Does Ketamine Really Modulate Glutamate?

Holden B Janssens, Lisa Yu, Harm Kooijker, Bryson Cwick, Nadege Morisot, Julien Roeser, Marieke van der Hart, Arash Rassoulpour Discovery, Charles River Laboratories, South San Francisco, CA 230.08

1 Introduction 3 Results (Rat) 4 Results (Mouse) 5 Summary/Conclusions Glutam ate in Rat Hipp Glutamate in Rat PFC Glutamate in Mouse PFC G lutam ate in M ouse H ipp 200 The noncompetitive NMDA receptor antagonist ketamine has been shown to 200 400 300

250 • Ketamine (20 mg/kg) does not increase the levels of elicit psychotic action in humans, exacerbate symptoms in schizophrenic 150 150 300 200 extracellular glutamate in either the PFC or hippocampus patients, and more recently has been shown to have potential in treating 100 100 150 of rats or mice depression. It is hypothesized that these actions may be regulated by 200 100 50 ketamine’s action on glutamate (glu), as has been previously demonstrated 50 Vehicle 100 50 Percent Baseline (%) Baseline Percent Percent Baseline (%) Baseline Percent Percent Baseline (%) Baseline Percent • Ketamine (20 mg/kg) does not increase the levels of using in vivo microdialysis in the prefrontal cortex of rodents (Lorrain et al. Ketamine HCl - 20 mg/kg (%) Baseline Percent 0 0 0 0 extracellular GABA in either the PFC or hippocampus of 2003, Moghaddam et al. 1997). In addition, it has also been demonstrated that -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 Tim e (m in) Tim e (m in) Tim e (m in) Tim e (m in) rats or mice ketamine increases (DA) activity (Moghaddam et al. 1997). Histamine in Rat PFC H istam ine in R at H ipp Histamine in Mouse PFC Histamine in Mouse Hipp However, we have historically been unable to attain a reproducible ketamine- 300 300 400 400 • Levels of histamine were significantly increased by 250 250 induced release of glutamate using both in vivo microdialysis and . 300 300 ketamine administration in the PFC and Hipp of both mice Thus, in the current set of experiments we utilized in vivo microdialysis to 200 200 150 200 and rats. examine the effects of different dose of ketamine on release 150 200 100 100 100 in the prefrontal cortex and hippocampus of mice and rats. Consistent with our 100 50

50 (%) Baseline Percent Percent Baseline (%) Baseline Percent • Dopamine, , and levels were Percent Baseline (%) Baseline Percent previous findings, we were unable to see an increase in glutamate levels of (%) Baseline Percent 0 0 0 0 differentially regulated in the rat PFC as compared to -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 either species using 10 and 20 mg/kg of ketamine. Interestingly, we were able -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 Tim e (m in) Tim e (m in) Tim e (m in) Tim e (m in) mouse PFC such that ketamine administration resulted in to observe a significant increase in dopamine levels, as previously reported as significant elevations of all 3 transmitters in the PFC of Norepinephrine in Rat PFC Norepinephrine in Rat Hipp well as an increase in both serotonin (5HT) and norepinephrine (NE). Some of Norepinephrine in Mouse PFC Norepinephrine in Mouse Hipp 300 300 rats with little to no effect on the PFC release of DA, NE, these monoaminergic changes were more pronounced in the hippocampus as 250 250 250 250 5HT in mice. compared to the prefrontal cortex. These results challenge existing reports of 200 200 200 200 the mechanism by which ketamine exerts its actions, and provide additional 150 150 150 150 • Monoamine levels (DA, 5HT, and NE) were elevated support for the neuroactive mechanisms of ketamine functioning through 100 100 100 100 following ketamine administration in the hippocampus of monoaminergic pathways. 50 50 50 50 Percent Baseline (%) Baseline Percent Percent Baseline (%) Baseline Percent Percent Baseline (%) Baseline Percent Percent Baseline (%) Baseline Percent both mice and rats

0 0 0 0 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 Tim e (m in) Tim e (m in) Tim e (m in) Tim e (m in) • Here we demonstrate that ketamine reproducibly

Dopamine in Rat PFC Dopamine in Rat Hipp increases the release of monoamines in the PFC and 2 Materials and Methods Dopamine in Mouse PFC Dopamine in Mouse Hipp 500 500 250 250 hippocampus in rats and mice whilst having no effect on

400 400 • Mice and Rats (n = 6/group) were anesthetized using isoflurane (2%, 800 mL/min 200 200 glutamate release. Therefore, when examining the

O2). 300 potential mechanism by which ketamine exerts its actions 300 150 150 • The animals underwent stereotaxic surgery where I-shaped microdialysis probes 200 one must look at the effects on monoamines compared to 200 100 100 (polyacrylonitril membrane, BrainLink, the Netherlands) were inserted into the PFC amino acids. The lack of a response on glutamate in our 100 100 50 50

and hippocampus (3 mm exposed surface for Hipp and 2 mm exposed surface for (%) Baseline Percent Percent Baseline (%) Baseline Percent Percent Baseline (%) Baseline Percent Percent Baseline (%) Baseline Percent hands could explain the variability reported on ketamine's the PFC). 0 0 0 0 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 actions in a variety of rodent behavior assays. These • Experiments were performed one day after surgery. Tim e (m in) Tim e (m in) Tim e (m in) Tim e (m in) • On the day of the experiment, the probes were perfused with aCSF containing 147 studies warrant further exploration into the discrepancies Serotonin in Rat PFC Serotonin in Mouse PFC Serotonin in Mouse Hipp on ketamine's in vivo effects. mM NaCl, 3.0 mM KCl, 1.2 mM CaCl2 and 1.2 mM MgCl2, at a flow rate of 1.5 Serotonin in Rat Hipp 250 250 µL/min. 300 300 • Microdialysis samples were collected for 30 minute periods by an automated 250 250 200 200

fraction collector into polystyrene mini-vials containing 15 µL of 0.02M formic acid 200 200 150 150

150 (FA) and 0.04% ascorbic acid in ultrapurified H2O. 150 100 100

100 • Ketamine (20 mg/kg, Sigma) was administered IP at time = 0. 100 50 50

• Microdialysis samples were derivatized using SymDaq® and analyzed for the levels 50 (%) Baseline Percent (%) Baseline Percent 50 (%) Baseline Percent Percent Baseline (%) Baseline Percent 0 0 0 of DA, NE, 5HT, Glu, GABA, and Gly by LC MS/MS. 0 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 -40 -20 0 20 40 60 80 100 120 140 160 180 Tim e (m in) Tim e (m in) Tim e (m in) Tim e (m in)