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. Author manuscript; available in PMC: 2021 Apr 26.
Published in final edited form as: Neuropharmacology. 2020 Jan 9;166:107947. doi: 10.1016/j.neuropharm.2020.107947

Ketamine increases vmPFC activity: Effects of (R)- and (S)-stereoisomers and (2R,6R)- hydroxynorketamine metabolite

Brendan D Hare 1, Santosh Pothula 1, Ralph J DiLeone 1, Ronald S Duman 1,2
PMCID: PMC8073170  NIHMSID: NIHMS1550120  PMID: 31926944

Abstract

Ketamine, an NMDA receptor antagonist and fast acting antidepressant, produces a rapid burst of glutamate in the ventral medial prefrontal cortex (mPFC). Preclinical studies have demonstrated that pyramidal cell activity in the vmPFC is necessary for the rapid antidepressant response to ketamine in rodents. We sought to characterize the effects of ketamine and its stereoisomers (R and S), as well as a metabolite, (2R,6R)-hydroxynorketamine (HNK), on vmPFC activity using a genetically encoded calcium indicator (GCaMP6f). Ratiometric fiber photometry was utilized to monitor GCaMP6f fluorescence in pyramidal cells of mouse vmPFC prior to and immediately following administration of compounds. GCaMP6f signal was assessed to determine correspondance of activity between compounds. We observed dose dependent effects with (R,S)-ketamine (3 to 100 mg/kg), with the greatest effects on GCaMP6f activity at 30 mg/kg and lasting up to 20 mins. (S)-ketamine (15 mg/kg), which has high affinity for the NMDA receptor channel produced similar effects to (R,S)-ketamine, but compounds with low NMDA receptor affinity, including (R)-ketamine (15 mg/kg) and (2R,6R)-HNK (30 mg/kg) had little or no effect on GCaMP6f activity. The initial response to administration of (R,S)-ketamine as well as (S)-ketamine is characterized by a brief period of robust GCaMP6f activation, consistent with increased activity of vmPFC pyramidal neurons. Because (2R,6R)-HNK and (R)-ketamine are reported to have antidepressant activity in rodent models the current results indicate that different initiating mechanisms could to lead to similar brain adaptive consequences that underlie the rapid antidepressant responses.

Keywords: Ketamine, Hydroxynorketamine, Depression, Frontal Cortex, NMDA

1. Introduction

Over 15% of the United States population is impacted by depression with a resultant economic burden of over $200 billion (Greenberg et al., 2015; Kessler et al., 2003). On a global scale, depression is categorized as one of our most devastating illnesses with burden peaking in early adulthood (Guilbert, 2003; Whiteford et al., 2013). Due to limited efficacy and time lag for therapeutic response of available medications, as well as difficulties in finding appropriate dose and drug combinations, current treatments may take months to years to improve symptoms, and one third of individuals are not responsive and are considered treatment resistant (Trivedi et al., 2006). Ketamine, an NMDA receptor antagonist, has recently produced great excitement in the field of depression as a rapid acting antidepressant. A single dose of ketamine produces an antidepressant response within hours of treatment that lasts for approximately one week (Berman et al., 2000; Zarate et al., 2006). The (S)-entaniomer, referred to as esketamine in the form of a nasal application has recently been approved for TRD by the USFDA. Studies of the factors that precipitate ketamine’s antidepressant response are ongoing, and could offer novel therapeutic targets that produce similar rapid antidepressant actions but without the dissociative and psychotomimetic side effects of ketamine and esketamine.

Studies investigating the mechanism of action of ketamine, as well as the pathophysiology of depression have focused on several cortical and limbic brain regions, and one that is particularly relevant is the PFC (Duman et al., 2019). Brain imaging studies report a reduction in PFC volume in depressed patients that is correlated with duration of depression (Savitz and Drevets, 2009), and postmortem studies report evidence of decreased expression of synaptic proteins and synaptic number in PFC subregions (Kang et al., 2012). Pre-clinical studies using stress to model depression have reported dendrite atrophy and loss of synaptic number and function in the vmPFC (Radley et al., 2006; Liu et al., 2008). In contrast, a single dose of ketamine increases synaptic number and function in the vmPFC and reverses the synaptic deficits caused by chronic stress exposure (Li et al., 2011). Pre-clinical studies have also demonstrated that infusions of ketamine into the vmPFC are sufficient to produce antidepressant behavioral responses and that vmPFC is required for the actions of systemic ketamine administration (Fuchikami et al., 2015). These findings support the hypothesis that ketamine-induced plasticity within the vmPFC is critical for antidepressant responses in depressed patients.

