A Method for Measuring Dissociative Effects of Sub-Anesthetic Doses of Ketamine in Juvenile Female Rats *

Abstract

Treatment-resistant juvenile depression is a globally prevalent issue, with a need for better pharmacotherapy. Typically, Selective Serotonin Reuptake Inhibitors are front line, but recently there is interest in examining using ketamine or esketamine adjunct in adolescents. In this study, a novel method is used to explore the adverse effects of sub-anesthetic doses of ketamine in a female pre-pubescent rat model, to assess its potential clinical implications. We assessed the severity and duration of dissociative behavior at three sub-anesthetic doses of ketamine (40, 50, and 60 mg•kg⁻¹). Dissociative behaviors cause a subject to feel apart from their reality and ketamine is often abused for such effects. Dissociative behaviors, such as circling and ataxia, were observed following ketamine administration, with higher doses leading to increased severity and duration of symptoms up to 120 min post-injection. Our findings revealed that increasing doses of ketamine intensified both the severity and duration of dissociative behaviors. In conclusion, these results suggest a correlation underlying ketamine's rapid-acting antidepressant effect, particularly with higher sub-anesthetic doses associated with more pronounced dissociative behaviors and a rapid-acting antidepressant effect. Further investigation into ketamine's impact on longer-term dissociative effects is warranted to deepen our understanding of its therapeutic risks of ketamine treatment of adolescent depression.

Introduction

There is an unmet need to develop better drugs for treating depression in adolescents. First line drugs for treating adolescent depression are the serotonin selective reuptake inhibitors (SSRIs), which have a delayed mechanism of action. Typically, it takes 4-6 weeks of chronic SSRI administration to observe an antidepressant effect in patients. Furthermore, there are also developmental differences in the responses to antidepressant drugs. Children respond to serotonin selective reuptake inhibitors (SSRIs),^1,2 but often inadequately. Children do not respond to norepinephrine reuptake inhibitors.^3,4 Indeed, only the SSRIs are approved for adolescent depression by the FDA. All the reuptake inhibitor drug classes have some efficacy in adults.

In 2000, considerable excitement arose from a clinical trial testing the antidepressant actions of sub-anesthetic doses of ketamine. ⁵ A robust antidepressant effect occurs within hours of ketamine infusion. This trial and subsequent trials established that ketamine, and its S enantiomer, Esketamine, are rapid acting antidepressants^6–8 culminating in the approval by the FDA of Esketamine) in 2019. Esketamine, formulated as a nasal spray, is approved for adjunct use with an SSRI in adults only with treatment resistant depression. There is a need to address whether ketamine has rapid acting antidepressant effect in adolescents and antidepressant-like effects in juvenile rats. There is data indicating in juvenile female rats, ketamine reduced depressive-like behavior (unpublished data, Brianna Stanfield). Sub-anesthetic doses of ketamine are seeing increasing use as a rapid-acting antidepressant adjunct with selective serotonin reuptake inhibitors (SSRIs).^9,10

However, there are concerns about the abuse potential and dissociative effects of subanesthetic ketamine.^9,11 In an initial study of sub-anesthetic doses of ketamine in healthy volunteers, it was found that lower doses produced behavioral effects similar to schizophrenia, whereas higher doses produced altered perception, derealization and depersonalization. ¹² These dissociative effects could pose a potential problem for ketamine's safety in juveniles, raising concerns for adolescent use of Ketamine for treating treatment resistant depression (TRD). Ketamine is used across many scopes of practice besides psychiatry, including surgery as an FDA approved anesthetic, the emergency room as an analgesic, and in veterinary medicine.^13,14 Ketamine, however, has side effects. Ketamine can cause a subject to temporarily lose sense of reality, be confused, or have poor control over voluntary movements shortly after administration (ataxia). These adverse dissociative symptoms occur rapidly following administration and subside after about two hours. ¹⁵ This information is well-studied in adult rats and humans, but not in adolescent rats. More research is needed for safe sub-anesthetic ketamine use in children with treatment-resistant depression. The underlying signaling pathways required for ketamine's mechanism of action are not completely established in both adult and juveniles. Based on other antidepressant actions, there could be differences in ketamine's action between adults and juveniles. There are developmental differences in the responses to reuptake inhibitors. Serotonin selective reuptake inhibitors (SSRIs) have efficacy,^1,2 but norepinephrine reuptake inhibitors lack demonstrated efficacy in adolescents.^3,4

