We are investigating the genes that control early serotonin neuron development and how they impact serotonergic modulation of behavior. Serotonin is an important modulator of neural circuitry that controls a wide range of behavioral and physiological processes including cognition, circadian rhythms, and mood. Defective serotonergic signaling has been implicated in numerous human behavioral disorders such as impulsive violence, anxiety, and depression. However, we know very little about the genes involved in vertebrate serotonin neuron development. We also know very little about how these genes function and interact with one another to govern the generation of these neurons and how they are linked to eventual serotonergic control of behavior after birth. Whatever those genes are we imagine they constitute a genetic program that operates to generate and maintain a certain number of serotonin neurons that function to modulate neuronal circuitry throughout the central nervous system. Although it is widely appreciated that understanding this genetic program is important little progress has been made because molecular genetic approaches aimed at serotonin neuron development have been difficult to apply. This is partly due to the small number of serotonin neurons in the brain and their scattered midbrain hindbrain distribution. It is also because of a lack of suitable serotonin neuron-specific (SNS) molecular markers at early embryonic stages. The research we have completed to date has opened up several new lines of investigation into the mechanisms of serotonin neuron development because we have identified an early marker of these cells that encodes a transcription factor.

The marker we discovered encodes an ETS protein, which we named, Pet-1 for PC12 ets. Expression of Pet-1 in the brain is restricted to developing and adult serotonin neurons. This suggested to us that Pet-1 is a key determinant of central serotonin neuron phenotype. To test this idea we prepared Pet-1 null mice. Analysis of these mice at the cellular, molecular, and behavioral levels led to a number of interesting results. Our findings indicated that most serotonin neurons fail to be generated in the null brain and the ones that remained were defective. These defects led to very low serotonin levels throughout the developing and adult brain. Significantly, our behavioral analyses indicated that the defective embryonic development of serotonin neurons is followed by aggressive and anxiety-like behavior in adults. These findings indicated that Pet-1 is a critical determinant of serotonin neuron identity and suggested the existence of a Pet-1 dependent transcriptional program that selectively couples early steps in serotonin neuron differentiation to serotonergic modulation of behavior in the adult.

A primary focus of the lab now is to determine the network of factors that operate in the Pet-1 dependent genetic program and determine the importance of these factors for serotonergic modulation of behavior. We decided very early on that in order to proceed further with our investigation of this genetic program we would benefit from identifying serotonin neuron-specific (SNS) transcriptional regulatory elements for mouse and human Pet-1. Identification of these elements would provide greater accessibility of serotonin neurons for molecular genetics studies by i) allowing us to begin to identify transcription factors that operate “upstream” of Pet-1 during serotonin neuron development, ii) use these elements to drive CRE or FLP recombinase expression to achieve spatial and temporal control of gene expression in these cells, and iii) SNS expression of GFP or its variants would allow us to sort these cells and then use them for studies in culture, preparation of SNS cDNA libraries or probes for microarray studies and SNS proteomic studies. Mike Scott is a graduate student in the lab and he has identified a fragment of BAC genomic DNA that drives correct SNS expression of lacZ in developing and adult transgenic mice. The SNS expression of lacZ is highly reproducible among different transgenic lines, which means we can easily use this fragment for recombinase or fluorescent reporter expression. So we are now poised to further investigate the mechanisms of serotonin neuron development using all of the modern molecular genetics approaches currently available. By coupling these approaches to analyses of various mouse behaviors and physiological processes we can determine how genes controlling serotonin neuron development and function impact models of mood and learning and memory to name a few of the possibilities.

Dr. Vittorio Erspamer was looking for substances capable of causing smooth muscle contraction and identified such a substance in an acetone extract of rabbit gastric mucosa in the 1930’s. He named this substance enteramine.

In the late 1940’s the laboratory of Dr Irving Page down the road at the Cleveland Clinic isolated a vasoconstricting substance in serum and named it serotonin. The structure of serotonin was reported in 1949. Around 1952 it was realized that enteramine and serotonin were the same substance.

In 1952 Dr Betty Twarog joined the Page lab at the Clinic to test the idea that invertebrate neurotransmitters might also be used as neurotransmitters in vertebrates. Her research resulted in the identification of serotonin in the brain, which was published in 1953.

The neurochemical anatomy of brain serotonin neurons was first studied by fluorescence histochemical detection of the indoleamine by Dahlstrom and Fuxe. In 1964 they reported the distribution of serotonin neuron cell bodies using the B nomenclature.

The system Dahlstrom and Fuxe reported consists of a relatively small population of morphologically diverse neurons whose cell bodies are present largely within the brainstem raphe nuclei and particular regions of the reticular formation. Raphe clusters of 5-HT neurons are found rostrally from the level of the interpeduncular nucleus in the midbrain to the level of the pyramidal decussation in the medulla. Although there are only about 20,000 serotonergic neurons in the rat brain (around 300K in humans) the extensive axonal projection system arising from these neurons bears a tremendous number of collateral branches so that the 5-HT system densely innervates nearly all regions of the CNS. The midline raphe nuclei consist of the caudal linear nucleus (CLi, B8), the dorsal raphe nucleus (DR, B6, B7), the median raphe nucleus (MnR, B5, B8), raphe magnus nucleus (RMg, B3), raphe pallidus nucleus (RPa, B1), and the raphe obscurus nucleus (ROb, B2). Outside the raphe nuclei there are collections of 5-HT containing cell bodies in a region adjacent to the medial lemniscus called the B9 cell cluster, in the ventrolateral medulla called the B3 cluster, and in the central gray of the medulla oblongata (B4). The B3 group of serotonergic neuron clusters are thought to be lateral extensions of serotonergic neuron clusters in midline raphe.

1. Erspamer, V. and Asero, B. (1952)
   Identification of enteramine, specific hormone of enterochromaffin cells, as 5-hydroxytryptamine. Nature. 169:800-801.
   
2. Rapport, MM, Green AA, Page, IH. (1948)
   Partial purification of the vasoconstrictor in beef serum. J. Biol. Chem. 174:735-741.
   
3. Rapport, MM. (1949)
   Serum vasoconstrictor (serotonin). V. The presence of creatinine in the complex: A proposed structure of the vasoconstrictor principle. J. Biol. Chem. 180:961-969.
   
4. Twarog, BM and Page IH. (1953)
   Serotonin content of some mammalian tissues and urine and a method for its determination. Am. J. Physiol. 175:157-161.
   
5. Dahlstrom, A., and Fuxe, K. (1964)
   Evidence for the existence of monamine-containing neurons in the central nervous system. I. Demonstration of monamines in the cell bodies of brainstem neurons. Acta Physiol Scand. 62: (Suppl. 232):1-55.