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Stress and glucocorticoids are among the strongest inhibitors of adult hippocampal neurogenesis. Despite the known role of the hippocampus in negative feedback regulation of the hypothalamo-pituitary adrenal axis, whether loss of hippocampal neurogenesis affects this inhibition has not been examined. Here we tested whether suppression of adult neurogenesis affected the hypothalamo-pituitary-adrenal axis response. Our results show that suppression of neurogenesis leads to a potentiated hypothalamo-pituitary-adrenal axis response following exposure to a mild stressor. This study suggests that suppressed neurogenesis directly regulates the hypothalamo-pituitary-adrenal axis response.
The hippocampus shows remarkable structural and functional plasticity even in the adult animal. A fundamental component of its structural plasticity is its capacity for adult neurogenesis. New neurons are generated in the brain throughout the life of many animal species including rodents, non-human primates and humans in the subgranular zone of the dentate gyrus in the hippocampus. Recent evidence has linked exposure to stressful life events to altered neurogenesis in the hippocampus [1–3].
Exposure to stressful events results in a series of responses that act to preserve or restore homeostasis. The key neuroendocrine response to stress is activation of the hypothalamo-pituitary-adrenal (HPA) axis, which triggers increased production of glucocorticoids (GC). Stress is a key etiological factor in depressive disorders; up to 50% of affected patients exhibit some form of HPA axis abnormality . GCs are potent factors in the regulation of both proliferation and differentiation of new neurons in the dentate gyrus [5,6]. Adrenal removal results in accelerated cell proliferation in the subgranular zone and increases the number of newly formed, surviving neurons. Conversely, corticosterone administration decreases the proliferation and survival of progenitor cells . Moreover, exposing animals to various forms of stress, a process that activates the adrenal glands and results in increased levels of corticosterone, has similar effects on hippocampal neurogenesis [1–3]. Importantly, it has been shown that this effect is dependent on corticosterone .
The hippocampus negatively regulates the HPA axis and this inhibitory feedback is altered by various forms of chronic stress [8,9]. As discussed, it is known that chronic stress results in significantly decreased rates of hippocampal neurogenesis . However, whether loss of neurogenesis itself regulates the HPA axis has not been studied. Thus, we wondered whether loss of neurogenesis in the hippocampus may lead to less efficient inhibitory control of hypothalamic cells that produce glucocorticotrophin-releasing hormone, with a resultant increased HPA-axis response.
Adult transgenic mice and control littermates were used for all experiments. hGFAPtk transgenic mice were generated as described below and backcrossed >10 times to a C57Bl/6J background. Animals were housed 4 per cage in a 12h (6am-6pm) light-dark colony room. The procedures described herein were conducted in accordance with the National Institutes of Health guidelines and were approved by the NIMH Institutional Animal Care and Use Committee.
To generate mice expressing Herpes-Simplex Virus Thymidine Kinase (HSV-tk) under the control of the human GFAP promoter, plasmid pGFA2-TK1 (a generous gift from Dr. Michael Brenner, UAB Department of Neurobiology, Birmingham, AL) was used. Transgenic mice were generated by microinjecting 2picoliters of a solution of plasmid DNA into the male pronucleus of fertilized oocytes from a mixed C57Bl/6J and DBA2 F1 background. Founder mice and subsequent offspring within lines were identified by PCR analysis of DNA extracted from tail snips.
Valganciclovir (VGCV, Roche, Indianapolis, IN) – the L-valyl ester of ganciclovir - was administered for 12 weeks through the animals chow at a concentration of 15mg/kg body weight/day. VGCV has a high (approx. 85%) bioavailability and after oral administration is rapidly converted into ganciclovir by intestinal and hepatic esterases. After phosphorylation by HSV-tk ganciclovir is toxic to proliferating cells in S-phase of mitosis. Since control mice do not express HSV-tk, VGCV administration does not suppress proliferation of GFAP-positive cells. To control for any possible effects of the drug, both control as well as hGFAPtk mice received VGCV-containing chow.
Animals were taken from their home cage and placed into a clean, standard mouse cage containing no bedding or nesting material in a brightly-lit procedure room for 15 minutes.
Mice were quickly decapitated for trunk blood collection. Plasma was isolated and blood levels of corticosterone were quantified using a corticosterone double antibody radio-immunoassay kit (MP Biomedicals) following the manufacturer's protocol.
