The novel findings of our study are that α2-adrenoceptor stimulation decreases adult hippocampal neurogenesis, while simultaneous α2-adrenoceptor blockade and antidepressant treatment hastens effects on hippocampal neurogenesis, plasticity-associated gene expression, and depression-related behavior. Our results highlight the importance of α2-adrenoceptors as targets for rapid-action antidepressants.
Adult hippocampal neurogenesis encompasses progenitor proliferation, survival and differentiation resulting in newborn neuron integration into hippocampal circuitry (Ming and Song, 2005
). The hippocampal neurogenic niche receives dense noradrenergic innervation, expresses all the different classes of adrenoceptors, and hippocampal progenitor proliferation is stimulated by norepinephrine (Nicholas et al., 1996
; Kulkarni et al., 2002
; Jhaveri and Bartlett, personal communication). Paradoxically, we found that α2
-adrenoceptor stimulation in vivo
decreases hippocampal progenitor proliferation. α2
-adrenoceptors exist both as heteroceptors on target neurons receiving noradrenergic innervation, and as α2
-autoreceptors that decrease norepinephrine release. Our in vitro
and in vivo
findings support a role for α2
-heteroceptors in mediating the effects on adult hippocampal progenitor turnover. First, we provide evidence that dispersed adult hippocampal progenitors in culture express the α2
-adrenoceptor subtypes, and exhibit decreased BrdU-incorporation following α2
-adrenoceptor stimulation. Second, α2
-adrenoceptor agonists significantly reduce neurosphere frequency in the neurosphere assay indicating a direct effect on the turnover of adult neural stem cells. Finally, our in vivo
finding that this decreased proliferation persists in Dbh
−/− mice indicates an effect on hippocampal progenitor proliferation via α2
-heteroceptors. While α2
-adrenoceptor stimulation selectively influences the proliferation, but not the survival or differentiation of hippocampal progenitors; α2
-adrenoceptor blockade does not influence hippocampal neurogenesis. Our results differ from a previous study (Rizk et al., 2006
) that reported enhanced progenitor survival following long duration treatment with the α2
-adrenoceptor antagonist dexefaroxan. This discrepancy may be a consequence of differences in drug choice, dose, route of administration or treatment duration. We also did not observe any change in progenitor proliferation or immature neuron number in α2A
-adrenoceptor knockout mice. Taken together, our results indicate that α2
-adrenoceptors exert a stimulation-induced, but not a basal tonic, inhibitory effect on adult hippocampal progenitor turnover.
-adrenoceptors are expressed within proliferative germinal zones, and their stimulation decreases DNA synthesis in the developing forebrain (Lidow and Rakic, 1994
; Kreider et al., 2004
) suggesting possible common mechanisms in the developing and adult brain. Given that cAMP-CREB signaling enhances adult hippocampal neurogenesis (Nakagawa et al., 2002
-adrenoceptor activation may decrease progenitor turnover through a Gi
-mediated inhibition of adenylate cyclase. The mechanistic underpinnings of the effects of α2
-adrenoceptors on adult hippocampal progenitors warrant further investigation.
Hippocampal volumetric loss is observed in recurrent major depression, and decreased neurogenesis reported in animal models of depression (Campbell et al., 2004
; Sahay and Hen, 2007
). Enhanced α2
-adrenoceptor density and mRNA expression within limbic brain regions is observed in animal models of depression (Ribas et al., 2001
; Flugge et al., 2003
). Further, increased hippocampal α2
-adrenoceptor binding (Meana et al., 1992
; González et al., 1994
) and prefrontal α2A
-adrenoceptor immunoreactivity (Garcia-Sevilla et al., 1999
) have been observed in postmortem studies of major depression. While it remains uncertain whether the changes in α2
-adrenoceptor binding reflect alterations in terminal α2
-autoreceptors or α2
-heteroceptors in target fields, the mRNA expression changes indicate enhanced expression of α2
-heteroceptors in limbic neurocircuitry following chronic stress. Our results suggest a possible role for α2
-heteroceptors in the neurogenic decline observed in animal models of depression.
The “neurotrophic” and “neurogenic” hypotheses of depression posit a role for decreased trophic support and neurogenesis in the hippocampal damage observed in depression, and a contribution of antidepressant induced trophic factor expression and hippocampal neurogenesis to their therapeutic action (Sahay and Hen, 2007
; Schmidt and Duman, 2007
). It is noteworthy that the neurotrophic and neurogenic changes exhibit a slow onset, and parallel the time course required for the behavioral benefits of sustained antidepressant administration (Sahay and Hen, 2007
; Schmidt and Duman, 2007
). One of our most interesting and novel findings is that α2
-adrenoceptor blockade accelerates the effects of chronic antidepressant treatment on hippocampal neurogenesis and trophic factor gene expression associated with faster behavioral effects.
