Lippincott Williams & Wilkins, Inc., Philadelphia
Enhancement of Hippocampal Neurogenesis by Lithium
Article first published online: 4 JAN 2002
Journal of Neurochemistry
Volume 75, Issue 4, pages 1729–1734, October 2000
How to Cite
Chen, G., Rajkowska, G., Du, F., Seraji-Bozorgzad, N. and Manji, H. K. (2000), Enhancement of Hippocampal Neurogenesis by Lithium. Journal of Neurochemistry, 75: 1729–1734. doi: 10.1046/j.1471-4159.2000.0751729.x
Abbreviations used: bcl-2, B-cell lymphoma protein-2; BrdU, 5-bromo-2-deoxyuridine; DG, dentate gyrus; MDI, manic-depressive illness; NeuN, neuron-specific nuclear protein; PBS, phosphate-buffered saline.
- Issue published online: 4 JAN 2002
- Article first published online: 4 JAN 2002
- Manic-depressive illness;
Abstract: Increasing evidence suggests that mood disorders are associated with a reduction in regional CNS volume and neuronal and glial cell atrophy or loss. Lithium, a mainstay in the treatment of mood disorders, has recently been demonstrated to robustly increase the levels of the cytoprotective B-cell lymphoma protein-2 (bcl-2) in areas of rodent brain and in cultured cells. In view of bcl-2's antiapoptotic and neurotrophic effects, the present study was undertaken to determine if lithium affects neurogenesis in the adult rodent hippocampus. Mice were chronically treated with lithium, and 5-bromo-2-deoxyuridine (BrdU) labeling of dividing cells was conducted over 12 days. Immunohistochemical analysis was undertaken 1 day after the last injection, and three-dimensional stereological cell counting revealed that lithium produced a significant 25% increase in the BrdU-labeled cells in the dentate gyrus. Double-labeling immunofluorescence studies were undertaken to co-localize BrdU-positive cells with neuron-specific nuclear protein and showed that ∼65% of the cells were double-labeled. These results add to the growing body of evidence suggesting that mood stabilizers and antidepressants exert neurotrophic effects and may therefore be of use in the long-term treatment of other neuropsychiatric disorders.
Manic-depressive illness (MDI) is a common, severe, chronic, and life-threatening disease. Despite the devastating impact that this illness has on the lives of millions, there is still a dearth of knowledge concerning its etiology and pathophysiology. In this context, it is noteworthy that although mood disorders have traditionally been conceptualized as neurochemical disorders, recent volumetric neuroimaging studies have demonstrated an enlargement of the third and lateral ventricles as well as reduced gray matter volumes in parts of the orbital and medial prefrontal cortex, the ventral stiatum, and the mesiotemporal cortex (Sheline et al., 1996, 1999; Drevets et al., 1997, 1999; Soares and Mann, 1997). In addition to the accumulating neuroimaging evidence, several postmortem brain studies are now providing direct evidence for reductions in regional CNS volume and cell number. Thus, studies have reported a layer-specific reduction in interneurons in the anterior cingulate cortex (Vincent et al., 1997) and reductions in nonpyramidal neurons (∼40% lower) in CA2 of the hippocampal formation in bipolar disorder subjects compared with control subjects (Benes et al., 1998). Furthermore, recent postmortem studies of the prefrontal cortex have also demonstrated reduced CNS volume and cell numbers (Ongur et al., 1998; Rajkowska et al., 1999; Rajkowska, 2000). Together, the preponderance of the data presents a convincing case that there is indeed a reduction in regional CNS volume accompanied by cell atrophy/loss in mood disorders; indeed, this has led to the formulation of a heuristic molecular and cellular model of depression (Duman et al., 1997).
Until recently, the loss or atrophy of large numbers of neurons has been accepted as an unavoidable fate, with neurogenesis being considered very implausible in the mature human brain. However, in addition to the identification of specific cellular pathways that regulate cell survival, it has recently been demonstrated that neurogenesis does occur in the adult human brain (Eriksson et al., 1998). The localization of pluripotent progenitor cells and neurogenesis occurs in restricted brain regions, in particular, the subventricular zone and the subgranular layer of the hippocampus (Gould et al., 1999; Kempermann and Gage, 1999). Additional studies have greatly enhanced our understanding of the genetic and environmental factors regulating neurogenesis in the adult mammalian brain (Kempermann et al., 1997; Gould et al., 1999; Kempermann and Gage, 1999; van Praag et al., 1999), leading to the exciting possibility that it may be possible to pharmacologically regulate neurogenesis in the brain to correct disease-related pathophysiological changes.
