Ketamine is a non-competitive NMDA antagonist that has been shown to have antidepressant properties both clinically as well as in preclinical studies when administered at a subanesthetic dose. In vivo electrophysiological recordings were carried in male Sprague Dawley rats 30 minutes following ketamine administration (10 mg/kg) to first assess its effects on monoaminergic firing. Whilst no change in the firing activity of serotonin (5-HT) neurons was observed in the dorsal raphe nucleus (DRN), an increase in the firing activity was observed for dopamine (DA) and noradrenergic (NE) neurons in the ventral tegmental area (VTA) and locus coeruleus (LC), respectively. The effect of ketamine on these electrophysiological parameters was prevented by pre-administration of the AMPA receptor antagonist NBQX 10 minutes prior to ketamine administration. In a second series of experiments, an increase in AMPA-evoked response was observed within 30 minutes in the CA3 layer of the hippocampus (HPC) following acute ketamine administration. These findings suggest that acute ketamine administration produces a prompt enhancement of AMPA transmission in the forebrain and also results in increased catecholaminergic activity. These effects may play a crucial role in the rapid antidepressant effects of ketamine observed shortly following its infusion in the clinic.

Objectives To compare the anaesthetic, analgesic and cardiorespiratory effects of intramuscular medetomidine and ketamine administered alone or combined with morphine or tramadol for orchiectomy in cats. Study design Randomised, blinded, prospective clinical study. Animals Thirty client owned healthy cats. Materials and methods Cats received a combination of medetomidine (60 μg kg-1) and ketamine (10 mg kg-1) alone (MedK) or combined with morphine (0.2 mg kg-1) (MedKM) or tramadol (2 mg kg-1) (MedKT) intramuscularly. Time of different events and physiological parameters were recorded by a blinded researcher. Pre-surgery arterial and venous blood gases were measured. Heart rate (HR), respiration rate (fR), systolic arterial blood pressure (SABP), peripheral haemoglobin saturation (SpO2) and end-tidal carbon dioxide tension (PETCO2) were recorded every 5 minutes of general anaesthesia and at each surgical stage. Post-operative analgesia was evaluated with a visual analogue scale, a multidimensional composite scoring system and a rigid tip von Frey mechanical threshold device every hour from 3 to 8 hours post-injection of the initial combination of drugs. Data were analysed with a linear mixed model, Kruskal Wallis or Chi-square tests (p < 0.05). Results Median (range) induction and recovery times (minutes) were not significantly (P = 0.125) different among the three combinations: 5.6 (2.7, 8.0), 7.4 (5.1, 9.6) and 8 (5.8, 14.9) for induction and 128 (95, 143), 166 (123, 210) and 143 (123, 180) for recovery, with MedK, MedKT and MedKM, respectively. All three combinations caused similar low partial pressure of arterial oxygen (PaO2) values (mean ± SD: 66.2 ± 1.7 mmHg). Surgery had a significant effect on SABP (p < 0.001), SpO2 (p < 0.001), fR (p = 0.003) and HR (p = 0.002), which increased; and PETCO2 (p = 0.003), which decreased, with all combinations. Non-significant differences were found in pain scores and von Frey results among treatments; however, the von Frey changes over time did vary by treatment (p < 0.001) with the MedK group returning to baseline values more rapidly than MedKM and MedKT. None of the cats required rescue analgesics. Conclusion and clinical relevance All three protocols provide adequate anaesthesia and analgesia for orchiectomy in cats. However, rescue intervention to maintain surgical anaesthesia such as isoflurane may be required in some cats. Oxygen should be supplemented. / Dissertation (MMedVet)--University of Pretoria, 2013 / gm2014 / Companion Animal Clinical Studies / unrestricted

