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Histamine has neurotransmitter/neuromodulator functions in the adult brain, but its role during CNS development has been elusive. We studied histamine effects on proliferation, cell death and differentiation of neuroepithelial stem cells from rat cerebral cortex in vitro. RT-PCR and Western blot experiments showed that proliferating and differentiated cells express histamine H1, H2 and H3 receptors. Treatments with histamine concentrations (100 nM–1 mM) caused significant increases in cell numbers without affecting Nestin expression. Cell proliferation was evaluated by BrdU incorporation; histamine caused a significant increase dependent on H2 receptor activation. Apoptotic cell death during proliferation was significantly decreased at all histamine concentrations, and cell death was promoted in a concentration-dependent manner by histamine in differentiated cells. Immunocytochemistry studies showed that histamine increased 3-fold the number of neurons after differentiation, mainly by activation of H1 receptor, and also significantly decreased the glial (astrocytic) cell proportion, when compared to control conditions. In summary, histamine increases cell number during proliferative conditions, and has a neuronal-differentiating action on neural stem cells, suggesting that the elevated histamine concentration reported during development might play a role in cerebrocortical neurogenesis, by activation of H2 receptors to promote proliferation of neural precursors, and favoring neuronal fate by H1-mediated stimulation.
Histamine (HA) is produced, stored, released and metabolized in the brain, filling the criteria for a neurotransmitter/neuromodulator (Schwartz et al. 1991; Hill et al. 1997). In the adult CNS, HA regulates pre- and post-synaptically a variety of functions, such as wakefulness, feeding, drinking, body temperature and motor activity (Schwartz et al. 1979; Knigge and Warberg 1991; Wada et al. 1991; Onodera et al. 1994; Haas and Panula 2003). These HA actions are mediated by the activation of three different histaminergic G protein-coupled receptors named H1R, H2R and H3R, which are widely distributed throughout the CNS (Hill et al. 1997), and have been cloned and characterized by their pharmacology and signal transduction mechanisms (Gantz et al. 1991; Yamashita et al. 1991; Lovenberg et al. 1999; Tardivel-Lacombe et al. 2000). Activation of H1R and H2R excites neurons or potentiates excitatory inputs (Haas and Panula 2003), while activation of H3R causes inhibition of synthesis and release of HA and other neurotransmitters (Clapham and Kilpatrick 1992; Schlicker et al. 1994; Molina-Hernandez et al. 2000, 2001). The affinity of HA for these receptors vary: H1R and H2R are activated at μmolar concentrations of HA (Garbarg and Schwartz 1987; Traiffort et al. 1994), whereas H3R respond to HA in the nM range (Rouleau et al. 2004).
During rat development, HA is one of the first neurotransmitters to be present in CNS, starting at embryonic day (E) 12, and reaching its maximum value at E14–E16, decreasing afterwards 5-fold to adult levels in the prosencephalic area (Vanhala et al. 1994). Between E14 and E18, fibers from transient histaminergic neurons in the mesencephalon can be detected, passing through the ventral tegmental area and within the medial forebrain bundle and the optic tract, reaching the frontal and the parietal cortex at E15, earlier than other monoaminergic systems (Specht et al. 1981; Lidov and Molliver 1982; Auvinen and Panula 1988; Reiner et al. 1988; Vanhala et al. 1994), which coincides with the period where neuronal differentiation is occurring in cerebral cortex (Sauvageot and Stiles 2002). Messenger RNA of H1R and H2R are widely distributed in the developing CNS, whereas H3R is present in spinal cord and mesencephalon, appearing in cerebral cortex at E19 (Kinnunen et al. 1998; Heron et al. 2001; Karlstedt et al. 2001a, 2003). The developmental role of HA in the nervous system, including the cerebral cortex is still unknown (Mezei and Mezei 1978; Happola et al. 1991; Vanhala et al. 1994; Nissinen and Panula 1995; Nissinen et al. 1995).
A correlation of neurogenesis and elevations of HA in cerebral cortex can be proposed from the above mentioned findings. However, a direct approach to test this link has not been reported. Neural stem cells (NSC) are key players in brain development (Temple 2001). This study was designed to establish the role of HA on NSC by exploring in vitro the effect of this biogenic amine on cell proliferation, apoptosis, and differentiation using rat cortical precursor cells from E14. We show that HA is a positive modulator in proliferation/expansion of NSC, and also a factor that promotes neuronal differentiation of neural precursors; in this study we identified the histaminergic receptors responsible for these effects.