It has recently been demonstrated that lithium (a mainstay in the treatment of recurrent mood disorders) robustly increases the levels of the cytoprotective B-cell lymphoma protein-2 (bcl-2) in areas of rodent brain and in cultured cells (Chen and Chuang, 1999; Chen et al., 1999; Manji et al., 1999, 2000a). Bcl-2 overexpression has been shown to protect neurons from a variety ofin vitro andin vivo insults (Adams and Cory, 1998, and references therein), and consistent with its effects on bcl-2, lithium has also been demonstrated to exert neuroprotective effects bothin vitro andin vivo (see Nonaka et al., 1998; Jope, 1999; Manji et al., 1999, 2000b). Bcl-2 is known to exert major antiapoptotic effects, and apoptosis has been postulated to play a major role in regulating the survival of newborn neurons in the adult rodent (see Kempermann et al., 1997). Furthermore, a growing body of data has shown that bcl-2 also exerts neurotrophic effects. Thus, bcl-2 enhances the regeneration of axons in the mammalian CNS (Chen et al., 1997), promotes neurite outgrowth (Oh et al., 1996), and increases axonal growth rate (Hilton et al., 1997), effects that may all be independent of its antiapoptotic effects. Additionally, recent studies have shown that neurotrophic factor signaling is mediated by both the phosphatidylinositol 3-kinase pathway as well as activation of the MAP (mitogen-activated protein) kinase cascade (Segal and Greenberg, 1996; Tao et al., 1998). Importantly for the present discussion, activation of the MAP kinase cascade increases the expression of bcl-2, an effect that likely involves the cyclic AMP response element binding protein (Bonni et al., 1999; Riccio et al., 1999). We have therefore undertaken the present study to determine if lithium, administered at therapeutically relevant concentrations, affects neurogenesis in the adult rodent hippocampus. In this article, we use the definition of “neurogenesis” proposed by Kempermann and Gage (1999). Thus, neurogenesis refers to a series of events (including proliferation of a neuronal precursor or stem cell) that results in the appearance of a new neuron.
MATERIALS AND METHODS
Animals and treatments
Adult male C57BL/6 mice (Harlan Sprague Dawley, Indianapolis, IN, U.S.A.) were housed two or three per cage with free access to food and water in a 12-h light/dark cycle animal facility. After ≥1-week accommodation period, the mice were treated with control or lithium chow (2.4 g/kg of Li2CO3) for 3-4 weeks. Saline was provided ad libitum to the lithium-treated mice to reduce potential toxicity. The mean pooled (due to the limited blood volume) serum lithium level was 0.97 ± 0.20 mM (therapeutic concentrations in humans, 0.6-1.2 mM). No significant differences in the body weights of the control and lithium-treated mice were observed. 5-Bromo-2-deoxyuridine (BrdU; Sigma, St. Louis, MO, U.S.A.) was dissolved in 0.9% NaCl and filtered sterilely at 0.2 mm. After treatment with lithium for 14 days, the mice received single doses of BrdU at 50 mg/g of body weight at a concentration of 10 mg/ml, one intraperitoneal injection per day for 12 successive days (van Praag et al., 1999). Lithium treatment continued throughout the 12 days of BrdU administration.
Immunoblotting for bcl-2
Immunoblotting of bcl-2 was conducted on hippocampal membrane fractions obtained from a separate group of mice chronically treated with lithium using anti-bcl-2 antibodies (N-19, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) (Chen et al., 1999).
Twenty-four hours after the last BrdU injection, mice were deeply anesthetized with chloral hydrate and perfused via the ascending aorta with saline until the outflow became clear, followed by 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde for 20 min. The brains were rapidly removed and postfixed in the same fixative plus 20% sucrose at 4°C for 48 h and then rapidly frozen and stored at -80°C. Serial sections (30 μm/section) were cut coronally through the entire anteroposterior extension of the hippocampi and were pretreated for DNA denaturation according to the methods of Kempermann et al. (1997). Sections were then incubated free-floating in phosphate-buffered saline (PBS; pH 7.4) containing an alkaline phosphatase-conjugated anti-BrdU antibody (anti-BrdU-AP, 1:800; Boehringer Mannheim) and 0.1% Triton X-100 for 3 days at 4°C. Following three washes in 0.05 M Tris buffer (pH 7.2), sections were incubated for 1 h at room temperature in 0.1 M Tris-HCl buffer (pH 9.5) containing 1% levamisole solution, 0.1% Tween 20, and the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) solutions from BCIP/NBT Alkaline Phosphatase Substrate Kit IV (Vector Labs, Burlingame, CA, U.S.A.; prepared according to the manufacturer's specifications). Subsequently, the sections were washed five times in distilled water and mounted on gelatin-coated slides. All sections were dehydrated in ethanol, cleared in xylene, and coverslipped in Permount (Fisher Scientific, Fair Lawn, NJ, U.S.A.). An unbiased stereological cell-counting method was used to count BrdU-positive cells (see below).
