In a previous study, we demonstrated that a mild reversible injury to the hippocampal CA1 subfield induced by a low concentration of DOM increases neurogenesis in both the dentate gyrus and the CA1 subfields of OHSC . Neuronal injury can lead to neural proliferation as a compensatory mechanism for cell death in the hippocampus [44, 45] and growth and mitogenic factors, such as BDNF, play a prominent role in proliferation and neurogenesis after excitotoxicity . In the present study, we investigated whether DOM alters BDNF expression after transient insult and explored the key intracellular signaling mechanisms by which DOM modulates neurogenesis. Our results showed that DOM insult upregulated BDNF expression by activation of both MAPK and PKA cascades and that these two pathways mediate, at least in part, the increased neural proliferation resulting after mild excitotoxicity.
Exposure to 2 μM DOM for 24 h (insult) followed by recovery induced a significant and long lasting increase in BDNF protein levels in OHSC. BDNF is a member of the neurotrophin family widely distributed in the brain with the highest levels in the hippocampus . It has been previously reported that excitotoxicity and seizure activity induce an overexpression of hippocampal BDNF at both protein and mRNA levels [48–51]. BDNF signals primarily through its high-affinity receptor TrkB that promotes neurogenesis, synaptic plasticity and cell survival [52, 53], and plays an important role in the development and plasticity of the brain . Consistent with the observed increase in BNDF expression, DOM insult also induced TrkB upregulation. Although TrkB phosphorylation, which was not assessed in the current study, is required for receptor-mediated signaling, a number of recent papers have reported that increases in both BDNF and TrkB expression correlate with functionally-relevant downstream effects both in vitro [55, 56] and in vivo [57, 58]. Thus, DOM-induced changes in growth factors and/or their receptors could stimulate the increased cell birth observed after excitotoxicity.
To determine the cellular source of increased BDNF we performed double-label immunohistochemistry in the CA1 hippocampal subfield. Although the response of progenitor cells in different hippocampal regions may vary (for review see ) we have shown previously that the CA1 region is particularly sensitive to both excitotoxic damage by DOM and shows robust microglial activation whereas other regions (eg. SGZ) do not . Our observation that BDNF is overexpressed in CA1 not only by neurons but also by microglial cells (Figure 2A) is in accordance with previous studies [13, 60–62], which highlights the importance of microglial cells as a source of BDNF following injury. Examination of the image presented in Figure 2A (Merge) shows clear double-labelling of BDNF and CD11b in the lower left quadrant while cells in the upper right quadrant (presumably neurons) express only BDNF. Further, the image shows that the two cell types are in very close proximity in this region. Therefore, we suggest that under mild excitotoxic conditions both neurons and microglia will respond with an increase in the production and release of BDNF.
Clinical and basic evidence supports the idea that abnormalities in brain neuronal regeneration assisted by BDNF are associated with a wide range of disorders such as neurodegenerative diseases and psychiatric or stress-related conditions (reviewed by [63, 64]). Our laboratory has reported previously that low concentrations of DOM administered in vivo during perinatal development cause permanent alterations in both behaviour and hippocampal structure consistent with many animal models of temporal lobe epilepsy as well as what is found in the human condition. Increased expression of both BDNF  and its corresponding TrkB receptor  were found in the hippocampus of DOM treated rats. Thus, the changes observed in OHSC in the current study are consistent with observations in vivo. The organotypic hippocampal slice culture system, however, provided us the means by which to evaluate the intracellular mechanism of enhanced BDNF expression initiated by transient DOM injury. Using immunobloting of specific signaling intermediates, we followed three important intracellular cascades: the MAPK, the PKA and the CaMKII pathways.
DOM insult led to increased p-ERK1/2 (p-ERK); two signaling proteins activated by the mitogen-activated protein kinase (MAPK) pathway. ERK1/2 (ERK) promote growth and modulate differentiation and survival via transcriptional regulation. ERK activation in OHSC was increased immediately following DOM exposure, reaching peak expression at 12 h post insult. DOM also caused a significant upregulation of p-PKA levels. Increases in intracellular Ca2+ by activation of NMDA receptors, AMPA/kainate receptors, or calcium channels increases intracellular cyclic AMP (cAMP) through activation of adenylyl cyclases that will result in the activation of PKA [65–67]. In addition to the increased p-ERK and p-PKA our results also demonstrated significant activation of CaMKII in OHSC. Other studies have reported a pivotal role for both PKA and CaMKII activation after long-lasting potentiation induced by a brief DOM treatment  and administration of DOM at doses that produce no major observable behavioral changes has been previously shown to increase significantly CaMKII phosphorylation . Therefore, these results suggest that alterations in intracellular signaling pathways might be a protective mechanism against DOM-induced excitotoxic damage.
