Effect of chronic morphine on the dentate gyrus neurogenic microenvironment

Abstract

Opiates, such as morphine, decrease neurogenesis in the postnatal hippocampal subgranular zone (SGZ) by inhibiting progenitor proliferation and maturation. However, it is not known how morphine influences the growth factors and vasculature that encompass the neurogenic SGZ microenvironment. We examined morphine's effect on pro- and anti-proliferative factors in the dentate gyrus (DG; Experiment 1) as well as the DG neurovasculature itself (Experiment 2). For Experiment 1, mice were implanted with subcutaneous sham or morphine pellets (0 and 48 h) and were decapitated 24 or 96 h later. One brain hemisphere was postfixed to examine proliferation by immunohistochemistry, and a DG-enriched sample was dissected from the other hemisphere to examine the neurogenic microenvironment via immunoblotting for known pro- and anti-proliferative factors. Consistent with previous results, morphine decreased the number of proliferating cells in the SGZ, as the number of Ki67-immunoreactive (IR) cells was decreased at 96 h. Morphine did not alter DG levels of the pro-proliferative factor brain-derived neurotrophic factor, anti-proliferative factor interleukin-1 beta, or their receptors TrkB and IL1R1 at either time point. However, morphine increased the pro-proliferative factor vascular endothelial growth factor (VEGF) at 96 h. Given that VEGF is also a potent angiogenic factor, Experiment 2 examined whether the morphine-induced increase in VEGF correlated with altered DG neurovasculature. Mice were implanted with morphine pellets as in Experiment 1, and 2 h before perfusion (24 or 96 h) were administered bromodeoxyuridine (BrdU; intraperitoneal, 150 mg/kg). Tissue was co-stained for BrdU and the endothelial cell marker endoglin to enable examination of DG vessels and proximity of BrdU-IR cells to endoglin-IR vessels. At 96 h, endoglin-IR vessel area and perimeter were increased, but proximity of BrdU-IR cells to endoglin-IR vessels remained unchanged. These data suggest that following chronic morphine exposure, factors within the neurogenic microenvironment are maintained or upregulated to compensate for decreased SGZ proliferation.

Figure 1. Chronic morphine inhibits the total population of SGZ proliferating cells

(A) C57BL/6J mice were implanted with s.c. sham or morphine pellets at 0 and 48 hrs and killed at 24 or 96 hrs via decapitation. (B) The number of Ki67-IR cells in the SGZ was not changed after 24 hrs. (C) The number of Ki67-IR cells was significantly decreased after 96 hrs of morphine exposure. (D) Representative IHC demonstrated decreased numbers of Ki67-IR cells in the SGZ. At all time points sham: n=4, morphine: n=4. Scale bar=100 μm, **p<0.01.

Figure 2. Chronic morphine does not alter DG levels of BDNF or IL1β

To determine if the microenvironment of the hippocampus was altered by chronic morphine, DG-enriched extracts were used to look for changes in protein levels via immunoblotting. (A–D) After 24 hrs of morphine exposure, dentate levels of IL1β, BDNF and their respective receptors, IL1R1 and TrkB remained unchanged. (E–H) After 96 hrs of morphine exposure, dentate levels of IL1β and BDNF and their respective receptors, IL1R1 and TrkB remained unchanged. At all time points sham: n=4–8, morphine: n=5–8.

Figure 3. Chronic morphine dynamically alters levels of VEGF in the DG

(A, B) After 24 hrs of morphine, DG protein levels of VEGF and VEGFR2 are unchanged as measured via immunoblotting. (C, D) After 96 hrs, VEGF protein levels are increased whereas receptor levels remain unchanged. At all time points sham: n=5–6, morphine: n=4–8, **p<0.01.

Figure 4. Endoglin expression in the hippocampal DG

(A) Low-power image shows that endoglin staining is present throughout the hippocampus and in the SGZ where the majority of proliferating BrdU-IR cells reside. Scale bar=100 um. (B–C) Confocal images show close proximity of BrdU-IR cells to endoglin-IR vessels. Scale bar=10 um.

Figure 5. Chronic morphine-induced increase in VEGF correlates with altered DG neurovasculature

(A)After 24 hrs of morphine exposure, various properties of endoglin-IR cells including vessel number, area and perimeter remain unchanged. (B) After 96 hrs of morphine exposure, the number of endoglin-IR vessels between sham and morphine groups did not differ. However the area and perimeter of endoglin-IR vessels are increased. (C) The proximity of BrdU-IR cells to endoglin-IR vessels, does not differ between sham and morphine treated groups. Proximity was measured as “touch”, touching or overlapping the vessel; “near”, within 10 μm of vessel, or “far”, beyond 10μm of vessel. For all time points sham: n=4–5, morphine: n=4–5, **p<0.01.