Review Article | Published:

Embracing diversity in the 5-HT neuronal system


Neurons that synthesize and release 5-hydroxytryptamine (5-HT; serotonin) express a core set of genes that establish and maintain this neurotransmitter phenotype and distinguish these neurons from other brain cells. Beyond a shared 5-HTergic phenotype, these neurons display divergent cellular properties in relation to anatomy, morphology, hodology, electrophysiology and gene expression, including differential expression of molecules supporting co-transmission of additional neurotransmitters. This diversity suggests that functionally heterogeneous subtypes of 5-HT neurons exist, but linking subsets of these neurons to particular functions has been technically challenging. We discuss recent data from molecular genetic, genomic and functional methods that, when coupled with classical findings, yield a reframing of the 5-HT neuronal system as a conglomeration of diverse subsystems with potential to inspire novel, more targeted therapies for clinically distinct 5-HT-related disorders.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Erspamer, V. & Asero, B. Identification of enteramine, the specific hormone of the enterochromaffin cell system, as 5-hydroxytryptamine. Nature 169, 800–801 (1952). This paper is the first to recognize that enteramine and 5-HT are the same chemical.

  2. 2.

    Twarog, B. M. & Page, I. H. Serotonin content of some mammalian tissues and urine and a method for its determination. Am. J. Physiol. 175, 157–161 (1953). This paper is the first to demonstrate that 5-HT is present in vertebrate brain extracts.

  3. 3.

    Vialli, M. & Erspamer, V. Ricerche sul secreto delle cellule enterocromaffini. Z. Zellforsch. Mikrosk. Anat. 27, 81–99 (1937). This paper presents the first reported discovery of ‘enteramine’ (5-HT), a chemical that was isolated from enterochromaffin cells in the rabbit gut mucosa and was shown to possess smooth-muscle-contracting properties.

  4. 4.

    Rapport, M. M., Green, A. A. & Page, I. H. Crystalline serotonin. Science 108, 329–330 (1948). This paper presents the first reported discovery of 5-HT, which was isolated from beef serum and shown to act as a vasoconstrictor.

  5. 5.

    Rapport, M. M. Serum vasoconstrictor (serotonin) the presence of creatinine in the complex; a proposed structure of the vasoconstrictor principle. J. Biol. Chem. 180, 961–969 (1949). This paper is the first to describe the chemical structure of 5-HT.

  6. 6.

    Halliday, G. M. et al. Distribution of monoamine-synthesizing neurons in the human medulla oblongata. J. Comp. Neurol. 273, 301–317 (1988). This article presents the first immunohistological characterization of 5-HT neurons in the human medulla.

  7. 7.

    Baker, K. G., Halliday, G. M. & Tork, I. Cytoarchitecture of the human dorsal raphe nucleus. J. Comp. Neurol. 301, 147–161 (1990). This paper presents one of the first immunohistological characterizations of 5-HT neurons in the human DR.

  8. 8.

    Baker, K. G. et al. Cytoarchitecture of serotonin-synthesizing neurons in the pontine tegmentum of the human brain. Synapse 7, 301–320 (1991). This article presents one of the first immunohistological characterizations of 5-HT neurons in the human MR.

  9. 9.

    Ishimura, K. et al. Quantitative analysis of the distribution of serotonin-immunoreactive cell bodies in the mouse brain. Neurosci. Lett. 91, 265–270 (1988).

  10. 10.

    Jacobs, B. L. & Azmitia, E. C. Structure and function aof the brain serotonin system. Physiol. Rev. 72, 165–229 (1992).

  11. 11.

    Calizo, L. H. et al. Raphe serotonin neurons are not homogenous: electrophysiological, morphological and neurochemical evidence. Neuropharmacology 61, 524–543 (2011).

  12. 12.

    Gaspar, P. & Lillesaar, C. Probing the diversity of serotonin neurons. Phil. Trans. R. Soc. B 367, 2382–2394 (2012).

  13. 13.

    Andrade, R. & Haj-Dahmane, S. Serotonin neuron diversity in the dorsal raphe. ACS Chem. Neurosci. 4, 22–25 (2013).

  14. 14.

    Lowry, C. A. Functional subsets of serotonergic neurones: implications for control of the hypothalamic-pituitary-adrenal axis. J. Neuroendocrinol. 14, 911–923 (2002). This influential perspective article hypothesizes the existence of topographically organized functionally distinct 5-HT subsystems.

  15. 15.

    Hale, M. W. & Lowry, C. A. Functional topography of midbrain and pontine serotonergic systems: implications for synaptic regulation of serotonergic circuits. Psychopharmacology 213, 243–264 (2011).

  16. 16.

    Tork, I. Anatomy of the serotonergic system. Ann. NY Acad. Sci. 600, 9–34; discussion 34–35 (1990).

  17. 17.

    Hannon, J. & Hoyer, D. Molecular biology of 5-HT receptors. Behav. Brain Res. 195, 198–213 (2008).

  18. 18.

    Hoyer, D. 5-HT receptor nomenclature: naming names, does it matter? A tribute to Maurice Rapport. ACS Chem. Neurosci. 8, 908–919 (2017).

  19. 19.

    Filip, M. & Bader, M. Overview on 5-HT receptors and their role in physiology and pathology of the central nervous system. Pharmacol. Rep. 61, 761–777 (2009).

  20. 20.

    Dahlstroem, A. & Fuxe, K. Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol. Scand. Suppl. 232, S231–S255 (1964). This paper is the first characterization of the distribution of 5-HT in cell bodies of the rodent brainstem, and classification of 5-HT neurons into nine distinct anatomical clusters, using the Falck–Hillarp histochemical fluorescence technique.

  21. 21.

    Alonso, A. et al. Development of the serotonergic cells in murine raphe nuclei and their relations with rhombomeric domains. Brain Struct. Funct. 218, 1229–1277 (2013). This paper proposes the most detailed anatomical subclassification of 5-HT neurons yet described, based in part on the inferred relationship between developmental rhombomeres and mature hindbrain anatomy.

  22. 22.

    Jensen, P. et al. Redefining the serotonergic system by genetic lineage. Nat. Neurosci. 11, 417–419 (2008). This paper is the first to iteratively apply intersectional genetics to fate map mouse 5-HT neurons arising from different rhombomerically defined lineages, demonstrating distinct but in some cases overlapping anatomical distributions in the mature brainstem. This paper also introduces the Pet1–Flpe driver line, allowing genetic access to 5-HT neurons, compatible with the use of Cre lines.

  23. 23.

    Hornung, J. P. The human raphe nuclei and the serotonergic system. J. Chem. Neuroanat. 26, 331–343 (2003).

  24. 24.

    Baker, K. G. et al. Distribution, morphology and number of monoamine-synthesizing and substance P-containing neurons in the human dorsal raphe nucleus. Neuroscience 42, 757–775 (1991).

  25. 25.

    Steinbusch, H. W. Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals. Neuroscience 6, 557–618 (1981). This article is the first comprehensive characterization of the rat 5-HT neuron system using serotonin immunohistology.

  26. 26.

    Lidov, H. G. & Molliver, M. E. Immunohistochemical study of the development of serotonergic neurons in the rat CNS. Brain Res. Bull. 9, 559–604 (1982).

  27. 27.

    Newton, B. W., Maley, B. & Traurig, H. The distribution of substance P, enkephalin, and serotonin immunoreactivities in the area postrema of the rat and cat. J. Comp. Neurol. 234, 87–104 (1985).

  28. 28.

    Lanca, A. J. & van der Kooy, D. A serotonin-containing pathway from the area postrema to the parabrachial nucleus in the rat. Neuroscience 14, 1117–1126 (1985).

  29. 29.

    Miller, R. L. & Loewy, A. D. 5-HT neurons of the area postrema become c-Fos-activated after increases in plasma sodium levels and transmit interoceptive information to the nucleus accumbens. Am. J. Physiol. Regul. Integr. Comp. Physiol. 306, R663–R673 (2014).

  30. 30.

    Azmitia, E. C. & Gannon, P. J. The primate serotonergic system: a review of human and animal studies and a report on Macaca fascicularis. Adv. Neurol. 43, 407–468 (1986).

  31. 31.

    Okaty, B. W. et al. Multi-scale molecular deconstruction of the serotonin neuron system. Neuron 88, 774–791 (2015). This study shows that molecular subtypes of 5-HT neurons identified by single-cell RNA-seq correspond to distinct rhombomerically defined sublineages and mature anatomical subdomains and show subtype-specific differences in electrophysiological properties, neuropeptide receptivity and in vivo behavioural functions, as revealed by conditional gene knockout and intersectional genetic neuron silencing experiments.

  32. 32.

    Simon, H. H., Scholz, C. & O’Leary, D. D. Engrailed genes control developmental fate of serotonergic and noradrenergic neurons in mid- and hindbrain in a gene dose-dependent manner. Mol. Cell Neurosci. 28, 96–105 (2005).

  33. 33.

    Konig, N., Wilkie, M. B. & Lauder, J. M. Tyrosine hydroxylase and serotonin containing cells in embryonic rat rhombencephalon: a whole-mount immunocytochemical study. J. Neurosci. Res. 20, 212–223 (1988).

  34. 34.

    Aitken, A. R. & Tork, I. Early development of serotonin-containing neurons and pathways as seen in wholemount preparations of the fetal rat brain. J. Comp. Neurol. 274, 32–47 (1988).

  35. 35.

    Hennessy, M. L. et al. Activity of tachykinin1-expressing Pet1 raphe neurons modulates the respiratory chemoreflex. J. Neurosci. 37, 1807–1819 (2017).

  36. 36.

    Niederkofler, V. et al. Identification of serotonergic neuronal modules that affect aggressive behavior. Cell Rep. 17, 1934–1949 (2016). This paper applies intersectional genetic 5-HT neuron silencing to show that behavioural aggression, a behavioural function previously associated with the 5-HT system in general, can be modulated by small subsets of 5-HT neurons in the DR marked by intersectional expression of Pet1 and two different dopamine receptor genes, but not to others. This study also introduced a dual-recombinase-responsive synaptophysin–GFP ROSA26 -knock-in mouse line for visualizing the synaptic terminals of intersectionally defined 5-HT neurons.

  37. 37.

    Teissier, A. et al. Activity of raphe serotonergic neurons controls emotional behaviors. Cell Rep. 13, 1965–1976 (2015).

