The results of the present study demonstrated the existence of important strain differences in the responses of mice to chronic administration of antidepressant drugs. MRL/MpJ mice demonstrated robust increases in hippocampal cell proliferation, increased cortico-limbic BDNF protein levels, and diminished reactions to novelty in the NIH test, whereas C57BL/6J mice did not show significant changes in these parameters after the same treatments. The background strain is critical for producing typical effects of antidepressants following acute (Lucki et al., 2001
; Crowley et al., 2005
) or chronic (Holick et al., 2008
; Miller et al., 2008
) treatment. The results of the present study emphasize the importance of strain differences for measuring the effects of antidepressants requiring chronic drug administration. MRL/MpJ mice were studied because they are known distinctively for enhanced wound healing and tissue regeneration after injury (Clark et al., 1998
; Leferovich et al., 2001
; Heber-Katz et al., 2004
). The present study demonstrated exaggerated neuroplastic responses in parallel with behavioral changes to chronic antidepressant drug treatments in these mice. Identification of genetic and neural substrates responsible for these responses in MRL/MpJ mice could provide important information concerning the presently unknown mechanisms that regulate the chronic effects of antidepressants, and that may be more closely associated with their ultimate clinical therapeutic effects.
The existence of neurogenesis throughout the mammalian lifespan in particular brain regions has now been accepted. Adult hippocampal neurogenesis has been associated with a number of diseases, such as depression, schizophrenia, epilepsy, Parkinson's disease, and Alzheimer's disease (Balu and Lucki, 2008
; Thompson et al., 2008
; Zhao et al., 2008
). As a result, there is much interest in studying the effects of various pharmacologic treatments on adult or developmental hippocampal neurogenesis. Hippocampal neurogenesis is regulated by a variety of neurotransmitters and hormones, and pharmacological regulation of neurogenesis can lead to the discovery of novel medications for these disorders. The most common method for measuring cell genesis in brain tissues involves marking DNA synthesis by administering the thymidine analog BrdU and then counting the number of BrdU-labeled cells using immunohistochemistry. However, the intensive labor and time required to perform these studies has made hippocampal neurogenesis an impractical target for screening novel compounds in animals. The present study used flow cytometry to quantify BrdU incorporation into hippocampal cells after administering chronic antidepressant drug treatments and demonstrated significant drug and strain differences in cell proliferation (see also (Bilsland et al., 2006
; Shankaran et al., 2006
). This technique circumvents many technical limitations associated with immunohistochemistry, and provides the speed and automated analysis necessary to facilitate drug discovery and understand the mechanisms underlying drug effects on neurogenesis. The enhanced analytical power of flow cytometry was associated with slightly inferior precision, as compared to immunohistochemistry, which may reflect the ability to topographically restrict counting labeled cells to the dentate gyrus. Future advances of cell phenotyping using flow cytometry, double-labeling BrdU-positive cells with antibodies for neural, glial and developmental markers, will enable more specific morphogenic changes produced by chronic antidepressant drug treatments to be identified rapidly. Nevertheless, rapid screening can still identify those conditions requiring more detailed anatomical analysis.
The value of a rapid quantitative screening approach for measuring hippocampal cell proliferation was demonstrated in the present study. Both fluoxetine and desipramine showed an inverted U-shaped dose-response curve for increased proliferative activity in MRL/MpJ mice following chronic treatment. The inverted U-shaped curve for these antidepressants could be caused by their affinity for receptors unrelated to their primary effect of selectively blocking serotonin and norepinephrine transporters, respectively (Richelson, 2003
). In contrast, C57BL/6J mice did not show an increase in cell proliferation. There have been mixed reports on the ability of antidepressants to regulate hippocampal neurogenesis in C57BL/6J mice. Other studies have also found that fluoxetine administered for 10 days (10 mg/kg i.p.) (Beauquis et al., 2006
) or for 24 days (10, 16, or 25 mg/kg in the drinking water) (Navailles et al., 2008
) failed to increase hippocampal cell proliferation. However, other studies reported that fluoxetine (10 mg/kg, i.p.) administered for 21 days (Lagace et al., 2007
) or amitriptyline (in drinking water) given for 28 days (Caldarone et al., 2004
) resulted in a 25% increase in hippocampal cell proliferation in C57BL/6J mice. It is unclear whether procedural differences in drug administration, BrdU loading protocols, or differences in levels of endogenous stress contributed to the different findings between laboratories. Differences in the response to chronic fluoxetine have been reported for other strains (c.f., Santarelli et al., 2003
; Holick et al., 2008
; Miller et al., 2008
), but no strain showed as large as a response as the MRL/MpJ mice. Most of the cells in the adult hippocampus generated from amplifying progenitor cells become neurons (Encinas et al., 2006
; Wang et al., 2008b
Although the regulation of hippocampal cell proliferation by chronic antidepressant treatments is established, their ability to enhance the long-term survival of newly born neurons is less well defined. Treatment with desipramine or fluoxetine for 21 days did not enhance the survival of newly born progenitors in either the MRL/MpJ or C57BL/6J strain in this study, when BrdU was administered before the antidepressant treatments. A previous study showed that hippocampal cell survival in rats was not altered by chronic treatment with fluoxetine (5 mg/kg) for 14 days (Malberg et al., 2000
). However, more recent studies demonstrated that fluoxetine given chronically to rats (5 mg/kg, i.p.) or 129SvEv mice (18 mg/kg, in drinking water) for 28 days was effective in prolonging cell survival (Nakagawa et al., 2002
; Wang et al., 2008a
). Treatments with other doses, longer durations, or BrdU-labeling protocols could show increased cell survival, and this remains to be determined. However, increased survival of newborn hippocampal cells was not produced under chronic treatment conditions that increased cell proliferation in MRL/MpJ mice.