The lack of data on both the antidepressant and dissociative effects on the use of ketamine in treating depression limits the use of ketamine in adolescents. In this study, the dose dependence and time course of ketamine's dissociative effects are investigated using a dissociative behavior observation method in juvenile PND 25 to 29 female Sprague-Dawley rats with a new analytic tool, using some established dissociative behaviors in adult rats. ¹⁶ Whether the data correlate well or not with data from adult patients and rats is discussed.

Materials and Methods

Animals

Female Sprague-Dawley rats (n = 72; ∼70 g, PND 25-29, Hilltop) were housed on-site in cages under regulated temperature (21 ± 1 °C) and light conditions (12-h light/dark cycle) at A.T. Still University – Kirksville College of Osteopathic Medicine's (ATSU-KCOM) animal care facility. Food and water were available ad libitum. All experimental protocols were approved by the KCOM Institutional Animal Care and Use Committee (IACUC), which maintains AAALAC accreditation and an assurance with OLAW.

Assessment of Dissociative Behaviors

Rats born in the same litter and approximately the same weight (∼70 g) were randomly divided into control and ketamine groups (n = 6 each) and studied independently at three different ketamine doses (40, 50, and 60 mg•kg⁻¹). Once divided into groups, rats were weighed and administered a single intraperitoneal (i.p.) injection in the lower right quadrant with either ketamine in saline (Vet One, Boise, ID) or 0.9% normal saline vehicle (Intermountain Life Sciences, West Jordan, UT). Doses of ketamine used were based on unpublished data (Brianna Stanfield) using the tail suspension test (TST) as a model of depressive-like behavior. Rapid-acting antidepressant effect were found with 40 and 60 mg•kg-1 ketamine compared to the saline vehicle. Rats were placed in a fresh cage with bedding from the animal care facility to mimic its daily environment and video recorded for five minutes at various time points (0, 20, 40, 60, and 120 min) post-injection.

After the video recording, each video was randomly assigned a number. Three observers, not involved in administering injections or video recording, were trained to identify dissociative behaviors (Figure 1). Dissociative behaviors include circling, head weaving, and temporary paralysis. ¹⁷ All observers blinded to the treatment conditions individually watched every recording and used a predefined tally sheet to record the occurrences of dissociative and non-dissociative (eg, grooming, burrowing, and rearing) behaviors. ¹⁷ To measure intra-rater reliability, one control and one ketamine video for each dose were duplicated and unknowingly re-watched by the observers. The provided tally sheets were used to summarize behavior occurrences for each rat at each time point post-injection, create a dissociative index, and information on inter-rater variability. The dissociative index was created by subtracting the total dissociative behaviors from the total non-dissociative behaviors a single observer saw in the video recording. The dissociative index was then normalized between +1.0 and −1.0.

Figure 1.

Dissociative behavior time course experimental timeline. The procedural timeline for conducting the dissociative behavior time course protocol.

Data Analysis

Statistical analyses were done using GraphPad Prism 9. During the dissociative time course assay, all observer data was compiled into Excel (Microsoft). The number of perceived behavior occurrences for each video was totaled as being either dissociative or non-dissociative. The total dissociative behaviors for the video were subtracted from the total non-dissociative behaviors during the video, creating a “dissociative index”. The dissociative index was then normalized by dividing the behavior category (dissociative or non-dissociative) with the highest frequency of behaviors for that video from the dissociative index. This normalized dissociative index was then compared between treatment groups in Prism using an unpaired two-tailed t-test at each time point and dose.

