A genetic approach to access serotonin neurons for in vivo and in vitro studies.
Journal: 2005/December - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
Serotonin (5HT) is a critical modulator of neural circuits that support diverse behaviors and physiological processes, and multiple lines of evidence implicate abnormal serotonergic signaling in psychiatric pathogenesis. The significance of 5HT underscores the importance of elucidating the molecular pathways involved in serotonergic system development, function, and plasticity. However, these mechanisms remain poorly defined, owing largely to the difficulty of accessing 5HT neurons for experimental manipulation. To address this methodological deficiency, we present a transgenic route to selectively alter 5HT neuron gene expression. This approach is based on the ability of a Pet-1 enhancer region to direct reliable 5HT neuron-specific transgene expression in the CNS. Its versatility is illustrated with several transgenic mouse lines, each of which provides a tool for 5HT neuron studies. Two lines allow Cre-mediated recombination at different stages of 5HT neuron development. A third line in which 5HT neurons are marked with yellow fluorescent protein will have numerous applications, including their electrophysiological characterization. To demonstrate this application, we have characterized active and passive membrane properties of midbrain reticular 5HT neurons, which heretofore have not been reported to our knowledge. A fourth line in which Pet-1 loss of function is rescued by expression of a Pet-1 transgene demonstrates biologically relevant levels of transgene expression and offers a route for investigating serotonergic protein structure and function in a behaving animal. These findings establish a straightforward and reliable approach for developing an array of tools for in vivo and in vitro studies of 5HT neurons.
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Proc Natl Acad Sci U S A 102(45): 16472-16477

A genetic approach to access serotonin neurons for <em>in vivo</em> and <em>in vitro</em> studies

Departments of Neurosciences and Genetics, Case School of Medicine, and Case Transgenic and Targeting Core Facility, Case Western Reserve University, Cleveland, OH 44106
To whom correspondence should be addressed. E-mail: ude.esac@dse.
Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved September 22, 2005
Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved September 22, 2005
Received 2005 Jun 1

Abstract

Serotonin (5HT) is a critical modulator of neural circuits that support diverse behaviors and physiological processes, and multiple lines of evidence implicate abnormal serotonergic signaling in psychiatric pathogenesis. The significance of 5HT underscores the importance of elucidating the molecular pathways involved in serotonergic system development, function, and plasticity. However, these mechanisms remain poorly defined, owing largely to the difficulty of accessing 5HT neurons for experimental manipulation. To address this methodological deficiency, we present a transgenic route to selectively alter 5HT neuron gene expression. This approach is based on the ability of a Pet-1 enhancer region to direct reliable 5HT neuron-specific transgene expression in the CNS. Its versatility is illustrated with several transgenic mouse lines, each of which provides a tool for 5HT neuron studies. Two lines allow Cre-mediated recombination at different stages of 5HT neuron development. A third line in which 5HT neurons are marked with yellow fluorescent protein will have numerous applications, including their electrophysiological characterization. To demonstrate this application, we have characterized active and passive membrane properties of midbrain reticular 5HT neurons, which heretofore have not been reported to our knowledge. A fourth line in which Pet-1 loss of function is rescued by expression of a Pet-1 transgene demonstrates biologically relevant levels of transgene expression and offers a route for investigating serotonergic protein structure and function in a behaving animal. These findings establish a straightforward and reliable approach for developing an array of tools for in vivo and in vitro studies of 5HT neurons.

Keywords: Cre recombinase, pet-1, transgenic, yellow fluorescent protein, monoamine
Abstract

Serotonin (5HT) is a transmitter of broad relevance to nervous system development and function (1-4). Serotonergic pathways innervate most cytoarchitectonic structures of the CNS, and accordingly they have been implicated in the modulation of circuitry involved in nearly all behaviors and physiological processes (3, 5-7). Additionally, 5HT neurotransmission is modulated through abundant afferent information arising from, for example, other monoaminergic systems (8, 9) and from orexinergic, glutamatergic, and GABAergic pathways (10-12). The remarkably expansive neuromodulatory influence of 5HT is the product of a complex transcriptional cascade that generates 5HT-synthesizing neurons in the ventral hindbrain (13-18). 5HT also figures prominently in mental health disorders as a number of lines of evidence provide strong support for the hypothesis that altered serotonergic signaling contributes to neurological and psychiatric pathogenesis (19-22). Despite considerable progress in understanding the importance of 5HT neurotransmission, however, the mechanisms governing 5HT neuron development and the precise physiological roles of 5HT in the modulation of CNS circuitry are not yet clear.