  38. 38.

    Jensen, P. & Dymecki, S. M. Essentials of recombinase-based genetic fate mapping in mice. Methods Mol. Biol. 1092, 437–454 (2014).

  39. 39.

    Brust, R. D., Corcoran, A. E., Richerson, G. B., Nattie, E. & Dymecki, S. M. Functional and developmental identification of a molecular subtype of brain serotonergic neuron specialized to regulate breathing dynamics. Cell Rep. 9, 2152–2165 (2014). This article presents the first study to use intersectional chemogenetics to study the in vivo physiological functions of rhombomerically defined subtypes of 5-HT neurons, demonstrating the importance of one particular subtype — r5 RMg– Pet1 neurons — in regulating the respiratory chemoreflex.

  40. 40.

    Bang, S. J., Jensen, P., Dymecki, S. M. & Commons, K. G. Projections and interconnections of genetically defined serotonin neurons in mice. Eur. J. Neurosci. 35, 85–96 (2012).

  41. 41.

    Dymecki, S. M., Ray, R. S. & Kim, J. C. Mapping cell fate and function using recombinase-based intersectional strategies. Methods Enzymol. 477, 183–213 (2010).

  42. 42.

    Sos, K. E. et al. Cellular architecture and transmitter phenotypes of neurons of the mouse median raphe region. Brain Struct. Funct. 222, 287–299 (2017).

  43. 43.

    Watson, C., Shimogori, T. & Puelles, L. Mouse Fgf8-Cre-LacZ lineage analysis defines the territory of the postnatal mammalian isthmus. J. Comp. Neurol. 525, 2782–2799 (2017).

  44. 44.

    Frasch, M., Chen, X. & Lufkin, T. Evolutionary-conserved enhancers direct region-specific expression of the murine Hoxa-1 and Hoxa-2 loci in both mice and Drosophila. Development 121, 957–974 (1995).

  45. 45.

    Awatramani, R., Soriano, P., Rodriguez, C., Mai, J. J. & Dymecki, S. M. Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation. Nat. Genet. 35, 70–75 (2003).

  46. 46.

    Di Bonito, M., Studer, M. & Puelles, L. Nuclear derivatives and axonal projections originating from rhombomere 4 in the mouse hindbrain. Brain Struct. Funct. 222, 3509–3542 (2017).

  47. 47.

    Walther, D. J. & Bader, M. A unique central tryptophan hydroxylase isoform. Biochem. Pharmacol. 66, 1673–1680 (2003).

  48. 48.

    Walther, D. J. et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299, 76 (2003). This paper reports the discovery of a second isoform of the tryptophan hydroxylase gene, expressed exclusively in the CNS, and used by 5-HT neurons to synthesize 5-HT.

  49. 49.

    Kuntzman, R., Shore, P. A., Bogdanski, D. & Brodie, B. B. Microanalytical procedures for fluorometric assay of brain DOPA-5HTP decarboxylase, norepinephrine and serotonin, and a detailed mapping of decarboxylase activity in brain. J. Neurochem. 6, 226–232 (1961).

  50. 50.

    Christenson, J. G., Dairman, W. & Udenfriend, S. On the identity of DOPA decarboxylase and 5-hydroxytryptophan decarboxylase (immunological titration-aromatic L-amino acid decarboxylase-serotonin-dopamine-norepinephrine). Proc. Natl Acad. Sci. USA 69, 343–347 (1972).

  51. 51.

    Albert, V. R., Allen, J. M. & Joh, T. H. A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues. J. Biol. Chem. 262, 9404–9411 (1987).

  52. 52.

    Erickson, J. D., Schafer, M. K., Bonner, T. I., Eiden, L. E. & Weihe, E. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter. Proc. Natl Acad. Sci. USA 93, 5166–5171 (1996).

  53. 53.

    Weihe, E., Schafer, M. K., Erickson, J. D. & Eiden, L. E. Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat. J. Mol. Neurosci. 5, 149–164 (1994).

  54. 54.

    Fon, E. A. et al. Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19, 1271–1283 (1997).

  55. 55.

    Lesch, K. P., Wolozin, B. L., Estler, H. C., Murphy, D. L. & Riederer, P. Isolation of a cDNA encoding the human brain serotonin transporter. J. Neural Transm. Gen. Sect. 91, 67–72 (1993).

  56. 56.

    Hoffman, B. J., Mezey, E. & Brownstein, M. J. Cloning of a serotonin transporter affected by antidepressants. Science 254, 579–580 (1991).

  57. 57.

    Wu, X. et al. Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain. J. Biol. Chem. 273, 32776–32786 (1998).

  58. 58.

    Baganz, N. L. et al. Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice. Proc. Natl Acad. Sci. USA 105, 18976–18981 (2008).

  59. 59.

    Zhou, M., Engel, K. & Wang, J. Evidence for significant contribution of a newly identified monoamine transporter (PMAT) to serotonin uptake in the human brain. Biochem. Pharmacol. 73, 147–154 (2007).

  60. 60.

    Engel, K., Zhou, M. & Wang, J. Identification and characterization of a novel monoamine transporter in the human brain. J. Biol. Chem. 279, 50042–50049 (2004).

  61. 61.

    Bogdanski, D. F., Weissbach, H. & Udenfriend, S. The distribution of serotonin, 5-hydroxytryptophan decarboxylase, and monoamine oxidase in brain. J. Neurochem. 1, 272–278 (1957).

  62. 62.

    Levitt, P., Pintar, J. E. & Breakefield, X. O. Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons. Proc. Natl Acad. Sci. USA 79, 6385–6389 (1982).

  63. 63.

    Richards, J. G., Saura, J., Ulrich, J. & Da Prada, M. Molecular neuroanatomy of monoamine oxidases in human brainstem. Psychopharmacology 106, S21–S23 (1992).

  64. 64.

    Luque, J. M., Kwan, S. W., Abell, C. W., Da Prada, M. & Richards, J. G. Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system. J. Comp. Neurol. 363, 665–680 (1995).

  65. 65.

    Saura, J., Kettler, R., Da Prada, M. & Richards, J. G. Quantitative enzyme radioautography with 3H-Ro 41–1049 and 3H-Ro 19–6327 in vitro: localization and abundance of MAO-A and MAO-B in rat CNS, peripheral organs, and human brain. J. Neurosci. 12, 1977–1999 (1992).

  66. 66.

    Vitalis, T. et al. Developmental expression of monoamine oxidases A and B in the central and peripheral nervous systems of the mouse. J. Comp. Neurol. 442, 331–347 (2002).

  67. 67.

    Lebrand, C. et al. Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 17, 823–835 (1996).

  68. 68.

    Lebrand, C. et al. Transient developmental expression of monoamine transporters in the rodent forebrain. J. Comp. Neurol. 401, 506–524 (1998).

  69. 69.

    Chen, X. et al. Disruption of transient serotonin accumulation by non-serotonin-producing neurons impairs cortical map development. Cell Rep. 10, 346–358 (2015).

  70. 70.

    Hansson, S. R., Mezey, E. & Hoffman, B. J. Serotonin transporter messenger RNA in the developing rat brain: early expression in serotonergic neurons and transient expression in non-serotonergic neurons. Neuroscience 83, 1185–1201 (1998).

  71. 71.

    Spencer, W. C. & Deneris, E. S. Regulatory mechanisms controlling maturation of serotonin neuron identity and function. Front. Cell Neurosci. 11, 215 (2017).

  72. 72.

    Kiyasova, V. & Gaspar, P. Development of raphe serotonin neurons from specification to guidance. Eur. J. Neurosci. 34, 1553–1562 (2011).

  73. 73.

    Fyodorov, D., Nelson, T. & Deneris, E. Pet-1, a novel ETS domain factor that can activate neuronal nAchR gene transcription. J. Neurobiol. 34, 151–163 (1998).

  74. 74.

    Hendricks, T., Francis, N., Fyodorov, D. & Deneris, E. S. The ETS domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes. J. Neurosci. 19, 10348–10356 (1999). This paper reports the discovery that Pet1 , a gene encoding an ETS transcription factor, is a marker of 5-HT neurons, expressed embryonically just prior to the onset of 5-HT production, and expressed throughout maturity.

  75. 75.

    Hendricks, T. J. et al. Pet-1 ETS gene plays a critical role in 5-HT neuron development and is required for normal anxiety-like and aggressive behavior. Neuron 37, 233–247 (2003). This article demonstrates that Pet1 not only is a marker of 5-HT neurons but also is required for the differentiation of the majority of 5-HT neurons.

  76. 76.

    Liu, C. et al. Pet-1 is required across different stages of life to regulate serotonergic function. Nat. Neurosci. 13, 1190–1198 (2010).

  77. 77.

    Wyler, S. C., Donovan, L. J., Yeager, M. & Deneris, E. Pet-1 controls tetrahydrobiopterin pathway and Slc22a3 transporter genes in serotonin neurons. ACS Chem. Neurosci. 6, 1198–1205 (2015).

  78. 78.

    Iyo, A. H., Porter, B., Deneris, E. S. & Austin, M. C. Regional distribution and cellular localization of the ETS-domain transcription factor, FEV, mRNA in the human postmortem brain. Synapse 57, 223–228 (2005).

  79. 79.

    Pelosi, B., Migliarini, S., Pacini, G., Pratelli, M. & Pasqualetti, M. Generation of Pet1210-Cre transgenic mouse line reveals non-serotonergic expression domains of Pet1 both in CNS and periphery. PLOS ONE 9, e104318 (2014).

  80. 80.

    Barrett, K. T. et al. Partial raphe dysfunction in neurotransmission is sufficient to increase mortality after anoxic exposures in mice at a critical period in postnatal development. J. Neurosci. 36, 3943–3953 (2016).

  81. 81.

    Haugas, M., Tikker, L., Achim, K., Salminen, M. & Partanen, J. Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe. Development 143, 4495–4508 (2016).

  82. 82.

    Kiyasova, V. et al. A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei. J. Neurosci. 31, 2756–2768 (2011). This paper presents an anatomical and hodological characterization of a unique subset of 5-HT neurons that persist in the absence of Pet1 expression.

  83. 83.

    Ding, Y. Q. et al. Lmx1b is essential for the development of serotonergic neurons. Nat. Neurosci. 6, 933–938 (2003).

  84. 84.

    Craven, S. E. et al. Gata2 specifies serotonergic neurons downstream of sonic hedgehog. Development 131, 1165–1173 (2004).