A separate group of animals (Control n=8, NG- n=8) was treated for 12 weeks with VGVC to control for suppression of adult hippocampal neurogenesis by immunohistochemistry against doublecortin. Mice were deeply anesthetized with isofluorane inhalation and transcardially perfused with 4% PFA, pH 7.4. Brains were dissected from their skull and postfixed in the same fixative overnight at 4°C. Brains were transferred to a 30% sucrose solution for cryopreservation and incubated at 4°C for 3 days. Brains were mounted on a freezing stage (Model BFS-MP30, Physitemp Instruments, Inc., Clifton, NJ) set to −25°C and coronal sections (40µm) were cut using a sliding microtome (LEICA, Germany). Sections were systematically sampled 480µm apart into 12 wells of a 24 well plate and stored in PBS, pH 7.4. Sections were immunofluorescently stained for doublecortin using a standard protocol. In brief, sections were washed for 15min in PBS, pH 7.4 containing 0.5% Tween20 and subsequently incubated for 30min at RT in blocking solution (PBS, pH 7.4, 0.5% Tween20, 3% normal donkey serum). Following blocking sections were incubated overnight at 4°C with the primary antibody (doublecortin, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA). Sections were washed for 15 min followed by incubation for 2h at RT with secondary antibodies (Alexa-555 donkey anti-goat IgG, 1:250, Invitrogen Corporation, Carlsbad, CA). Sections were counterstained using DAPI (Sigma-Aldrich Corporation, St. Louis, MO) and subsequently mounted on glass-slides and coverslipped with aqueous hardening mounting medium (Immu-Mount, Thermo-Fisher Scientific, Inc., Waltham, MA). Fluorescently immunolabed sections were analyzed on a Nikon Eclipse 800 fluorescent microscope (Nikon Instruments, Inc., Melville, NY). In brief, every twelfth section through the hippocampal dentate gyrus was identified and all doublecortin positive cell bodies containing a DAPI positive nucleus were counted. A total cell count was determined by multiplying by 12 to calculate the total number of doublecortin positive cells per dentate gyrus. For photomicrographs showing the dentate gyrus of Control and NG- animals, images were captured on a Zeiss Meta confocal microscope (Model LSM 510; Carl Zeiss MicroImaging, Inc., Thornwood, NY).
After collection of trunk blood, brains were immediately extracted on ice and the hippocampus was quickly dissected. Hippocampi were minced and placed directly into RNALater (Ambion) where they were kept at 4°C until RNA extraction.
Total RNA was isolated and extracted from the hippocampus using Trizol (Invitrogen) according to the manufacturer's instructions. RNA was subsequently purified using an RNease mini column (Qiagen) with on-column DNase removal (Qiagen). RNA was quantified using a NanoDrop spectrophotometer (ThermoScientific) and 2ug of total RNA was reverse-transcribed into single-stranded cDNA using Superscipt III (Invitrogen) following the manufacturer's protocol. Quantitative RT-PCR was performed in triplicate using the IQ5 iCycler (Bio-Rad) using iQ SYBR Greener supermix (Invitrogen) with 20ng of synthesized cDNA/reaction. PCR efficiencies of primers were examined by standard curve of serial-diluted cDNA and melting curve functionality. The sequence mRNA levels were normalized for each well to the actin mRNA levels. Sequences of primers used were as follows:
In order to study a potential role for adult neurogenesis in the regulation of HPA axis activity we generated transgenic mice in which we could conditionally suppress adult neurogenesis. In these mice the viral thymidine kinase (HSV-tk) is expressed under control of the mouse GFAP promoter. Expression of HSV-tk allows for a nearly complete reduction of proliferating GFAP-positive neural progenitor cells by administration of the antiviral drug Valganciclovir (VGCV). HSV-tk phosphorylates the prodrug ganciclovir into a toxic compound that selectively kills cells undergoing S-phase of mitosis. Adult progenitors in the subgranular zone express GFAP and thus administration of VGCV to transgenic mice results in the suppression of neurogenesis. Twelve weeks of VGCV treatment results in complete suppression of doublecortin-positive neuroblasts in the dentate gyrus of transgenic mice (Figure 1). The general health of transgenic mice with suppressed neurogenesis (NG- group) does not appear to be compromised by effects of either the transgene alone or prolonged VGCV treatment. No changes in body weight, reflexes or motor behavior were observed after up to 12 weeks of treatment (data not shown).
In order to study the stress-induced response of the HPA axis in mice without neurogenesis, we measured plasma concentrations of corticosterone in mice in their home cage environment or after exposure to a mild stressor consisting of introduction to a novel environment. As expected, exposure to a novel environment resulted in a significant increase in corticosterone levels. However, comparison of groups by a one-way ANOVA revealed significantly higher corticosterone levels in NG- mice as compared to WT animals (Figure 2).
