Dynorphin up-regulation in the dentate granule cell mossy fiber pathway following chronic inhibition of GluN2B-containing NMDAR is associated with increased CREB (Ser 133) phosphorylation, but is independent of BDNF/TrkB signaling pathways

Abstract

Emerging evidence suggests that neuronal responses to N-methyl-d-aspartate (NMDAR) activation/inactivation are influenced by subunit composition. For example, activation of synaptic NMDAR (comprised of GluN2A>GluN2B) phosphorylates cAMP-response-element-binding protein (CREB) at Ser 133, induces BDNF expression and promotes neuronal survival. Activation of extrasynaptic NMDAR (comprised of GluN2B>GluN2) dephosphorylates CREB (Ser 133), reduces BDNF expression and triggers neuronal death. These results led us to hypothesize that chronic inhibition of GluN2B-containing NMDAR would increase CREB (Ser 133) phosphorylation, increase BDNF levels and subsequently alter downstream dynorphin (DYN) and neuropeptide Y (NPY) expression. We focused on DYN and NPY because these neuropeptides can decrease excitatory neurotransmission and seizure occurrence and we reported previously that seizure-like events are reduced following chronic treatment with GluN2B antagonists. Consistent with our hypothesis, chronic treatment (17-21days) of hippocampal slice cultures with the GluN2B-selective antagonists ifenprodil or Ro25,6981 increased both CREB (Ser 133) phosphorylation and granule cell mossy fiber pathway DYN expression. Similar treatment with the non-subtype-selective NMDAR antagonists d-APV or memantine had no significant effect on either CREB (Ser 133) phosphorylation or DYN expression. In contrast to our hypothesis, BDNF levels were decreased following chronic treatment with Ro25,6981, but not ifenprodil, d-APV or memantine. Blockade of BDNF actions and TrkB activation did not significantly augment hilar DYN expression in vehicle-treated cultures and had no effect in Ro25,6981 treated cultures. These findings suggest that chronic exposure to GluN2B-selective NMDAR antagonists increased DYN expression through a putatively pCREB-dependent, but BDNF/TrkB-independent mechanism.

Keywords: Hippocampus; Ifenprodil; Memantine; N-methyl-d-aspartate; Neuropeptide Y.

Fig. 1

CREB (Ser 133) phosphorylation was increased following chronic treatment with GluN2B-selective, but not non-subunit-selective NMDAR antagonists. Western blots probed for CREB, pCREB (Ser133) and NSE (A) or β-tubulin (E) were generated using (A-C, E) homogenates prepared from hippocampal slice cultures treated with vehicle or the stated NMDAR antagonists for the entire 17-21 DIV culture period or (D) purchased extracts prepared from untreated SK-N-MC cells or SK-N-MC cells treated with forskolin and IBMX as described in the Materials and Methods. A) shows a representative Western blot for pCREB, CREB, and neuron specific enolase (NSE) (all obtained from the same blot). B) quantitative Western blot analysis showed increased CREB levels relative to NSE loading controls in organotypic hippocampal slice cultures treated chronically with the GluN2B-selective NMDAR antagonist Ro25,6981, but not ifendprodil, as well as the non-subtype-selective NMDAR antagonist, APV. C) quantitative Western blot analysis showed increased CREB (Ser133) phosphorylation relative to CREB levels in slice cultures treated with the GluN2B-selective NMDAR antagonists Ro25,6981 or ifenprodil, but not in cultures treated with the non-subtype-selective NMDAR antagonists APV or memantine. D) Specificity of the anti-pCREB antibody for pCREB is illustrated by increased pCREB following forskolin and IBMX treatment compared to untreated controls. E) The specificities of anti-pCREB and anti-CREB antibodies are shown by peptide block. β-tubulin was used as a loading control. Bars represent the mean ± SEM (normalized to vehicle mean/experiment). X-axis label in C is for A-C. Numbers in parentheses indicate the number of slice cultures. *, p<0.05, different than vehicle; #, p<0.001, different than vehicle, ifenprodil, APV; ANOVA with Holm-Sidak posthoc comparison.