  85. 85.

    Zhao, Z. Q. et al. Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity. J. Neurosci. 26, 12781–12788 (2006).

  86. 86.

    Yan, R. et al. Lmx1b controls peptide phenotypes in serotonergic and dopaminergic neurons. Acta Biochim. Biophys. Sin. 45, 345–352 (2013).

  87. 87.

    Scott, M. M. et al. A genetic approach to access serotonin neurons for in vivo and in vitro studies. Proc. Natl Acad. Sci. USA 102, 16472–16477 (2005). This paper describes the development of two Pet1–Cre driver mouse lines, allowing for genetic access to 5-HT neurons, either constitutively or in a tamoxifen-inducible manner.

  88. 88.

    Wyler, S. C. et al. Pet-1 switches transcriptional targets postnatally to regulate maturation of serotonin neuron excitability. J. Neurosci. 36, 1758–1774 (2016).

  89. 89.

    Domonkos, A. et al. Divergent in vivo activity of non-serotonergic and serotonergic VGluT3-neurones in the median raphe region. J. Physiol. 594, 3775–3790 (2016).

  90. 90.

    Dulcis, D., Jamshidi, P., Leutgeb, S. & Spitzer, N. C. Neurotransmitter switching in the adult brain regulates behavior. Science 340, 449–453 (2013).

  91. 91.

    Guemez-Gamboa, A., Xu, L., Meng, D. & Spitzer, N. C. Non-cell-autonomous mechanism of activity-dependent neurotransmitter switching. Neuron 82, 1004–1016 (2014).

  92. 92.

    Spitzer, N. C. Neurotransmitter switching? No surprise. Neuron 86, 1131–1144 (2015).

  93. 93.

    Dulcis, D. et al. Neurotransmitter switching regulated by miRNAs controls changes in social preference. Neuron 95, 1319–1333 (2017).

  94. 94.

    Spitzer, N. C. Neurotransmitter switching in the developing and adult brain. Annu. Rev. Neurosci. 40, 1–19 (2017).

  95. 95.

    Malek, Z. S., Sage, D., Pevet, P. & Raison, S. Daily rhythm of tryptophan hydroxylase-2 messenger ribonucleic acid within raphe neurons is induced by corticoid daily surge and modulated by enhanced locomotor activity. Endocrinology 148, 5165–5172 (2007).

  96. 96.

    Malek, Z. S., Pevet, P. & Raison, S. Circadian change in tryptophan hydroxylase protein levels within the rat intergeniculate leaflets and raphe nuclei. Neuroscience 125, 749–758 (2004).

  97. 97.

    Malek, Z. S., Dardente, H., Pevet, P. & Raison, S. Tissue-specific expression of tryptophan hydroxylase mRNAs in the rat midbrain: anatomical evidence and daily profiles. Eur. J. Neurosci. 22, 895–901 (2005).

  98. 98.

    Donner, N. C., Montoya, C. D., Lukkes, J. L. & Lowry, C. A. Chronic non-invasive corticosterone administration abolishes the diurnal pattern of tph2 expression. Psychoneuroendocrinology 37, 645–661 (2012).

  99. 99.

    Donner, N. & Handa, R. J. Estrogen receptor beta regulates the expression of tryptophan-hydroxylase 2 mRNA within serotonergic neurons of the rat dorsal raphe nuclei. Neuroscience 163, 705–718 (2009).

  100. 100.

    Hiroi, R. & Handa, R. J. Estrogen receptor-beta regulates human tryptophan hydroxylase-2 through an estrogen response element in the 5’ untranslated region. J. Neurochem. 127, 487–495 (2013).

  101. 101.

    Givan, S. A. & Cummings, K. J. Intermittent severe hypoxia induces plasticity within serotonergic and catecholaminergic neurons in the neonatal rat ventrolateral medulla. J. Appl. Physiol. 120, 1277–1287 (2016).

  102. 102.

    Gardner, K. L. et al. Adverse experience during early life and adulthood interact to elevate tph2 mRNA expression in serotonergic neurons within the dorsal raphe nucleus. Neuroscience 163, 991–1001 (2009).

  103. 103.

    Donner, N. C. et al. Elevated tph2 mRNA expression in a rat model of chronic anxiety. Depress. Anxiety 29, 307–319 (2012).

  104. 104.

    Lukkes, J. L., Kopelman, J. M., Donner, N. C., Hale, M. W. & Lowry, C. A. Development x environment interactions control tph2 mRNA expression. Neuroscience 237, 139–150 (2013).

  105. 105.

    Fox, J. H. et al. Preimmunization with a heat-killed preparation of Mycobacterium vaccae enhances fear extinction in the fear-potentiated startle paradigm. Brain Behav. Immun. 66, 70–84 (2017).

  106. 106.

    Belin, M. F. et al. Immunohistochemical evidence for the presence of gamma-aminobutyric acid and serotonin in one nerve cell. A study on the raphe nuclei of the rat using antibodies to glutamate decarboxylase and serotonin. Brain Res. 275, 329–339 (1983).

  107. 107.

    Johansson, O. et al. Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord. Neuroscience 6, 1857–1881 (1981).

  108. 108.

    Steinbusch, H. W., Verhofstad, A. A. & Joosten, H. W. Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications. Neuroscience 3, 811–819 (1978).

  109. 109.

    Chan-Palay, V. Evidence for the coexistence of serotonin and substance P in single raphe cells and fiber plexuses: combined immunocytochemistry and autoradiography. Adv. Exp. Med. Biol. 133, 81–97 (1981).

  110. 110.

    Chan-Palay, V., Jonsson, G. & Palay, S. L. Serotonin and substance P coexist in neurons of the rat’s central nervous system. Proc. Natl Acad. Sci. USA 75, 1582–1586 (1978).

  111. 111.

    Wylie, C. J. et al. Distinct transcriptomes define rostral and caudal serotonin neurons. J. Neurosci. 30, 670–684 (2010).

  112. 112.

    Dougherty, J. D. et al. The disruption of Celf6, a gene identified by translational profiling of serotonergic neurons, results in autism-related behaviors. J. Neurosci. 33, 2732–2753 (2013).

  113. 113.

    Farago, A. F., Awatramani, R. B. & Dymecki, S. M. Assembly of the brainstem cochlear nuclear complex is revealed by intersectional and subtractive genetic fate maps. Neuron 50, 205–218 (2006).

  114. 114.

    Spaethling, J. M. et al. Serotonergic neuron regulation informed by in vivo single-cell transcriptomics. FASEB J. 28, 771–780 (2014).

  115. 115.

    Storm-Mathisen, J. et al. First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301, 517–520 (1983).

  116. 116.

    Nicholas, A. P., Cuello, A. C., Goldstein, M. & Hokfelt, T. Glutamate-like immunoreactivity in medulla oblongata catecholamine/substance P neurons. Neuroreport 1, 235–238 (1990).

  117. 117.

    Nicholas, A. P., Pieribone, V. A., Arvidsson, U. & Hokfelt, T. Serotonin-, substance P and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primate. Neuroscience 48, 545–559 (1992).

  118. 118.

    Kaneko, T., Akiyama, H., Nagatsu, I. & Mizuno, N. Immunohistochemical demonstration of glutaminase in catecholaminergic and serotoninergic neurons of rat brain. Brain Res. 507, 151–154 (1990).

  119. 119.

    Fremeau, R. T. Jr. et al. The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate. Proc. Natl Acad. Sci. USA 99, 14488–14493 (2002).

  120. 120.

    Gras, C. et al. A third vesicular glutamate transporter expressed by cholinergic and serotoninergic neurons. J. Neurosci. 22, 5442–5451 (2002).

  121. 121.

    Ni, B., Rosteck, P. R. Jr., Nadi, N. S. & Paul, S. M. Cloning and expression of a cDNA encoding a brain-specific Na(+)-dependent inorganic phosphate cotransporter. Proc. Natl Acad. Sci. USA 91, 5607–5611 (1994).

  122. 122.

    Takamori, S., Rhee, J. S., Rosenmund, C. & Jahn, R. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407, 189–194 (2000).

  123. 123.

    Takamori, S., Rhee, J. S., Rosenmund, C. & Jahn, R. Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2). J. Neurosci. 21, RC182 (2001).

  124. 124.

    Herzog, E. et al. Localization of VGLUT3, the vesicular glutamate transporter type 3, in the rat brain. Neuroscience 123, 983–1002 (2004).

  125. 125.

    Hioki, H. et al. Chemically specific circuit composed of vesicular glutamate transporter 3- and preprotachykinin B-producing interneurons in the rat neocortex. Cereb. Cortex 14, 1266–1275 (2004).

  126. 126.

    Hioki, H. et al. Vesicular glutamate transporter 3-expressing nonserotonergic projection neurons constitute a subregion in the rat midbrain raphe nuclei. J. Comp. Neurol. 518, 668–686 (2010).

  127. 127.

    Commons, K. G. Locally collateralizing glutamate neurons in the dorsal raphe nucleus responsive to substance P contain vesicular glutamate transporter 3 (VGLUT3). J. Chem. Neuroanat. 38, 273–281 (2009).

  128. 128.

    Prouty, E. W., Chandler, D. J. & Waterhouse, B. D. Neurochemical differences betweentarget-specific populations of rat dorsal raphe projection neurons. Brain Res. 1675, 28–40 (2017).

  129. 129.

    Grimes, W. N., Seal, R. P., Oesch, N., Edwards, R. H. & Diamond, J. S. Genetic targeting and physiological features of VGLUT3+amacrine cells. Vis. Neurosci. 28, 381–392 (2011).

  130. 130.

    Ren, J. et al. Anatomically defined and functionally distinct dorsal raphe serotonin sub-systems. Cell 175, 472–487 (2018). This study shows that DR 5-HT neurons can be broadly subdivided into (partially overlapping) cortical-projecting and subcortical-projecting populations, with cell body distributions preferentially localized to ventral and dorsal DR, respectively. Furthermore, the authors use fibre photometry, chemogenetics and conditional gene knockout to demonstrate that these distinct DR 5-HT neuron subpopulations show opposite in vivo activity responses to aversive stimuli and differentially promote active coping versus anxiety-like behaviours.

  131. 131.

    Stornetta, R. L. et al. Coexpression of vesicular glutamate transporter-3 and gamma-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column. J. Comp. Neurol. 492, 477–494 (2005).

