Yukari Takahashi

NMDA receptor-independent synaptic plasticity in the central amygdala in the rat model of neuropathic pain

Abstract Neurons in the latero‐capsular part of the central nucleus of the amygdala (CeA), a region now called the “nociceptive amygdala”, receive predominantly nociceptive information from the dorsal horn through afferent pathways relayed at the nucleus parabrachialis (PB). Excitatory synaptic transmission between the PB afferents and these neurons is reported to become potentiated within a few hours of the establishment of arthritic or visceral pain, making it a possible mechanism linking chronic pain and unpleasant negative emotional experiences. However, it remains unknown whether such synaptic potentiation is consolidated or becomes adaptively extinct in the longer‐lasting form of chronic pain, such as neuropathic pain, an as yet serious and frequent type of pain of important clinical concern. To address this issue, we recorded postsynaptic currents in CeA neurons evoked by PB tract stimulation in acute brain slices from young rats with neuropathic pain, as evaluated by tactile allodynic responses, following unilateral spinal nerve ligature made 1 week earlier. CeA neurons contralateral to the nerve ligation showed significantly larger‐amplitude postsynaptic currents than those in the ipsilateral CeA and sham‐ and non‐operated groups. The degree of synaptic potentiation, as compared between two sides, was positively correlated to that of tactile allodynia responses. In addition, blockade of NMDA receptors did not affect this potentiation. We conclude that potentiation of the PB‐CeA synapse is consolidated in long‐lasting neuropathic pain but that this potentiation results from a molecular mechanism distinct from that in arthritic and visceral pain.

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.

Synaptic potentiation in the nociceptive amygdala following fear learning in mice

BackgroundPavlovian fear conditioning is a classical form of associative learning, which depends on associative synaptic plasticity in the amygdala. Recent findings suggest that the central amygdala (CeA) plays an active role in the acquisition of fear learning. However, little is known about the synaptic properties of the CeA in fear learning. The capsular part of the central amygdala (CeC) receives direct nociceptive information from the external part of the lateral parabrachial nucleus (lPB), as well as highly processed polymodal signals from the basolateral nucleus of the amygdala (BLA). Therefore, we focused on CeC as a convergence point for polymodal BLA signals and nociceptive lPB signals, and explored the synaptic regulation of these pathways in fear conditioning.ResultsIn this study, we show that fear conditioning results in synaptic potentiation in both lPB-CeC and BLA-CeC synapses. This potentiation is dependent on associative fear learning, rather than on nociceptive or sensory experience, or fear memory retrieval. The synaptic weight of the lPB-CeC and BLA-CeC pathways is correlated in fear-conditioned mice, suggesting that fear learning may induce activity-dependent heterosynaptic interactions between lPB-CeC and BLA-CeC pathways. This synaptic potentiation is associated with both postsynaptic and presynaptic changes in the lPB-CeC and BLA-CeC synapses.ConclusionsThese results indicate that the CeC may provide an important locus of Pavlovian association, integrating direct nociceptive signals with polymodal sensory signals. In addition to the well-established plasticity of the lateral amygdala, the multi-step nature of this association system contributes to the highly orchestrated tuning of fear learning.

Overlapping memory trace indispensable for linking, but not recalling, individual memories

Unrelated memories get blurred together If one retrieves two memories around the same time, a small number of neurons will become involved in both memories. Yokose et al. investigated the cellular ensemble mechanisms underlying the association between two such memories. In mice, a small population of neurons mediates the association. Memory traces for two independent emotional memories in the brain partially overlapped when the two memories were retrieved synchronously to create a linkage. Suppressing the activity of the overlapping memory trace interrupted the linkage without damaging the original memories. Science, this issue p. 398 In mice, repeated simultaneous reactivation of two initially separated memory traces links them together. Memories are not stored in isolation from other memories but are integrated into associative networks. However, the mechanisms underlying memory association remain elusive. Using two amygdala-dependent behavioral paradigms—conditioned taste aversion (CTA) and auditory-cued fear conditioning (AFC)—in mice, we found that presenting the conditioned stimulus used for the CTA task triggered the conditioned response of the AFC task after natural coreactivation of the memories. This was accompanied through an increase in the overlapping neuronal ensemble in the basolateral amygdala. Silencing of the overlapping ensemble suppressed CTA retrieval-induced freezing. However, retrieval of the original CTA or AFC memory was not affected. A small population of coshared neurons thus mediates the link between memories. They are not necessary for recalling individual memories.

Cellular tagging as a neural network mechanism for behavioural tagging

Behavioural tagging is the transformation of a short-term memory, induced by a weak experience, into a long-term memory (LTM) due to the temporal association with a novel experience. The mechanism by which neuronal ensembles, each carrying a memory engram of one of the experiences, interact to achieve behavioural tagging is unknown. Here we show that retrieval of a LTM formed by behavioural tagging of a weak experience depends on the degree of overlap with the neuronal ensemble corresponding to a novel experience. The numbers of neurons activated by weak training in a novel object recognition (NOR) task and by a novel context exploration (NCE) task, denoted as overlapping neurons, increases in the hippocampal CA1 when behavioural tagging is successfully achieved. Optical silencing of an NCE-related ensemble suppresses NOR–LTM retrieval. Thus, a population of cells recruited by NOR is tagged and then preferentially incorporated into the memory trace for NCE to achieve behavioural tagging.

