Changes in gut microbiota composition have been linked to anxiety behavior in rodents. However, the underlying neural circuitry linking microbiota and their metabolites to anxiety behavior remains unknown. Using male C57BL/6J germ-free (GF) mice, not exposed to live microbes, increased anxiety-related behavior was observed correlating with a significant increase in the immediate early c-Fos gene in the basolateral amygdala (BLA). This phenomenon coincided with increased intrinsic excitability and spontaneous synaptic activity of BLA pyramidal neurons associated with reduced small conductance calcium-activated potassium (SK) channel currents. Importantly, colonizing GF mice to live microbes or the microbial-derived metabolite indoles reverted SK channel activities in BLA pyramidal neurons and reduced the anxiety behavioral phenotype. These results are consistent with a molecular mechanism by which microbes and or microbial-derived indoles, regulate functional changes in the BLA neurons. Moreover, this microbe metabolite regulation of anxiety links these results to ancient evolutionarily conserved defense mechanisms associated with anxiety-related behaviors in mammals.
Number of ligations made in the chronic constriction injury (CCI) neuropathic pain model has raised serious concerns. We compared behavioural responses, nerve morphology and expression of pain marker, c-fos among CCI models developed with one, two, three and four ligations. The numbers of ligation(s) on sciatic nerve shows no significant difference in displaying mechanical and cold allodynia, and mechanical and thermal hyperalgesia throughout 84 days. All groups underwent similar levels of nerve degeneration post-surgery. Similar c-fos level in brain cingulate cortex, parafascicular nuclei and amygdala were observed in all CCI models compared to sham-operated group. Therefore, number of ligations does not impact intensity of pain symptoms, pathogenesis and neuronal activation. A single ligation is sufficient to develop neuropathic pain, in contrast to the established model of four ligations. This study dissects and characterises the CCI model, ascertaining a more uniform animal model to surrogate actual neuropathic pain condition.