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Keywords:

  • VIP;
  • stem cells;
  • neurogenesis;
  • cell fate;
  • cell death;
  • symmetric cell division

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The controlled production of neurons in the postnatal dentate gyrus and thoughout life is important for hippocampal learning and memory. The mechanisms underlying the necessary coupling of neuronal activity to neural stem/progenitor cell (NSPC) function remain poorly understood. Within the dentate subgranular stem cell niche, local interneurons appear to play an important part in this excitation-neurogenesis coupling via GABAergic transmission, which promotes neuronal differentiation and integration. Here we show that vasoactive intestinal polypeptide, a neuropeptide coreleased with GABA under specific firing conditions, is uniquely trophic for proliferating postnatal nestin-positive dentate NSPCs, mediated via the VPAC2 receptor. We also show that VPAC2 receptor activation shifts the fate of symmetrically dividing NSPCs toward a nestin-only phenotype, independent of the trophic effect. In contrast, selective VPAC1 receptor activation shifts NSPC fate toward granule cell neurogenesis without any trophism. We confirm a trophic role for VPAC2 receptors in vivo, showing reduced progeny survival and dentate neurogenesis in adult Vipr2−/− mice. We also show a specific reduction in type 2 nestin-positive precursors in vivo, consistent with a role for VPAC2 in maintaining this cell population. This work provides the first evidence of differential fate modulation of neurogenesis by neurotransmitter receptor subtypes and extends the fate-determining effects of neurotransmitters to maintaining the nestin-positive pool of NSPCs. This differential receptor effect may support the independent pharmacological manipulation of precursor pool expansion and neurogenic instruction for therapeutic application in the treatment of cognitive deficits associated with a decline in neurogenesis. STEM CELLS 2009;27:2539–2551


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Given the emerging roles of postnatal neurogenesis in hippocampal-dependent learning [1–6] and behavior [7], as well as possible roles of abnormal neurogenesis in the pathophysiology of disease [8], much research has concentrated on elucidating the function of hippocampal neurogenesis and the determining factors that influence it [9]. Central to these questions are the mechanisms whereby neurogenesis is linked to neuronal activity. Although the principal neural inputs into the dentate gyrus have been well defined, there is much evidence to suggest that local networks, largely subserved by GABAergic-releasing interneurons, significantly modulate activity and are thus ideally placed to signal neuronal activity to the local stem cell niche (for review see [10]). Tonic GABA release promotes neuronal differentiation of neural stem/precursor cells (NSPCs) [11] and the integration of their progeny [12] in the adult dentate gyrus. We have shown that NPY, a neuropeptide coreleased from GABAergic interneurons under high-frequency firing conditions, is a potent proliferative factor for NSPCs in the postnatal and adult dentate [13, 14]. With the demonstration that net dentate neurogenesis is largely determined by trophic rather than proliferative modulation [15], and the emerging importance of alterations in the proliferation pattern and fate choices of nestin NSPCs in response to physiological [16] and pathological stimuli [17], we wondered if other neuropeptides released by interneurons might be trophic for neuronal precursor cells and/or influence their fate choice.

Vasoactive intestinal polypeptide (VIP), a peptide neurotransmitter released by GABAergic interneurons in the dentate gyrus, is well positioned for a role in modulating NSPCs. VIP is a potent growth factor in early postimplantation embryos [18], shortens the cell cycle of embryonic neuroepithelial cells [19], and promotes neuronal differentiation of embryonic hippocampal neurons in culture [20]. VIP and its receptors (VPAC1 and VPAC2) are expressed in developing and adult dentate gyrus, and VIP blockade causes impairments in associative learning abilities [21].

To determine the role of VIP in dentate neurogenesis, we examined the effects of VIP on dissociated cultures of postnatal rat hippocampus and dentate gyrus, when dentate neurogenesis is at its peak, and in adult Vipr2−/− mice. We show that VPAC2 receptor activation in vitro expands the pool of proliferating nestin-expressing dentate NSPCs, by preventing either a neuronal or glial fate choice and by separately supporting their survival, while selective VPAC1 receptor activation promotes a neurogenic granule cell fate. Adult Vipr2−/− mice show a corresponding reduction in the survival of newly generated neurons, significantly fewer total granule cell neurons, and a specific reduction in the number of nestin-positive type 2 precursors. Our results demonstrate a unique modulation of NSPC survival and fate determination via differential VPAC receptor activation in the postnatal and adult dentate gyrus.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In Vitro Experiments

Monolayer Cell Culture and Quantification

Rat hippocampal NSPC cultures were generated from postnatal Wistar rat hippocampi (P8–P10) as described elsewhere [22] (see supporting information).

Quantification was performed using unbiased methods as previously reported [13, 14] and further described in the supporting information.

Pharmacology

To examine VPAC1 and VPAC2 mediation of VIP effects in NSPCs, we used the selective VPAC1 receptor agonist [Lys15, Arg16, Leu27]-VIP (1pdash)7)-GRF (8-27) (Phoenix Pharmaceutical, Inc., Burlingame, CA, http://www.phoenixpeptide.com) at 30 nM or 1 μM, the VPAC1 selective antagonist [Ac-His1, D-Phe2, Lys15, Arg16, Leu27]-VIP (3-7)-GRF (8-27) (Phoenix Pharmaceutical, Inc.) at 1 μM, and the VPAC1/VPAC2 nonselective antagonist [D-p-Cl-Phe6, Leu17]-VIP (Sigma-Genosys, Cambridge, U.K., http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html) at 10 nM.

Assessment of Cell Proliferation

To study cell proliferation, we used the thymidine analog BrdU and the cell cycle marker Ki-67 as detailed in the supporting information.

