Interview with Professor Donald Kuhn
Dr. Peter Boyd, a project manager at genOway, spoke with Professor Kuhn about how the Tph2 KO mouse model we provided him with in 2008 has performed.
5-hydroxytryptamine (5-HT), more commonly known as serotonin, is produced entirely by tryptophan hydroxylase, which converts tryptophan to serotonin. Serotonin is produced independently in the peripheral tissues and the brain by two isozymes: Tph1 & 2. There is no transfer of peripheral serotonin to the brain, as serotonin can not cross the blood-brain barrier. The Tph2 constitutive KO model was generated in C57BL/6N mice by removing exon 1, including the ATG sequence, resulting in complete absence of tryptophan hydroxylase 2 expression. The model exhibits a complete absence of serotonin in the brain while retaining normal serotonin levels in the blood and peripheral tissues.
Professor Donald M. Kuhn
Department of Psychiatry &
School of Medicine
Wayne State University
PB: "Professor Kuhn, thank you for taking the time to speak with me. I have searched the literature and found 10 published articles which use this model. Would you mind talking to me about your research with these mice?"
DK: "Peter, thank you for calling. I would be happy to talk to you. The model has worked very well. We have observed a variety of interesting and surprising phenotypes, despite significant difficulty with the breeding."
PB: "That is very good to hear. Can you comment on the phenotypes you observed?"
DK: "First of all, we were surprised that these mice were even viable at homozygous status, since they are entirely without brain serotonin. We observed several gross phenotypes. These mice are very aggressive. To demonstrate: in wild-type mice, aggressive behavior can be measured by placing an intruder male into another male’s cage. Extreme aggressive behavior starts on average after around four weeks of habituation to the cage. In the Tph2 KO mice, extreme aggressive behavior can be observed without any habituation at all. These mice also exhibit OCD-like behavior, with mice neglecting to build nests or rear young. Pups also tend not to thrive, and a visible difference in size can allow tentative identification of homozygous Tph2 KO mice at 20 days. We are also observing some other very surprising phenotypes which will form the basis for much of our ongoing research."
PB: "Could you tell me a little about the breeding issues?"
DK: "Certainly. The behavioral phenotypes greatly reduce the number of viable offspring in homozygous Tph2 KO mice. Firstly, the aggression regularly manifests as cannibalization of pups. Those pups that do survive often die from neglect due to the mothers’ OCD-like behavior. This was largely circumvented by breeding heterozygous females with either heterozygous or wildtype males to maintain the colony and/or produce homozygous animals for study. This has made the maintenance of a colony rather cumbersome."
PB: "Perhaps you could describe how you have characterized the line?"
DK: "As I mentioned, we were very surprised by the viability and seemingly normal development of the homozygous KO mice. We validated a total absence of brain Tph2, which was accompanied by a concomitant lack of serotonin in brain homogenates. Moreover, no serotonin was able to be produced by adding tryptophan directly to brain homogenates. This is an important distinction, as some other published Tph2 KO lines demonstrate some residual serotonin levels, which suggests incomplete KO. We performed some phenotype rescue experiments by introducing serotonin directly to the brains of homozygous KO mice, which exhibited an exaggerated response similar to serotonin syndrome, indicating increased sensitivity to serotonin in the KO animals. Similar effects were also obtained by implanting slow-release pellets subcutaneously, which release 5-hydroxytryptophan, the immediate precursor to serotonin, and restore brain levels of the neurotransmitter to normal."
PB: "Lastly, what do you see as being your next steps for further investigating the Tph2 gene?"
DK: "As I mentioned, we are currently investigating some fascinating phenotypes which really raise very interesting questions about some truly unexpected functions of serotonin. It could also be interesting to develop an inducible KO model to allow for investigation of the loss of brain serotonin in developed animals."
PB: "Professor Kuhn, thank you very much for your time and for sharing these truly fascinating insights."
A list of publications with this model:
Thomas, D., Pérez, M., Francescutti-Verbeem, D., Shah, M. and Kuhn, D. (2010). The role of endogenous serotonin in methamphetamine-induced neurotoxicity to dopamine nerve endings of the striatum. Journal of Neurochemistry, 115(3), pp. 595-605.
Angoa-Pérez, M., Kane, M., Briggs, D., Sykes, C., Shah, M., Francescutti, D., Rosenberg, D., Thomas, D. and Kuhn, D. (2012). Genetic depletion of brain 5HT reveals a common molecular pathway mediating compulsivity and impulsivity. Journal of Neurochemistry, 121(6), pp. 974-984.
Kane, M., Angoa-Peréz, M., Briggs, D., Sykes, C., Francescutti, D., Rosenberg, D. and Kuhn, D. (2012). Mice Genetically Depleted of Brain Serotonin Display Social Impairments, Communication Deficits and Repetitive Behaviors: Possible Relevance to Autism. PLoS ONE, 7(11), p. e48975.
Angoa-Pérez, M., Kane, M., Briggs, D., Francescutti, D. and Kuhn, D. (2013). Marble Burying and Nestlet Shredding as Tests of Repetitive, Compulsive-like Behaviors in Mice. Journal of Visualized Experiments, (82).
Hickner, S., Hussain, N., Angoa-Perez, M., Francescutti, D., Kuhn, D. and Mateika, J. (2014). Ventilatory long-term facilitation is evident after initial and repeated exposure to intermittent hypoxia in mice genetically depleted of brain serotonin. Journal of Applied Physiology, 116(3), pp. 240-250.
Angoa-Pérez, M., Kane, M., Briggs, D., Herrera-Mundo, N., Sykes, C., Francescutti, D. and Kuhn, D. (2014). Mice Genetically Depleted of Brain Serotonin Do Not Display a Depression-like Behavioral Phenotype. ACS Chemical Neuroscience, 5(10), pp. 908-919.
Angoa-Pérez, M., Kane, M., Sykes, C., Perrine, S., Church, M. and Kuhn, D. (2014). Brain serotonin determines maternal behavior and offspring survival. Genes, Brain and Behavior, 13(7), pp. 579-591.
Solarewicz, J., Angoa-Pérez, M., Kuhn, D. and Mateika, J. (2015). The sleep-wake cycle and motor activity, but not temperature, are disrupted over the light-dark cycle in mice genetically depleted of serotonin. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 308(1), pp. R10-R17.
Angoa-Pérez, M., Herrera-Mundo, N., Kane, M., Sykes, C., Anneken, J., Francescutti, D. and Kuhn, D. (2015). Brain Serotonin Signaling Does Not Determine Sexual Preference in Male Mice. PLoS ONE, 10(2), p. e0118603.
Komnenov, D., Solarewicz, J., Afzal, F., Nantwi, K., Kuhn, D. and Mateika, J. (2016). Intermittent hypoxia promotes recovery of respiratory motor function in spinal cord-injured mice depleted of serotonin in the central nervous system. Journal of Applied Physiology, 121(2), pp. 545-557.