Combined 7 day yohimbine and imipramine (Y+I) treatment significantly improved behavioral outcome in the NSF test, which is normally sensitive only to longer periods (3 weeks) of antidepressant treatment (Santarelli et al., 2003
). Y+I treatment also increased hippocampal progenitor proliferation and NeuroD-positive neuroblast number within 7 days, an effect not observed with imipramine treatment alone. Further, a striking shift in the distribution of DCX-positive cells to those bearing complex, tertiary dendritic morphology was observed in the 7 day Y+I group. Enhanced BrdU/calretinin percent colocalization observed at 7 days in the Y+I group suggests an influence on the developmental progression of immature neurons. Taken together, our results indicate that simultaneous α2
-adrenoceptor blockade hastens effects of antidepressant treatment on distinct aspects of adult hippocampal neurogenesis.
It is unlikely that increased progenitor proliferation contributes to the behavioral effects observed with 7 day Y+I treatment; rather our observation of effects on the morphological complexity of DCX-positive cells suggest that the integration of immature neurons into hippocampal neurocircuitry may be accelerated. It is tempting to speculate that this component of the neurogenic effects of 7 day Y+I treatment may contribute to the rapid rate of behavioral response. Future experiments are required to address whether 7 day Y+I treatment accelerates the differentiation, integration and recruitment of newborn neurons into hippocampal circuitry. Interestingly, 21 day Y+I treatment increased progenitor proliferation and DCX-positive cell number to a similar extent as imipramine treatment alone, suggesting that combined treatment may largely involve acceleration, rather than augmentation, of the neurogenic effects of imipramine.
Seven day Y+I treatment increased BDNF, VEGF and FGF-2 mRNA expression in the DG subfield, an effect not observed with imipramine treatment. The combined treatment also enhanced VGF expression, an effect also observed with imipramine treatment. The effects of Y+I do not appear simply additive, and suggest the possible recruitment of distinct signaling mechanisms. The robust increase in the expression of the activity-dependent gene Arc (Bramham et al., 2008
) in the DG subfield indicates enhanced neuronal activity following Y+I treatment. It is interesting to note that enhanced DG activity as measured by voltage sensitive dye imaging has been previously strongly linked to antidepressant-induced neurogenic and behavioral responses (Airan et al., 2007
). Neuronal activity is known to recruit latent stem/progenitor cells in the adult hippocampus, and enhance trophic factor expression (Gall and Lauterborn, 1997
; Walker et al., 2008
). Trophic factors could serve to link neuronal activity to structural plasticity in the hippocampal neurogenic niche. Our results also raise the strong possibility that non-cell autonomous effects via enhanced neuronal activity and trophic factor release may also contribute to the neurogenic effects of Y+I treatment. Further, BDNF, FGF-2, VEGF and VGF are implicated as key mediators of both the neurogenic and behavioral effects of antidepressants (Palmer et al., 1999
; Warner-Schmidt et al., 2007
; Bachis et al., 2008
; Li et al., 2008
; Thakker-Varia et al., 2008; Perez et al., 2009
), and may play a critical role in mediating faster effects on neurogenesis and behavior with 7 day Y+I treatment.
Prior clinical literature has suggested the coadministration of α2
-adrenoceptor antagonists with antidepressants as a therapeutic strategy to hasten antidepressant responses (Blier, 2003
). The mode of action for these faster effects has been unclear. Our results suggest that adjunct α2
-adrenoceptor antagonists accelerate the effects of antidepressant treatment on the slow-onset adaptive changes in neurogenesis and trophic factor expression that are thought to underlie the behavioral effects of antidepressants. Our findings support the view that structural remodeling and enhanced trophic support may be key events determining the latency in onset of antidepressant action. At present, we cannot distinguish whether blockade of α2
-autoreceptors or heteroceptors, or a combination of both, contribute to the faster effects of Y+I treatment. It has been suggested that concomitant blockade of inhibitory presynaptic α2
-autoreceptors would increase noradrenergic neurotransmission, whereas in the absence of such a blockade a desensitization/downregulation of presynaptic α2
-adrenoceptors would have to precede an increase in noradrenergic neurotransmission, thus contributing to the delay in antidepressant action (Blier, 2003
). It is possible that we observed faster effects with Y+I treatment due to the blockade of inhibitory α2
-autoreceptors, or heteroceptors on serotonergic neurons, thus enhancing noradrenergic and serotonergic neurotransmission. Our failure to observe a change in hippocampal norepinephrine levels following 7 day Y+I treatment does not rule out possible autoreceptor effects, particularly since we measured tissue norepinephrine levels, which are not always correlated with extracellular norepinephrine that is available for signaling. Future studies using genetic perturbation approaches will be required to dissect out the contribution of α2
-autoreceptors and heteroceptors to the faster effects of adjunct α2
-adrenoceptor antagonist and antidepressant treatment.
In conclusion, we demonstrate that α2-adrenoceptor stimulation decreases adult hippocampal neurogenesis, while concomitant α2-adrenoceptor blockade accelerates the neurogenic, neurotrophic and behavioral effects of chronic antidepressant treatment. The delayed onset of action of antidepressants is a major drawback for both treatment and compliance. Our results highlight the importance of α2-adrenoceptors in modulating the speed of antidepressant response, and motivate future research in the development of rapid-action antidepressants that target the α2-adrenoceptor.