Traditional antidepressants, which act on the serotonin, dopamine, and norepinephrine systems, require many weeks to produce a therapeutic effect and are not effective for every patient. A sub-anesthetic dose of the anesthetic agent ketamine, a glutamate N-methyl-D-aspartate receptor antagonist, has been shown to produce a rapid and robust antidepressant effect in treatment-resistant major depressive disorder (MDD). As depressive symptoms typically return after one week following a single infusion, recent work has begun to focus on methods for prolonging the effects. Repeated infusions on a specific dosing schedule are being explored, however, the early identification of treatment responders and non-responders would be beneficial for optimized treatment selection within this population. The mechanisms underlying ketamine’s rapid effects conceivably involve the regulation of altered glutamatergic signaling in MDD, though this is not yet completely understood. Understanding of the central mechanisms mediating ketamine’s rapid antidepressant effects may be increased through the use of non-invasive electroencephalographic measures, including resting electroencephalography (EEG) and the mismatch negativity (MMN) event-related potential. These measures have been shown to be altered in depressed individuals and are sensitive to ketamine administration. The primary objectives of this study were to 1) examine acute changes in EEG- and MMN-derived indices, immediately post- and two hours postinfusion, with a sub-anesthetic ketamine dose in comparison to an active placebo (midazolam), and 2) to examine their relationships with early and sustained antidepressant treatment response to ketamine within an eight week clinical trial involving three study phases. Ketamine decreased measures of scalp-level alpha and theta resting activity, immediately postinfusion, and increased gamma immediately and two hours postinfusion. An increase in source-localized anterior cingulate activity two hours postinfusion was also observed. Regarding the MMN, ketamine reduced frontal amplitudes as well as theta event-related oscillations and source-localized peak frontal generator activity. Measures of resting theta and change in gamma, as well as left frontal MMN amplitude, theta event-related oscillations, baseline left phase locking factor, and baseline right inferior temporal lobe activity were predictive of decreases in depressive symptoms at both early and sustained treatment time points. Alpha power was predictive of decrease in suicidal ideation, though the relationship with baseline and early change in symptoms was stronger. These findings contribute to our understanding of the role of baseline and ketamine-induced changes in both resting and task-evoked electrophysiological measures, and may have the potential to act as non-invasive biomarkers of antidepressant response prediction to glutamatergic agents.

Background: A single infusion of ketamine has rapid antidepressant properties, although the drawback is a lack of sustained effect. A previous study showed a rapid enhancement (within 2 hours) in ventral tegmental area (VTA) dopamine (DA) neuron population and locus coeruleus (LC) norepinephrine (NE) firing and bursting activity following a single ketamine administration. The current study investigated whether these changes are present 24 hours after a single administration and if they are maintained with repeated administration. Additionally, we examined dorsal raphe nucleus (DRN) serotonin (5-HT) neurons to assess the effects of single and repeated ketamine administration on these neurons. Methods: Ketamine (10 mg/kg, i.p.) was administered to male Sprague Dawley rats once or repeatedly (3 times/week) for 2 weeks. After single and repeated administration of ketamine, electrophysiological recordings were done in the VTA, LC and DRN in anesthetized rats, 24 hrs, 3 or 7 days post-administration. Spike frequency, bursting, and for VTA neurons, spontaneously active neurons/trajectory were assessed. Results: In the VTA, LC and DRN, 24 hrs after ketamine was injected acutely there was no significant difference between controls and treated animals in all parameters assessed. However, after repeated administration, there was an increase in bursting and number of spontaneously discharging neurons per tract of VTA DA neurons as well as an increase in frequency of discharge of LC NE neurons. While the increased number of spontaneously discharging neurons per tract had dissipated after 3 days, the enhanced bursting was still present but dissipated after 7 days. As for LC NE neurons, the increased frequency of discharge was no longer present after 3 days. No significant differences in the firing of DRN 5-HT neurons were observed between controls and treated animals even after ketamine was administered repeatedly. Conclusion: These results indicate that repeated but not acute administration of ketamine maintained the increase in population activity of DA neurons and firing activity of NE neurons.