Time-Lapse Video-Microscopy and Cell-Fate Studies

Time-lapse microscopy was performed on live cultures as detailed in the supporting information.

To examine the survival of proliferating cells, the resulting time-lapse movies were carefully studied for mitosis. Each newly born cell was tracked to determine whether it survived or died, and the numbers of newly born cells that either survived or died were thus quantified under control and VIP conditions.

To examine the effect of VIP on cell fate, the time-lapse movies were examined from separate experiments to identify divisions where both daughter cells survived and thus correct for any trophic effects of VIP. These surviving daughter pairs were then immunostained and imaged using confocal microscopy for cell fate markers (nestin and TUJ1) or markers of symmetric division (NUMB and nestin).

Immunohistochemistry

Immunofluorescent staining was performed on 4% paraformaldehyde (PFA)-fixed cells using antibodies against BrdU, nestin, glial fibrillary acidic protein (GFAP), TUJ1, VPAC1 and VPAC2 receptors, Prox-1, NUMB, Ki-67, and cleaved caspase-3 as previously described [14, 22] and further detailed in the supporting information.

Western Blot Analysis for VPAC2 Receptor Protein

See supporting information.

Elimination of Cell Proliferation in Culture

We used (β-D-arabinofuranosyl)cytosine (Ara-c) (Sigma) to abolish cell proliferation and to eliminate the actively dividing NSPCs. Ara-C was applied to cells at 0.2 μM at 24 hours after initial plating to allow for the cells to settle in the in vitro environment [25]. See supporting information for details.

Quantification of Cell Death

Cell death in live cultures was quantified using the cell death marker propidium iodide and the nuclear stain 4'-6-diamidino-2-phenylindole (DAPI) (see supporting information for details). In separate experiments, activated caspase-3 immunostaining was also used to identify apoptotic cells in PFA-fixed cultures.

PCR Assay

For PCR, total RNA was extracted from cultured cells and directly reverse-transcribed to complementary DNA (cDNA) by using SuperScript™ III Cells Direct cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The cDNA was then amplified by one-step polymerase chain reaction (PCR) kit (rat custom real-time PCR assay for use with SYBRgreen chemistry) (PrimerDesign Ltd, Southampton, U.K., http://www.primerdesign.co.uk) in a real-time thermocycler (Corbett Robotics Rotor-Gene 6000; Qiagen, Hilden, Germany, http://www1.qiagen.com). See supporting information for further details.

In Vivo Experiments

Animals

All experimentation was conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986. Every effort was made to minimize the number of animals used and their suffering. The Vipr2−/− mice and the age-, sex-, and generation-matched wild-type controls used in these experiments have been previously reported [26].

BrdU Injection and Tissue Preparation

Five 8-week-old male Vipr2−/− and five age- and generation-matched male wild-type controls received once daily intraperitoneal injections of BrdU at 50 mg/kg body weight (10 mg/mL stock, dissolved in normal saline (0.9% NaCl)) for 5 successive days, and were then allowed to survive for 4 weeks before being sacrificed via terminal anesthesia. Five 12-week-old male Vipr2−/− and control mice received a single intraperitoneal injection of BrdU and were sacrificed via terminal anesthesia after 4 hours for proliferation studies. Animals were then perfused transcardially with saline and 4% PFA, and the brains removed and postfixed in 4% PFA. Coronal sections containing hippocampus were cut at 40 μm on a VT100M Vibratome (Leica, Heerbrugg, Switzerland, http://www.leica.com).

Immunohistochemistry

Every twelfth systematically randomly sampled section was stained. Floating sections were then processed for standard immunohistochemistry against BrdU, NeuN, Prox-1 nestin (See supporting information for details).

Quantification of BrdU and BrdU/NeuN-Positive Cells, Prox-1-Positive Cells, and Nestin-Positive Cells in Vipr2−/− Mice

Nonbiased stereological methods were used to quantify the numbers of BrdU-, NeuN-, nestin-, and Prox-1–positive cells in adult Vipr2−/− animals and wild-type littermate controls as described previously [14] and specifically detailed in the supporting information. Nestin-positive cells in the granule cell layer were classified purely by morphology into type 1 (cells with a radial process) and type 2 (no radial process). All data were collected with the investigator blinded to the animals' status.

Data Analysis

Data points were plotted using GraphPad Prism data analysis software (GraphPad, Inc., San Diego, CA, http://www.graphpad.com). Statistical significance was assessed with either Students' t test for single comparisons or with ANOVA followed by appropriate post hoc tests for multiple comparisons (p < .05 considered significant).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

VPAC1 and VPAC2 Receptors Are Expressed in Hippocampal Progenitor Cell Cultures

VPAC1 and VPAC2 immunoreactivity was consistently observed on nestin-positive cells (Fig. 1A–1F) as well as on TUJ1-positive cells with a neuronal morphology (data not shown). Antibody-negative controls showed no staining, and positive staining was abolished by coapplication of receptor-specific blocking peptides (data not shown). Western blot analysis for the VPAC2 receptor protein revealed a specific band of staining at 65 kDa as previously reported [27] (Fig. 1G).

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Figure 1. VPAC1 and VPAC2 Receptors and their mRNAs are expressed in hippocampal NSPC cultures, which were generated from postnatal day [8–10] rats. Cells were then processed for nestin and either VPAC1 or VPAC2 receptor immunoexpression, Western blot analysis for VPAC2 protein, or cDNA extraction for PCR assay. VPAC1(A–C) and VPAC2(D–F) are coexpressed by nestin-positive cells at 1 day in vitro (DIV). Scale bar (A–C), 40 μm; (D–F), 20 μm. (G): Western blot analysis revealed a specific band at 65 kDa. (H): Nonquantitative PCR gels demonstrate the presence of VPAC1 and VPAC2 mRNAs in hippocampal NSPC cultures, as well as in whole brain tissue extracts. (I): Graph shows the folds of expression of each receptor transcript in dentate NSPC cultures as determined with quantitative PCR relative to a reference sample of cDNA extracted from different types of rat tissues (including brain). Abbreviations: 1DIV, XXXX; NSPC, neural stem/progenitor cell; PCR, polymerase chain reaction.