Studies of 5HT neurons are hindered by their small numbers and scattered distributions in the brain. Moreover, experimental perturbation of 5HT neuron function has relied almost exclusively on the use of dietary or pharmacological means to either deplete 5HT or alter 5HT neurotransmission (4). Although these approaches have contributed significantly to our understanding of the 5HT transmitter system they are often complicated by peripheral effects, variable levels of depletion, lack of target specificity, effects unrelated to depletion of 5HT, and in some instances unclear mechanisms of action (23-26). An additional critical shortcoming is the inability to selectively alter 5HT neuron gene expression. Recent gene-targeting studies of 5HT neurons are also limited in that most of the targeted genes reported to date are ubiquitously expressed and play essential roles in early development. Consequently, propagation of their homozygous null alleles in the mouse results in embryonic or early postnatal death (13, 17). Thus, an approach to conveniently alter gene expression in a 5HT neuron-specific manner would greatly facilitate studies of the 5HT transmitter system.

Here, we present a general strategy for manipulating 5HT neuron-specific gene expression by using a transgenic-based approach. This approach relies on the 5HT neuron-specific expression pattern of the Pet-1 ETS gene. Pet-1 is expressed in all mid-hindbrain 5HT-synthesizing neurons, and its embryonic expression precedes the appearance of serotonergic-specific traits. Moreover, in contrast to other serotonergic-related genes, such as the 5HT transporter (27), Pet-1 transcription in the brain is restricted to 5HT neurons and their postmitotic precursors (28). We recently identified an enhancer region upstream of mouse Pet-1-transcribed sequences that is able to accurately recapitulate the spatiotemporal pattern of Pet-1 expression in the brain. Importantly, the Pet-1 enhancer (ePet) region can direct highly reproducible 5HT neuron-specific transgene expression with little or no ectopic expression among independent founder lines (29). We describe several transgenic lines that demonstrate the reliability and general utility of the ePet region for development of serotonergic tools. The tools described here and additional ones that can now be generated will greatly increase the accessibility of 5HT neurons for investigations of 5HT neuron development, function, and plasticity in culture and behaving animals.

Data were obtained from surveys of individual adult organs and E16 embryonic sagittal sections.

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Acknowledgments

We thank Dr. Jerry Silver for important discussions of potential applications and Dr. Stephen O'Gorman for the ROSAR26R strain and Cre recombinase cDNA. This research was supported by National Institutes of Health Grants NS047752 (to S.H.), NS33590 (to B.W.S.), and MH62723 (to E.S.D.).

Acknowledgments

Notes

Author contributions: M.M.S., C.J.W., J.K.L., S.H., B.W.S., and E.S.D. designed research; M.M.S., C.J.W., J.K.L., R.M., K.L., W.J., R.A.C., and B.W.S. performed research; M.M.S., C.J.W., J.K.L., B.W.S., and E.S.D. analyzed data; and M.M.S. and E.S.D. wrote the paper.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: 5HT, serotonin; ePet, Pet-1 enhancer; EYFP, enhanced yellow fluorescent protein; TPH, tryptophan hydroxylase; E(n), embryonic day (n).

Notes
Author contributions: M.M.S., C.J.W., J.K.L., S.H., B.W.S., and E.S.D. designed research; M.M.S., C.J.W., J.K.L., R.M., K.L., W.J., R.A.C., and B.W.S. performed research; M.M.S., C.J.W., J.K.L., B.W.S., and E.S.D. analyzed data; and M.M.S. and E.S.D. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: 5HT, serotonin; ePet, Pet-1 enhancer; EYFP, enhanced yellow fluorescent protein; TPH, tryptophan hydroxylase; E(n), embryonic day (n).

