Although histamine can induce both

Although histamine can induce both proliferation and differentiation in vitro (Molina-Hernandez and Velasco, 2008), the promotion of neurogenesis conferred by H3R antagonism here does not seem to be caused by either the proliferation or the reduction of apoptosis of NSCs (Figure S3). Furthermore, H3R antagonism enhances differentiation of neuroblasts with an elaborated morphology, having longer leading processes and longer total branches (Figures 4A and 4B). An effective neurogenesis relies not only on the eventual differentiation into mature neural cells, but also on the right composition of differentiated Phenyl sulfate receptor and functional integration (Li and Chen, 2016). Here, we found that those newly generated neuroblasts can differentiate into mature neural cells, including neurons, astrocytes, and oligodendrocytes (Figure 5C). Importantly, H3R antagonism elevated the number of each type of cell, without affecting their composition, which was comparable to that of the sham group (Figures 5C, 5D, and 5F). Such a normal cell composition may be critical for neurological repair, since excessive generation of one cell type may result in tumorigenesis (Buffo et al., 2008, Niu et al., 2013). Moreover, the simultaneous generation of excitatory and inhibitory neurons in the right composition could potentially balance excitation and inhibition (Guo et al., 2014). We further found that the composition of newly generated CaMKIIα+ glutamatergic neurons and PV+ inhibitory neurons, after H3R antagonist treatment, resembles that in the sham group (Figures 5E and 5G), suggesting that the H3R antagonism induces differentiation of the right neurons to achieve effective neurogenesis. In addition, the integrated interactions between functional neurons are fundamental for the recovery of neurological functions (Hagg, 2005). Indeed, we observed synaptic connections with surrounding neurons, functional responses, and the capacity to fire action potentials in newly generated neurons, which implies that functional integration can be established in those cells. H3 antagonism thus boosts neurogenesis by promotion of NSC differentiation into mature and functional neurons to provide neurological recovery following TBI. In addition, this promotion of differentiation is manipulated by the H1R in NSCs, since the increased differentiation is abrogated in Hrh1;Nestin mice (Figures 4C, 4D, and 5K). It has been suggested that transcription of pro-neurogenic genes such as Mash1, Dlx2, and Neurogenin1 is related to the neurogenic effect of H1R in vitro (Bernardino et al., 2012, Rodriguez-Martinez et al., 2012). However, the determination of whether these proteins are involved in the action of H1R in this process requires further study since different NSC differentiation patterns have been observed between the present study and previous in vitro research. For example, in cultured NSCs, histamine reportedly favors neuron commitment during differentiation through H1R (Molina-Hernandez and Velasco, 2008), although we found the H1R in NSCs does not skew the differentiation pattern, but preserves a close-to-natural cell composition that could be critical for neurological recovery.
Experimental Procedures
Author Contributions
Acknowledgments This work was supported by the National Natural Science Foundation of China (81473186, 81673405, 81722045), the Zhejiang Provincial Natural Science Foundation of China under grants LR17H310001 and LY18H310003, Fundamental Research Funds for the Central Universities (2018XZZX002-13), and the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2017PT31038 and 2018PT31041). We are very grateful to Dr. John Hugh Snyder for language editing.
Introduction We have previously reported that moderate prenatal alcohol exposure (PAE) produced long-lasting deficits in dentate gyrus long-term potentiation (LTP) (Sutherland et al., 1997, Varaschin et al., 2010) as well as deficits in learning tasks sensitive to hippocampal damage (Savage et al., 2002, Staples et al., 2013, Sutherland et al., 2000, Weeber et al., 2001). Subsequently, we demonstrated that the histamine H3 receptor antagonist ABT-239 ameliorated both the LTP deficits (Varaschin et al., 2010) as well as spatial navigation and contextual fear-conditioning deficits caused by PAE (Savage et al., 2010). Curiously, ABT-239 did not enhance LTP or learning in control rats. Further, the administration of the selective H3 receptor agonist methimepip in control rats mimicked the LTP deficits observed in saline-treated PAE rats (Varaschin, Rosenberg, Hamilton, & Savage, 2014). These results suggested that PAE elevates histaminergic H3 receptor-mediated inhibition of glutamatergic neurotransmission, an effect that leads to increased responsiveness to histamine H3 receptor antagonists. More recently, we examined this hypothesis by measuring histamine H3 receptor number and function in PAE rats. While the density of histamine H3 receptors was not different in a variety of brain regions examined, histamine H3 receptor-effector coupling, as measured by methimepip-stimulated [35S]-GTPγS binding, was significantly elevated in cerebral cortex, dentate gyrus, and cerebellum of PAE rats compared to controls (Varaschin, Allen, Rosenberg, Valenzuela, & Savage, 2018). Further, the same range of methimepip concentrations that significantly elevated H3 receptor-effector coupling also inhibited paired-pulse plasticity in dentate gyrus to a greater extent in PAE rats than in controls (Varaschin et al., 2018).