Double-labeling of BrdU and neuron-specific nuclear protein (NeuN)
For the double-labeling of BrdU and NeuN, sections that had been pretreated for DNA denaturation were first incubated free-floating in 0.01 M PBS containing 1% normal donkey serum, 0.3% Triton X-100, and a mouse anti-NeuN monoclonal antibody (Chemicon, Temecula, CA, U.S.A.; 1:2,000) for 24 h at 4°C. This was followed by incubation of sections in PBS containing rhodamine Redô-X-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Labs, West Grove, PA, U.S.A.; 1:100) for 1 h at room temperature and then in 10% normal mouse serum for another hour. After thorough washes, the sections were incubated in 0.01 M PBS containing 0.3% Triton X-100 and a biotinylated monoclonal mouse anti-BrdU antibody (Zymed Labs, South San Francisco, CA, U.S.A.; 1:250) for 3 days at 4°C. Subsequently, the sections were incubated with fluorescein streptavidin (Vector Labs) for 1 h at room temperature. After several washes in PBS, sections were mounted on gelatin-coated slides with Vectashield Æ (Vector Labs). Fluorescent signals were detected using a confocal laser scanning microscope (Leica TCS), and confocal images were produced at a fixed laser power setting with a 40× oil-immersion objective. Separate optical images of BrdU and NeuN immunoreactivity were captured from the same optical section. The captured images were then pseudocolored green or red. A digital overlay was generated and companion images were superimposed. Regions of colocalization were reflected by the additive effect of superimposed green and red pixels, appearing in yellow. Image analysis was performed by using the standard system operating software provided with the confocal microscope (Version 1.6). Fifty double-labeled cells per animal were analyzed from six animals in each group.
BrdU-positive cells were counted in a one-in-six series of sections (180 μm apart) throughout the rostrocaudal extent of the granule cell layer of hippocampus using unbiased stereological three-dimensional cell-counting methods with a computer-assisted image analysis system (Williams and Rakic, 1988). This counting method is based on the optical dissector method and estimates the number of cells directly in the optically defined volume of tissue independently of section thickness, cell shape, or orientation. BrdU-positive cells were counted within the counting box, ignoring cells in the upper-most focal plane and focusing through the thickness of the section (optical dissector principle) to avoid oversampling errors. Only profiles of cell nuclei with a complete nuclear contour were counted at the focal plane in which they were maximum in size. The granule cell area was traced in a blinded manner using the NIH Image software. The granule cell reference volume was determined by summing the traced granule cell areas for each section and multiplying by the distance between sections sampled. The number of BrdU-labeled cells was then related to granule cell layer sectional volume and multiplied by the reference volume to estimate total number of BrdU-positive cells.
RESULTS AND DISCUSSION
It has been previously demonstrated that chronic lithium administration increases bcl-2 levels in Wistar rats (Chen et al., 1999; Manji et al., 1999). Recent genetic studies suggest that C57BL/6 mice are very suitable for adult neurogenesis studies (Kempermann et al., 1997). We therefore first undertook a study to determine if lithium also increases bcl-2 levels in the hippocampus of C57BL/6 mice. Similar to what has been observed in Wistar rats and cells in culture, chronic lithium administration significantly increased bcl-2 levels in hippocampus of the C57BL/6 mice (Fig. 1).
We next investigated the effects of lithium administration on neurogenesis in the hippocampus of C57BL/6 mice. In adulthood, precursor cells in the hilus and subgranular zone of the dentate gyrus (DG) divide and produce daughter cells that are capable of ultimately differentiating into mature granule neurons (Tanapat et al., 1998). Lithium's effects on BrdU incorporation were therefore investigated in the subgranular zone. In all the mice (control and lithium treated), BrdU-labeled cells were observed in the DG (Fig. 2). In particular, BrdU-positive cells were observed in the subventricular zone lining the wall of the lateral ventricle. BrdU labeling of dividing cells was conducted over 12 days and immunohistochemical analysis undertaken 1 day after the last injection (van Praag et al., 1999). Stereological three-dimensional unbiased cell counting revealed that lithium treatment produced a significant 25% increase in the BrdU-labeled cells in the DG (Fig. 3). Lithium did not significantly affect the volume of either the right or the left DG and produced a significant increase in the density of BrdU-positive cells in the DG (Fig. 3).