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Semiquantitative PCR demonstrated VPAC1 and VPAC2 mRNA expression in whole hippocampal cultures (Fig. 1H), and quantitative PCR showed that VPAC1 and VPAC2 mRNA were equally expressed in the dentate-specific progenitor cell cultures (Fig. 1I).

VIP Enhances Cell Survival in Postnatal Hippocampal Cultures

Total cell counts of primary hippocampal cultures grown under VIP-treated conditions (1 nM–1 μM) were significantly higher than under control conditions (Fig. 2A) down to concentrations as low as 1 nM. Because VIP binds with different affinities to VPAC1, VPAC2, and PAC1 receptors [28], we chose the concentration of 30 nM to elucidate VPAC1 and VPAC2 effects on hippocampal NSPCs. This is also close to the physiological concentration of VIP (10 nM) in human cerebral spinal fluid [29].

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Figure 2. VIP enhances the survival of hippocampal NSPCs mainly through trophic activity. Hippocampal NSPCs were grown for 5DIV under control or VIP (1 nM–1 μM) conditions before they were processed for cell proliferation or survival. (A): 1 nM–1 μM VIP over 5 days increases the total number of live cells in culture. (B): VIP has no effect on the number of proliferating cells in the S-phase of the cell cycle at concentrations <1 μM. VIP is purely trophic at 30 nM, as it did not affect indices of cell proliferation, such as the labeling index (C) and growth fraction (D). (E): Nanomolar concentrations of VIP decreased the proportions of propidium iodide-positive (dead/dying) cells imaged in live cultures from 36% ± 1% (under control conditions) to 14% ± 6% (Student's t test: p < .05). Data represent mean ± standard error based on a sample that represents at least 12 wells per condition from three different experiments. For comparisons between different conditions, a one-way ANOVA with Dunnett's multiple comparison test was used in panels A, B, and E, while Student's t test was used in panels C, D. *, p < .05; **, p < .01; and ***, p < .001 considered significant. Abbreviations: 5DIV, XXXX; DAPI, 4'-6-diamidino-2-phenylindole; NSPCs, neural stem progenitor cells; PI, propidium iodide; VIP, vasoactive intestinal polypeptide.

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To address VIP effects on cell proliferation, cultures were pulsed with the S-phase marker BrdU for the terminal 4 hours before fixation, and the mitotic index (BrdU/DAPI) was determined. At concentrations less than 1 μM, VIP had no effect on cell proliferation (Fig. 2B). At peptide concentration of 30 nM, VIP affected neither the labeling index (cell cycle speed) nor the growth fraction (recruitment of quiescent G0 cells) (Fig. 2C, 2D). This was confirmed under time-lapse live cell imaging, which showed no significant difference in the number of cells generated by mitosis in this period (42 vs. 46 cells in 18 high power fields per condition) (supporting information). However, at a pharmacological concentration of 1 μM VIP, a proliferative effect was observed (Fig. 2B) consistent with activation of the PAC1 receptor [28].

We next examined for trophic effects on cells using propidium iodide imaging of live cell cultures. VIP at nanomolar concentrations decreased cell death over 5 days in culture (Fig. 2E), confirming a trophic mechanism of action.

VIP Selectively Increases the Overall Proportion of Nestin-Positive Cells and the Proportion of Cycling Cells with a Nestin-Positive Phenotype

Having demonstrated a trophic effect of VIP, we next sought to determine the phenotype of VIP-responsive cells in culture. Interestingly, we found that while VIP enhanced the survival of the other cell subpopulations, it enhanced a proportional increase only in nestin-positive cells (Fig. 3A–3C). Because nestin-positive postnatal hippocampal NSPCs consist of distinct subpopulations on the basis of the presence or absence of TUJ1 or GFAP colocalized immunostaining [13, 30, 31], we sought to further determine which subpopulation of nestin cells responds specifically to VIP.

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Figure 3. VIP selectively increases the overall proportion of nestin-positive cells and the proportion of cycling cells with a nestin-positive phenotype. At 5DIV under either control or 30 nM VIP conditions, NSPCs were processed for the expression of nestin, TuJ1, and GFAP. (A): The numbers and proportions of nestin-expressing cells were exquisitely responsive to 30 nM VIP. (B, C): VIP increased the raw numbers, but not the proportions of cells expressing TuJ1 and GFAP, respectively. (D, E): VIP enhanced the proportions of nestin-positive TuJ1-negative and nestin-positive GFAP-negative cells with respect to total cell counts, compared with control conditions. (F): VIP shifted the Ki-67-positive cell population towards a nestin-expressing phenotype (from 0.67 ± 0.01 under control conditions to 0.80 ± 0.02 under 30 nM VIP). Data represent mean ± SE based on a sample that represents at least 12 wells per condition from three different experiments. Comparisons between different conditions are analyzed using Student's t test. *, p < .05; **, p < .01; and ***, p < .001 considered significant. Abbreviation: GFAP, glial fibrillary acidic protein.

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Nestin and TUJ1 immunostaining was specific and the proportion of TUJ1-positive nestin-positive cells decreased under FGF2 and EGF mitogen conditions (supporting information). At 5DIV, VIP selectively increased the proportion of nestin positive TUJ1-negative cells (Fig. 3D), typical of the transient amplifying cells found in the dentate subgranular zone (SGZ) [30] and subventricular zone [32]. This VIP-responsive population of nestin-expressing cells was also GFAP-negative (Fig. 3E and supporting information).