References

  • 1. Gaspar, P., Cases, O. &amp; Maroteaux, L. (2003) Nat. Rev. Neurosci.4, 1002-1012. [[PubMed]
  • 2. Mason, P(2001) Annu. Rev. Neurosci.24, 737-777. [[PubMed][Google Scholar]
  • 3. Richerson, G. B. (2004) Nat. Rev. Neurosci.5, 449-461. [[PubMed]
  • 4. Sodhi, M. S. &amp; Sanders-Bush, E. (2004) Int. Rev. Neurobiol.59, 111-174. [[PubMed]
  • 5. Barnes, N. M. &amp; Sharp, T. (1999) Neuropharmacology38, 1083-1152. [[PubMed]
  • 6. Jacobs, B. L. &amp; Azmitia, E. C. (1992) Physiol. Rev.72, 165-229. [[PubMed]
  • 7. Vinay, L., Brocard, F., Clarac, F., Norreel, J. C., Pearlstein, E. &amp; Pflieger, J. F. (2002) Brain Res. Brain Res. Rev.40, 118-129. [[PubMed]
  • 8. Baraban, J. M. &amp; Aghajanian, G. K. (1980) Neuropharmacology19, 355-363. [[PubMed]
  • 9. Baraban, J. M. &amp; Aghajanian, G. K. (1981) Brain Res.204, 1-11. [[PubMed]
  • 10. Brown, R. E., Sergeeva, O. A., Eriksson, K. S. &amp; Haas, H. L. (2002) J. Neurosci.22, 8850-8859.
  • 11. Liu, R., Ding, Y. &amp; Aghajanian, G. K. (2002) Neuropsychopharmacology27, 329-340. [[PubMed]
  • 12. Levine, E. S. &amp; Jacobs, B. L. (1992) J. Neurosci.12, 4037-4044.
  • 13. Pattyn, A., Simplicio, N., Van Doorninck, J. H., Goridis, C., Guillemot, F. &amp; Brunet, J. F. (2004) Nat. Neurosci.7, 589-595. [[PubMed]
  • 14. Hendricks, T. J., Fyodorov, D. V., Wegman, L. J., Lelutiu, N. B., Pehek, E. A., Yamamoto, B., Silver, J., Weeber, E. J., Sweatt, J. D. &amp; Deneris, E. S. (2003) Neuron37, 233-247. [[PubMed]
  • 15. Pattyn, A., Vallstedt, A., Dias, J. M., Samad, O. A., Krumlauf, R., Rijli, F. M., Brunet, J. F. &amp; Ericson, J. (2003) Genes Dev.17, 729-737.
  • 16. Cheng, L., Chen, C. L., Luo, P., Tan, M., Qiu, M., Johnson, R. &amp; Ma, Q. (2003) J. Neurosci.23, 9961-9967.
  • 17. Ding, Y. Q., Marklund, U., Yuan, W., Yin, J., Wegman, L., Ericson, J., Deneris, E., Johnson, R. L. &amp; Chen, Z. F. (2003) Nat. Neurosci.6, 933-938. [[PubMed]
  • 18. Craven, S. E., Lim, K. C., Ye, W., Engel, J. D., De Sauvage, F. &amp; Rosenthal, A. (2004) Development (Cambridge, U.K.)131, 1165-1173. [[PubMed]
  • 19. Kinney, H. C., Filiano, J. J. &amp; White, W. F. (2001) J. Neuropathol. Exp. Neurol.60, 228-247. [[PubMed]
  • 20. Gordon, J. A. &amp; Hen, R. (2004) Annu. Rev. Neurosci.27, 193-222. [[PubMed]
  • 21. Mann, J. J. (2003) Nat. Rev. Neurosci.4, 819-828. [[PubMed]
  • 22. Scott, M. M. &amp; Deneris, E. S. (2005) Int. J. Dev. Neurosci.23, 277-285. [[PubMed]
  • 23. Choi, S., Jonak, E. &amp; Fernstrom, J. D. (2004) Brain Res.1007, 19-28. [[PubMed]
  • 24. Chang, N., Kaufman, S. &amp; Milstien, S. (1979) J. Biol. Chem.254, 2665-2668. [[PubMed]
  • 25. Knuth, E. D. &amp; Etgen, A. M. (2004) Brain Res. Dev. Brain Res.151, 203-208. [[PubMed]
  • 26. Stokes, A. H., Xu, Y., Daunais, J. A., Tamir, H., Gershon, M. D., Butkerait, P., Kayser, B., Altman, J., Beck, W. &amp; Vrana, K. E. (2000) J. Neurochem.74, 2067-2073. [[PubMed]
  • 27. Hansson, S. S., Mezey, E. &amp; Hoffman, B. J. (1998) Neuroscience83, 1185-1201. [[PubMed]
  • 28. Hendricks, T., Francis, N., Fyodorov, D. &amp; Deneris, E. (1999) J. Neurosci.19, 10348-10356.