The consistency between the observers (Inter-rater variability) was measured by conducting a Two-Way Repeated Measures ANOVA with a Tukey post hoc. Intra-rater reliability was measured using a Wilcoxon Signed-Rank test to compare the difference in dissociative index the observer perceived the first time watching the video versus the second time watching the same video. For each dose, there were one saline, and one ketamine video replicated for the observer to watch. The observers were not aware of this control in the study.

The prevalence of each behavior among ketamine and control rats was totaled at each time point and categorized as ketamine or saline control in Excel. The most prevalent behavior for the ketamine and the control category was used as the denominator and every other behavior in that testing group for a single dose category was divided by that denominator and normalized to 100%. The behavior prevalence analysis was conducted to get a better understanding of the most common dissociative behaviors in juvenile rats under the influence of ketamine.

Results

Dissociative Behavior Time Course

To determine how long dissociative behaviors last in a juvenile model a time course of dissociative behavior was conducted at three doses (40, 50, and 60 mg•kg⁻¹) of ketamine and compared to saline vehicle control at five time points (0, 20-, 40-, 60-, and 120-min. post-injection). 40 and 50 mg•kg⁻¹ ketamine (Figure 2(A) & (B)) induced significant dissociative symptoms, which started to fade at the 60-min time point post-injection. At 120 min post-injection, was not different from the saline vehicle control, thus indicating the dissociative symptoms had worn off. The data for the 60 mg•kg⁻¹ ketamine dose (Figure 2(C)) was significantly different when compared to saline at all five time points: 0, 20-, 40-, 60-, and 120 min post-injection. The average dissociative index did not appear to fade throughout the entire 120-min time course. The 60 mg•kg⁻¹ dose was different from both the 40 and 50 mg•kg⁻¹ doses, as the dissociative symptoms had worn off by the 120-min time-point in the 40 and 50 mg•kg⁻¹ doses. To test whether the dissociative effects are still relatively short lived even with a 60 mg•kg⁻1 dose, an experiment was done testing. 60 mg•kg⁻¹ dose at 1 and 4 h. The data contrasting 60 mg•kg⁻¹ ketamine dose was significantly different when compared to saline at 60-min post-injection, but not at the 240-min post-injection time point (data not shown) confirming the dissociative effects are still relatively short lived.

Figure 2.

Dissociative behavior time course. Dissociative index over time for saline and ketamine treated rats. Ketamine dosages were 40 mg•kg⁻¹ (A), 50 mg•kg⁻¹ (B), and 60 mg•kg⁻¹ (C) shown as a bar graph N = 6 per group # = p < .05 versus saline at each time point. Two-Way ANOVA test was done by averaging dissociative indexes (n = 6) at every timepoint for each observer thus ending with n = 3. The ANOVA test revealed an interaction of treatment over time at 40, 50, and 60 mg/kg⁻¹. The Sidak correction test found a point of significant difference at 0 min. post injection between the ketamine and saline treatment group at 40, 50, and 60 mg/kg⁻¹ indicated by a ^# symbol.

Inter-Rater Variability

To test for consistency between the three observers, an inter-rater variability test was conducted to compare each observer's mean normalized dissociative index score at a single time point. At all three doses (40, 50, and 60 mg•kg⁻¹) of ketamine and all five time points (0, 20-, 40-, 60-, and 120 min) (Figure 3(A) to (C)), there were no points of significant difference between any of the three observers (A, B, C). This outcome indicated that all three observers had a similar interpretation of the rat behaviors.

Figure 3.

Inter-Rater variability. Dissociative index over time for saline and ketamine treated rats. Ketamine dosages were 40 mg•kg⁻¹ (A), 50 mg•kg⁻¹ (B), and 60 mg•kg⁻¹ (C) shown as a bar graph. Dissociative indexes for the same dose, treatment, and time were compared between the three observers. N = 6 per group. Two-way ANOVA with Tukey post-hoc.

