Habenula activation patterns in a preclinical model of neuropathic pain accompanied by depressive-like behaviour

Abstract

Pain and depression are complex disorders that frequently co-occur, resulting in diminished quality of life. The habenula is an epithalamic structure considered to play a pivotal role in the neurocircuitry of both pain and depression. The habenula can be divided into two major areas, the lateral and medial habenula, that can be further subdivided, resulting in 6 main subregions. Here, we investigated habenula activation patterns in a rat model of neuropathic pain with accompanying depressive-like behaviour. Wistar rats received active surgery for the development of neuropathic pain (chronic constriction injury of the sciatic nerve; CCI), sham surgery (surgical control), or no surgery (behavioural control). All animals were evaluated for mechanical nociceptive threshold using the paw pressure test and depressive-like behaviour using the forced swimming test, followed by evaluation of the immunoreactivity to cFos-a marker of neuronal activity-in the habenula and subregions. The Open Field Test was used to evaluate locomotor activity. Animals with peripheral neuropathy (CCI) showed decreased mechanical nociceptive threshold and increased depressive-like behaviour compared to control groups. The CCI group presented decreased cFos immunoreactivity in the total habenula, total lateral habenula and lateral habenula subregions, compared to controls. No difference was found in cFos immunoreactivity in the total medial habenula, however when evaluating the subregions of the medial habenula, we observed distinct activation patterns, with increase cFos immunoreactivity in the superior subregion and decrease in the central subregion. Taken together, our data suggest an involvement of the habenula in neuropathic pain and accompanying depressive-like behaviour.

Fig 1. Methods of study.

A. Experimental design. After habituation to the animal facility, Wistar rats habituated to the paw pressure test (PPT) and, on the following day, were evaluated for baseline measures of mechanical nociceptive threshold. Animals were then randomly allocated into three groups (i.e. naive [no surgery], false-operated, and chronic constriction injury [active surgery]), followed by the assigned surgery. Thirteen days after the baseline measure, animals were habituated to the Forced Swimming Test (FST). On the following day, animals were tested in the Open Field Test (OFT) and final measures of the PPT and FST were taken. B. Photomicrography of a Nissl-stained coronal slice, showing the lateral and medial habenula and its subdivisions. Abbreviations: LHbL: Lateral subdivision of the lateral habenula. LHbM: Medial subdivision of the lateral habenula. MHbS: Superior subdivision of the medial habenula. MHbI: Inferior subdivision of the medial habenula. MHbL: Lateral subdivision of the medial habenula. MHbC: Central subdivision of the medial habenula. sm: stria medullaris.

Fig 2. Behavioural results.

A. Mechanical Nociceptive Threshold (g) in the paw pressure test before randomization (baseline measure) and after 14 days (final measure). A significant reduction in mechanical nociceptive thresholds represents neuropathic pain. B. Immobility time (s) in the Forced Swimming Test, evaluated 14 days after group allocation. Increased time spent immobile characterized depressive-like behaviour. Values are presented as mean ± SEM. ***p < 0.001. Abbreviations: FOP: false-operated, CCI: chronic constriction injury.

Fig 3. cFos immunoreactivity (cFos-IR) pattern in the habenula.

A. Total Habenula (tHb) cFos-IR. B. Total Lateral Habenula (tLHb) cFos-IR. C. cFos-IR in the lateral subregion of the LHb (LHbL). D. cFos-IR in the medial subregion of the LHb (LHbM). E. cFos-IR in the central subregion of the medial habenula (MHbC). F. cFos-IR in the superior subregion of the medial habenula (MHbS). Abbreviations: FOP: false-operated group, CCI: chronic constriction injury group. Values are presented as normalized mean±SEM. *p<0.05, **p<0.01, ***p<0.001.