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Neural stem cells are important elements of the developing nervous system that can be isolated and grown in vitro to study the role of a number of factors that might affect proliferation and cell fate. In these cells, we show here that HA induce the following effects: (i) expansion of NSC numbers due to an increase in proliferation caused by activation of H2R; (ii) decreased apoptosis in NSC stimulated with bFGF; (iii) a concentration-dependent induction of TUNEL-positive cells in the differentiation phase, and (iv) higher number of neurons after differentiation of NSC, an effect due to H1R activation.
The biogenic amine HA, which acts as a neurotransmitter/neuromodulator in the adult rat CNS (Schwartz et al. 1991; Hill et al. 1997), is present in high concentrations (five times higher than those found in adult brain) in the prosencephalic area at E14, and these levels remain elevated until E17 (Vanhala et al. 1994) suggesting a role of HA in neurogenesis occurring in that period. Histaminergic receptors must be in place for HA to act. Expression of histaminergic receptors at these stages of development has been reported. Hybridization studies show the distribution of mRNA for HA receptors in different brain regions, but no information about the cell types expressing the receptors is provided (Kinnunen et al. 1998; Heron et al. 2001; Karlstedt et al. 2001b, 2003). It has been shown that the cerebral cortical area expresses H1R at E14 (Kinnunen et al. 1998). Little is known about H2R expression before E15, but after this age it can be clearly detected in the cerebral cortex (Karlstedt et al. 2001a). Messenger RNA for H3R is detected in E19 in the cortical area (Karlstedt et al. 2003). There are no reports showing the expression of HA receptors in NSC. We show in this study that cortical NSC express H1R, H2R and all reported isoforms of H3R before and after differentiation, at the mRNA level. Based on hybridization studies, expression of H1R and H2R was expected in NSC, but the presence of H3R was not anticipated, due to the fact that it is expressed from E19 onwards. H1R and H2R were detected by immunoblot as single bands; however, H3R presented several bands that varied in size between NSC and adult cerebral cortex. Similar differences in abundance and masses have been reported between embryonic and adult brown adipose tissue (Karlstedt et al. 2003). Bands below 35 kDa are not compatible with expected sizes of G-protein coupled receptors, and might result from active H3R degradation processing, as suggested earlier (Karlstedt et al. 2003).
In the present study, we found that HA did not modify NSC identity during proliferation, since a high proportion of cells continue to express the intermediate filament protein Nestin, and therefore, HA caused a significant expansion of NSC. Interestingly, HA could induce the appearance of a few cells positive to differentiated cell markers, although these cells are still Nestin-positive. This suggests that HA could promote premature differentiation under proliferating conditions in a discrete population. It remains to be investigated whether a combination of HA with other signals could promote differentiation, even in the presence of bFGF.
Based on HA concentrations that have evident effects in this study, we though that either H1R (Traiffort et al. 1994) or H2R (Garbarg and Schwartz 1987) could be implicated, since these two receptors are activated with micromolar concentrations of HA, while H3R is activated in the nanomolar range (Chen et al. 2003; Rouleau et al. 2004) and suffer desensitization at μM concentrations (Perez-Garcia et al. 1998). We choose 100 μM HA to further study if H1R, H2R or H3R were responsible for the effects seen on NSC proliferation and differentiation caused by HA. The increase of bFGF-induced proliferation caused by HA was due to the activation of H2R, as demonstrated by the reversion of BrdU incorporation when cells were incubated with 100 μM HA + 30 μM cimetidine. Although there is no report of the effect of HA and the pathways that are stimulated by this biogenic amine on NSC, there are evidences that H2 receptor activation is linked to different signaling systems: 1. Stimulation of cAMP formation in brain slices (Al-Gadi and Hill 1985), vascular smooth muscle and neutrophils (Hill 1990). 2. Stimulation of phospholipid methylation in rat mast cells (Tolone et al. 1982). 3. Increases in the slow inward Ca2+current in several models such as guinea pig ventricular myocytes, via cAMP formation (Hill 1990). 4. Inhibition of Cl−-mediated K+ conductance in hippocampal pyramidal cells (Haas and Greene 1986). 5. Increases of [Ca2+]i mobilization in a human lymphocytic cell line (HL-60) (Mitsuhashi and Payan 1991). Multiple reports demonstrated that single receptors may be associated with more than one G protein, and thus to multiple intracellular signaling systems (Vallar et al. 1990; Van Sande et al. 1990; Gudermann et al. 1992; Raymond 1995; Arai and Charo 1996). Our results open two possibilities by which HA can be regulating cell proliferation by H2R activation: a) Via phosphoinositide/protein kinase C signal transduction cascade (Del Valle and Gantz 1997) or b) By increasing cAMP, since this cyclic molecule stimulate proliferation in many cell types, an effect that is largely attributed to cross-talk from cAMP and the mitogen-activated protein kinase pathway (Dumaz and Marais 2005). The effect of cimetidine on decreasing cell proliferation is in agreement with a study made by Finn et al., in which this H2R blocker also inhibited proliferation in three out of five glial cell lines (Finn et al. 1996). Pharmacological blockade of HA effects rules out the possibility that this amine could be acting on the polyamine site of the NMDA receptor, because such interaction is not susceptible to be interrupted by histaminergic H1 and H2 receptor antagonists (Bekkers 1993; Vorobjev et al. 1993).