Mental illness has been a perplexing mystery for centuries, inciting both fear and stigmatization. Yet, the knowledge that the brain gives rise to the mind transformed the field of psychiatry; biological studies of aberrant human behavior has revealed that mental disorders are rooted in physical abnormalities that may be targeted to alleviate symptoms. Even with recent progress, there remains many open questions, one of which is: how exactly are some individuals more susceptible to developing these disorders than others? Excess, or traumatic, stress can lead to the onset of maladaptive disorders such as major depressive disorder (MDD) and post-traumatic stress disorder (PTSD). But what if it were possible to prevent these diseases from occurring in the first place? What if there was a prophylactic or vaccine-like approach to increase resilience against environmental stressors? Would we be able to target susceptible populations and administer this prophylactic? In this thesis, I present our work demonstrating the potential for prophylactic pharmaceuticals to enhance stress resilience and protect against stress-induced psychopathology. (R,S)-ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been demonstrated to be a viable candidate drug to administer as a prophylactic against stress-induced psychopathology. It was serendipitously discovered to rapidly (in as little as a half hour) and persistently (up to 2 weeks) alleviate depressive symptoms in patients with MDD. Since its discovery as an effective antidepressant, research has been focused on its mechanism of action with the goal of ultimately developing more efficacious, rapid-acting, long-lasting antidepressant drugs. However, in our lab, we made a truly unexpected discovery in 2016, described in Chapter 2: (R,S)-ketamine prevents the development of psychiatric symptoms when administered before a stressor. We found that prophylactic (R,S)-ketamine is effective against behavioral despair and buffers against learned fear in a time- and dose-specific manner, described in Chapter 3. This was the first indication that a drug can be administered before stress to prevent stress-induced psychopathology, opening a novel field of preventative psychopharmaceuticals. Follow-up studies in our and other labs have consistently replicated this effect using different stressors and mouse strains, in rats, and in both males and females. These data demonstrate that (R,S)-ketamine can effectively enhance resilience pre-clinically. To address how (R,S)-ketamine is inducing long-lasting protection, in Chapter 4, I describe a study that used a metabolomics platform to uncover the long-term effects of (R,S)-ketamine in buffering against learned fear. We found that (R,S)-ketamine alters purine and pyrimidine metabolism in brain and, most notably, the periphery. These data suggest the potential to conduct a simple blood test to screen for biomarkers of prophylactic efficacy in the clinic. However, while these data revealed the end-products of therapeutic efficacy, it was unknown what brain mechanisms may mediate such long-lasting protection against a psychological stressor. In a separate study, the ventral CA3 (vCA3) region of the hippocampus was uncovered to be necessary for (R,S)-ketamine’s prophylactic fear buffering effects, and that targeting this region both mimics and occludes its effects. It was then discovered that 1 week after a single administration of (R,S)-ketamine or FENM, AMPA bursts were attenuated in vCA3. These data reveal vCA3 a central node for prophylactic (R,S)-ketamine efficacy. The biggest limitation of these preliminary studies is that they each only assessed changes at single timepoints rather than mapped out what occurs throughout treatment, during stress, and during recall of a stressor. It remained unknown whether (R,S)-ketamine alters the experience or recollection of a stressor to induce long-lasting protection. The next goal was to use in vivo technologies such as 1-photon Ca2+ imaging in freely-moving mice to develop a more thorough understanding of how exactly (R,S)-ketamine is acting on vCA3 to confer its prophylactic fear buffering effects, which is outlined in Chapter 5. Mice were imaged in the ventral hippocampus throughout a prophylactic (R,S)-ketamine administration paradigm. We found that prophylactic (R,S)-ketamine administration buffered against the experience of the stressor specifically in vCA3 and reduced ventral hippocampal correlated network activity to ultimately buffer against learned fear. These data indicate that (R,S)-ketamine actively buffers against learned fear in the ventral hippocampal at the time of stress. The promise of (R,S)-ketamine is that it is also beneficial as a prophylactic in other settings beyond MDD and PTSD, such as in patients with traumatic brain injury (TBI). In Chapter 6, I describe a study that sought to determine whether (R,S)-ketamine can be useful as a prophylactic for TBI-induced neuropsychiatric effects. Here, TBI mice developed fear generalization, or the inability to distinguish between fear-inducing and neutral stimuli. To understand how TBI alters fear memory traces to promote fear generalization, we used the ArcCreERT2 x enhanced yellow fluorescent protein (eYFP) activity-dependent memory tagging strategy developed by Dr. Christine Ann Denny and found that TBI-induced fear generalization is partially mediated by dentate gyrus (DG) memory trace dysregulation. To reverse this fear generalization phenotype, a single administration of (R,S)-ketamine 1 hour after a TBI prevented the fear generalization phenotype. These data reveal the possibility of administering (R,S)-ketamine or other prophylactics in the clinic as part of post-operative care for TBI patients to prevent long-term fear generalization deficits. Altogether, this thesis demonstrates the potential for pharmacotherapies for stress resilience enhancement and reveals potential targets for prophylactic drug development. We have uncovered the long-term metabolomic changes that occur after a single dose of (R,S)-ketamine, revealed a central node for prophylactic efficacy, mapped the dynamic changes that occur throughout treatment, and applied the prophylactic paradigm to a model of TBI to demonstrate the broad range of applications of this approach. This work paves the way for the novel field of preventative psychiatry and opens new avenues to explore ways to reduce the devastating impact of mental illness on individuals and society.

