Like a thorny vine climbing along a wrought iron gate, pain and anxiety are inextricably intertwined. Anxiety disorders often develop in people with chronic pain, and being anxious worsens pain. Now, research led by Min Zhuo at the University of Toronto, Canada, indicates that a novel form of synaptic plasticity in the anterior cingulate cortex (ACC) may account for anxiety associated with chronic pain. The work was published online December 31 in Neuron.
“The study moves us one step closer to understanding the interaction between pain and anxiety in the brain,” write David Seminowicz, University of Maryland, Baltimore, US, and Marta Ceko, National Center for Complementary and Integrative Health, Bethesda, US, in a commentary for PRF. Seminowicz and Ceko were not involved in the work.
The ACC plays a critical role in chronic pain, and human studies suggest that it might be a hub for anxiety as well. In people with chronic pain, anxiety levels predict the extent of pain, and in imaging studies, people with anxiety show increased activation in the ACC. Zhuo and colleagues had previously shown that changes in synaptic strength in the ACC contribute to the genesis of neuropathic pain, through a form of long-term potentiation (LTP), in which AMPA-type glutamate receptors are increased on the postsynaptic membrane.
In the new study, Zhuo and colleagues looked at a different kind of LTP, generated by increased glutamate release from presynaptic terminals onto pyramidal neurons of the ACC. Their results show that this presynaptic LTP (pre-LTP) is induced after nerve injury or inflammation, and contributes not only to pain, but also to anxiety in a mouse model of neuropathic pain.
To characterize presynaptic LTP in the ACC, first author Kohei Koga induced pre-LTP with a low-frequency stimulation protocol in brain slices from adult mice. As expected based on previous work in hippocampus and amygdala, pre-LTP did not require NMDA-type glutamate receptors, postsynaptic calcium, G protein-coupled receptors, or protein kinase M zeta (PKMζ), all of which participate in postsynaptic LTP. Pre-LTP did involve kainate-type glutamate receptors (KAR), the investigators showed. Adenylyl cyclase 1 (AC1), the enzyme that produces the second messenger cyclic AMP, and protein kinase A (PKA) were also required for pre-LTP, as they are for post-LTP in the ACC.
Pre-LTP in the hippocampus involves hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels. Using electron microscopy of gold-particle immunolabeling and electrophysiological recordings, the researchers found HCN channels and currents in both pre- and postsynaptic terminals in the ACC and, importantly, in thalamic neurons that project to the ACC. Block of HCN channels in the ACC prevented pre-LTP and reversed established pre-LTP, indicating the channels are required for induction and maintenance of plasticity. That result suggests that HCN antagonists might selectively dampen pre-LTP and the behaviors it underlies.
To better understand the functional physiological consequences of pre-LTP, the researchers turned to animal models of pain. Acute pain did not induce pre-LTP, but in mice with chronic inflammation or nerve injury, the synaptic amplification was saturated, the researchers found. They used transgenic mice with a fluorescent reporter protein to show which specific ACC neurons had been activated by nerve injury. “The ACC lights up like a Christmas tree?we see a lot of neurons activated by injury,” Zhuo said. In those injury-activated neurons, blocking HCN channels significantly decreased their activity, demonstrating that pre-LTP was present in the mice with chronic pain.
The ACC is known to play a role in providing emotional context to experiences, so the authors next investigated how anxiety impacted pre-LTP. Their experiments showed that pre-LTP was induced in normal, uninjured mice by exposure to a maze designed to amp up anxiety. In mice allowed to recover from the anxiety-provoking experience, however, pre-LTP had diminished, indicating that an anxiety-producing situation can evoke transient pre-LTP. Interestingly, classical fear conditioning did not produce pre-LTP in the mice, indicating that pre-LTP in the ACC contributes to anxiety but not fear.
The researchers next tested the idea that pre-LTP in the ACC underlies the anxiety that accompanies chronic pain. Compared to sham-operated mice, mice with nerve injury displayed significantly more anxiety-like behaviors, but those behaviors were dampened by bilateral injection of an HCN blocker into the ACC. Importantly, the blocker injection also attenuated mechanical allodynia in nerve-injured mice, suggesting that dampening pre-LTP could reduce neuropathic pain behaviors. Blocking activity of PKMζ to prevent post-LTP reduced allodynia but had no effect on anxiety behavior in nerve-injured mice, confirming that pre-LTP is specifically tethered to anxiety.
Studies have shown that ACC activity is heightened not only in people experiencing physical pain, but psychological pain such as divorce, the death of a loved one?basically with suffering, Zhuo said. Much like pain patients, “we know those people suffer tremendous anxiety. We think our study will really link pain of all sorts with anxiety at the level of the ACC.” If scientists could one day find a way to safely prevent pre-LTP in the ACC, perhaps by blocking HCN channels, it might alleviate these forms of anxiety as well as in chronic pain.
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