In general, stem cell pools result from the contribution of various factors, i.e. rate of proliferation, time of exponential expansion of cell number, ratio of asymmetric to symmetric cell divisions (Caviness and Takahashi 1995), and apoptotic cell death (Blaschke et al. 1996). In the present study, we measured apoptotic cell death levels, in order to estimate the contribution of this factor on the effect of HA increasing cell number during the proliferation phase. A low proportion of TUNEL-positive cells was found in control NSC cultures. These results are in accordance with a study in cortical stem cells, showing low numbers of apoptotic cells (Chang et al. 2004). Our data show that HA was able to further decrease the proportion of TUNEL-positive NSC in a H2R-dependent manner, suggesting that HA is acting both as a proliferating and an anti-apoptotic factor for NSC in the presence of bFGF. The effect of HA increasing cell number during the proliferation phase was not observed after 6 days of differentiation. This could be due to the concentration-dependent increased in the number of TUNEL-positive cells in HA-treated cells which, together with the HA-induced increase in cell number during proliferation, might account for the similar number of cells found in control and HA-exposed cultures. The increased cell death during differentiation could contribute to the neurogenic effect of HA if glial progenitors/cells are induced to undergo cell death, and this will be certainly interesting to investigate further.
About differentiation, our results show that NSC treated daily with micromolar or low millimolar concentrations of HA during the proliferation and differentiation stages generate more neurons and less GFAP-positive cells. Regulation of cell fate acquisition in the vertebrate CNS is dependent on the stage of development. In the rat cerebral cortex, neurogenesis begins at E12, peaks at E14, and recedes by E17 (Sauvageot et al. 2005). In vivo, neurons are generated first, followed by astrocytes, and later by oligodendrocytes, and this behavior is mimicked by cultured NSC, that first generate neurons and then glial progeny (Qian et al. 2000; Morrow et al. 2001; Panchision and McKay 2002; Sauvageot and Stiles 2002). HA concentration peaks in prosencephalon from E14 to E17 (Vanhala et al. 1994), suggesting a role of this biogenic amine in neuronal differentiation. The marked shift in the proportion of neurons induced by HA is consistent with this idea. Although HA could contribute to neuronal differentiation in vivo, it is important to mention that knockout mice for the HA synthesizing enzyme, histidine decarboxylase, do not show any evident alteration in brain development (Watanabe and Yanai 2001). These results are not necessarily opposed to our findings, since there might be redundant mechanisms for neuronal differentiation in the cerebral cortex.
To study which histaminergic receptor is responsible for the increase on the number of neurons in culture, we performed experiments with H1R, H2R and H3R antagonists and 100 μM HA. Our results show that HA increase neuronal differentiation due to activation of H1R. Activation of this receptor leads to production of IP3 and diacylglycerol, that in turn promote an increase on [Ca2+]i due to activation of IP3 receptors in the endoplasmic reticulum, and the activation of protein kinase C. Calcium release from intracellular stores into the cytosol is a critical component during ontogenesis and contributes particularly to the formation and maintenance of dendritic structures (Lohmann et al. 2002, 2005). Regarding astrocyte production, it is interesting to note that neither chlorpheniramine, nor cimetidine, nor thioperamide were able to revert HA effect on decreasing glial differentiation. Notwithstanding, the overall effect of antagonizing H1R in the presence of HA is to block its neuronal-promoting effect.
There are only a few examples of other neurotransmitters having effects on proliferation and differentiation of NSC, and some of them are conflicting. GABA and glutamate decrease cortical precursors proliferation (LoTurco et al. 1995; Antonopoulos et al. 1997), acetylcholine increases cell proliferation (Ma et al. 2000) and dopamine has been shown to promote (Hoglinger et al. 2004) or inhibit cell proliferation (Kippin et al. 2005) of adult NSC. Regarding neurogenesis, a recent report shows that GABA has an effect promoting this process in NSC derived from adult brain (Tozuka et al. 2005). Thus, HA is the first neurotransmitter showing a positive effect on both NSC proliferation and in the proportion of neurons derived from cortical NSC, that correlate with increased HA levels during neuronal differentiation in the cerebral cortex.