Cell Proliferation Is Essential for VIP Trophic Effects on Nestin-Positive Cells

Because all nestin-positive cells in culture were proliferating ([13] and data not shown), our finding of a significant increase in the proportion of proliferating (Ki-67+) cells exhibiting a nestin-positive phenotype, without any alteration in growth fraction under VIP conditions (Fig. 3F), suggested to us that VIP might be trophic for proliferating cells. To test this, we abolished cell proliferation in culture with the antimitogenic agent Ara-c [25]. We found that 0.2 μM Ara-c abolished cell proliferation (mean BrdU cell count 10.2 ± 1.2 per mm2 in control cultures to zero with 0.2 μM Ara-c) without increasing cell death in our cultures (Fig. 4C).

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Figure 4. Cell proliferation is essential for VIP-trophic effects on nestin-positive cells. To address VIP effects on cell survival in the absence of cell proliferation, hippocampal cells were grown for 3 days under control and VIP conditions with or without the coapplication of 0.2 μM ara-C. (A): Ara-C cotreatment significantly, but not completely, reduced the survival effect of VIP as indicated by DAPI cell counts. (B): Abolishing cell proliferation via ara-C completely blocked the survival effects of VIP on nestin-expressing NSPCs. (C): Ara-C did not induce apoptosis of nestin-positive cells as evidenced by colabeling for activated caspase-3, while VIP treatment significantly increased the numbers of nonapoptotic nestin-positive cells. (D): Micrographs of cultures stained for DAPI, activated caspase-3, and nestin. Note both the caspase-3–positive (white arrow) and caspase-3–negative (pink arrow) nestin cells. Scale bar = 20 μm. Values represent mean ± SE based on a sample that represent at least eight wells per condition from three experiments. Comparisons between different conditions, one-way ANOVA with Bonferroni's multiple comparison test. *, p < .05; **, p < .01; and ***, p < .001 considered significant. Abbreviation: Act, activated.

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Ara-C significantly attenuated the VIP induced increase in total cell counts (Fig. 4A), and completely blocked the increase in nestin-positive cell counts (Fig. 4B), over 3 days in vitro, suggesting that VIP is a trophic neuropeptide that supports the survival of proliferating nestin-positive cells and their progeny, and that cell proliferation is essential for the mediation of the VIP effect on these cells. This was confirmed by activated caspase-3 (an apoptotic marker) and nestin coimmunostaining, which showed that VIP increased the number of nestin-positive activated caspase-3–negative cells, whereas neither Ara-C nor Ara-C + VIP altered apoptotic nestin cell death compared with control conditions (Fig. 4C, 4D).

Interestingly, Ara-C also significantly decreased total cell numbers and nestin-positive cell counts under control conditions; consistent with blocking, the endogenous proliferation was seen under normal conditions in our cultures [22] (Fig. 4A).

This purely trophic effect on proliferating cells was confirmed under time-lapse live cell imaging, which showed that 29% of newly born cells died over a 48-hour period under control conditions compared to only 5% under VIP treatment (p ≤ .01), whereas there was no difference in the number of nonmitotic cells that died in this period (supporting information).

VIP Shifts Symmetrically Proliferating Cells Toward a Primitive Nestin Phenotype

The observed proportional increase in nestin cells in the absence of a proliferative effect of VIP raised the question of how this shift toward the nestin phenotype was achieved, as a purely trophic effect on proliferating nestin-positive precursor cells and their progeny would increase the numbers but not the proportions of each phenotype.

To address the mechanism of the VIP effect on cell fate, we first quantified the pattern of cell division (symmetric vs. asymmetric) in our cultures using NUMB expression and distribution after division [33]. Using time-lapse live imaging microscopy, we followed NUMB expression in the newly born cells of our cultures as these cells divided. We found that virtually the entire nestin-expressing newly born cell population expressed NUMB (Fig. 5A), suggesting that these cells underwent predominantly symmetric rather than asymmetric cell divisions. We further tracked these dividing cells for 48 hours and then stained them for nestin and the neuronal marker TuJ1. We identified three types of symmetric cell divisions: type I symmetric cell divisions yielding two nestin-positive, TuJ1-negative daughter cells (Fig. 5B), type II symmetric cell divisions producing two neuronal lineage–committed progeny cells (nestin-positive, TuJ1-positive) daughter cells (Fig. 5C), and type III where a mother cell divides to give two mature neuroblasts (nestin-negative, TuJ1-positive) (not shown).

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Figure 5. VIP shifts symmetrically proliferating cells toward a pure nestin-expressing phenotype. Time-lapse contrast-phase imaging of dividing hippocampal NSPCs, followed by immunohistochemical localization of nestin (red) and either NUMB or TuJ1 (green). (A): A dividing cell yielding two daughter cells (over 18 hours); the immunostaining supports a symmetric cell division as both cells coexpress nestin and NUMB. (B): A dividing cell that produced two cells that are nestin-positive but TuJ1-negative (type I symmetric cell division). Note the nondividing TUJ1-positive and nestin-negative cell in the lower right hand corner. (C): A dividing cell that yielded two daughter cells that coexpress nestin and TuJ1 (type II symmetric cell division). Scale bar, 20 μm. (D): Summary of the effects of VIP on the fate of newly born nestin-expressing cells. The number of type I symmetric cell divisions were significantly greater under VIP conditions compared with controls, the number of type II symmetric divisions were less under VIP conditions (not significant), and the number of type III symmetric divisions (both daughter cells TUJ1-positive but nestin-negative) did not exist under VIP conditions. The overall comparisons between control and VIP conditions are χ2 with Z test comparing the number of type I or type II divisions under control and VIP conditions, and Fisher exact test for type III cell divisions. *, p < .05; **, p < .01.