  • 29. Scott, M. M., Krueger, K. C. &amp; Deneris, E. S. (2005) J. Neurosci.25, 2628-2636.
  • 30. Helms, A. W., Abney, A. L., Ben-Arie, N., Zoghbi, H. Y. &amp; Johnson, J. E. (2000) Development (Cambridge, U.K.)127, 1185-1196. [[PubMed]
  • 31. Monani, U. R., Sendtner, M., Coovert, D. D., Parsons, D. W., Andreassi, C., Le, T. T., Jablonka, S., Schrank, B., Rossol, W., Prior, T. W., et al. (2000) Hum. Mol. Genet.9, 333-339. [[PubMed]
  • 32. Frengen, E., Weichenhan, D., Zhao, B., Osoegawa, K., van Geel, M. &amp; de Jong, P. J. (1999) Genomics58, 250-253. [[PubMed]
  • 33. Branda, C. S. &amp; Dymecki, S. M. (2004) Dev. Cell6, 7-28. [[PubMed]
  • 34. Soriano, P(1999) Nat. Genet.21, 70-71. [[PubMed][Google Scholar]
  • 35. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K. &amp; Miyawaki, A. (2002) Nat. Biotechnol.20, 87-90. [[PubMed]
  • 36. Vertes, R. P. &amp; Crane, A. M. (1997) J. Comp. Neurol.378, 411-424. [[PubMed]
  • 37. Chen, H., Lun, Y., Ovchinnikov, D., Kokubo, H., Oberg, K. C., Pepicelli, C. V., Gan, L., Lee, B. &amp; Johnson, R. L. (1998) Nat. Genet.19, 51-55. [[PubMed]
  • 38. Li, Y., Gao, X. B., Sakurai, T. &amp; van den Pol, A. N. (2002) Neuron36, 1169-1181. [[PubMed]
  • 39. Liu, R. J., van den Pol, A. N. &amp; Aghajanian, G. K. (2002) J. Neurosci.22, 9453-9464.
  • 40. Sakurai, T., Nagata, R., Yamanaka, A., Kawamura, H., Tsujino, N., Muraki, Y., Kageyama, H., Kunita, S., Takahashi, S., Goto, K., et al. (2005) Neuron46, 297-308. [[PubMed]
  • 41. Braz, J. M., Rico, B. &amp; Basbaum, A. I. (2002) Proc. Natl. Acad. Sci. USA99, 15148-15153.
  • 42. Zhuang, X., Masson, J., Gingrich, J. A., Rayport, S. &amp; Hen, R. (2005) J. Neurosci. Methods143, 27-32. [[PubMed]
  • 43. Marek, K. W. &amp; Davis, G. W. (2003) Curr. Opin. Neurobiol.13, 607-611. [[PubMed]
  • 44. Miyawaki, A(2003) Curr. Opin. Neurobiol.13, 591-596. [[PubMed][Google Scholar]
  • 45. Gossen, M. &amp; Bujard, H. (2002) Annu. Rev. Genet.36, 153-173. [[PubMed]
  • 46. Vallier, L., Mancip, J., Markossian, S., Lukaszewicz, A., Dehay, C., Metzger, D., Chambon, P., Samarut, J. &amp; Savatier, P. (2001) Proc. Natl. Acad. Sci. USA98, 2467-2472.
  • 47. Vandermaelen, C. P. &amp; Aghajanian, G. K. (1983) Brain Res.289, 109-119. [[PubMed]
  • 48. Aghajanian, G. K. &amp; Vandermaelen, C. P. (1982) J. Neurosci.2, 1786-1792.
  • 49. Beck, S. G., Pan, Y. Z., Akanwa, A. C. &amp; Kirby, L. G. (2004) J. Neurophysiol.91, 994-1005.
  • 50. El Yacoubi, M., Bouali, S., Popa, D., Naudon, L., Leroux-Nicollet, I., Hamon, M., Costentin, J., Adrien, J. &amp; Vaugeois, J. M. (2003) Proc. Natl. Acad. Sci. USA100, 6227-6232.
  • 51. Trulson, M. E. &amp; Frederickson, C. J. (1987) Brain Res. Bull.18, 179-190. [[PubMed]
  • 52. Froger, N., Gardier, A. M., Moratalla, R., Alberti, I., Lena, I., Boni, C., De Felipe, C., Rupniak, N. M., Hunt, S. P., Jacquot, C., et al. (2001) J. Neurosci.21, 8188-8197.
  • 53. Kirby, L. G., Pernar, L., Valentino, R. J. &amp; Beck, S. G. (2003) Neuroscience116, 669-683.
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