After peripheral injury, ACC cells become more excitable

After nerve injury, mice showed a lower mechanical withdrawal threshold and shorter heat-withdrawal latency. ACC c-Fos labeling increased, while glutamatergic cells produced more spikes under matched current input and required less current to begin firing.

In vivo calcium signals distinguished spontaneous activity from evoked responses. Selected mild stimuli produced stronger behavioral and calcium responses after injury than in controls, whereas an even weaker condition elicited neither response in either group. Increased sensitivity therefore remained conditional rather than universal.

Original figure 2ACC activity and sensitization

Lower cellular activity accompanies reduced sensitivity

If heightened cortical activity merely accompanied pain, changing it might leave behavior unaffected. Suppressing ACC glutamatergic cells increased mechanical thresholds and heat-withdrawal latencies after injury. Conversely, activating these cells in uninjured mice increased mechanical and thermal sensitivity.

These complementary findings place the population within a functional sensitization pathway. The measured nociceptive behaviors do not cover every affective, cognitive or spontaneous aspect of chronic pain. They sharpen the next question: what makes these cells more excitable?

Original figure 3Changing activity alters mechanical and thermal responses

LCN2 is elevated before the two-week time point

LCN2 participates in inflammation and neural plasticity. Its ACC protein levels were elevated two weeks after injury and were already higher at an earlier three-day time point. Changes at both stages identified a continuing lead for investigation.

Increased expression alone does not establish whether it is cause or consequence. Peripheral injury, cortical activity and inflammatory signaling can influence each other. Altering LCN2 and following both cells and behavior was therefore necessary to test its contribution.

Original figure 4LCN2 expression early and two weeks after injury

The increase is concentrated in neuron-associated signals

ACC LCN2 signals predominantly overlapped neuronal markers, with less overlap in microglia and astrocytes. After injury, neuron-associated colabeled area increased, whereas the corresponding glial measures did not change significantly, highlighting regional and cellular context.

The LCN2 receptor 24p3R also predominantly localized to neurons and overlapped substantially with glutamatergic labeling. Its protein level did not increase significantly after injury. Increased LCN2 therefore did not coincide with increased receptor abundance, and tissue colocalization did not directly establish the source of secretion.

Original figure 5Cellular distribution of LCN2 and its receptor

Lowering LCN2 before injury attenuates later sensitization

Lowering ACC LCN2 before injury increased subsequent mechanical thresholds and heat-withdrawal latencies in injured animals. Evoked firing decreased and the current required to initiate firing increased. Concordant cellular and behavioral changes extended the finding beyond expression alone.

Corresponding mechanical and thermal measures did not change significantly in sham controls. LCN2 therefore needs to be considered in the context of nerve injury; a role in pathological sensitivity does not establish general sensory suppression across all states.

This comparison addressed outcomes after a change made before injury. Whether an intervention could also affect established sensitization required a separate comparison with a different temporal sequence.

Original figure 6Behavior and firing after prior LCN2 reduction

Neutralizing LCN2 also changes established sensitization

After sensitization had developed, local LCN2 neutralization likewise increased mechanical thresholds and heat-withdrawal latencies and reduced evoked firing. This complemented the earlier expression-reduction experiment, connecting LCN2 with persistent sensitization.

Adding LCN2 in uninjured mice produced changes in the opposite direction in sensitivity and excitability. Together these findings support an ACC contribution while leaving receptor necessity, downstream inflammatory signaling and relevance to human chronic pain unresolved. The result identifies a research target rather than an established clinical treatment.

Original figure 7Responses after local LCN2 neutralization