Fig. 2

DYN expression in the dentate granule cell mossy fiber pathway was increased following chronic treatment with GluN2B-selective, but not non-subtype-selective NMDAR antagonists. Hippocampal slice cultures treated for the entire 17-21 day culture period with different classes of NMDAR antagonists were stained immunohistochemically with anti-DYN A antiserum using the ABC method and analyzed as described in the Materials and Methods. A) Representative hippocampal slice cultures as well as B) subjective analysis and C) quantitative densitometry revealed increased mossy fiber DYN immunoreactivity associated with the dentate granule cell mossy fiber pathway in organotypic hippocampal slice cultures treated with the GluN2B-selective NMDAR antagonists Ro25,6981 or ifenprodil, but not in cultures treated with the non-subtype-selective NMDAR antagonists APV or memantine. C inset) Hilar DYN immunoreactivity density (white box, 200 × 100 μm) was subtracted from background density in CA1 (black box, 200 × 100 μm). Abbreviations: g, dentate granule cell layer; h, hilus; m, molecular layer; sl, stratum lucidum; p, pyramidal cell layer. Arrowheads in A point to DYN-IR puncta. Bars indicate B, percentages or C, means ± SEM. The number of slice cultures is indicated in parentheses. Scale bar is for all panels in A, 500 μm. *, different than vehicle, p<0.05, ANOVA with Holm-Sidak post hoc comparison; **, different than vehicle, p<0.001, z-test.

Fig. 3

NPY expression was not significantly affected by chronic treatment with NMDAR antagonists. Organotypic hippocampal slice cultures treated for the entire 17-21 day culture period with different classes of NMDAR antagonists were stained immunohistochemically with anti-NPY IgG using the ABC method and analyzed as described in the Materials and Methods. A) Representative low power images of hippocampal slice cultures showed NPY immunoreactivity in scattered neurons and neuropil throughout the slice culture. B) Representative higher power images revealed NPY immunoreactivity in neuronal somata (open arrow), dendrites (arrows) and thin neuronal processes (arrowheads). C) Quantitative neuron counts in the granule cell layer and hilus showed that chronic treatment with D-APV decreased the number of NPY-IR neurons. Quantitative densitometry D) in the hilus and E) in the entire hippocampal slice culture illustrated no effect of chronic NMDAR antagonist treatment on NPY immunoreactivity. Abbreviations: g, dentate granule cell layer. Bars indicate means ± SEM. The number of slice cultures is indicated in parentheses. Scale bar in A applies to all panels in A, 500 μm; in B1, 500 μm; in B2, B3, 100 μm. *, p<0.05, different than vehicle, ANOVA on Ranks with Dunn’s posthoc comparison.

Fig. 4

BDNF levels were unaltered by chronic treatment with GluN2B-selective NMDAR antagonists. ELISA was used to assay BDNF levels in hippocampal slice cultures treated for the entire 17-21 day culture period with different classes of NMDAR antagonists as described in the Materials and Methods. BDNF levels were decreased in organotypic hippocampal slice cultures treated with the GluN2B-selective NMDAR antagonist Ro25,6981, but not in cultures treated with the GluN2B-selective NMDAR antagonist ifenprodil or the non-subtype-selective NMDAR antagonists APV or memantine. Bars represent mean ± SEM. Numbers in parentheses indicate the number of slice cultures. *, p<0.05, different than vehicle, ANOVA on Ranks with Dunn’s posthoc comparison.

Fig 5

Increased DYN expression following chronic treatment with GluN2B-selective NMDAR antagonists was independent of TrkB-mediated signaling pathways. Organotypic hippocampal slice cultures were treated for the entire 17-21 day culture period with either vehicle or the GluN2B-selective NMDAR antagonist Ro25,6981 alone or in combination with the TrkB/human IgG Fc region chimera TrkB-Fc, the tyrosine kinase inhibitor K252a or their respective human IgG-Fc or DMSO controls. Cultures were then stained immunohistochemically with anti-DYN A antiserum using the ABC method and analyzed as described in the Materials and Methods and depicted in Fig. 2. A) Representative hippocampal slice cultures as well as B), C) quantitative densitometry show increased hilar DYN immunoreactivity following chronic Ro25,6981 treatment (p=0.012, Mann Whitney Rank Sum test), but no significant effect of K252a or TrkB-Fc in either vehicle- or Ro25,6981-treated slice cultures. Bars indicate means ± SEM. Numbers in parentheses indicate the number of slice cultures.