The early appearance of the serotonergic system in the fetal brain [1-3] together with the well known effects of serotonin (5-HT) during the morphogenesis of the central nervous system (CNS) [4] support that these early functions of 5-HT are exerted through a system of axon terminals in the target regions of the fetal brain, derived of neuronal somata exclusively located in the brainstem. A series of studies support 5-HT as an early appearing molecule (E11) during CNS development in the laboratory rat which has a neurotrophic role in advance to its neurotransmitter and neuromodulator role in the adult brain. Both 5-HT and its specific receptors bound to ionic channels or to metabotropic cascades have, at these early periods of brain development, various functions [5, 6]. We obtained in our laboratory complementary information on the functional role of 5-HT in neuroblastic structures like the axonal growth cones from rat E17 encephala, and the presence of the 5-HT_1A and 5-HT_1B receptors [3,7]; the 5-HT synthetic path as a well as a 5-HT releasing system K⁺ and Ca⁺⁺ dependent and a Na⁺ dependent uptake system [3,8].

A transitory increase in the expression of SERT and 5-HT nerve terminals in sensory areas of the cerebral cortex, has also been described [9,10]. The early appearance of serotonergic terminals in the maturating cerebral cortex is particularly intense especially in the sensory areas: visual, somatosensory and auditory [9,11,12]. Mazer et al., [13] reported that experimental decrease of serotonergic signals alters the normal development and maturation of the cerebral cortex, decreasing the synaptic density with a consecutive damage of cognitive development. It is also known that 5-HT has a regulatory role on various cortical areas through axonic terminals of serotonergic neurons located in the mesencephalic raphe nuclei, and not observed, to our knowledge, in the developing cerebral cortex. Lebrand [14] proposed that regulation of corticogenesis by 5-HT, takes place through thalamo-cortical fibers, supported by the presence of 5-HT_1A and 5-HT_1B receptors and the serotonin transporter (SERT) in this system, also shown the presence of serotonergic receptors in fetal rat brain [15].

There are other types of early appearance neurons in the growing cerebral cortex like Cajal-Retzius in the marginal zone, related to cell migration and to the insideout cell development pattern of the cortex [16]. As well as gabaergic and tyrosine hydroxylase (TH) immunopositive neurons in the rat dorsolateral and medial cortex [17] prenatally and posnatally [15]. Probably their expression is epigenetically regulated, like in the peripheral nervous system [19,20], so that cortical neurons may transitorily express some enzyme molecules like TH [18]. Here we propose that the same may happen with the serotonin synthesizing enzyme Tryptophan-5-hidroxylase (T5-H), and other key 5-HT markers. So, in the present work we describe the presence of 5-HT cells in the fetal neopallium in coculture with the mesencephalon, and in also not cultured cortices in the same developmental stage, and besides the molecular signaling path of the 5-HT_1A receptor, which is presented in the fetal cortical target area.

Experimental Animals: Nuliparous female Wistar rats, 200 - 300 g body weight (bw), were used. Kept during two weeks before experiments under standard conditions: 12 hours light periods; 20˚C ± 1.5˚C temperature and 55% - 65%, humidity; fed water and Purina Chaw pellets ad-libitum. 250 g bw males were employed for mating and the presence of a vaginal plug was taken as the beginning of gestation. Crown-rump length was assessed to confirm gestational age. At E13, E16 or E17, a cesarean section was practiced under pentobarbital anesthesia (40 mg/Kg, ip), then the fetal encephala were dissected out and kept in a Petri dish in PBS at 4˚C. The experimental protocol was approved by The Internal Committee for the Use of Experimental Animals, according to the Official Mexican Norm (NPM-062-ZOO-1999) and to the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publ. No. 8023, 1978).

Organotypic Cultures: The Stoppini method was generally followed, briefly: the E-17 encephala were included in low melting point agarose (8˚C - 17˚C, Sigma, 6% in PBS) [21]. 300 μm slices were obtained with a Vibratome (Pellco101, series 1000) in the area of the mesencephalon (Me), following the coordinates of Foster and Lavdas, where serotonergic cells are located [22,23] (Figures 1(a) and (b)). Neopallium (Np) fragments (0.5

to 1.0 mm) from E13 or E16 encephala were dissected out (Figure 1(c)). Slices were set on culture wells on a permeable membrane (0.4 μm pore, Falcon), close to each other with 1.2 ml culture medium: Sigma MEMEagle, added with 24% of inactivated horse serum (By Products); 0.2 mM Hepes (Sigma); 2% glutamax (Gibco); 0.07% gentamicin (Sigma). Both regions were put in close contact (Figures 1(d) and (e)). Cultures were incubated at 37˚C in 5% CO₂ (Hotpack, Phil., PA, USA) during 12 days and 50% of medium was changed every 48 hours.