  132. 132.

    Nakamura, K. et al. Identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions. J. Neurosci. 24, 5370–5380 (2004).

  133. 133.

    Samano, C., Cifuentes, F. & Morales, M. A. Neurotransmitter segregation: functional and plastic implications. Prog. Neurobiol. 97, 277–287 (2012).

  134. 134.

    Zhang, S. et al. Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons. Nat. Neurosci. 18, 386–392 (2015).

  135. 135.

    Nusbaum, M. P., Blitz, D. M., Swensen, A. M., Wood, D. & Marder, E. The roles of co-transmission in neural network modulation. Trends Neurosci. 24, 146–154 (2001).

  136. 136.

    Shutoh, F., Ina, A., Yoshida, S., Konno, J. & Hisano, S. Two distinct subtypes of serotonergic fibers classified by co-expression with vesicular glutamate transporter 3 in rat forebrain. Neurosci. Lett. 432, 132–136 (2008).

  137. 137.

    Voisin, A. N. et al. Axonal segregation and role of the vesicular glutamate transporter VGLUT3 in serotonin neurons. Front. Neuroanat. 10, 39 (2016).

  138. 138.

    El Mestikawy, S., Wallen-Mackenzie, A., Fortin, G. M., Descarries, L. & Trudeau, L. E. From glutamate co-release to vesicular synergy: vesicular glutamate transporters. Nat. Rev. Neurosci. 12, 204–216 (2011).

  139. 139.

    Soiza-Reilly, M. & Commons, K. G. Glutamatergic drive of the dorsal raphe nucleus. J. Chem. Neuroanat. 41, 247–255 (2011).

  140. 140.

    Gagnon, D. & Parent, M. Distribution of VGLUT3 in highly collateralized axons from the rat dorsal raphe nucleus as revealed by single-neuron reconstructions. PLOS ONE 9, e87709 (2014).

  141. 141.

    Amilhon, B. et al. VGLUT3 (vesicular glutamate transporter type 3) contribution to the regulation of serotonergic transmission and anxiety. J. Neurosci. 30, 2198–2210 (2010).

  142. 142.

    Johnson, M. D. Synaptic glutamate release by postnatal rat serotonergic neurons in microculture. Neuron 12, 433–442 (1994). This paper is the first to show synaptic co-release of glutamate and 5-HT by 5-HT neurons in ex vivo microculture.

  143. 143.

    Johnson, M. D. Electrophysiological and histochemical properties of postnatal rat serotonergic neurons in dissociated cell culture. Neuroscience 63, 775–787 (1994).

  144. 144.

    Johnson, M. D. & Yee, A. G. Ultrastructure of electrophysiologically-characterized synapses formed by serotonergic raphe neurons in culture. Neuroscience 67, 609–623 (1995).

  145. 145.

    Varga, V. et al. Fast synaptic subcortical control of hippocampal circuits. Science 326, 449–453 (2009).

  146. 146.

    Liu, Z. et al. Dorsal raphe neurons signal reward through 5-HT and glutamate. Neuron 81, 1360–1374 (2014). This paper is the first to show synaptic co-release of glutamate and 5-HT by genetically targeted 5-HT neurons in vivo using optogenetics and to further show that some of the behavioural functions served by 5-HT neurons, such as signalling reward, depend on glutamate co-release, as demonstrated by conditional loss of Vglut3 in Pet1 neurons.

  147. 147.

    Qi, J. et al. A glutamatergic reward input from the dorsal raphe to ventral tegmental area dopamine neurons. Nat. Commun. 5, 5390 (2014).

  148. 148.

    McDevitt, R. A. et al. Serotonergic versus nonserotonergic dorsal raphe projection neurons: differential participation in reward circuitry. Cell Rep. 8, 1857–1869 (2014).

  149. 149.

    Zhuang, X., Masson, J., Gingrich, J. A., Rayport, S. & Hen, R. Targeted gene expression in dopamine and serotonin neurons of the mouse brain. J. Neurosci. Methods 143, 27–32 (2005).

  150. 150.

    Sengupta, A., Bocchio, M., Bannerman, D. M., Sharp, T. & Capogna, M. Control of amygdala circuits by 5-HT neurons via 5-HT and glutamate cotransmission. J. Neurosci. 37, 1785–1796 (2017). This study reports stimulation frequency-dependent co-transmitter release from 5-HT neuron synaptic terminals in the basal amygdala, with glutamate being released at low optogenetic stimulation frequencies (≤1 Hz) and 5-HT being released at higher stimulation frequencies (10–20 Hz), suggesting that some 5-HT neurons can quickly switch their neurotransmitter output depending on activity.

  151. 151.

    Kapoor, V., Provost, A. C., Agarwal, P. & Murthy, V. N. Activation of raphe nuclei triggers rapid and distinct effects on parallel olfactory bulb output channels. Nat. Neurosci. 19, 271–282 (2016).

  152. 152.

    Zhao, S. et al. Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Methods 8, 745–752 (2011).

  153. 153.

    Stornetta, R. L. & Guyenet, P. G. Distribution of glutamic acid decarboxylase mRNA-containing neurons in rat medulla projecting to thoracic spinal cord in relation to monoaminergic brainstem neurons. J. Comp. Neurol. 407, 367–380 (1999).

  154. 154.

    Fu, W. et al. Chemical neuroanatomy of the dorsal raphe nucleus and adjacent structures of the mouse brain. J. Comp. Neurol. 518, 3464–3494 (2010).

  155. 155.

    Fernandez, S. P. et al. Multiscale single-cell analysis reveals unique phenotypes of raphe 5-HT neurons projecting to the forebrain. Brain Struct. Funct. 221, 4007–4025 (2016). This study combines retrograde projection mapping, slice electrophysiology and post-recording single-cell RT-PCR of DR and MR 5-HT neurons and finds consistent relationships between hodological, electrophysiological and molecular phenotypes, suggestive of functionally distinct 5-HT neuron subtypes.

  156. 156.

    Shikanai, H. et al. Distinct neurochemical and functional properties of GAD67-containing 5-HT neurons in the rat dorsal raphe nucleus. J. Neurosci. 32, 14415–14426 (2012).

  157. 157.

    Wu, Y., Wang, W., Diez-Sampedro, A. & Richerson, G. B. Nonvesicular inhibitory neurotransmission via reversal of the GABA transporter GAT-1. Neuron 56, 851–865 (2007).

  158. 158.

    Tritsch, N. X., Ding, J. B. & Sabatini, B. L. Dopaminergic neurons inhibit striatal output through non-canonical release of GABA. Nature 490, 262–266 (2012).

  159. 159.

    Chiba, T. & Masuko, S. Coexistence of varying combinations of neuropeptides with 5-hydroxytryptamine in neurons of the raphe pallidus et obscurus projecting to the spinal cord. Neurosci. Res. 7, 13–23 (1989).

  160. 160.

    Appel, N. M., Wessendorf, M. W. & Elde, R. P. Thyrotropin-releasing hormone in spinal cord: coexistence with serotonin and with substance P in fibers and terminals apposing identified preganglionic sympathetic neurons. Brain Res. 415, 137–143 (1987).

  161. 161.

    Hokfelt, T. et al. Multiple messengers in descending serotonin neurons: localization and functional implications. J. Chem. Neuroanat. 18, 75–86 (2000).

  162. 162.

    Groenewegen, H. J., Ahlenius, S., Haber, S. N., Kowall, N. W. & Nauta, W. J. Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat. J. Comp. Neurol. 249, 65–102 (1986).

  163. 163.

    Bowker, R. M., Westlund, K. N., Sullivan, M. C., Wilber, J. F. & Coulter, J. D. Transmitters of the raphe-spinal complex: immunocytochemical studies. Peptides 3, 291–298 (1982).

  164. 164.

    Bowker, R. M., Steinbusch, H. W. & Coulter, J. D. Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method. Brain Res. 211, 412–417 (1981).

  165. 165.

    Chan-Palay, V. Combined immunocytochemistry and autoradiography after in vivo injections of monoclonal antibody to substance P and 3H-serotonin: Coexistence of two putative transmitters in single raphe cells and fiber plexuses. Anat. Embryol. 156, 241–254 (1979).

  166. 166.

    Chan-Palay, V. The paratrigeminal nucleus. II. Identification and inter-relations of catecholamine axons, indoleamine axons, and substance P immunoreactive cells in the neuropil. J. Neurocytol. 7, 419–442 (1978).

  167. 167.

    Kachidian, P., Poulat, P., Marlier, L. & Privat, A. Immunohistochemical evidence for the coexistence of substance P, thyrotropin-releasing hormone, GABA, methionine-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: a dual labeling in the rat. J. Neurosci. Res. 30, 521–530 (1991).

  168. 168.

    Sergeyev, V., Hokfelt, T. & Hurd, Y. Serotonin and substance P co-exist in dorsal raphe neurons of the human brain. Neuroreport 10, 3967–3970 (1999).

  169. 169.

    Bright, F. M., Byard, R. W., Vink, R. & Paterson, D. S. Normative distribution of substance P and its tachykinin neurokinin-1 receptor in the medullary serotonergic network of the human infant during postnatal development. Brain Res. Bull. 137, 319–328 (2018).

  170. 170.

    Rood, B. D. et al. Dorsal raphe serotonin neurons in mice: immature hyperexcitability transitions to adult state during first three postnatal weeks suggesting sensitive period for environmental perturbation. J. Neurosci. 34, 4809–4821 (2014).

  171. 171.

    Challis, C. et al. Raphe GABAergic neurons mediate the acquisition of avoidance after social defeat. J. Neurosci. 33, 13978–13988 (2013).

  172. 172.

    Crawford, L. K., Craige, C. P. & Beck, S. G. Increased intrinsic excitability of lateral wing serotonin neurons of the dorsal raphe: a mechanism for selective activation in stress circuits. J. Neurophysiol. 103, 2652–2663 (2010).

  173. 173.

    Beck, S. G., Pan, Y. Z., Akanwa, A. C. & Kirby, L. G. Median and dorsal raphe neurons are not electrophysiologically identical. J. Neurophysiol. 91, 994–1005 (2004). This study is one of the earliest to show the electrophysiological diversity of 5-HT neurons using patch clamp recordings in acute brain slices.

  174. 174.

    Kirby, L. G., Pernar, L., Valentino, R. J. & Beck, S. G. Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and immunohistochemical studies. Neuroscience 116, 669–683 (2003).