Synaptic and network consequences of monosynaptic nociceptive inputs of parabrachial nucleus origin in the central amygdala

A large majority of neurons in the superficial layer of the dorsal horn projects to the lateral parabrachial nucleus (LPB). LPB neurons then project to the capsular part of the central amygdala (CeA; CeC), a key structure underlying the nociception-emotion link. LPB-CeC synaptic transmission is enhanced in various pain models by using electrical stimulation of putative fibers of LPB origin in brain slices. However, this approach has limitations for examining direct monosynaptic connections devoid of directly stimulating fibers from other structures and local GABAergic neurons. To overcome these limitations, we infected the LPB of rats with an adeno-associated virus vector expressing channelrhodopsin-2 and prepared coronal and horizontal brain slices containing the amygdala. We found that blue light stimulation resulted in monosynaptic excitatory postsynaptic currents (EPSCs), with very small latency fluctuations, followed by a large polysynaptic inhibitory postsynaptic current in CeC neurons, regardless of the firing pattern type. Intraplantar formalin injection at 24 h before slice preparation significantly increased EPSC amplitude in late firing-type CeC neurons. These results indicate that direct monosynaptic glutamatergic inputs from the LPB not only excite CeC neurons but also regulate CeA network signaling through robust feed-forward inhibition, which is under plastic modulation in response to persistent inflammatory pain.

Essential role of endogenous calcitonin gene-related peptide in pain-associated plasticity in the central amygdala

The role of the neuropeptide calcitonin gene‐related peptide (CGRP) is well established in nociceptive behaviors. CGRP is highly expressed in the projection pathway from the parabrachial nucleus to the laterocapsular region of the central amygdala (CeC), which plays a critical role in relaying nociceptive information. The CeC is a key structure in pain behavior because it integrates and modulates nociceptive information along with other sensory signals. Previous studies have demonstrated that blockade of the amygdalar CGRP‐signaling cascade attenuates nociceptive behaviors in pain models, while CGRP application facilitates amygdalar synaptic transmission and induces pain behaviors. Despite these lines of evidence, it remains unclear whether endogenous CGRP is involved in the development of nociceptive behaviors accompanied with amygdalar plasticity in a peripheral inflammation model in vivo. To directly address this, we utilized a previously generated CGRP knockout (KO) mouse to longitudinally study formalin‐induced plasticity and nociceptive behavior. We found that synaptic potentiation in the right PB‐CeC pathway that was observed in wild‐type mice was drastically attenuated in the CGRP KO mice 6 h post‐inflammation, when acute nociceptive behavior was no longer observed. Furthermore, the bilateral tactile allodynia 6 h post‐inflammation was significantly decreased in the CGRP KO mice. In contrast, the acute nociceptive behavior immediately after the formalin injection was reduced only at 20–25 min post‐injection in the CGRP KO mice. These results suggest that endogenous CGRP contributes to peripheral inflammation‐induced synaptic plasticity in the amygdala, and this plasticity may underlie the exaggerated nociception–emotion linkage in pain chronification.

Enhanced long-term potentiation in mature rats in a model of epileptic spasms with betamethasone-priming and postnatal N-methyl-D-aspartate administration.

Patients with epileptic spasms are at high risk for learning and memory impairment later in life. We examined whether synaptic plasticity is affected in the adult hippocampus, a structure responsible for learning and memory, using an animal model of epileptic spasms of unknown cause.

Potentiation of NMDA receptor-mediated synaptic transmission at the parabrachial-central amygdala synapses by CGRP in mice

The capsular part of the central amygdala (CeC) is called the “nociceptive amygdala,” as it receives nociceptive information from various pathways, including monosynaptic input from the lateral part of the parabrachial nucleus (LPB), a major target of ascending neurons in the spinal and medullary dorsal horn. LPB-CeC synaptic transmission is mediated by glutamate but the fibers from the LPB also contain calcitonin gene-related peptide (CGRP) and the CeC is rich in CGRP-binding sites. CGRP might be released in response to strong nociception and activate these CGRP receptors. Though it has been shown that CGRP affects the excitatory postsynaptic current (EPSC) amplitude at this synapse in a manner sensitive to NMDA receptor (NMDA-R) blockers, the effect of CGRP on postsynaptic NMDA-R-mediated current recorded in isolation has never been directly examined. Thus, we evaluated the effects of CGRP on NMDA-R-mediated EPSCs that were pharmacologically isolated in brain slices from naïve mice. CGRP significantly increased the amplitude of EPSCs mediated by NMDA-Rs in a manner dependent on protein kinase A activation, but not that mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, in concentration-dependent and antagonist-sensitive manners. This CGRP-induced potentiation of synaptic NMDA-R function would have a potent impact on the strengthening of the nociception-emotion link in persistent pain.