According to the World Health Organization (WHO) depression is the leading cause of disability worldwide with more than 300 million patients affected. Current antidepressants have a delayed onset of action and moreover, only two-thirds of patients suffering from depressive disorder respond to antidepressant drug treatment. The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of this thesis was to investigate the molecular mechanism of ketamine and its major metabolites at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). As the pathophysiology of depression correlates with decreased adult neurogenesis, I aimed to investigate the molecular effects of ketamine on neural progenitor cell proliferation using a human-based iPSC-model. The findings from this thesis substantially contribute to an enhanced understanding of the molecular mode of action of ketamine as a novel signaling pathway involved in ketamine-induced effects was identified. Ketamine induced proliferation of human iPSC-derived NPCs and bioinformatic analysis of RNA-Seq data revealed significant upregulation of insulin-like growth factor2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 hours after ketamine treatment. In line with this, ketamine dependent proliferation was significantly impaired after IGF2 knockdown. Moreover, ketamine was able to enhance cAMP signaling in NPCs and both, cell proliferation as well as IGF2 expression, were reduced after protein kinase A (PKA)-inhibition. Noteworthy, the Nestin-expressing NPCs do not express functional NMDA receptors, suggesting that the proproliferative effect of ketamine in NPCs is NMDA receptor-independent. Furthermore, 24 hours post administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

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Numerous studies show that a low level of response to the intoxicating effects of alcohol is considered a risk factor for future alcoholism. However, assessing this sensitivity usually requires administering a controlled dose of alcohol, which has a number of inherent problems. Early observations in our lab suggest that the response to anesthetics that show cross tolerance with alcohol, like ketamine, are blunted in nonhuman primates at risk for high alcohol intake, and may be a viable measure of future alcohol consumption. This study was designed to test potential predictors of future alcohol consumption using the change in ketamine across repeated exposures (i.e., tolerance). In addition, potential mediating factors of alcohol consumption, including early temperament and behavior, were assessed. Subjects were 16 three-year-old, alcohol naïve rhesus macaque males raised by their biological mothers. Ketamine Exposure-Each subject was exposed to three 10.0 mg/kg intramuscular doses of ketamine. The time from injection to recovery from anesthetic was recorded for each dose, to be used as a measure of subject's sensitivity and developed tolerance. Alcohol Intake Assessment-Two weeks after the final ketamine dose, subjects were allowed ad libitum access to a palatable 8.4% alcohol solution for two-hours a day, five days a week, for six weeks. During the Two-Choice phase of testing, subjects were simultaneously given ad libitum access to the 8.4% alcohol solution and to a sweetened solution for two-hours a day, five days a week, for four weeks. Solution consumption was recorded daily and averaged across the weeks for each phase of alcohol testing. Temperament and Behavior-As infants, all subjects participated in a bio-behavioral assessment (BBA), when they were between 90 and 120 days of age. Data collected during the BBA on subjects' temperament (Vigilance, Gentleness, Confidence, and Nervousness) and Behavior (Activity and Emotionality) were used in analyses. Results showed a relationship between the tolerance developed between ketamine doses and average alcohol consumption during the Alcohol-Only phase (r = 0.61, R2 = 0.372, F (1,14) = 8.300, p = 0.012). Average alcohol consumption during the Alcohol-Only phase was also related to ratings of Confidence (r = 0.499, R2=0.249, F(1,14)=4.647, p = 0.049), Activity (Day 1: r = 0.503, R2 = 0.253, F(1,14) = 4.732, p = 0.047; Day 2: r = 0.455, R2 = 0.207, F(1,14) = 3.652, p = 0.077), and Emotionality (r = 0.466, R2 = 0.217, F(1,14) = 3.885, p=0.069). The results of this study suggest that change in ketamine recovery time and early life temperament and behaviors may be measures of future risk for alcohol abuse disorders. This data is limited by the small sample size and future study is necessary to further tease out the relationships between these variables and alcohol consumption.

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