  175. 175.

    Severson, C. A., Wang, W., Pieribone, V. A., Dohle, C. I. & Richerson, G. B. Midbrain serotonergic neurons are central pH chemoreceptors. Nat. Neurosci. 6, 1139–1140 (2003).

  176. 176.

    Washburn, C. P., Sirois, J. E., Talley, E. M., Guyenet, P. G. & Bayliss, D. A. Serotonergic raphe neurons express TASK channel transcripts and a TASK-like pH- and halothane-sensitive K+ conductance. J. Neurosci. 22, 1256–1265 (2002).

  177. 177.

    Baccini, G., Mlinar, B., Audero, E., Gross, C. T. & Corradetti, R. Impaired chemosensitivity of mouse dorsal raphe serotonergic neurons overexpressing serotonin 1A (Htr1a) receptors. PLOS ONE 7, e45072 (2012).

  178. 178.

    Mulkey, D. K. et al. TASK channels determine pH sensitivity in select respiratory neurons but do not contribute to central respiratory chemosensitivity. J. Neurosci. 27, 14049–14058 (2007).

  179. 179.

    Sargin, D., Oliver, D. K. & Lambe, E. K. Chronic social isolation reduces 5-HT neuronal activity via upregulated SK3 calcium-activated potassium channels. eLife 5, e21416 (2016).

  180. 180.

    Tye, K. M. Neural circuit motifs in valence processing. Neuron 100, 436–452 (2018).

  181. 181.

    Winston, J. S., Gottfried, J. A., Kilner, J. M. & Dolan, R. J. Integrated neural representations of odor intensity and affective valence in human amygdala. J. Neurosci. 25, 8903–8907 (2005).

  182. 182.

    Anderson, A. K. et al. Dissociated neural representations of intensity and valence in human olfaction. Nat. Neurosci. 6, 196–202 (2003).

  183. 183.

    Descarries, L., Watkins, K. C., Garcia, S. & Beaudet, A. The serotonin neurons in nucleus raphe dorsalis of adult rat: a light and electron microscope radioautographic study. J. Comp. Neurol. 207, 239–254 (1982).

  184. 184.

    Kohler, C. & Steinbusch, H. Identification of serotonin and non-serotonin-containing neurons of the mid-brain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain. Neuroscience 7, 951–975 (1982).

  185. 185.

    Liu, R. J., Lambe, E. K. & Aghajanian, G. K. Somatodendritic autoreceptor regulation of serotonergic neurons: dependence on L-tryptophan and tryptophan hydroxylase-activating kinases. Eur. J. Neurosci. 21, 945–958 (2005).

  186. 186.

    Mlinar, B., Montalbano, A., Piszczek, L., Gross, C. & Corradetti, R. Firing properties of genetically identified dorsal raphe serotonergic neurons in brain slices. Front. Cell Neurosci. 10, 195 (2016).

  187. 187.

    Trulson, M. E. & Trulson, V. M. Activity of nucleus raphe pallidus neurons across the sleep-waking cycle in freely moving cats. Brain Res. 237, 232–237 (1982).

  188. 188.

    Trulson, M. E., Crisp, T. & Trulson, V. M. Activity of serotonin-containing nucleus centralis superior (Raphe medianus) neurons in freely moving cats. Exp. Brain Res. 54, 33–44 (1984).

  189. 189.

    Trulson, M. E., Preussler, D. W. & Trulson, V. M. Differential effects of hallucinogenic drugs on the activity of serotonin-containing neurons in the nucleus centralis superior and nucleus raphe pallidus in freely moving cats. J. Pharmacol. Exp. Ther. 228, 94–102 (1984).

  190. 190.

    Trulson, M. E. & Jacobs, B. L. Raphe unit activity in freely moving cats: correlation with level of behavioral arousal. Brain Res. 163, 135–150 (1979). This study is one of the earliest to record single-unit activity of neurons of the DR in an awake and freely behaving vertebrate, showing that the activity of putative 5-HT neurons correlated with the level of behavioural arousal.

  191. 191.

    Rasmussen, K., Heym, J. & Jacobs, B. L. Activity of serotonin-containing neurons in nucleus centralis superior of freely moving cats. Exp. Neurol. 83, 302–317 (1984).

  192. 192.

    Auerbach, S., Fornal, C. & Jacobs, B. L. Response of serotonin-containing neurons in nucleus raphe magnus to morphine, noxious stimuli, and periaqueductal gray stimulation in freely moving cats. Exp. Neurol. 88, 609–628 (1985).

  193. 193.

    Fornal, C., Auerbach, S. & Jacobs, B. L. Activity of serotonin-containing neurons in nucleus raphe magnus in freely moving cats. Exp. Neurol. 88, 590–608 (1985).

  194. 194.

    Wilkinson, L. O. & Jacobs, B. L. Lack of response of serotonergic neurons in the dorsal raphe nucleus of freely moving cats to stressful stimuli. Exp. Neurol. 101, 445–457 (1988).

  195. 195.

    Jacobs, B. L., Fornal, C. A. & Wilkinson, L. O. Neurophysiological and neurochemical studies of brain serotonergic neurons in behaving animals. Ann. NY Acad. Sci. 600, 260–268; discussion 268–271 (1990).

  196. 196.

    Veasey, S. C., Fornal, C. A., Metzler, C. W. & Jacobs, B. L. Response of serotonergic caudal raphe neurons in relation to specific motor activities in freely moving cats. J. Neurosci. 15, 5346–5359 (1995).

  197. 197.

    Jacobs, B. L., Martin-Cora, F. J. & Fornal, C. A. Activity of medullary serotonergic neurons in freely moving animals. Brain Res. Brain Res. Rev. 40, 45–52 (2002).

  198. 198.

    Martin-Cora, F. J., Fornal, C. A. & Jacobs, B. L. Single-unit responses of serotonergic medullary raphe neurons to cardiovascular challenges in freely moving cats. Eur. J. Neurosci. 22, 3195–3204 (2005).

  199. 199.

    Aghajanian, G. K., Rosecrans, J. A. & Sheard, M. H. Serotonin: release in the forebrain by stimulation of midbrain raphe. Science 156, 402–403 (1967).

  200. 200.

    Aghajanian, G. K. & Haigler, H. J. L-Tryptophan as a selective histochemical marker for serotonergic neurons in single-cell recording studies. Brain Res. 81, 364–372 (1974).

  201. 201.

    Aghajanian, G. K. & Vandermaelen, C. P. Intracellular recordings from serotonergic dorsal raphe neurons: pacemaker potentials and the effect of LSD. Brain Res. 238, 463–469 (1982).

  202. 202.

    Aghajanian, G. K. & Vandermaelen, C. P. Intracellular recording in vivo from serotonergic neurons in the rat dorsal raphe nucleus: methodological considerations. J. Histochem. Cytochem. 30, 813–814 (1982).

  203. 203.

    Hajos, M., Gartside, S. E. & Sharp, T. Inhibition of median and dorsal raphe neurones following administration of the selective serotonin reuptake inhibitor paroxetine. Naunyn Schmiedebergs Arch. Pharmacol. 351, 624–629 (1995).

  204. 204.

    Sharp, T., Umbers, V. & Gartside, S. E. Effect of a selective 5-HT reuptake inhibitor in combination with 5-HT1A and 5-HT1B receptor antagonists on extracellular 5-HT in rat frontal cortex in vivo. Br. J. Pharmacol. 121, 941–946 (1997).

  205. 205.

    Allers, K. A. & Sharp, T. Neurochemical and anatomical identification of fast- and slow-firing neurones in the rat dorsal raphe nucleus using juxtacellular labelling methods in vivo. Neuroscience 122, 193–204 (2003).

  206. 206.

    Varga, V., Kocsis, B. & Sharp, T. Electrophysiological evidence for convergence of inputs from the medial prefrontal cortex and lateral habenula on single neurons in the dorsal raphe nucleus. Eur. J. Neurosci. 17, 280–286 (2003).

  207. 207.

    Hajos, M. et al. Neurochemical identification of stereotypic burst-firing neurons in the rat dorsal raphe nucleus using juxtacellular labelling methods. Eur. J. Neurosci. 25, 119–126 (2007). This study is one of the first to demonstrate distinct subtypes of 5-HT neurons on the basis of in vivo firing properties.

  208. 208.

    Cohen, J. Y., Amoroso, M. W. & Uchida, N. Serotonergic neurons signal reward and punishment on multiple timescales. eLife 4, e06346 (2015).

  209. 209.

    Kocsis, B., Varga, V., Dahan, L. & Sik, A. Serotonergic neuron diversity: identification of raphe neurons with discharges time-locked to the hippocampal theta rhythm. Proc. Natl Acad. Sci. USA 103, 1059–1064 (2006).

  210. 210.

    Li, Y. et al. Serotonin neurons in the dorsal raphe nucleus encode reward signals. Nat. Commun. 7, 10503 (2016).

  211. 211.

    Matias, S., Lottem, E., Dugue, G. P. & Mainen, Z. F. Activity patterns of serotonin neurons underlying cognitive flexibility. eLife 6, e20552 (2017).

  212. 212.

    Zhong, W., Li, Y., Feng, Q. & Luo, M. Learning and stress shape the reward response patterns of serotonin neurons. J. Neurosci. 37, 8863–8875 (2017).

  213. 213.

    Luo, M., Li, Y. & Zhong, W. Do dorsal raphe 5-HT neurons encode “beneficialness. Neurobiol. Learn. Mem. 135, 40–49 (2016).

  214. 214.

    Azmitia, E. C. & Segal, M. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Comp. Neurol. 179, 641–667 (1978).

  215. 215.

    Muzerelle, A., Scotto-Lomassese, S., Bernard, J. F., Soiza-Reilly, M. & Gaspar, P. Conditional anterograde tracing reveals distinct targeting of individual serotonin cell groups (B5-B9) to the forebrain and brainstem. Brain Struct. Funct. 221, 535–561 (2016). This study employs conditional expression of anterogradely transported AAV virus encoding Cre-dependent GFP to comprehensively characterize the projection targets of separately transfected 5-HT ( Sert–Cre ) neuron subpopulations with cell bodies occupying distinct raphe nuclei. The authors demonstrate that 5-HT neurons residing in distinct nuclei mostly project to non-overlapping targets, arguing for topographic organization of 5-HT neuron efferent pathways.