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To correct for trophic effects, we counted 52 divisions whose progeny survived under both VIP and control conditions (Fig. 5D). Interestingly, and in agreement with our early findings, our time-lapse data demonstrated that, out of 52 survived cell divisions, there was a significant increase in type I symmetric cell divisions under VIP treatment with a concomitant decrease in type II divisions. This suggests that, in addition to its trophic effect, VIP enhanced a shift towards the generation of nestin-positive TuJ1-negative progenitor cells by enhancing type I symmetric cells divisions without affecting the rate of cell proliferation. While the number of divisions yielding nestin-positive TuJ1-positive (type II) were far less under VIP treatment, this change was statistically insignificant. Interestingly, 8% (4 of 52 imaged cell pairs) of the divisions produced two mature neurons (type III) under control conditions, whereas none of this type of division occurred under VIP treatment.

VPAC2 Mediates the Trophic and Nestin Fate-Determining Effects of VIP, Whereas VPAC1 Mediates Neuronal Differentiation

The increases in total cell counts and the proportional increase in nestin-positive cells observed under 30 nM VIP treatment were unaffected by the coapplication of the VPAC1 antagonist, but these increases were abolished once VIP treatment was combined with 10 nM VPAC1/VPAC2 antagonist (Fig. 6A, 6B), suggesting that the trophic effects and the phenotypic shift toward a nestin phenotype were mediated by the VPAC2 receptor. This specificity was confirmed by the observation that the VPAC1 agonist had no effect on the total number of DAPI cells or the proportions of nestin cells in culture (Fig. 6A, 6B).

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Figure 6. VPAC2 mediates the trophic and nestin fate-determining effects of VIP while VPAC1 mediates neuronal differentiation. (A–D): Cell cultures were grown under control conditions or (1) 30 nM VIP, (2) 1 μM VPAC1 agonist, (3) 1 μM VPAC1 antagonist, (4) 1 μM VPAC1 antagonist and 30 nM VIP, (5) 10 nM VPAC1/VPAC2 antagonist, or (6) 10 nM VPAC1/VPAC2 antagonist and 30 nM VIP. Cells were then stained for either nestin or TuJ1 and counterstained with DAPI. (A, B): The combined VPAC1/VPAC2 antagonist abolished the VIP increase in total cell counts and the proportion of nestin-positive cells, while a selective VPAC1 antagonist did not. In addition, VPAC1 agonist application had no effect on the total DAPI cell counts or the proportions of nestin-expressing cells. (C, D): A selective VPAC1 agonist increased both the total number and proportion of TuJ1-positive cells, while VIP increased only the raw numbers of TuJ1 cells without a proportional change. (E): To further delineate the pharmacology of the neurogenic effect, the VPAC1 agonist was used at a concentration of 30 nM, which does not affect the PAC1 receptor. Cells were cultured as for panels (A–D) either under (C) control, (I) 30 nM VPAC1 agonist, (II) 30 nM VPAC1 agonist and 1 μM VPAC1 antagonist, or (III) 1 μM VPAC1 antagonist alone. In these studies, cells were also stained for the dentate granule cell specific marker Prox-1 as well as TuJ1. The VPAC1 agonist at 30 nM enhanced a proportional increase in Prox-1–positive (dentate specific) TuJ1-expressing cells, an effect that was completely blocked by a selective VPAC1 antagonist. Data represent mean ± SE based on a sample of at least 12 wells per condition from three different experiments. (F): To investigate the mechanism of the dominant VPAC2 effect of VIP, quantitative PCR was performed on control cultures and those exposed to 30 nM VIP for three hours at 3DIV. VIP increased VPAC2 mRNA transcription 120-fold compared to only an eight-fold increase in VPAC1 mRNA. Note the log scale. (G): NSPC cultures were generated from postnatal day 8–10 rats and grown under control conditions for 5 days. Cells were then processed for TUJ1 (green) and Prox-1 (red) immunoexpression and counterstained with DAPI (blue). Scale bar, 40 μm. Comparisons between different conditions are based on either a one-way ANOVA with Dunnett's multiple comparison test (A–E) or Student's t test (F). *, p < .05; **, p < .01; ***, p < .001).

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Interestingly, the selective VPAC1 agonist at 1 μM increased both the absolute number (Fig. 6C) and the proportions of TuJ1-expressing cells compared with control conditions (Fig. 6D). This neurogenic effect of the VPAC1 agonist was replicated at a concentration of 30 nM, which does not affect PAC1 receptors [34], and was blocked by the specific VPAC1 antagonist. This appears to be a pure fate determination effect as the VPAC1 agonist had no trophic effect on total cell counts (Fig. 6A).

As VIP did not exhibit a neuronal differentiation effect itself, suggesting that the VPAC2 effect was dominant, we sought to investigate the mechanism of this dominance and wondered if VIP might selectively enhance VPAC2 receptor expression. Quantitative PCR indeed demonstrated that 3 hours of VIP treatment selectively enhanced VPAC2 mRNA expression (121-fold increase (p < .01)) over VPAC1 mRNA expression (6-fold (p > .05) (Fig. 6F).

