGlu3-mediated neuronal plasticity leads to a reduced ability to execute motivation-dependent tasks following stress exposure   [M. E. Joffe, et al. Mol. Psychiatry, (2017) published online Dec 21, DOI: 10.1038/s41380-017-0015-z].


The excitatory effects of glutamate result from activating one or more of three ionotropic receptors (AMPA, NMDA, and kainate) that respond to glutamate binding by opening an ion channel (Figure 1). The resulting flow of Na+ and Ca2+ into the cell, and K+ out of the cell leads to an excitatory post-synaptic potential (EPSP) that if strong enough, initiates an action potential in the post-synaptic neuron. In contrast, the metabotropic glutamate receptors comprise a group of G-protein-coupled receptors that modulate levels of cyclic AMP and Ca2+, thereby regulating the response of the synapse to incoming signals. Metabotropic glutamate receptors may be expressed by the pre- or post-synaptic neuron.



FIGURE 1. Schematic of a hypothetical glutamatergic synapse. The presynaptic neuron stores glutamate (Glu) in vesicles near the synaptic membrane. Upon stimulation, the vesicles fuse with the membrane, releasing glutamate into the synaptic cleft. The release of glutamate may be regulated by one or more metabotropic glutamate receptors present on the membrane of the presynaptic neuron. Depending on the subclass, binding of glutamate to these receptors leads to signaling that either promotes or suppresses synaptic transmission. The postsynaptic neuron may express one or more of three ionotropic glutamate receptors (NMDA, AMPA, or kainate). These are ion channels that open upon binding of glutamate. All of the channels allow inflow of Na+ and outflow of K+ ions, and the NMDA and AMPA receptors also allow the inflow of Ca2+ ions. Glutamate may also bind to metabotropic glutamate receptors on the postsynaptic membrane. Shown here are receptors that modulate postsynaptic signaling by reducing the concentration of cAMP in the cell.



Prior evidence had suggested that mGlu3 modulates glutamatergic neurotransmission in the PFC, where it is expressed by large pyramidal neurons that are found in layer 5. These neurons, which serve as the primary output from the PFC play a major role in the selection and execution of complex, goal-oriented tasks. The importance of mGlu3 in cortical function is supported by the fact that inactivating mutations are associated with schizophrenia and cognitive impairments, whereas receptor activation plays a role in working memory and extinction learning. In PFC pyramidal cells, binding of glutamate to mGlu3 leads to reductions in the levels of cAMP and increased intracellular Ca2+, resulting in long-term depression (LTD) of excitatory transmission. LTD is characterized by an extended period of decreased synaptic responsiveness to new stimuli.


To better understand the role of mGlu3 in modulating the synaptic responses of PFC pyramidal cells, the investigators began their study by monitoring the electrophysiological responses of single neurons in cortical slices obtained from mice. Their method enabled them to measure EPSCs that resulted from incoming glutamate-dependent signals at the cell's synapses. Consistent with prior observations, they found that upon addition of an agonist of both mGlu2 and mGlu3, the cells exhibited LTD. The agonist had no effect on signaling of nearby interneurons, and it did not alter inhibitory post-synaptic potentials in the pyramidal cells. More sophisticated electrophysiological examination indicated that the LTD was not likely due to a decrease in the size or number of glutamate packets released by the presynaptic cell. An mGlu3-specific negative allosteric modulator (NAM) blocked the agonist-mediated LTD. Together, these results suggested that the observed LTD was the result of mGlu3 activation at the level of the post-synaptic pyramidal cell.


The investigators next studied pyramidal cell EPSPs under conditions that enabled them to specifically monitor the response of NMDA receptors. They found that the mGlu2/3 agonist had no effect on NMDA receptor-mediated EPSPs, suggesting that the LTD was modulating the activity of AMPA receptors. Prior work had shown that metabotropic glutamate receptors can induce LTD by promoting internalization of AMPA receptors via dynamin-dependent endocytosis. Consistently, treatment of the cells with a dominant negative peptide that blocks dynamin function blocked the development of LTD in response to the mGlu2/3 agonist. These findings supported the conclusion that the LTD observed in the PFC pyramidal cells was due to AMPA receptor internalization (Figure 2).




FIGURE 2. Mechanism of mGlu3-mediated LTD. Binding of glutamate (Glu) to mGlu3 leads to selective internalization of AMPA receptors.



The PFC receives glutamatergic input from neuronal tracts within the cerebral cortex, but also via tracts from the basolateral amygdala (BLA) and the ventral hippocampus (VH). Signaling from the BLA modulates emotional state, whereas that from the VH is associated with memory. Both regions play a role in motivation-dependent tasks. To identify the source of signals that lead to LTD in the PFC, the investigators injected an expression vector encoding channelrhodopsin-2 (ChR2) into either the BLA or the VH of mice. Then, after allowing sufficient time for ChR2 expression, they sacrificed the mice and prepared cortical slices for electrophysiological evaluation. ChR2 expression enables selective excitation of the expressing neurons by exposure to light. This experimental approach demonstrated that excitatory input from the BLA – but not the VH – leads to mGlu3-mediated LTD, suggesting that the modulation of glutamatergic signaling is more important with regard to emotional state than memory. This led the investigators to hypothesize that it might play a role in the response to stress.


To test their hypothesis, the researchers exposed mice to a 20-minute period of restraint as a stressor. They then sacrificed the mice 30 minutes later and prepared brain slices for electrophysiological examination. They found that slices from mice exposed to stress exhibited an impaired ability to develop LTD in response to mGlu3 stimulation. This finding suggested the hypothesis that excessive mGlu3 activation during the stress might lead to subsequent receptor dysfunction. To test this hypothesis, they treated mice with an mGlu3-selective NAM prior to stress exposure. Slices from mice treated in this way showed no reduction in their ability to develop mGlu3-mediated LTD, indicating that the NAM had protected the receptor from inactivation (Figure 3).




FIGURE 3. Effect of stress on mGlu3-mediated LTD. (Top) Stress causes increased firing of glutamatergic neurons (left) resulting in inactivation of mGlu3 (right). Thus, mGlu3 is not capable of mediating LTD. (Bottom) Treatment with an mGlu3-selective NAM under stress conditions (left) leads to preservation of receptor function, and its ability to generate LTD is retained after stress is removed.



Next, the researchers hypothesized that mGlu3-mediated LTD modulates the ability to execute motivation-dependent tasks, and that this modulation might be affected by stress. To test this hypothesis, they trained mice to poke holes in a device to obtain a reward. They then changed the number of holepokes required to obtain the reward. An animal's ability to adjust to this change depends on a functioning PFC. The investigators found that exposure to restraint stress reduced the animal's ability to adjust the number of holepokes in this task. However, treatment with an mGlu3 NAM prior to stress exposure increased success in the task to the level observed in unstressed mice.


Together the results suggest that stress leads to loss of mGlu3-mediated neuronal plasticity, and that this, in turn, impairs the PFC-dependent ability to select and carry out tasks requiring motivation. This provides an important new mechanism by which stress can lead to decreased cognitive function and exacerbate symptoms associated with various psychiatric disorders. The findings also suggest a potential role for mGlu3-targeted therapy for stress-associated abnormalities of higher cognitive functions.




View Molecular Psychiatry article: Metabotropic glutamate receptor subtype 3 gates acute stress-induced dysregulation of amygdalo-cortical function







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