Cerebrospinal fluid-contacting (CSF-c) cells containing monoamines such as dopamine (DA) and serotonin (5-HT) occur in the periventricular zones of the hypothalamic region of most vertebrates except for placental mammals. Here we compare the organization of the CSF-c cells in chicken, Xenopus, and zebrafish, by analyzing the expression of synthetic enzymes of DA and 5-HT, respectively, tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), and draw an evolutionary scenario for this cell population. Due to the lack of TH immunoreactivity in this region, the hypothalamic CSF-c cells have been thought to take up DA from the ventricle instead of synthesizing it. We demonstrate that a second TH gene (TH2) is expressed in the CSF-c cells of all the three species, suggesting that these cells do indeed synthetize DA. Furthermore, we found that many CSF-c cells coexpress TH2 and TPH1 and contain both DA and 5-HT, a dual neurotransmitter phenotype hitherto undescribed in the brain of any vertebrate. The similarities of CSF-c cells in chicken, Xenopus, and zebrafish suggest that these characteristics are inherited from the common ancestor of the Osteichthyes. A significant difference between tetrapods and teleosts is that teleosts possess an additional CSF-c cell population around the posterior recess (PR) that has emerged in specific groups of Actinopterygii. Our comparative analysis reveals that the hypothalamus in mammals and teleosts has evolved in a divergent manner: placental mammals have lost the monoaminergic CSF-c cells, while teleosts have increased their relative number.

Although the simultaneous presence of tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) is considered as a phenotypic signature of dopamine (DA) neurons, it has been suggested that they are not uniformly expressed in all dopaminergic brain nuclei. Moreover, in nonmammalian vertebrates, two tyrosine hydroxylase genes (TH1 and TH2) are found, and they exhibit different expression patterns in zebrafish brains. Here we present a detailed description of the distribution of TH1, TH2, AADC, DAT, and VMAT2 transcripts, in relation to TH and DA immunoreactivity to better characterize dopaminergic nuclei in the adult zebrafish forebrain. TH2-positive cells in the hypothalamus are strongly DA immunoreactive (DAir), providing direct evidence that they are dopaminergic. DAir cells are also found in most TH1-positive or TH-immunoreactive (THir) nuclei. However, the DAir signal was weaker than THir in the olfactory bulb, telencephalon, ventral thalamus, pretectum, and some posterior tubercular and preoptic nuclei. These cell populations also exhibited low levels of VMAT2 transcripts, suggesting that low DA is due to a lower vesicular DA accumulation. In contrast, cell populations with low levels of AADC did not always have low levels of DA. DAT transcripts were abundantly expressed in most of the TH1- or TH2-positive territories. In addition, DAT and/or VMAT2 transcripts were found in some periventricular cell populations such as in the telencephalon without TH1 or TH2 expression. Thus, expression patterns of dopaminergic cell markers are not homogeneous, suggesting that the gene regulatory logic determining the dopaminergic phenotype is unexpectedly complex.