VPAC1 and VPAC2 Effects Are Dentate Gyrus-Specific, and Vipr2−/− Mice Demonstrate Reduced Neurogenesis due to Reduced Survival of NSPC Progeny and a Smaller Type 2 Nestin-Positive Precursor Pool In Vivo

To examine whether these VIP effects are dentate-specific or not, we used microdissection to split the hippocampus of postnatal rats into dentate gyrus and the hippocampal subventricular zones as previously described [13]. Precursor cells from the two areas were cultured under control and 30-nM VIP conditions. Our findings demonstrated that, although VIP increased the proportions of nestin-positive TuJ1-negative cells in the dentate microdissected cell cultures, it had no effect on cells dissociated from the hippocampal subventricular zones (Fig. 7A, 7B), suggesting that the VIP effect is specific to the dentate gyrus in vitro. Interestingly, the pure neurogenic VPAC1 effect was also dentate-specific because selective VPAC1 activation increased the proportion of TUJ1 cells that expressed the granule cell–specific marker Prox-1 [35] (Fig. 6E, 6G).

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Figure 7. The effects of VPAC1 and VPAC2 are dentate gyrus-specific, and Vipr2−/− mice demonstrate reduced neurogenesis as a result of reduced survival of neural stem/progenitor cell progeny and a smaller type 2 nestin-positive precursor pool in vivo. Dentate gyrus–specific and hippocampal subventricular zone microdissected cultures [13] were grown individually for 5DIV under control or 30 nM VIP conditions. (A, B): VIP enhanced the proportion of nestin-positive TuJ1-negative cells in the dentate gyrus microdissected but not in HSVZ hippocampal subventricular zone cell cultures. (C, D): Stereological analysis of the left GCL in adult 12-week-old Vipr2−/− mice and controls, sacrificed 4 hours after a single BrdU injection, showed no difference between the total number of BrdU-positive cells or nestin- and BrdU-colabeled cells. (E): Stereological analysis revealed significantly lower numbers of BrdU-positive cells in the GCL (4 weeks after pulse and chase BrdU) in Vipr2−/− mice compared with wild-type animals, consistent with a reduced survival of newly born cells. (F): Quantitative analysis of BrdU NeuN-positive cells (new neurons) revealed a significant drop in the number of surviving newly-born neurons in the GCL. The proportion of BrdU-positive cells that were NeuN positive did not differ between Vipr2−/− and controls (data not shown). (G): The total number of Prox-1–positive granule cell neurons in the left GCL was measured using nonbiased stereology, and, on the basis of the systematic random sampling of four sections per animal spanning the entire left hippocampus from five animals per group, the total number of Prox-1–positive granule cell neurons was significantly (45%) lower in adult Vipr2−/− mice (p < .05 Student's t-test; CE (coefficient of error) 3.6%–6.8%). (H, I): Micrographs of the subgranular zone and GCL stained for Prox-1 (red) and nestin (green) showing type 1 nestin cells with a radial process extending up into the GCL. Scale bars, 15 μm. (J): Stereological quantification revealed significantly less GCL nestin-positive cells in Vipr2−/− mice. This was due to a specific reduction in type 2 nonradial nestin-positive cells (K–N). Scale bars, 20 μm. (O, P): Stereological quantification of the number of type 1 (radial) and type 2 (nonradial) nestin cells that were BrdU-positive after a 4 h pulse revealed a significant increase in the mitotic index of both cell types in Vipr2−/− animals compared with controls, in agreement with the unchanged total number of nestin-positive and BrdU-positive cells seen in (C) and (D) above and consistent with a compensatory proliferative response to a trophic loss of type 2 cells. Values are means ± SE. Comparisons were made using Student's t test with *, p < .05 and **, p < .01 considered significant. Abbreviations: GCL, granule cell layer; HSVZ, hippocampal subventricular zones.

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Because our data demonstrated a predominant VPAC2 mediation of the VIP effect in vitro, we used adult Vipr2−/− mice to examine for VPAC2 involvement in ongoing dentate neurogenesis in vivo. Point proliferation of dentate NSPCs and the survival and neuronal differentiation of their progeny in adult Vipr2−/− mice and wild-type littermate controls was examined using a pulse and chase BrdU labeling paradigm with sacrifice 4 h or 4 weeks after BrdU injection, respectively. Consistent with our in vitro findings, the total number of BrdU cells and the number of nestin-positive BrdU-positive cells in the SGZ 4 h after BrdU were unchanged between adult Vipr2−/− mice and controls (Fig. 7C, 7D), suggesting that VPAC2 deletion had no effect on the numbers of proliferating NSPCs. However, the number of proliferating (BrdU-positive) cells that survived in the granule cell layer after 4 weeks was significantly lower in adult Vipr2−/− mice compared with the wild-type animals (Fig. 7E). This resulted in a significant reduction in net neurogenesis in the 4-week period examined (Fig. 7F) without a significant alteration in the proportion of BrdU cells exhibiting a neuronal phenotype (data not shown). This reduction in dentate gyrus neurogenesis also appeared to be structurally relevant, as the total number of granule cell neurons in the dentate granule cell layer was significantly lower in Vipr2−/− mice (Fig. 7G–7I). There was a nonsignificant decrease in dentate granule cell layer volumes in Vipr2−/− mice (10.06 ± 0.56 × 104 μm3) compared to controls (12.09 ± 0.16 × 104 μm3). These findings confirm a survival role for VPAC2 in ongoing adult dentate neurogenesis.