Immunolabeling and Microscopic Fluorescence: Tissues were washed in cold phosphate buffer saline (PBS), pH 7.4 and fixed with 4% paraformaldehyde. Immunolabeling was carried out with polyclonal antibodies for 5-HT_1A, 5-HT, serotonin transporter (SERT), MAPK, ERK1 or Vimentin (1:1000), or with monoclonal antibodies for T5-H, GFAP, Giα-1, MAPKK, Enolase or NeuN. Fluorescent secondary antibodies (1:500) were used for FITC (Jackson IR), Fluorescein (Pierce), Alexa 568 (Molecular Probes) and Lisamine-Rhodamine (LRSC, Jackson IR). Epifluorometry (Carl Zeiss, axioscope) or Confocal microscopy (Leica, TCS SP2) were performed to identify and locate the various labeled molecules. Appropiate negative controls omitting the primary antibodies were included in each experiment.

In Figure 2(a) there is a control that confirms the presence of 5-HT cells in the Me area in coculture. Figure 2(b) shows immunopositive cells for 5-HT in the Np. The rate limiting enzyme T5H, (Figure 2(c)) is in a not cultured Np. In Figure 2(d), 5HT_1A Immunopositive cells also in the Np are shown.  

Immunopositive cells to T5H and 5-HT coexisting in the same cell were also observed in coculture or in not cultured Np (Figures 3(a) and (b)). Neuronal markers Enoalse or NeuN were positive together with 5-HT, in culture or in not cultured Np (Figures 4 (a) and (b)), SERT colocalizes with T5H or 5-HT in cells of the Np (Figures 5(a) and (b)), GFAP label also did coexist with the serotonin marker (T5H) in cells of cultured or in not cultured Np (Figures 6(a) and (b)), GFAP coexist with 5-HT in cells of not cultured Np (Figure 6(c)). The signaling molecules (Giα-1, MAPK, MAPKK and ERK1) also do colocalize with their receptor 5-HT_1A, in the cocultured as well as in not cultured Np (Figures 7-11).

There are cells with a neurochemical phenotype that appears early in the primitive cerebral cortex, like gabaergic neurons probably implicated in the processes of corticogenesis [17,24]. Catecholaminergic cells also appear in the prenatal period probably implied too in neurogenesis [18,25,26]. Very early in the rombencephalon area (E11-E12) serotonergic neurons are already presented, they will form later the mesencephalic raphe nuclei, also observed in some early hypothalamic nuclei

[14]. However, the early serotonergic cells do differ with

the catecholaminergic cells in that they have not been observed in the early cerebral cortex (neopallium) of the fetal brain, neither in the adult cortex.

Early serotonergic cells appearing at the mesencephalic area, are the sources of a wide axonal system afferent to multiple brain regions including the maturing cerebral cortex, particularly the sensory cortex, or areas like the marginal zone, septal, piriform and the cortical plate. Up to now it is accepted that regulatory serotonergic messages are displayed through this neuronal axon fiber system, widely distributed [13,27-29]. These elements of the serotonergic system can increase their expression and function during some periods of cortical development [11] as it is the case of SERT, the serotonin transporter, in the visual and in the somatosensory cortices [7,12,30].  

Considering the preceding reference framework, the observations made in the present work may take relevance concerning the unexpected presence of serotonergic cells in the rat fetal neopallium in the culture conditions here described, with the 5-HT label coexisting in the same cells with T5-H, the main enzyme in the serotonin synthesizing path. Their presence has been also found in the fetal Np (E16) not cultivated (in situ), suggesting that they appear earlier. It is also interesting that serotonin markers did also coexist with Vimentin and with GFAP, strongly suggesting that young and mature neuroglia are also expressing the serotonergic phenotype in the maturing cortex. The morphology of the cells, excludes the possibility of serotonergic terminals.

In conclusion it seems possible that as it happens with serotonergic fibers that augment significantly during certain periods of corticogénesis and after some time they decrease to normal, the serotonergic cells here described in the fetal neopallium in culture or in situ, could transiently express the serotonergic phenotype that would possibly allow them to participate directly in the structuration processes of the cerebral cortex, to disappear afterwards, as it happens with the mentioned TH positive cells [18,31].

Results of the present study also show the presence of a molecular signaling system that coexists with 5-HT_1A receptors in cells of the Np after 12 days in cocultures and also in situ. So it may well be that these molecules (Giα-1, ERK1, MAPK and MAPKK) are involved in a signaling cascade initiated by the serotonergic input on the 5-HT_1A receptor.

It is also known that elements of the serotonergic system may increase their expression during some periods of cortical development [11] as it is the case of SERT in the visual and somatosensory cortex [7,12,30,32]. The presence of SERT in the Np both in-situ or cultured, would reinforce the hypothesis that there are serotonin recognizing target cells in the fetal Np. We are currently examining how long their presence could last and how could we study their possible functional role.

We are grateful for excellent technical assistance to T. I. Ignacio Vargas Martínez and T. I. Marisol Bautista Flores, Lab. de Neurontogenia, Departamento de Fisiología, Biofísica y Neurociencias, and M.V.Z. Rafael Leyva Muñoz, Unidad de Producción y Experimentación de Animales de Laboratorio, Cinvestav-IPN.