Central sensitization of the spino-parabrachial-amygdala pathway that outlasts a brief nociceptive stimulus

Abstract

Key points: Chronic pain is disabling because sufferers form negative associations between pain and activities, such as work, leading to the sufferer limiting these activities. Pain information arriving in the amygdala is responsible for forming these associations and contributes to us feeling bad when we are in pain. Ongoing injuries enhance the delivery of pain information to the amygdala. If we want to understand why chronic pain can continue without ongoing injury, it is important to know whether this facilitation continues once the injury has healed. In the present study, we show that a 2 min noxious heat stimulus, without ongoing injury, is able to enhance delivery of pain information to the amygdala for 3 days. If the noxious heat stimulus is repeated, this enhancement persists even longer. These changes may prime this information pathway so that subsequent injuries may feel even worse and the associative learning that results in pain-related avoidance may be promoted.

Abstract: Pain is an important defence against dangers in our environment; however, some clinical conditions produce pain that outlasts this useful role and persists even after the injury has healed. The experience of pain consists of somatosensory elements of intensity and location, negative emotional/aversive feelings and subsequent restrictions on lifestyle as a result of a learned association between certain activities and pain. The amygdala contributes negative emotional value to nociceptive sensory information and forms the association between an aversive response and the environment in which it occurs. It is able to form this association because it receives nociceptive information via the spino-parabrachio-amygdaloid pathway and polymodal sensory information via cortical and thalamic inputs. Synaptic plasticity occurs at the parabrachial-amygdala synapse and other brain regions in chronic pain conditions with ongoing injury; however, very little is known about how plasticity occurs in conditions with no ongoing injury. Using immunohistochemistry, electrophysiology and behavioural assays, we show that a brief nociceptive stimulus with no ongoing injury is able to produce long-lasting synaptic plasticity at the rat parabrachial-amygdala synapse. We show that this plasticity is caused by an increase in postsynaptic AMPA receptors with a transient change in the AMPA receptor subunit, similar to long-term potentiation. Furthermore, this synaptic potentiation primes the synapse so that a subsequent noxious stimulus causes prolonged potentiation of the nociceptive information flow into the amygdala. As a result, a second injury could have an increased negative emotional value and promote associative learning that results in pain-related avoidance.

Keywords: Synaptic plasticity; ampa receptor; amygdala; pain; parabrachial nucleus.

Figure 1. A brief nociceptive stimulus without inflammation or ongoing activation of the spino‐parabrachio‐amygdaloid pathway

A, nociceptive stimulus procedure. Both hindpaws were immersed in a water bath at 44°C for 30 s. This was repeated four times with an interstimulus interval of 2 min. B, nociceptive stimulus does not cause immediate or early‐onset peripheral inflammation measured by paw volume displacement. Paw volume was not increased at 2 min and decreased 3 h after 44°C nociceptive stimulus, whereas paw volume was significantly increased 2 min and 3 h after the positive control treatment at 52°C. Statistical significance was tested with one‐way repeated measures ANOVA followed by a Sidak post hoc test. C, nociceptive stimulus does not cause late‐onset peripheral inflammation. There were no differences in footpad histology of rat hindpaws: Ca, 1 day after treatment with a control temperature of 33°C and Cb, 1 day after treatment with the nociceptive stimulus. Left images: 1× magnification images of hindpaw showing haired skin (A), bone (B), muscle (C) and non‐haired skin (D). Middle and right images: 10× and 20× magnification images of the boxed area on the 1× images showing some small blood vessels (arrows) throughout the superficial dermis. The small blood vessels were present in samples taken from control animals and nociceptive stimulus‐treated animals. Scale bars = 500, 50 and 20 μm, respectively (n = 4 for each group). D, confocal images of cFos‐immunoreactive (‐IR) neurons (arrow) in the PB following heat treatment. cFos‐IR neurons were counted in the external lateral portion of the PB (outlined). The rostrocaudal location of sections was: 33°C at 3 h = −9.68 mm from Bregma; 44°C at 3 h = −9.8, 33°C at 1 day = −9.8; 44°C at 1 day = −9.68. Scale bars = 100 μm. E, number of cFos‐IR neurons in the parabrachial, spinal cord and CeLC following heat treatment. Ea, in the external lateral parabrachial nucleus, the nociceptive stimulus significantly increased the number of cFos‐IR neurons after 3 h but had returned to baseline levels after 1 day. Eb, the nociceptive stimulus did not alter cFos‐IR in the spinal cord. Ec, the nociceptive stimulus did not alter cFos‐IR in the CeLC. Statistical significance was tested with an unpaired Student's t test. Dots show data from individual animals and the bar chart shows the mean ± SD. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2. A brief nociceptive stimulus induces long lasting synaptic plasticity specifically at the PB‐CeLC synapse