Given that the VPAC2 receptor also had a fate-determining effect to promote the generation of nestin-positive TUJ1-negative cells, we counted the number of SGZ nestin cells and found that Vipr2−/− mice have significantly fewer nestin cells due to a specific significant reduction of type 2 nonradial nestin cells (Fig. 7J–7N), confirming our in vitro effect. Interestingly, both type 1 and type 2 nestin cells showed a significant increase in their mitotic index in Vipr2−/− mice compared with controls (Fig. 7O, 7P), and analysis of their ratio of BrdU incorporation showed no significant change, suggesting that the proliferation rate or growth fraction of both subtypes were equally increased to restore the size of the proliferating pool of progenitors.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The mechanisms underlying excitation neurogenesis coupling in the postnatal and adult hippocampus are incompletely understood. Glutamate promotes neuronal differentiation in cultures of hippocampal precursor cells [36], whereas GABA promotes neuronal differentiation of type 2 SGZ precursors in vivo [11], yet N-methyl-D-aspartic acid blockade in vivo increases SGZ proliferation [37]. A recent report has demonstrated that latent stem cells in the hippocampus are activated by neural excitation via L-type Ca2+ channels [38] but not via glutamate or GABA. Here we show that differential VPAC receptor activation by VIP, a peptide neurotransmitter released by interneurons, promotes the survival of proliferating precursors and shifts their fate toward a nestin-expressing state via VPAC2 receptor activation, yet also promotes a neurogenic fate without a trophic effect via the VPAC1 receptor.

We confirm a continuing trophic role for VPAC2 receptors in vivo, showing reduced progeny survival and adult dentate neurogenesis, as well as fewer granule cell neurons in adult Vipr2−/− mice. We also show a specific reduction in type 2 nestin-positive precursors in vivo, consistent with a role for VPAC2 in maintaining this cell population. These results identify a key role for VIP and VPAC receptors in the survival, fate specification, and maintenance of the nestin-positive precursor pool in the postnatal and adult dentate gyrus.

Differential VPAC Receptor Activation Has Distinct Trophic and Fate Mapping Effects on Precursor Cells

At nanomolar concentrations VIP had no effect on the rate of cell proliferation or the recruitment of quiescent cells. Although a simple trophic effect on proliferating cells would explain the significant increases in the numbers of neurons, astrocytes, and nestin-positive cells, it cannot explain the specific proportional increase in nestin cells observed under VIP. Even a specific trophic effect on proliferating nestin-positive but both TUJ1- and GFAP-negative cells cannot explain the raw increases in all three phenotypes with a proportional increase solely in the purely nestin-expressing population, without a concomitant fate-determining effect.

We therefore postulated that VIP had an additional phenotype specification effect on proliferating nestin-positive cells by maintaining them in a nestin-positive TUJ1-negative state. This was confirmed with time-lapse microscopy with lineage tracing of equal numbers of divisions to control for a selective trophic effect on proliferating nestin-positive TUJ1-negative precursors. Interestingly, this effect was mediated on symmetric cell divisions of nestin-positive cells, and there was no evidence of a shift from asymmetric to symmetric divisions of nestin-positive cells either by NUMB segregation [33] or phenotypic identification of mitotic daughter pairs. Indeed, asymmetric divisions in culture were rare as judged by both methods (data not shown). There was further evidence of downstream block in differentiating divisions between nestin-positive TUJ1-positive cells and nestin-negative TUJ1-positive cells under VIP conditions, but this may simply be a consequence of the more proximal effect as some cells divided twice in the time lapse series over 48 h. We therefore conclude that VPAC2 receptor activation expands the pool of nestin cells by both maintaining proliferating nestin-positive NSPCs in an undifferentiated state and separately supporting their survival, without affecting their proliferation rate or recruitment.

In contrast, selective VPAC1 receptor activation had no trophic effect on cells in culture but significantly increased the proportion of TUJ1-positive neurons generated, consistent with a neurogenic fate-determining effect. This effect was present at the VPAC1 selective agonist concentration of 30 nM [39] and was blocked by the specific VPAC1 antagonist, neither of which affects the PAC1 receptor [34]. Indeed, VPAC1 receptor activation increased granule cell (Prox-1–positive) neurogenesis consistent with a dentate specific effect. VIP (30 nM) increased the number but not the proportion of new neurons in culture over 5 days, consistent with a dominant effect on nestin precursors mediated via the VPAC2 receptor, with subsequent differentiation divisions resulting in an increased number but not proportion of neurons, because VIP did not completely abolish differentiating neuronal divisions. This dominance is supported by our finding that exposure to VIP dramatically increases VPAC2 mRNA transcription (120-fold) but not VPAC1 mRNA transcription.

Although previous studies have shown that 3 μM VIP is both mitotic and partially (50%–60%) trophic for sympathetic neuroblasts in rats at a specific stage of embryogenesis (E15.5) [40, 41], these findings have subsequently been suggested to be mediated via the high-affinity pituitary adenylate cyclase activating polypeptide (PACAP) receptor PAC1 [42]. Significantly, their trophic effect was confined to embryonic neuroblasts with no evidence of a VIP-induced maintenance of a more primitive precursor type. At micromolar concentrations, VIP stimulates PAC1 receptors [28, 43], and indeed we found a proliferative effect at 1 μM but not at nanomolar concentrations of VIP. At 30 nM, VIP has high affinity for VPAC1 and VPAC2 receptors but does not stimulate PAC1 [28, 43]. Taken together with our selective pharmacology data, these results show that the observed trophic and fate-determining effects are mediated via VPAC1 and VPAC2 receptors and not via PAC1. Although Scharf et al. [44] have recently reported that PACAP downregulated TUJ1 and GFAP expression in cultures of neurosphere-derived whole-brain NSPCs acting via the PAC1 receptor, their findings could also be mediated by the VPAC2 receptor, as this was expressed by the NSPCs and they did not investigate receptor pharmacology. Their findings are completely consistent with our hypothesis, as the endogenous ligand for VPAC1/VPAC2 receptors could be either VIP or PACAP or both, because both have an equal affinity for these receptors [45].