Another important marker of chronic antidepressant treatments is their ability to increase levels of BDNF protein or mRNA, particularly in the hippocampus and frontal cortex (Duman and Monteggia, 2006
). Although similar findings have been reported in the mouse (Conti et al., 2002
; Song et al., 2006
; Tsankova et al., 2006
), BDNF was measured only at the mRNA level. In MRL/MpJ mice, chronic administration of fluoxetine increased BDNF protein levels in the hippocampus, frontal cortex, and amygdala, while desipramine elevated BDNF levels specifically in the frontal cortex, which is similar to effects reported recently in rats (Balu et al., 2008
). In contrast, the chronic antidepressant treatments did not increase BDNF levels in the frontal cortex or hippocampus in C57BL/6J mice. Fluoxetine (10 mg/kg) increased BDNF in the amygdala and decreased BDNF in the hippocampus, while desipramine dose-dependently and selectively, reduced BDNF protein levels in the brain stem. Mobilization of neurotrophins following chronic antidepressant drug treatments has been considered to play a key role in promoting neuroplasticity and behavioral change (Duman and Monteggia, 2006
; Sairanen et al., 2005). In the present study, the increased BDNF levels in MRL/MpJ mice was associated with increased hippocampal cytogenesis and changes in behavior, and is a candidate for driving these changes. In addition, the augmentation of other forms of synaptic plasticity following chronic antidepressant treatments, such as enhanced LTP (Wang et al., 2008b
), circuit level activity (Airan et al., 2007
) in the dentate gyrus of the hippocampus, and increased dendritic arborization of adult-born hippocampal neurons (Wang et al., 2008b
), could be mediated, in part, by BDNF mobilization (Elmariah et al., 2005; Mamounas et al., 1995).
The differential effects of fluoxetine and desipramine in neuroplasticity and behavior between the two mouse strains could result from differences in pharmacokinetics. However, since these effects were produced by two pharmacologically distinct antidepressants, this explanation is unlikely.
Reduction of hyponeophagia is one of the few behavioral responses produced by chronic antidepressant drug treatments (Merali et al., 2003
; Dulawa and Hen, 2005), and was used to compare behavioral effects between these mouse strains. Chronic administration of fluoxetine or desipramine to MRL/MpJ mice, but not C57BL/6J mice, reduced their latency to consume food in the NIH paradigm. The divergent behavioral responses of the mouse strains paralleled their response to chronic antidepressant treatments on hippocampal cell proliferation and cortio-limbic BDNF levels, although the link between these factors is unclear (Dranovsky and Hen, 2006
). Hippocampal neurogenesis appeared to be required for the behavioral effects of chronic antidepressant treatments in the NSF paradigm in 129SvEvTac mice because cessation of neurogenesis by x-irradiation prevented chronic antidepressant behaviors (Santarelli et al., 2003
). However, hippocampal neurogenesis did not correlate with the ability of chronic fluoxetine to mediate NIH behavior in unperturbed BALB/cJ mice (Holick et al., 2007
). Nevertheless, a causal link may still exist when changes in hippocampal neurogenesis occur and the moderation of NIH behavior following chronic antidepressant drug treatments, and future studies are needed to assess this relationship in the MRL/MpJ strain. Taken together with neurogenesis and BDNF mobilization, these results using markers of chronic, rather than acute, antidepressant drug activity highlight the potential utility of the MRL/MpJ mouse to test novel compounds for potential antidepressant activity.