It has been demonstrated that differences in the levels of glucocorticoid receptor (GR) expression in the hippocampus are correlated with the extent of GC feedback sensitivity and the HPA response to stress [10, 11]. These studies have suggested that differences in hippocampal GR expression may serve as a mechanism for the development of individual differences in HPA responses to stress. In particular, it has been demonstrated in rats that expression of GR mRNAs specifically containing exon 17 is decreased in offspring from dams giving poor maternal care as compared to those showing better maternal care . As adults offspring arising out of poorer maternal care also show potentiated HPA-axis responses after exposure to stress [10, 11]. Considering the importance of the hippocampal GR system for negative-feedback regulation of HPA activity, we examined GR mRNA expression in both wild type and NG- mice. The existence of an exon 17 splice variant has been postulated in mice based on sequence homology, but has not been reported definitively in the literature. We confirmed the production of this transcript via sequencing of the cDNA amplified with a forward primer in exon 17 and a reverse primer in exon 2. We observed no difference in the level of total GR receptor expression or in the exon 17 splice variant within the hippocampus (Figure 3), suggesting that loss of new neurons in the hippocampus affects HPA-axis inhibition via an alternative mechanism.
We observed that mice with suppressed neurogenesis show an increased HPA-axis response after exposure to a stressful situation, suggesting that newly formed neurons in the dentate gyrus are important for the known inhibitory function of the hippocampus over the HPA axis. It is well accepted that both stress and GCs are amongst the most potent inhibitors of neurogenesis. However, to our knowledge, studies into whether intact neurogenesis may be important for the hippocampus in providing its inhibitory influence over the HPA axis have not yet been reported.
It has been demonstrated in rats that adverse early life experiences, including maternal deprivation, result in decreased rates of hippocampal neurogenesis in the adult animal that do not appear to result directly from increased GCs . Maternally deprived rats also demonstrate impaired negative feedback of the HPA axis in response to stressful situations [14, 15]. It was suggested that the decrease in adult-generated neurons observed with maternal deprivation could also contribute to the altered negative feedback of the HPA axis in the adult . Our data support this notion by showing that newly born neurons in the hippocampus do indeed contribute to the hippocampus' ability to mediate proper inhibitory regulation over the HPA axis.
Antidepressant therapies, including SSRIs, tricyclics, MAO inhibitors, tianeptine and electroconvulsive shock have been shown to stimulate adult neurogenesis. A functional role for this increase in neurogenesis is supported by experiments showing that the success of antidepressant treatment in a rodent model of depression was dependent on intact neurogenesis . Successful antidepressant treatment is associated with resolution of impairments in HPA axis negative feedback [17, 18]. Recent reports have also suggested limited efficacy of SSRIs in patients with abnormal HPA axis function, which has prompted the assertion that concurrent manipulation of the HPA system with antidepressant therapy may improve antidepressant efficacy. Monoaminergic antidepressants do not directly target the HPA axis; however, they may interrupt this circuit by stimulating neurogenesis. In theory, this could compensate for neuronal atrophy in the hippocampus, thus allowing for normal hippocampal functioning, including inhibition of the HPA axis. Support for this idea comes from experiments showing that therapeutic effects can be achieved even after suppression of hippocampal neurogenesis via manipulations that directly target the HPA axis.
Evidence points to changes in hippocampal GR expression in some models as a mediator of HPA axis inhibition efficiency [10, 11]. Previous reports have shown that regulated expression of hippocampal GR mRNA is restricted to the dentate gyrus and the CA1 subfield . Decreased GR mRNA and hence receptor production, would be expected to decrease sensitivity to negative feedback by GCs on the HPA axis. Our data demonstrate that the HPA axis hyperactivity seen in response to stress in animals lacking adult neurogenesis is not a result of altered GR expression in the hippocampus. Thus, we favor the idea that the incorporation of new neurons may alter the electrophysiological properties at CA1 synapses, the only subregion in the hippocampus containing neurons that project to the hypothalamus.
We conclude that newly born neurons in the adult play a role in the functional inhibition of the HPA axis by the hippocampus.
This research was supported in whole by the National Institutes of Health, National Institute of Mental Health (Bethesda, MD). We thank Diane Venable for outstanding technical assistance.
Funding was provided by the National Institute of Mental Health.
The authors declare no conflicts of interest.