Intra-Rater Reliability

Another test for observer consistency was conducted, called an intra-rater reliability test. In this single-blind experiment, each observer was given a repeat recording of a single ketamine and saline vehicle video recording at a single time point. Repeated video recordings were conducted once for each dose of ketamine. The difference in normalized dissociative index scores between the first and second viewing, regardless of treatment, was compared to an expected no difference to test for statistical significance. This test determined if the observer was consistent in identifying dissociative or non-dissociative behaviors. Among the three observers (Figure 4) there were no differences between the first and second viewing of the same video recordings. This data illustrated the consistency of each observer in scoring the same video recording the second time that is similar to the first time they watched the video. Observer C had the highest difference in normalized dissociative index scores between the first and second viewing of the video recordings (Figure 4). Observer C miscategorized saline-treated rats as being under paralysis while watching saline-treated rats compared to the 40 mg•kg⁻¹ ketamine-treated rats. This video session was right after the initial training of the observers to identify behaviors, meaning the observers were inexperienced at the start of the 40 mg•kg⁻¹ video recording session. An additional behavior, burrowing, was added to the 50 and 60 mg•kg⁻¹ session recordings and will be discussed in the “behavior prevalence” section.

Figure 4.

Intra-Rater reliability. Dissociative index difference score for each observer: A, B, and C are shown as a box and whisker plot. N = 6 per group. Wilcoxon Signed-Rank test.

Dissociative and Non-Dissociative Behavior Prevalence

To determine the frequency of which behaviors occur in saline-treated, and ketamine treated juvenile rats, the occurrence of behaviors were calculated as a percentage of the most prevalent behavior in each behavioral category, dissociative or non-dissociative, at each dose. The most prevalent dissociative behavior at 40 mg•kg⁻¹ was circling followed by ataxia (Figure 5). The most prevalent non-dissociative behavior at 40 mg•kg⁻¹ was rearing. The miscategorized saline-treated rats as being under paralysis as discussed in the section above was due in part to not being trained to identify the resting non-dissociative behavior. Thus, a third non-dissociative behavior, “burrowing”, was added to the 50 and 60 mg•kg⁻¹ ketamine treatment as described previously. The observers were trained to categorize any sleeping/resting behavior as burrowing. Following this addition, the rate of paralysis in saline-treated rats went from 3% at 40 mg•kg^−1% to 0% at 50 and 60 mg•kg⁻¹ (Figure 5). The most prevalent dissociative behavior at 50 mg•kg⁻¹ was head weaving followed by circling (Figure 5). The most prevalent dissociative behavior at 60 mg•kg⁻¹ was circling followed by ataxia (Figure 5). As the dose of ketamine increased by 10 mg•kg⁻¹ the rate of paralysis increased by about two-fold (Figure 5).

Figure 5.

Dissociative and Non-dissociative behavior frequency. Prevalence of both dissociative and non-dissociative behaviors for saline and ketamine treated rats. Ketamine dosages were 40 mg•kg⁻¹ (A), 50 mg•kg⁻¹ (B), and 60 mg•kg⁻¹ (C) shown as individual tables. Prevalence normalized to a max of 100%.

The most common non-dissociative behaviors for observers to look for after a saline i.p. injection are rats rearing on their hind legs and burrowing in the bedding or sleeping. The most common dissociative behaviors for rats having received a ketamine i.p. injection are circling, ataxia, and head weaving respectively.

