Ketamine decreases NSC viability in a dose-dependent manner.

<p>NSCs were plated as single cells treated with 50 μM, 200 μM, 400 μM and 1 mM ketamine. To measure cell viability, intracellular ATP levels were measured after 24 hours. Data are shown as mean ±SEM (n = 22–35). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P <0.05. * denotes P<0.05 compared with control.</p

Ketamine impairs NSC viability <i>via</i> AMPA receptor activation.

<p>NSCs were plated as single cells and treated with 400 μM ketamine or pre-treated with 10 μM NBQX prior to 400 μM ketamine exposure. After 24 hour incubation, intracellular ATP levels were measured. Data are shown as mean ±SEM (n = 23–39). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P<0.05. * denotes P<0.05 compared with control, # denotes P<0.05 compared to ketamine.</p

Ketamine-induced cell death is counteracted by PACAP <i>via</i> PAC-1 receptor activation.

<p>NSCs were plated as single cells. Cells were incubated with 400 μM ketamine and PACAP (100 nM) or agonist for PAC-1; Max-4 (30 nM). After 24 hour incubation, intracellular ATP levels were measured for cell viability. Data are shown as mean ±SEM (n = 13–39). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P<0.05. * denotes P<0.05 compared with control, # denotes P<0.05 compared to 400 μM ketamine.</p

A schematic diagram showing a proposed signaling pathway for PACAP mediated suppression of ketamine induced neurotoxicity of NSCs <i>in vitro</i>.

<p>A schematic diagram showing a proposed signaling pathway for PACAP mediated suppression of ketamine induced neurotoxicity of NSCs <i>in vitro</i>.</p

PACAP Protects Adult Neural Stem Cells from the Neurotoxic Effect of Ketamine Associated with Decreased Apoptosis, ER Stress and mTOR Pathway Activation

<div><p>Ketamine administration is a well-established approach to mimic experimentally some aspects of schizophrenia. Adult neurogenesis dysregulation is associated with psychiatric disorders, including schizophrenia. The potential role of neurogenesis in the ketamine-induced phenotype is largely unknown. Recent results from human genetic studies have shown the pituitary adenylate cyclase-activating polypeptide (PACAP) gene is a risk factor for schizophrenia. Its potential role on the regulation of neurogenesis in experimental model of schizophrenia remains to be investigated. We aimed to determine whether ketamine affects the viability of adult neural stem cells (NSC). We also investigated whether the detrimental effect mediated by ketamine could be counteracted by PACAP. NSCs were isolated from the subventricular zone of the mouse and exposed to ketamine with/without PACAP. After 24 hours, cell viability, potential involvement of apoptosis, endoplasmic reticulum (ER) stress, mTOR and AMPA pathway activation were assessed by quantitative RT-PCR and Western blot analysis. We show that ketamine impairs NSC viability in correlation with increased apoptosis, ER stress and mTOR activation. The results also suggest that the effect of ketamine occurs <i>via</i> AMPA receptor activation. Finally, we show that PACAP counteracted the decreased NSC viability induced by ketamine <i>via</i> the specific activation of the PAC-1 receptor subtype. Our study shows that the NSC viability may be negatively affected by ketamine with putative importance for the development of a schizophrenia phenotype in the ketamine induced animal model of schizophrenia. The neuroprotective effect via PAC-1 activation suggests a potentially novel pharmacological target for the treatment of schizophrenia, <i>via</i> neurogenesis normalization.</p></div

PACAP counteracts ketamine induced mTOR activation.

<p>NSCs were plated as single cells and treated with 400 μM alone or with 100 nM PACAP and 400 μM ketamine. Cells were treated as indicated and after 1, 2, 6 and 24 hour incubation cells were harvested for Western blot experiments <b>(A)</b>. Cells were treated as indicated and incubated for 1 and 2 hours and harvested for Western blot experiments <b>(B)</b>. To obtain quantitative measurements p-mTOR were normalized against total-mTOR. Data are shown as mean ±SEM (A, n = 4–8; B, n = 3–5). Kruskal-Wallis followed by Dunn’s test or Fisher LSD test was used. Differences were considered significant at P<0.05. * denotes P<0.05 compared with control, # denotes P<0.05 compared to ketamine + 1 hour or ketamine + 2 hours.</p

GLP-1R activation by Ex-4 did not protect NSCs from hypoglycaemic <i>milieu</i>.

<p>NSCs were plated as single cells. Prior to 2.5mM glucose addition cells were incubated with Ex-4 (10 nM) or PACAP (100 nM) for 10 min. After 24 hours incubation intracellular ATP levels were measured. Data are shown as mean ±SEM (n = 17–25). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P<0.05. * denotes P <0.05 compared with control, # denotes P<0.05 compared to 2.5mM glucose.</p

A hypoglycaemic <i>milieu</i> decreases NCS viability in a dose-dependent manner.

<p>NSCs were plated as single cells and treated with 20 mM (control) 10, 5, 2.5, 0 mM glucose. To measure cell viability, intracellular ATP levels were measured after 24 hours. Values are shown as mean ±SEM (n = 14–25). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P <0.05. * denotes P <0.05 compared with control, # denotes P<0.05 compared to 2.5mM glucose.</p

PAC-1 activation counteracts impaired NSCs under hypoglycaemic <i>milieu via</i> the PKA-dependent pathway.

<p><b>(A, B)</b> NSCs were plated as single cells. Prior to 2.5mM glucose addition cells were incubated with PACAP (100 nM), Max-4 (30 nM), H89 (1 uM) and Gö6976 (1 uM) for 10 min. After 24 hours incubation, intracellular ATP levels were measured. Data are shown as mean ±SEM (A, n = 15–50; B, n = 16–28). Kruskal-Wallis followed by Dunn’s test was used. Differences were considered significant at P<0.05. * denotes P <0.05 compared with control, # denotes P<0.05 compared to 2.5mM glucose.</p

PACAP increases NSC viability in response to hypoglycaemic <i>milieu</i> in correlation with a downward trend in apoptosis.

<p>NSCs were plated as single cells. Prior to 2.5mM glucose addition cells were incubated with PACAP (100 nM) for 10 min. After 24 hours incubation cells were harvested for Western blot experiments <b>(A, B)</b> or intracellular ATP determination <b>(C)</b> or NSC manual counting <b>(D)</b>. To obtain quantitative measurements Bcl-2 protein levels and cleaved caspase 3 were normalized against β-actin. Data are shown as mean ±SEM (A, n = 6–7; B, n = 5–8, C, n = 30–50; D, n = 3–4). Kruskal-Wallis followed by Dunn’s test or Fisher LSD test was used. Differences were considered significant at P<0.05. * denotes P <0.05 compared with control, # denotes P<0.05 compared to 2.5mM glucose.</p