A, nociceptive stimulus potentiates the PB‐CeLC synapse. Aa, schematic diagram of stimulation and recording site. Stimulating electrodes were placed dorsomedial to the CeA to stimulate PB fibers. The response of the CeLC neurons to this stimulation was recorded. Ab, example traces of EPSCs from control and nociceptive treated rats 1 day after treatment. The amplitude of the AMPAR component of the EPSC was measured at the peak current recorded at −70 mV and the amplitude of the NMDAR component of the EPSC was measured as the average amplitude 70–90 ms after stimulation recorded at +40 mV (grey box). B, nociceptive stimulus does not potentiate the mixed synaptic input. Ba, schematic diagram of stimulation and recording site. Stimulating electrodes were placed dorsal to CeA to stimulate fibres coming from but not limited to the cortex, hypothalamus, thalamus and PB. The response of the CeLC neurons to this stimulation was recorded. Bb, example traces of EPSCs from control and nociceptive treated rats 1 day after treatment. The amplitude of AMPAR and NMDAR EPSCs was measured as above. C, scatter plots of AMPA/NMDA ratio at the PB‐CeLC and mixed‐CeLC synapses for individual neurons. Nociceptive stimulus increased the AMPA/NMDA ratio at the PB‐CeLC synapse but not at the mixed input‐CeLC synapse. D, nociceptive stimulus causes long‐lasting changes at the PB‐CeLC synapse. Scatter plot showing AMPA/NMDA over the 2 weeks following the nociceptive stimulus. The nociceptive stimulus increased the AMPA/NMDA ratio for at least 3 days. Statistical significance was tested with two‐tailed Mann–Whitney test. Each circle shows the data from an individual neuron and the graph also shows the median ± interquartile range. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3. A brief nociceptive stimulus produces a transient change in AMPAR subunit composition at the PB‐CeLC synapse

A, nociceptive stimulus increases inward rectification of AMPAR EPSCs at 1 day. Aa, example traces of AMPAR EPSCs recorded at −60 mV and +40 mV after control or nociceptive treatment. Recordings were made in the presence of APV (100 μm) and spermine (100 μm) was included in the internal solution. Ab, I–V plot showing greater inward rectification of AMPAR EPSCs 1 day after the nociceptive stimulus. Ac, rectification index (I _+40 mV/I _−60 mV) showing a significantly lower rectification index in nociceptive stimulus group at 1 day. Rectification index returns to control levels at 3 days. B, AMPAR‐mediated EPSCs have faster decay kinetics in nociceptive group at 1 day and return to control levels after 3 days. Ba, normalized example traces of AMPAR EPSCs after control and nociceptive treatments. Bb, weighted time constants showing that the nociceptive stimulus speeds decay of the AMPAR synaptic at 1 day. Bc, PPR was not changed by the nociceptive stimulus. Example traces of two consecutive EPSCs of identical intensity (interstimulus interval of 30 ms) showing that the PPR was not changed by the nociceptive stimulus. PPR was calculated by dividing the second EPSC amplitude by the amplitude of the first. Statistical significance was tested using a two‐tailed unpaired Student's t test. Dots show data from individual neurons and the bar chart shows the mean ± SD. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4. PB‐CeLC synapse undergoes metaplastic‐like changes following synaptic plasticity

A, timeline of treatment (above) and example traces of EPSCs (below) in the control two stimuli 7 days group (44°C/33°C) and increased EPSC in the nociceptive two stimuli 8 days group (44°C/44°C). The amplitude of the AMPA and NMDA EPSC was measured as in Fig. 2. B, AMPA/NMDA ratio showing the increase in AMPA/NMDA ratio in the nociceptive two stimuli 8 days group. C, consecutive treatment with the nociceptive stimulus protocol on days 1 and 2 does not cause peripheral damage. The timeline of treatment (above) and histology images of (below) the three groups. Paw volume displacement measurements and histological samples were taken on day 7 for the 44°C/33°C at 7 days group (Ca), day 8 for the 44°C/44°C at 8 days group (Cb) and immediately after the completion of the second nociceptive stimulus protocol on day 2 for the 44°C/44°C group (Cc). Similar to single treatment with the nociceptive stimulus protocol, small blood vessels were found throughout the superficial dermis in all groups (arrows). Scale bar = 20 μm (n = 4 for each group). Statistical significance was tested using a two‐tailed Mann–Whitney test. Dots show data from individual neurons and the graph also shows the median ± interquartile range. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5. Nociceptive stimulus produces mechanical but not thermal hyperalgesia

A, mechanical threshold showing that the single nociceptive stimulus (44°C) causes a reduction in mechanical threshold at 1 day. B, PWL following the Hargreaves test showing that the single nociceptive stimulus (44°C) does not produce thermal hyperalgesia at any time point. Statistical significance was tested using a two‐tailed unpaired Student's t test. Dots show data from individual neurons and the bar chart shows the mean ± SD. [Color figure can be viewed at http://wileyonlinelibrary.com]