  216. 216.

    Kast, R. J., Wu, H. H., Williams, P., Gaspar, P. & Levitt, P. Specific connectivity and unique molecular identity of MET receptor tyrosine kinase expressing serotonergic neurons in the caudal dorsal raphe nuclei. ACS Chem. Neurosci. 8, 1053–1064 (2017).

  217. 217.

    Descarries, L., Riad, M. & Parent, M. Ultrastructure of the serotonin innervation in the mammalian central nervous system-CHAPTER 1.4. Handb. Behav. Neurosci. 21, 65–101 (2010).

  218. 218.

    Moukhles, H. et al. Quantitative and morphometric data indicate precise cellular interactions between serotonin terminals and postsynaptic targets in rat substantia nigra. Neuroscience 76, 1159–1171 (1997).

  219. 219.

    Dinopoulos, A., Dori, I. & Parnavelas, J. G. Serotonergic innervation of the mature and developing lateral septum of the rat: a light and electron microscopic immunocytochemical analysis. Neuroscience 55, 209–222 (1993).

  220. 220.

    Papadopoulos, G. C., Parnavelas, J. G. & Buijs, R. M. Light and electron microscopic immunocytochemical analysis of the serotonin innervation of the rat visual cortex. J. Neurocytol. 16, 883–892 (1987).

  221. 221.

    Muller, J. F., Mascagni, F. & McDonald, A. J. Serotonin-immunoreactive axon terminals innervate pyramidal cells and interneurons in the rat basolateral amygdala. J. Comp. Neurol. 505, 314–335 (2007).

  222. 222.

    Ridet, J. L., Rajaofetra, N., Teilhac, J. R., Geffard, M. & Privat, A. Evidence for nonsynaptic serotonergic and noradrenergic innervation of the rat dorsal horn and possible involvement of neuron-glia interactions. Neuroscience 52, 143–157 (1993).

  223. 223.

    Bunin, M. A. & Wightman, R. M. Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission. J. Neurosci. 18, 4854–4860 (1998).

  224. 224.

    Bunin, M. A. & Wightman, R. M. Paracrine neurotransmission in the CNS: involvement of 5-HT. Trends Neurosci. 22, 377–382 (1999).

  225. 225.

    Agnati, L. F. et al. A correlation analysis of the regional distribution of central enkephalin and beta-endorphin immunoreactive terminals and of opiate receptors in adult and old male rats. Evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission. Acta Physiol. Scand. 128, 201–207 (1986).

  226. 226.

    Fuxe, K. et al. From the Golgi-Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: wiring and volume transmission. Brain Res. Rev. 55, 17–54 (2017).

  227. 227.

    Mlinar, B. et al. Nonexocytotic serotonin release tonically suppresses serotonergic neuron activity. J. Gen. Physiol. 145, 225–251 (2015).

  228. 228.

    Colgan, L. A., Cavolo, S. L., Commons, K. G. & Levitan, E. S. Action potential-independent and pharmacologically unique vesicular serotonin release from dendrites. J. Neurosci. 32, 15737–15746 (2012).

  229. 229.

    Eulenburg, V. & Gomeza, J. Neurotransmitter transporters expressed in glial cells as regulators of synapse function. Brain Res. Rev. 63, 103–112 (2010).

  230. 230.

    Hrdina, P. D., Foy, B., Hepner, A. & Summers, R. J. Antidepressant binding sites in brain: autoradiographic comparison of [3H]paroxetine and [3H]imipramine localization and relationship to serotonin transporter. J. Pharmacol. Exp. Ther. 252, 410–418 (1990).

  231. 231.

    Sur, C., Betz, H. & Schloss, P. Immunocytochemical detection of the serotonin transporter in rat brain. Neuroscience 73, 217–231 (1996).

  232. 232.

    Brown, P. & Molliver, M. E. Dual serotonin (5-HT) projections to the nucleus accumbens core and shell: relation of the 5-HT transporter to amphetamine-induced neurotoxicity. J. Neurosci. 20, 1952–1963 (2000).

  233. 233.

    Kosofsky, B. E. & Molliver, M. E. The serotoninergic innervation of cerebral cortex: different classes of axon terminals arise from dorsal and median raphe nuclei. Synapse 1, 153–168 (1987).

  234. 234.

    Mamounas, L. A. & Molliver, M. E. Evidence for dual serotonergic projections to neocortex: axons from the dorsal and median raphe nuclei are differentially vulnerable to the neurotoxin p-chloroamphetamine (PCA). Exp. Neurol. 102, 23–36 (1988).

  235. 235.

    Molliver, M. E. Serotonergic neuronal systems: what their anatomic organization tells us about function. J. Clin. Psychopharmacol. 7, 3S–23S (1987).

  236. 236.

    Mamounas, L. A., Mullen, C. A., O’Hearn, E. & Molliver, M. E. Dual serotoninergic projections to forebrain in the rat: morphologically distinct 5-HT axon terminals exhibit differential vulnerability to neurotoxic amphetamine derivatives. J. Comp. Neurol. 314, 558–586 (1991).

  237. 237.

    Jin, Y. et al. Regrowth of serotonin axons in the adult mouse brain following injury. Neuron 91, 748–762 (2016). Building off the work of Molliver and colleagues, this study uses in vivo two-photon microscopy in the neocortex of Sert –eYFP mice to study 5-HT neuron axon degeneration and subsequent regeneration over a period of months following exposure to amphetamine.

  238. 238.

    Kajstura, T. J., Dougherty, S. E. & Linden, D. J. Serotonin axons in the neocortex of the adult female mouse regrow after traumatic brain injury. J. Neurosci. Res. 96, 512–526 (2017).

  239. 239.

    Weissbourd, B. et al. Presynaptic partners of dorsal raphe serotonergic and GABAergic neurons. Neuron 83, 645–662 (2014). This is one of three concurrently released articles applying virally mediated monosynaptic retrograde circuit mapping to characterize brain-wide afferent inputs to 5-HT neurons of the rostral brainstem.

  240. 240.

    Ogawa, S. K., Cohen, J. Y., Hwang, D., Uchida, N. & Watabe-Uchida, M. Organization of monosynaptic inputs to the serotonin and dopamine neuromodulatory systems. Cell Rep. 8, 1105–1118 (2014). This is one of three concurrently released articles applying virally mediated monosynaptic retrograde circuit mapping to characterize brain-wide afferent inputs to 5-HT neurons of the rostral brainstem.

  241. 241.

    Pollak Dorocic, I. et al. A whole-brain atlas of inputs to serotonergic neurons of the dorsal and median raphe nuclei. Neuron 83, 663–678 (2014). This is one of three concurrently released articles applying virally mediated monosynaptic retrograde circuit mapping to characterize brain-wide afferent inputs to 5-HT neurons of the rostral brainstem.

  242. 242.

    Zhang, T. et al. ON and OFF retinal ganglion cells differentially regulate serotonergic and GABAergic activity in the dorsal raphe nucleus. Sci. Rep. 6, 26060 (2016).

  243. 243.

    Commons, K. G. Two major network domains in the dorsal raphe nucleus. J. Comp. Neurol. 523, 1488–1504 (2015).

  244. 244.

    Commons, K. G. Ascending serotonin neuron diversity under two umbrellas. Brain Struct. Funct. 221, 3347–3360 (2016).

  245. 245.

    Zhou, L. et al. Organization of functional long-range circuits controlling the activity of serotonergic neurons in the dorsal raphe nucleus. Cell Rep. 18, 3018–3032 (2017).

  246. 246.

    Kirby, L. G., Rice, K. C. & Valentino, R. J. Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus. Neuropsychopharmacology 22, 148–162 (2000).

  247. 247.

    Audero, E. et al. Suppression of serotonin neuron firing increases aggression in mice. J. Neurosci. 33, 8678–8688 (2013).

  248. 248.

    Espallergues, J. et al. HDAC6 regulates glucocorticoid receptor signaling in serotonin pathways with critical impact on stress resilience. J. Neurosci. 32, 4400–4416 (2012).

  249. 249.

    Richardson-Jones, J. W. et al. Serotonin-1A autoreceptors are necessary and sufficient for the normal formation of circuits underlying innate anxiety. J. Neurosci. 31, 6008–6018 (2011).

  250. 250.

    Ray, R. S. et al. Impaired respiratory and body temperature control upon acute serotonergic neuron inhibition. Science 333, 637–642 (2011). This is the first published study to use chemogenetics to study the in vivo functions of 5-HT neurons.

  251. 251.

    Muller, J. M., Morelli, E., Ansorge, M. & Gingrich, J. A. Serotonin transporter deficient mice are vulnerable to escape deficits following inescapable shocks. Genes Brain Behav. 10, 166–175 (2011).

  252. 252.

    Cummings, K. J., Hewitt, J. C., Li, A., Daubenspeck, J. A. & Nattie, E. E. Postnatal loss of brainstem serotonin neurones compromises the ability of neonatal rats to survive episodic severe hypoxia. J. Physiol. 589, 5247–5256 (2011).

  253. 253.

    Richardson-Jones, J. W. et al. 5-HT1A autoreceptor levels determine vulnerability to stress and response to antidepressants. Neuron 65, 40–52 (2010).

  254. 254.

    Cummings, K. J., Li, A., Deneris, E. S. & Nattie, E. E. Bradycardia in serotonin-deficient Pet-1−/− mice: influence of respiratory dysfunction and hyperthermia over the first 2 postnatal weeks. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298, R1333–R1342 (2010).

  255. 255.

    Hodges, M. R. et al. Defects in breathing and thermoregulation in mice with near-complete absence of central serotonin neurons. J. Neurosci. 28, 2495–2505 (2008).

  256. 256.

    Mosienko, V. et al. Life without brain serotonin: reevaluation of serotonin function with mice deficient in brain serotonin synthesis. Behav. Brain Res. 277, 78–88 (2015).

  257. 257.

    Jennings, K. A. et al. Increased expression of the 5-HT transporter confers a low-anxiety phenotype linked to decreased 5-HT transmission. J. Neurosci. 26, 8955–8964 (2006).

  258. 258.

    Maddaloni, G. et al. Serotonin depletion causes valproate-responsive manic-like condition and increased hippocampal neuroplasticity that are reversed by stress. Sci. Rep. 8, 11847 (2018).

  259. 259.

