Norepinephrine Directly Activates Adult Hippocampal Precursors via β₃-Adrenergic Receptors

We appreciate the thoughtful comments from Christ et al. in their letter regarding the conclusion of our paper in which we had demonstrated that norepinephrine directly activated a hippocampal cell population consisting of stem and precursor cells via beta3-adrenergic receptors (ARs). Specifically, the question was raised as to whether we had truly ruled out a role for beta1-ARs in the norepinephrine-mediated activation of a hippocampal precursor population. They questioned the selectivity and efficacy of beta3-AR agonist and antagonist used in our study based on previous human studies. Here it is important to note that despite 82% amino acid sequence homology between human and murine beta3-ARs, their pharmacological properties are quite distinct (Mersmann, 1998). BRL37344, a selective beta3-AR agonist that we have shown can activate adult hippocampal precursor population in mouse brain, was in fact demonstrated to have higher selectivity and efficacy in rodents compared to humans (Arch et al., 1984; Blin et al., 1994, also reviewed in Vrydag and Michel, 2007; Michel et al., 2010). Therefore, making a direct comparison of the pharmacological properties of various beta3-ARs agonists and antagonist between species is very difficult. Nevertheless, since the publication of our paper, we have conducted further experiments, which provide further support to our original conclusion and address the questions raised by Christ and colleagues.

We have examined the efficacy of other selective beta3-AR agonists in activating a latent population of stem and precursor cells in the hippocampus of the adult mouse. We used L755,507, the most potent and selective beta3-AR agonist developed by Merck Research Laboratories, which is reported to have over 440-fold selectivity for beta3-AR over both beta1 - and beta2-ARs (Parmee et al., 1998). Moreover, it has been shown to be only a weak partial agonist at beta1-ARs with >1300-fold selectivity for beta3 agonist activity over beta1 agonist activity. The addition of L755,507 at both 100 nM and 1 uM led to a significant increase, of 174.9+/ -10.1% and 180.2+/-0.4%, respectively (n=2 experiments, p<0.001), in the total number of neurospheres compared to the control (EGF+bFGF) group. This increase in neurosphere number was comparable to that obtained by 10 uM norepinephrine treatment (192.7+/-4.6%). More importantly, several large neurospheres (>200 uM in diameter) were obtained in L755,507- treated cultures, similar to that obtained in the presence of norepinephrine, indicating an activation of a stem cell pool (100 nM: 4.5+/-1.5 neurospheres; 1 uM: 9.5+/-5.5 neurospheres). Similarly, the addition of another selective beta3-AR agonist, ZD7114, developed by Astra Zeneca also led to a significant increase in neurosphere numbers compared to the control (100 nM: 182.1+/-3.0%, 1 uM: 167.9+/-3.7%, 10 uM: 158.4+/- 4.3%; n=2 experiments, p<0.001). Together, these results again confirmed our previous findings and indicated that stimulation of beta3- ARs is sufficient to activate hippocampal stem and precursor cell population residing in the adult mouse brain.

In addition, we have carried out experiments using 100 nM CGP20712A, a selective beta1-AR antagonist, and found that even at higher concentrations to that used in our original study, beta1 blockade fails to inhibit the norepinephrine response. CGP20712A treatment did not significantly decrease the total number of neurospheres obtained in the presence of norepinephrine nor did it block the generation of large neurospheres measuring >200 um. These results complement our recent findings that showed no significant change in the total neurosphere numbers in the presence of either the selective beta1/beta2-AR agonist dobutamine or the selective beta2-AR agonist salbutamol, when compared to the control (Dobutamine- 100 nM: 115.6+/-9.3%, 1 uM: 121.5+/-15.2%; Salbutamol- 100 nM: 97.6+/- 4.7%, 1 uM: 114.1+/-19.1%, 10 uM: 118.5+/- 6.3%; n=3 experiments, p>0.05). Neither of these agonists generated large neurospheres, which are indicative of stem cell activation. These results suggest that stimulation of either beta1 or beta2-ARs is not sufficient to activate the hippocampal precursor population.

Although, propranolol has been shown to be only a partial agonist to human receptors, it still remains a potent antagonist to mouse beta3-ARs (Blin et al., 1994). Similarly, recent studies have suggested that SR59230A may be a partial agonist to human beta3-ARs, however, it is a potent antagonist of mouse beta3-ARs (reviewed in Ursino et al., 2009). Furthermore, evidence is emerging that suggests that antagonist properties of SR59230A may be dependent on the level of beta3-ARs expression on a given cell, with SR59230A exhibiting complete antagonism in low-expressing cells (Sato et al., 2007). In agreement, we have shown that beta3-ARs is expressed at low levels in the hippocampal precursor cells, requiring ~45 PCR cycles for their detection (Jhaveri et al., 2010). Moreover, we have performed additional experiments using higher concentrations of SR59230A (50 nM and 100 nM) and find that these concentrations similarly block the norepinephrine response. This inhibitioin is similar to that seen with 10 nM SR59230A, both in terms of the total number of neurospheres formed and the fraction of these which contained very large neurospheres.

Taken together, these results indicate that norepinephrine primarily signals via beta3-ARs to activate the precursor cell population in the adult mouse hippocampus. Moreover, it once again highlights that the efficacy and selectivity of agonists and antagonists to these receptors may differ between the species and the cell or tissue type used.

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Vrydag W, Michel MC (2007) Tools to study beta3-adrenoceptors. Naunyn Schmiedebergs Arch Pharmacol 374:385-398

Conflict of Interest:

None declared