Ca2+-mediated signaling pathways tightly modulate BDNF expression mainly through the transcription factor CREB [38, 70, 71]. In conjunction with the observed increase in BDNF and TrkB, DOM insult was found to stimulate activation of CREB in hippocampal cultures. Several studies have proven that CREB activation requires serine-133 phosphorylation, which can be mediated by PKA, MAPK pathway or CaMKs, among others, depending on the activating signal and cell type [15–17]. In the current experiments, inhibitors of both MEK and PKA attenuated the DOM-stimulated activation of CREB as well as upregulation of BDNF. In contrast, the CaMKII inhibitor failed to prevent or significantly decrease any of the protein changes observed. These data strongly suggest that transient DOM exposure in hippocampal cultured slices upregulates CREB-dependent transcription of BDNF by activating the MAPK and PKA pathways rather than the CaMKII cascade. ERK activation has been previously associated with the transcription factor CREB in cultured hippocampal neurons and brain slices [72, 73] and as MAPK signaling is required for prolonged CREB phosphorylation [74, 75], it has been suggested that MAPK signalling might be highly relevant for the activation of CREB-dependent transcription. It has also been reported that PKA regulation of transcription via CREB is implicated in brain plasticity, learning and memory [76–79]. Our results showed that the DOM-induced increases in BDNF expression and CREB phosphorylation were completely blocked with concurrent exposure to PKA and MEK inhibitors. We further explored whether crosstalk between the PKA and ERK pathways might also play a role in the observed activation of CREB following DOM insult. Although evidence of coupling between these signaling pathways has been provided previously in vivo and in vitro [71, 80–82] no evidence was found in OHSC after DOM insult; namely, the MEK inhibitor PD98059 failed to modulate PKA pathway activation and no significant changes were found in p-ERK levels after concurrent exposure to the PKA inhibitor H89 and DOM compared to exposure to DOM alone. Together, these pieces of evidence suggest that the PKA- and MEK-activated pathways are operating in parallel in this system and converge upon CREB, leading to BDNF overexpression. An interesting but currently unexplained finding from our experiments was that the DOM-induced increase in CaMKII was attenuated with MEK inhibition. It has been previously described that CaMKII, as an upstream kinase, interacts with Raf, modulating the activation of ERK proteins [83–85] but, to our knowledge, there is no previous evidence of ERK acting as an upstream regulator of CaMKII phosphorylation in the CNS. The observed phenomenon and its implications should be investigated further in a future study.
A major goal of this study was to elucidate the relationship between PKA/ MAPK pathways and the increased neurogenesis we reported previously in OHSC using both immunostaining and DCX positive cell counts . As shown in Figure 7B, analysis by Western Blot revealed that concurrent chemical inhibition of PKA and MEK activation specifically attenuated the increase in the neuroblast cell marker DCX. In accordance with the results obtained in the present study, these kinases have been reported to mediate growth factor-induced neurogenesis and neuroprotection . The extracellular signal-regulated kinase (ERK) is activated by MEK in response to growth stimuli  and much evidence exists that the ERK pathway plays a role in progenitor cell proliferation or differentiation in a number of model systems. For example, the ERK pathway is involved in neurogenesis, neurite outgrowth, and neuronal survival induced by either neurotrophic factors [20, 21] or pharmacological agents such as valproate  or lithium  and it has been proven that ERK activation promotes hippocampal neurogenesis in vivo [26, 27] and in vitro[28, 29]. Similarly, PKA regulation of transcription via CREB has been associated with growth factor-dependent neurogenesis, cell survival, synaptic transmission and cognitive function in the nervous system [23–25].
Phosphorylation of CREB and overexpression of BDNF have been implicated in the regulation of the expression of many genes and cellular processes important in brain function  and the up-regulation of hippocampal cell proliferation [40, 41]. We have previously shown that neurogenesis after DOM insult in OHSC occurred primarily during the first week of exposure in both the subgranular zone of the hippocampus and in the CA1 hippocampal subfield, with a decreasing tendency clearly observed over the next days . In the current study, DOM insult induced a significant long lasting increase in BDNF protein levels in OHSC that was sustained throughout the 14 day period, although in the current study we did not determine if this effect was regionally selective. BDNF is one of the most studied extrinsic factors that not only promotes neurogenesis, but also regulates dendrite outgrowth [87–92], increases proximal dendrite growth and number in pyramidal neurons [89, 91, 93] and promotes synaptogenesis [94–96] and neuronal survival during development . Our results suggest that BDNF in OHSC may be promoting neurogenesis as well as maturation and integration of new neurons after DOM insult, although the specific hippocampal regions at which these neurons originate, whether they in fact migrate to, or originate in, areas of transient damage, and whether they are capable of restoring normal function to the resulting circuitry needs to be determined.