Discussion

Sub-anesthetic doses of ketamine led to dissociative behaviors in female juvenile rats, manifested as circling, head weaving, ataxia, and an increase in time paralyzed with increasing dosage of ketamine. The dissociative symptoms wore off by 60 min post-injection for the lower two sub-anesthetic doses (40, and 50 mg•kg⁻¹) of ketamine tested. Dissociative symptoms were still present at 120 min post-injection for the 60 mg•kg⁻¹ dose but wore off by 240 min. This correlates closely to the time course of Ketamine's dissociative effects in humans, which start within 10 min of infusion initiation and wear off within about an hour. ¹⁸ Indeed, the FDA mandates stay in the clinic for 2 h after nasal administration of esketamine to let the dissociative effects wear off. To summarize, ketamine elicits dissociative effects at sub-anesthetic doses in female juvenile rats that are similar to adult rats and humans, but this does little to address whether there are differences in the mechanism of ketamine's dissociative action between juveniles and adults.

One study investigated the dissociative stereotypy of adult male Sprague-Dawley rats receiving a single i.v. infusion of ketamine. ¹⁹ Radford et al found that there were dose-dependent differences in the prevalence of dissociative behaviors during the last 10 min of the infusion. It was found that there were increased dissociative behaviors and a decrease in non-dissociative behaviors at 20 mg/kg/h ketamine infusion. ¹⁹ On the other hand, rats receiving 5 and 10 mg/kg/h showed similar behavioral patterns as to the saline controls, except for a reduction in rearing and grooming behavior frequency. ¹⁹ This finding is both similar and contradictory to what was discovered administering a single i.p. injection of ketamine to juvenile female rats. Our results were similar by indicating a dose-dependent nature to the prevalence of dissociative behaviors following administration of ketamine. We found as the dose of ketamine increased, there was also an increase in dissociative symptoms as well as a decrease or complete absence of non-dissociative symptoms. However, at lower doses we still saw dissociative effects of ketamine, which is contradictory to Radford et al when administering the 5 mg/kg/h i.v. infusion of ketamine. We also found that as the dose of ketamine increased, so did the severity and longevity of the dissociative symptoms. At the highest sub-anesthetic dose of ketamine, there were still dissociative symptoms present at 120 min post-injection which was unlike both the 40 and 50 mg•kg-1 doses that wore off by 120 min post-injection. This time course indicates that the lower two doses have a different dissociative behavior time course than the 60 mg•kg-1 dose of ketamine. The drugs used in our study were delivered via an i.p. injection with a lower bioavailability than an i.v, infusion^19–21 The lower bioavailability in the i.p. injection may account for the difference in the manifestations of the dissociative behaviors between Radford et al's study and our own.

The mechanism of ketamine's dissociative action is likely blockade of N-methyl-D-aspartate (NMDA) receptors. NMDAR blockade is thought to underlie the dissociative anesthetic and amnesic effects of ketamine. ²² The S enantiomer of ketamine is more potent than the R enantiomer at altering sensory perception, which matches affinity for NMDA receptors suggesting NMDA receptor blockade underlies ketamine's dissociative effects. ²³ There is little evidence that the ketamine dissociative mechanism is different between juveniles and adults, however, other antidepressant drug classes do have developmental differences. Escitalopram, a SSRI, but not desipramine, a noradrenergic reuptake inhibitor, induce brain derived neurotrophic factor (BDNF) expression in the juvenile rat's hippocampus.^24,25 Furthermore, fluoxetine, a SSRI, but not desipramine induces neurogenesis in juvenile rats. ²⁶

It is not clear whether dissociative effects are required for ketamine's rapid acting antidepressant effect. A thorough review of the literature found the data is mixed on whether dissociation is required for an antidepressant effect ⁹ However, Luckenbaugh et al found, in a dose dependent fashion, that “only dissociative side effects predicted a more robust and sustained antidepressant” response.

In conclusion, the findings in this study indicate that increasing doses of ketamine increases both the severity and duration of dissociative behaviors in juvenile rats up to 120 min post-injection. Investigations are underway to determine the time course and dose response of ketamine's antidepressant-like effects in juvenile rats, and whether ketamine has analgesic effects in juvenile rats. This work builds upon increasing clinical research that ketamine and esketamine may be useful for treating adolescent depression.