    Fischer, A. G. & Ullsperger, M. An update on the role of serotonin and its interplay with dopamine for reward. Front. Hum. Neurosci. 11, 484 (2017).

  260. 260.

    Hu, H. Reward and aversion. Annu. Rev. Neurosci. 39, 297–324 (2016).

  261. 261.

    Teixeira, C. M. et al. Hippocampal 5-HT input regulates memory formation and Schaffer collateral excitation. Neuron 98, 992–1004 (2018).

  262. 262.

    Walsh, J. J. et al. 5-HT release in nucleus accumbens rescues social deficits in mouse autism model. Nature 560, 589–594 (2018).

  263. 263.

    Dolen, G., Darvishzadeh, A., Huang, K. W. & Malenka, R. C. Social reward requires coordinated activity of nucleus accumbens oxytocin and serotonin. Nature 501, 179–184 (2013).

  264. 264.

    Miyazaki, K. W. et al. Optogenetic activation of dorsal raphe serotonin neurons enhances patience for future rewards. Curr. Biol. 24, 2033–2040 (2014).

  265. 265.

    Xu, S., Das, G., Hueske, E. & Tonegawa, S. Dorsal raphe serotonergic neurons control intertemporal choice under trade-off. Curr. Biol. 27, 3111–3119 (2017).

  266. 266.

    Nectow, A. R. et al. Identification of a brainstem circuit controlling feeding. Cell 170, 429–442 (2017).

  267. 267.

    Fernandez, S. P. et al. Constitutive and acquired serotonin deficiency alters memory and hippocampal synaptic plasticity. Neuropsychopharmacology 42, 512–523 (2017).

  268. 268.

    Pobbe, R. L., Zangrossi, H. Jr., Blanchard, D. C. & Blanchard, R. J. Involvement of dorsal raphe nucleus and dorsal periaqueductal gray 5-HT receptors in the modulation of mouse defensive behaviors. Eur. Neuropsychopharmacol. 21, 306–315 (2011).

  269. 269.

    McNaughton, N. & Corr, P. J. A two-dimensional neuropsychology of defense: fear/anxiety and defensive distance. Neurosci. Biobehav Rev. 28, 285–305 (2004).

  270. 270.

    Millan, M. J. The neurobiology and control of anxious states. Prog. Neurobiol. 70, 83–244 (2003).

  271. 271.

    Graeff, F. G. On serotonin and experimental anxiety. Psychopharmacology 163, 467–476 (2002).

  272. 272.

    Blanchard, D. C., Griebel, G., Rodgers, R. J. & Blanchard, R. J. Benzodiazepine and serotonergic modulation of antipredator and conspecific defense. Neurosci. Biobehav Rev. 22, 597–612 (1998).

  273. 273.

    Commons, K. G., Cholanians, A. B., Babb, J. A. & Ehlinger, D. G. The rodent forced swim test measures stress-coping strategy, not depression-like behavior. ACS Chem. Neurosci. 8, 955–960 (2017).

  274. 274.

    Roche, M., Commons, K. G., Peoples, A. & Valentino, R. J. Circuitry underlying regulation of the serotonergic system by swim stress. J. Neurosci. 23, 970–977 (2003).

  275. 275.

    Kim, J. C. et al. Linking genetically defined neurons to behavior through a broadly applicable silencing allele. Neuron 63, 305–315 (2009). This is the first study to develop an intersectional genetic strategy for investigating the in vivo behavioural functions of 5-HT neuron subtypes by restricting the expression of a synaptic-silencing effector transgene (a ROSA26 knock-in allele) to neuron subsets defined by the intersectional expression of two genes.

  276. 276.

    Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).

  277. 277.

    Ansorge, M. S., Zhou, M., Lira, A., Hen, R. & Gingrich, J. A. Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice. Science 306, 879–881 (2004).

  278. 278.

    Kim, Y. et al. Whole-brain mapping of neuronal activity in the learned helplessness model of depression. Front. Neural Circuits 10, 3 (2016).

  279. 279.

    Meloni, E. G., Reedy, C. L., Cohen, B. M. & Carlezon, W. A. Jr. Activation of raphe efferents to the medial prefrontal cortex by corticotropin-releasing factor: correlation with anxiety-like behavior. Biol. Psychiatry 63, 832–839 (2008).

  280. 280.

    Johnson, P., Lowry, C., Truitt, W. & Shekhar, A. Disruption of GABAergic tone in the dorsomedial hypothalamus attenuates responses in a subset of serotonergic neurons in the dorsal raphe nucleus following lactate-induced panic. J. Psychopharmacol. 22, 642–652 (2008).

  281. 281.

    Takase, L. F. et al. Inescapable shock activates serotonergic neurons in all raphe nuclei of rat. Behav. Brain Res. 153, 233–239 (2004).

  282. 282.

    Grahn, R. E. et al. Activation of serotonin-immunoreactive cells in the dorsal raphe nucleus in rats exposed to an uncontrollable stressor. Brain Res. 826, 35–43 (1999).

  283. 283.

    Ohmura, Y., Tanaka, K. F., Tsunematsu, T., Yamanaka, A. & Yoshioka, M. Optogenetic activation of serotonergic neurons enhances anxiety-like behaviour in mice. Int. J. Neuropsychopharmacol. 17, 1777–1783 (2014).

  284. 284.

    Andrade, T. G., Zangrossi, H. Jr & Graeff, F. G. The median raphe nucleus in anxiety revisited. J. Psychopharmacol. 27, 1107–1115 (2013).

  285. 285.

    Nishitani, N. et al. Manipulation of dorsal raphe serotonergic neurons modulates active coping to inescapable stress and anxiety-related behaviors in mice and rats. Neuropsychopharmacology 44, 721–732 (2018).

  286. 286.

    Urban, D. J. et al. Elucidation of the behavioral program and neuronal network encoded by dorsal raphe serotonergic neurons. Neuropsychopharmacology 41, 1404–1415 (2016).

  287. 287.

    Marcinkiewcz, C. A. et al. Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala. Nature 537, 97–101 (2016).

  288. 288.

    Correia, P. A. et al. Transient inhibition and long-term facilitation of locomotion by phasic optogenetic activation of serotonin neurons. eLife 6, e20975 (2017).

  289. 289.

    Chamberlain, B., Ervin, F. R., Pihl, R. O. & Young, S. N. The effect of raising or lowering tryptophan levels on aggression in vervet monkeys. Pharmacol. Biochem. Behav. 28, 503–510 (1987).

  290. 290.

    Mosienko, V. et al. Exaggerated aggression and decreased anxiety in mice deficient in brain serotonin. Transl Psychiatry 2, e122 (2012).

  291. 291.

    Alenina, N. et al. Growth retardation and altered autonomic control in mice lacking brain serotonin. Proc. Natl Acad. Sci. USA 106, 10332–10337 (2009).

  292. 292.

    Richerson, G. B. Response to CO2 of neurons in the rostral ventral medulla in vitro. J. Neurophysiol. 73, 933–944 (1995).

  293. 293.

    Iceman, K. E., Richerson, G. B. & Harris, M. B. Medullary serotonin neurons are CO2 sensitive in situ. J. Neurophysiol. 110, 2536–2544 (2013).

  294. 294.

    Pilowsky, P. M., de Castro, D., Llewellyn-Smith, I., Lipski, J. & Voss, M. D. Serotonin immunoreactive boutons make synapses with feline phrenic motoneurons. J. Neurosci. 10, 1091–1098 (1990).

  295. 295.

    Depuy, S. D., Kanbar, R., Coates, M. B., Stornetta, R. L. & Guyenet, P. G. Control of breathing by raphe obscurus serotonergic neurons in mice. J. Neurosci. 31, 1981–1990 (2011).

  296. 296.

    Buchanan, G. F. & Richerson, G. B. Central serotonin neurons are required for arousal to CO2. Proc. Natl Acad. Sci. USA 107, 16354–16359 (2010).

  297. 297.

    Hodges, M. R., Wehner, M., Aungst, J., Smith, J. C. & Richerson, G. B. Transgenic mice lacking serotonin neurons have severe apnea and high mortality during development. J. Neurosci. 29, 10341–10349 (2009).

  298. 298.

    Dosumu-Johnson, R. T., Cocoran, A. E., Chang, Y., Nattie, E. & Dymecki, S. M. Acute perturbation of Pet1-neuron activity in neonatal mice impairs cardiorespiratory homeostatic recovery. eLife 7, e37857 (2018).

  299. 299.

    Donnelly, W. T., Xia, L., Bartlett, D. & Leiter, J. C. Activation of serotonergic neurons in the medullary caudal raphe shortens the laryngeal chemoreflex in anaesthetized neonatal rats. Exp. Physiol. 102, 1007–1018 (2017).

  300. 300.

    Duncan, J. R. et al. Brainstem serotonergic deficiency in sudden infant death syndrome. JAMA 303, 430–437 (2010).

  301. 301.

    Paterson, D. S. et al. Multiple serotonergic brainstem abnormalities in sudden infant death syndrome. JAMA 296, 2124–2132 (2006).

  302. 302.

    Kinney, H. C. et al. Serotonergic brainstem abnormalities in Northern Plains Indians with the sudden infant death syndrome. J. Neuropathol. Exp. Neurol. 62, 1178–1191 (2003).

  303. 303.

    Bright, F. M., Byard, R. W., Vink, R. & Paterson, D. S. Medullary serotonin neuron abnormalities in an Australian cohort of sudden infant death syndrome. J. Neuropathol. Exp. Neurol. 76, 864–873 (2017).

  304. 304.

    Yu, Q. et al. Dopamine and serotonin signaling during two sensitive developmental periods differentially impact adult aggressive and affective behaviors in mice. Mol. Psychiatry 19, 688–698 (2014).

  305. 305.

    Olivier, B. Serotonergic mechanisms in aggression. Novartis Found. Symp. 268, 171–183 (2005).

  306. 306.

    Graeff, F. G., Viana, M. B. & Mora, P. O. Dual role of 5-HT in defense and anxiety. Neurosci. Biobehav Rev. 21, 791–799 (1997).

  307. 307.

    Blanchard, D. C., Sakai, R. R., McEwen, B., Weiss, S. M. & Blanchard, R. J. Subordination stress: behavioral, brain, and neuroendocrine correlates. Behav. Brain Res. 58, 113–121 (1993).

  308. 308.