The mechanisms governing neurogenesis in the late postnatal period are highly conserved in adulthood, with similar expression patterns of basic-helix-loop-helix mRNAs in precursor cells during the development of the dentate gyrus as well as in the mature dentate gyrus [35]. By P8–P10, the time at which our cultures were harvested, the neurogenic zone in the SGZ has been fully established [46], and the first functional adult-like granule cells are present at P7 [47]. We have previously shown that NPY, another neuropeptide transmitter, has the same proliferative effect on dentate NSPCs isolated at P8–P10 as on adult dentate NSPCs in vivo [13, 14, 22]. The behavior of dentate NSPCs isolated in the late postnatal period (P8–P10) is therefore highly relevant to adult dentate neurogenesis in comparison to those isolated at P0–P2 [38, 48, 49], which may not be relevant because the SGZ is not formed at this time [46]. However, the behavior of these postnatal NSPCs might be different than NSPCs isolated from the adult hippocampus.

There is considerable evidence supporting the hypothesis of an early neurodevelopmental origin for later psychopathological vulnerabilities, identifying the postnatal period when the dentate is developing as a period particularly sensitive to environmental change [50]. The inhibition of hippocampal neurogenesis by postnatal irradiation [51] impairs hippocampal-dependent spatial memory [52] and behavior [51] in adulthood. Alterations in postnatal neurogenesis are therefore critical to the levels and normal function of adult neurogenesis, and the effect of VIP on NSPCs in the postnatal period is likely to be relevant to adult hippocampal function.

VPAC2 mRNA distribution has been found in the granular layer of the adult rat dentate gyrus [53], where it overlaps with VPAC1 mRNA distribution [54], and we demonstrate a continuing trophic role for VIP in the adult dentate gyrus of adult Vipr2−/− mice. Because the total number of proliferating BrdU-positive cells after 4 h is unaltered in adult Vipr2−/− mice, our finding of a reduced total number of BrdU-positive cells and BrdU-positive/NeuN-positive cells in the dentate gyrus after 4 weeks implies a pure defect in cell survival, which is ongoing in the adult animal and is consistent with the trophic VPAC2 effect on proliferating cells demonstrated in vitro.

Our finding of a specific reduction in the number of type 2 nonradial nestin-positive cells in the SGZ in Vipr2−/− mice supports our other in vitro finding of a role for the VPAC2 receptor in maintaining the pool of nestin-positive but GFAP- and TUJ1-negative stem/progenitor cells. Interestingly, Vipr2−/− mice appeared to compensate for the reduction in the number of these nestin cells by increasing the proportion of them in the S-phase of the cell cycle to restore the levels of proliferating NSPCs to normal. However, they were unable to completely compensate for the loss of the VPAC2 receptor–mediated trophic effect, resulting in a net reduction in neurogenesis. Interestingly, the proportion of 4-week-old BrdU-positive cells that expressed a neuronal phenotype (Neu-N-positive) did not differ significantly between Vipr2−/− and wild-type controls, as might have been expected with unopposed VPAC1 stimulation. However, this demonstrates that VPAC2 does not modulate terminal fate specification, consistent with its role in maintaining stemness of the nestin-positive NSPC. It may be that the VPAC1 receptor is involved in determining neuronal fate in NSPCs downstream of those affected by VPAC2 stimulation, and a VPAC1 effect was not seen because these cells did not survive.

The significant reduction in total granule cell neurons in Vipr2−/− mice is consistent with an ongoing deficit in subgranular neurogenesis throughout postnatal development and adult life, and this may be functionally relevant because VIP blockade in adulthood causes impairments in associative learning abilities [21]. However, Vipr2−/− mice also exhibit disturbed circadian rhythms [55], which may affect learning and memory.

VPAC Receptors as Key Regulators of Precursor Dynamics and Potential Therapeutic Targets in Cognitive Dysfunction and Disordered Mood

The dynamic subgranular niche can differentially alter the pattern as well as the rate of precursor proliferation in response to physiological stimuli like running or an enriched environment [17] and pathological stimuli such as status epilepticus [17, 56, 57]. Of relevance to neural control of the niche has been the demonstration of “excitation-neurogenesis coupling” [36] by the synaptic contact of GABAergic interneurons on nestin-positive type 2 precursors and the promotion of their subsequent neuronal differentiation by GABA [11]. VIP is coreleased from GABAergic interneurons under specific firing conditions and therefore may be a mechanism by which neural activity can modulate the size of the transiently amplifying pool of nestin-positive type 2a precursors [30, 31] via VPAC2 stimulation, or direct their fate towards a neuronal lineage via stimulation of the VPAC1 receptor. To our knowledge this is the first demonstration of such a role for a synaptically released neurotransmitter.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This study identifies a key role for VPAC receptors as dynamic regulators of the proliferation pattern and survival of NSPCs in the postnatal and adult dentate gyrus. These findings also highlight VPAC receptors as potential targets for the therapeutic modification of hippocampal neurogenesis in disorders of cognition and possibly mood, offering the advantage of selectively expanding and maintaining a diminished pool of precursors as well as independently directing them down a neuronal lineage.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

The authors acknowledge the staff at the University of Southampton Biomedical Imaging Unit, particularly Dr. Peter Lackie, David Johnston, and Dr. Hans Schuppe for their assistance with the time lapse and confocal imaging, and Dr. Sandrine Williame-Morawek for helping with the figures. We also thank Prof. Charles ffrench-Constant, University of Edinburgh, for helpful discussions. This study was supported by a Medical Research Council strategic grant (G0300356) to W.P.G. and a Medical Research Council Programme Grant (G9719726) to A.J.H.

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  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional supporting information available online.

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STEM_184_sm_SuppTable1.doc30KSupporting Information Table 1.
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STEM_184_sm_SuppMaterials.doc71KSupporting Information Materials.

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