The phenotype of the BrdU-positive cells was next examined (Fig. 4) by double-labeling studies with BrdU and the neuronal marker NeuN (Mullen et al., 1992; Kempermann and Gage, 1999). In the control mice, 68 ± 6% of the BrdU-positive cells double-stained with NeuN; following chronic lithium treatment, the percentage of cells double-staining with BrdU and NeuN was virtually identical (66 ± 6%). These results suggest that even at this early time point, approximately two-thirds of the newborn cells detected after chronic lithium exhibited a neuronal phenotype. In this context, it is noteworthy that bcl-2 has also recently been proposed to accelerate the maturation of neurons (Middleton et al., 1998). The percentage of double-labeled cells remained unchanged despite the overall 25% increase in the number of BrdU-labeled cells. These results suggest that lithium produces an equivalent increase in non-NeuN-immunoreactive cells. It is now known that although NeuN labels neuronal cells almost exclusively, there are several neuronal subtypes that do not express NeuN (Mullen et al., 1992; Davenne et al., 1999; Peissner et al., 1999). In addition to potentially increasing the number of non-NeuN-containing neurons, it is possible that chronic lithium is also increasing the number of nonneuronal cells including progenitor cells and glia. In view of the reports of reductions in the numbers of both neurons and glia in postmortem brain from MDI patients (Ongur et al., 1998; Rajkowska et al., 1999; Rajkowska, 2000), these findings require further study.
As discussed in the introductory section, neurogenesis in the present context refers to a series of events that result in the appearance of a new cell with a neuronal phenotype (and includes both proliferation and survival) (Kempermann and Gage, 1999). Given the short time frame of the present studies in which BrdU immunolabeling was conducted 24 h after 12 daily BrdU injections, it is likely that increased proliferation plays a role in lithium's effects. However, in view of bcl-2's major antiapoptotic effects and the postulated role of apoptosis in regulating the survival of newborn neurons in the adult rodent (see Kempermann et al., 1997), it is possible that enhanced survival of the newborn cells also plays a role in lithium's effects. Additional studies will be necessary to more fully delineate lithium's effects on the proliferation and survival of newborn neurons in the adult brain.
In conclusion, we report here the novel observation that chronic lithium increases neurogenesis in the DG of adult rodents. It is clear that in addition to its effects on bcl-2, lithium affects a variety of signaling pathways and transcription factors (Klein and Melton, 1996; Jope, 1999), some of which may also play a role in regulating the proliferation and survival of newborn neurons. Studies currently underway using bcl-2 transgenic and knockdown mice should serve to more fully elucidate the role of bcl-2 in mediating lithium's effects on newborn neurons. These novel findings add to the growing body of recent data demonstrating that mood stabilizers and antidepressants may exert major neurotrophic/neuroprotective effects (Duman et al., 1997; Nonaka et al., 1998; Wang et al., 1999; Manji et al., 2000b). Most recently, human studies have demonstrated that chronic lithium not only increases the levels of N-acetylaspartate (a putative marker of neuronal viability) (Tsai and Coyle, 1995) but also increases brain gray matter volumes in MDI patients (Moore et al., 2000a,b). Interestingly, recent studies by Duman and Malberg (1999) have demonstrated that the chronic administration of a variety of antidepressants also increases hippocampal neurogenesis. Although much research is required to elucidate the functional significance of the newborn neurons, these results suggest that lithium and antidepressants may exert some of their beneficial effects by regulating hippocampal neurogenesis and may thus also have utility in the treatment of other neuropsychiatric disorders.
The authors' research is supported by the National Institute of Mental Health (RO1-MH57743, RO1-MH 59107), Theodore and Vada Stanley Foundation, NARSAD, and Joseph Young Sr. research grants. The authors thank Gregory Kapatos for his very thoughtful critique of the manuscript and P. S. Goldman-Rakic, Ph.D., and P. Rakic, Ph.D., from Yale University School of Medicine for sharing software for three-dimensional cell counting. Outstanding technical assistance was provided by Li-Dong Huang.
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