Exploring Itch Related Brain Areas In Mice Using C-fos Protein As A Marker

Itch is an uncomfortable sensation that causes a desire to scratch. It results from physical or chemical stimulations of skin or Mucosa. Itch serves to prevent our body from further injury by harmful stimulus, such as mosquito bites and chemicals. It also signals potential lesions of our body, allowing a timely medical treatment. Studies on itch transmission have mainly focused on its peripheral and spinal mechanisms. Thus, much progress has been made in the past decade in the understanding of itch receptors in periphery and specific itch cells/pathways in the spinal cord. However, little is known about the transmission and processing of itch signals in the brain at cellular and network level. A few studies reported itch-activated brain areas using brain imaging techniques with limited spatial resolution. This study sought to explore the distribution of itch-activated brain areas with cellular resolution by detecting the expression of c-fos protein, a marker for neural activity following itch stimulation in mice.Experiments were performed in ICR mice (n=6, male,30-40g). Intradermal injection of Chloroquine (10%,2μ1) to the dorsal surface of left hind paws caused itch-like behaviors in mice. Mice displayed licking and biting at injected site, which lasted for 30-40 min with a peak at 20 min following Chloroquine injection.Our results showed that strong fluorescent signals of Chloroquine-induced c-fos protein expression were mainly located in three brain regions, including paraventricular thalamic nucleus (PVA), periventricular hypothalamic nucleus (Pe), and suprachiasmatic nucleus (Sch). These three areas showed different distribution patterns of activated neurons. Thus, the densities of activated neurons in paraventricular thalamic nucleus and suprachiasmatic nucleus were significantly higher than that in periventricular hypothalamic nucleus. The intensity of fluorescence signals in the activated neurons in suprachiasmatic nucleus were significantly weaker than paraventricular thalamic nucleus and periventricular hypothalamic nucleus.To investigate if the distribution of pain related neurons overlaps with itch-related neurons in these three brain regions, we examined c-fos expression following intradermal injection of paraformaldehyde (4%,2μl) to the dorsal surface of a left hindpaw. We found that, in paraventricular thalamic nucleus and periventricular hypothalamic nucleus, itch-and pain-related neurons showed a similar pattern in both distribution and intensity of fluorescence. In the suprachiasmatic nucleus, fewer number of activated neurons and weaker fluorescence signals were observed following nociceptive stimulus, compared with the itch stimulation.In summary, our results indicate that paraventricular thalamic nucleus, periventricular hypothalamic nucleus and suprachiasmatic nucleus are likely involved in the transmission and coding of itch-related information in the CNS. Paraventricular thalamic nucleus and periventricular hypothalamic nucleus receive both itch-and pain-related information, suggesting a function of sensory convergence. However, whether itch and pain signals are actually processed by the same neuronal populations in these nuclei remains explicated. On the other hand, suprachiasmatic nucleus seems more selective to itch signals. The functional role of these three brain areas in the transmission and processing of itch signals requires further study.