    Raleigh, M. J., McGuire, M. T., Brammer, G. L., Pollack, D. B. & Yuwiler, A. Serotonergic mechanisms promote dominance acquisition in adult male vervet monkeys. Brain Res. 559, 181–190 (1991).

  309. 309.

    Rozeske, R. R. et al. Uncontrollable, but not controllable, stress desensitizes 5-HT1A receptors in the dorsal raphe nucleus. J. Neurosci. 31, 14107–14115 (2011).

  310. 310.

    Maier, S. F. & Watkins, L. R. Stressor controllability and learned helplessness: the roles of the dorsal raphe nucleus, serotonin, and corticotropin-releasing factor. Neurosci. Biobehav Rev. 29, 829–841 (2005).

  311. 311.

    Hammack, S. E. et al. Corticotropin releasing hormone type 2 receptors in the dorsal raphe nucleus mediate the behavioral consequences of uncontrollable stress. J. Neurosci. 23, 1019–1025 (2003).

  312. 312.

    Hammack, S. E., Pepin, J. L., DesMarteau, J. S., Watkins, L. R. & Maier, S. F. Low doses of corticotropin-releasing hormone injected into the dorsal raphe nucleus block the behavioral consequences of uncontrollable stress. Behav. Brain Res. 147, 55–64 (2003).

  313. 313.

    Bland, S. T. et al. Stressor controllability modulates stress-induced dopamine and serotonin efflux and morphine-induced serotonin efflux in the medial prefrontal cortex. Neuropsychopharmacology 28, 1589–1596 (2003).

  314. 314.

    Hammack, S. E. et al. The role of corticotropin-releasing hormone in the dorsal raphe nucleus in mediating the behavioral consequences of uncontrollable stress. J. Neurosci. 22, 1020–1026 (2002).

  315. 315.

    Ettenberg, A. et al. Inactivation of the dorsal raphe nucleus reduces the anxiogenic response of rats running an alley for intravenous cocaine. Pharmacol. Biochem. Behav. 97, 632–639 (2011).

  316. 316.

    Corrodi, H., Fuxe, K. & Hokfelt, T. Central serotonin neurons and thermoregulation. Adv. Pharmacol. 6, 49–54 (1968).

  317. 317.

    Magnusson, J. & Cummings, K. J. Plasticity in breathing and arterial blood pressure following acute intermittent hypercapnic hypoxia in infant rat pups with a partial loss of 5-HT neurons. Am. J. Physiol. Regul. Integr. Comp. Physiol. 309, R1273–R1284 (2015).

  318. 318.

    Fonseca, M. S., Murakami, M. & Mainen, Z. F. Activation of dorsal raphe serotonergic neurons promotes waiting but is not reinforcing. Curr. Biol. 25, 306–315 (2015).

  319. 319.

    Lottem, E. et al. Activation of serotonin neurons promotes active persistence in a probabilistic foraging task. Nat. Commun. 9, 1000 (2018).

  320. 320.

    Dugue, G. P. et al. Optogenetic recruitment of dorsal raphe serotonergic neurons acutely decreases mechanosensory responsivity in behaving mice. PLOS ONE 9, e105941 (2014).

  321. 321.

    Wong-Lin, K., Wang, D. H., Moustafa, A. A., Cohen, J. Y. & Nakamura, K. Toward a multiscale modeling framework for understanding serotonergic function. J. Psychopharmacol. 31, 1121–1136 (2017).

  322. 322.

    Tremblay, R., Lee, S. & Rudy, B. GABAergic interneurons in the neocortex: from cellular properties to circuits. Neuron 91, 260–292 (2016).

  323. 323.

    Puissant, M. M., Mouradian, G. C. Jr., Liu, P. & Hodges, M. R. Identifying candidate genes that underlie cellular pH sensitivity in serotonin neurons using transcriptomics: a potential role for Kir5.1 channels. Front. Cell Neurosci. 11, 34 (2017).

  324. 324.

    Maddaloni, G. et al. Development of serotonergic fibers in the post-natal mouse brain. Front. Cell Neurosci. 11, 202 (2017).

  325. 325.

    Deneris, E. & Gaspar, P. Serotonin neuron development: shaping molecular and structural identities. Wiley Interdiscip. Rev. Dev. Biol. 7, e301 (2018).

  326. 326.

    Waddington, C. H. The Strategy of The Genes: A Discussion of Some Aspects of Theoretical Biology (Allen & Unwin, 1957).

  327. 327.

    Tononi, G., Sporns, O. & Edelman, G. M. Measures of degeneracy and redundancy in biological networks. Proc. Natl Acad. Sci. USA 96, 3257–3262 (1999).

  328. 328.

    Edelman, G. M. & Gally, J. A. Degeneracy and complexity in biological systems. Proc. Natl Acad. Sci. USA 98, 13763–13768 (2001).

  329. 329.

    Weber, T. et al. Inducible gene manipulations in serotonergic neurons. Front. Mol. Neurosci. 2, 24 (2009).

  330. 330.

    Gong, S. et al. Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. J. Neurosci. 27, 9817–9823 (2007).

  331. 331.

    Hainer, C. et al. Beyond gene inactivation: evolution of tools for analysis of serotonergic circuitry. ACS Chem. Neurosci. 6, 1116–1129 (2015).

Download references


The authors are grateful to the peer reviewers for their thoughtful input and to members of the Dymecki laboratory for discussions related to this article. The work of the authors is supported by US National Institutes of Health grants (P01 HD036379 to B.W.O., K.G.C. and S.M.D.; R01 DA034022 to B.W.O. and S.M.D.; R21 DA036056 to S.M.D.; and R01 DA 021801 to K.G.C.), the Blavatnik Biomedical Accelerator (grant to B.W.O. and S.M.D.) and the GVR Khodadad Family Foundation (grant to S.M.D.).

Reviewer information

Nature Reviews Neuroscience thanks P. Gaspar, C. Lowry and T. Sharp for their contribution to the peer review of this work.

Author information

The authors all researched data for the article, provided substantial contributions to discussion of content, wrote the article and reviewed and edited the manuscript before submission.

Competing interests

The authors declare no competing interests.

Correspondence to Benjamin W. Okaty or Susan M. Dymecki.

Supplementary information

Supplementary Information



Transiently appearing segmented divisions of the developing embryonic hindbrain of vertebrates that are distinguishable morphologically. Rhombomeres function as molecularly discrete, transverse progenitor domains, each giving rise to cell populations with some restriction in fates.


The most rostral transverse segment of the developing embryonic hindbrain. It abuts the midbrain at its rostral border and rhombomere 1 at its caudal border and thus is often referred to as the midbrain–hindbrain junction. The isthmus gives rise to select neurons.

Fate mapping

Indelibly marking a regionally or temporally restricted cohort of cells so as to be able to link labelled mature cells to their developmental origin. Genetic fate mapping involves the use of gene-enhancer-driven Cre or Flp recombinase expression to ‘capture’ cells for constitutive reporter expression. Reporter expression results from Cre-mediated or Flp-mediated recombination of an otherwise ‘silent’ transgene.

Intersectional genetics

Here refers specifically to the use of gene-enhancer-driven Cre and Flp recombinase expression in conjunction with expression of a dual-recombinase-responsive reporter or effector transgene to afford precise labelling or manipulation of cells defined by a history of both Cre and Flp expression.

Transcriptomic technologies

High-throughput approaches, such as microarray gene chip and next-generation RNA sequencing, that allow measurement of the relative abundances of all transcripts (including mRNAs, non-coding RNAs and microRNAs) expressed by a tissue, a pool of cells or a single cell or contained in an isolated cellular compartment, such as the nucleus or synaptosome.


Release of more than one neurotransmitter from a single neuron, either from the same or segregated release sites.


The use of photoactivable ion channel proteins cloned from algae to acutely excite or inhibit transfected cells using light.

Intrinsic electrical properties

The set of electrical characteristics a neuron possesses by virtue of its membrane permeability and capacitance, morphology and its intracellular ionic content and the ionic driving forces acting upon on it. Typically, intrinsic properties are divided into passive and active properties.

Fibre photometry

The use of a brain-implanted optical fibre to visualize and record local Ca2+ fluctuations, a proxy for neuroelectrical activity, as revealed by a genetically encoded calcium indicator, such as GCaMP6. Typically, this method does not provide single-neuron activity resolution but reflects aggregate Ca2+ fluxes coming from populations of cells.

Volume transmission

Neuronal release of neurotransmitter in a diffuse manner, either in the absence of a closely apposed postsynaptic site or in the absence of an efficient local reuptake mechanism, such that cells and synapses distal to the locus of release may ultimately bind the released transmitter.

Selective serotonin reuptake inhibitors

(SSRIs). SSRIs are a class of pharmacological compounds that block the reuptake of extracellular 5-HT by the serotonin transporter, frequently prescribed to treat major depressive disorder and increasingly prescribed to treat other affective disorders such as anxiety and obsessive–compulsive disorder.

Prepulse inhibition

(PPI). The tendency for an organism to display an attenuated response to a given sensory stimulus if it is preceded by a (typically weaker) prestimulus; for example, as observed in the blunting of the acoustic startle reflex to a sudden unexpected loud noise by a preceding quieter tone.


An acronym that stands for designer receptors exclusively activated by designer drugs, an approach that deploys metabotropic receptors that have been re-engineered to respond preferentially to designer compounds (which are otherwise biologically inert) in order to manipulate the activity of transfected cells.

Clozapine N-oxide

An engineered ligand, derived from the antipsychotic psychoactive drug clozapine, that activates DREADD (designer receptors exclusively activated by designer drugs) receptors but is otherwise inert (in its unmetabolized form).


The use of DREADD (designer receptors exclusively activated by designer drugs) technology to acutely manipulate the activity of a population of cells, typically to investigate its in vivo functions.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: Overview of 5-HT neuron diversity.
Fig. 2: Sagittal cross-sectional representation of the mature mouse brainstem with labelled raphe subdomains and corresponding genetically fate-mapped cohorts of Pet1 neurons.
Fig. 3: 5-HT synthesis pathway and core 5-HT neuron molecular phenotype.
Fig. 4: Constellations of differentially expressed genes distinguish Pet1 neuron subtypes defined by lineage and anatomy.
Fig. 5: Summary of Drd2–Pet1 neuron phenotype, integrating across domains.
Fig. 6: An ‘epigenetic landscape’ model of hypothesized 5-HT neuron subtypes.