Recent studies demonstrate rapid antidepressant effects in response to the (R)- and (S)-stereoisomers of ketamine, with (R)-ketamine having the potential for a reduced side effect profile compared to (S)-ketamine, based on rodent studies (Chang et al., 2019; Fukumoto et al., 2017; Shirayama and Hashimoto, 2017; Yang et al., 2015). This is thought to be related to the four-fold lower affinity of (R)- vs. (S)-ketamine for the NMDA receptor channel (White et al., 1985). In addition, a metabolite of ketamine, (2R,6R)-HNK, which also has a reduced side effect profile, produces rapid antidepressant actions in rodent models at concentrations that do not interact with the NMDA receptor (Lumsden et al., 2019; Morris et al., 2017; Zanos et al., 2019; Zanos et al., 2016) but see (Kavalali and Monteggia, 2018). Detailed ligand binding and electrophysiological studies demonstrate that (R,S)-ketamine is greater than 50 fold more potent than (2R,6R)-HNK at blocking NMDA receptors (Lumsden et al., 2019).The antidepressant actions of these agents, with reduced or very low affinity for the NMDA receptor indicates that (R)-ketamine and (2R,6R)-HNK have different initial targets than (S)-ketamine (Hashimoto, 2019; Lumsden et al., 2019; Zanos et al., 2019; Zanos et al., 2016).

Microdialysis studies report that ketamine produces a rapid but transient (30 to 80 min) increase in extracellular glutamate in the mPFC (Moghaddam et al., 1997). This finding is consistent with other work indicating that NMDA receptors on GABA interneurons are primarily affected by low, subanesthetic doses of NMDA receptor antagonists like ketamine, leading to increased glutamate transmission and increased pyramidal cell activity immediately following administration (Homayoun and Moghaddam, 2007; Jackson et al., 2004). To our knowledge the immediate effects of the individual ketamine enantiomers and (2R,6R)-HNK on mPFC activity have not been examined.

Recent advances in genetically encoded calcium indicators (e.g. GCaMP), and techniques to record GCaMP associated fluorescence from the brain, allow in-vivo monitoring of cellular responses in freely behaving animals (Lerner et al., 2015). These techniques can be leveraged to monitor cellular activity in genetically defined populations of neurons in the period surrounding experimental manipulations. Given the known increase in glutamatergic transmission following (R,S)-ketamine we sought to examine whether GCaMP activity in vmPFC pyramidal neurons could be monitored, and compared (R)- and (S)-ketamine as well as (2R,6R)-HNK. To achieve this aim we implemented a viral vector strategy to target GCaMP6f to vmPFC Camk2α expressing pyramidal cells. Subsequent to viral expression we utilized ratiometric fiber-photometry to monitor fluorescence activity through a vmPFC targeted indwelling optical fiber in the period immediately after rapid acting antidepressant administration.

2. Methods

2.1. Animals

Experiment 1 utilized Camk2a-Cre recominbase mice (8–12 weeks) (n=19) originally obtained from Günter Shütz (German Cancer Research Center, Heidelberg, Germany) and bred on a C57BL/6J background. Remaining experiments used male C57BL/6J mice (8–12 weeks) (n=26; Jackson Laboratories, Bar Harbor ME). Mice were housed under 12 h light-dark cycle with ad libitum access to water and rodent chow. Animals were group housed until surgery after which they were single housed to prevent dislodging of cannula. Housing was maintained in a ventilated rack, and contained corn cob bedding and a nestlet. Animal use and procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Yale University Animal Care and Use Committees.

2.2. Surgical procedures

For all stereotaxic surgeries, animals were anesthetized with a ketamine/xylazine (120mg/kg/10mg/kg) cocktail. Animals were administered a subcutaneous injection of carprofen (5mg/kg, Zoetis, Parsippany, NJ) prior to surgery, and received additional injections for two days following surgery. Once anaesthetized, fur at the incision site was removed and eyes were coated with ophthalmic ointment. Next, animals were headfixed in a stereotaxic apparatus (David Kopf, Tujunga, CA) and the incision site was sterilized. A single craniotomy was made and a Hamilton syringe (Reno, NV) fitted with a 28 gauge needle was used to place a viral bolus (500nL) at the following coordinates in mm within the mPFC (AP: 1.9, ML: 0.4, DV −2.8). Fiber optic cannula (400uM core, 0.48NA, Ceramoptec, Germany) were implanted at the same locations as the viral bolus. Cannula were fixed to the skull using dental cement and a pair of skull screws.

2.3. Virus

For experiment 1 utilizing Camk2a-Cre mice a Cre-dependent GCaMP6f was employed (AAV9.Syn.Flex.GCaMP6f.WPRE.SV40, Addgene, Watertown, MA, United States). For the subsequent experiments C57BL6J mice were infused with a viral vector with GCaMP6f under the control of a Camk2a promotor (AAV5.CamKIIa.GCaMP6f.WPRE.SV40, Addgene) or GFP control virus (AAV5.CamKIIa.GFP, Addgene). Viruses were injected at a titer of ~2X1012 GC/per mL.

2.4. Drugs

(R,S)-ketamine (Sigma Aldrich, St. Louis, MO, United States) was administered at various doses (3 mg/kg, 10 mg/kg, 30 mg/kg, 100 mg/kg, i.p.). (S)-ketamine (Sigma Aldrich) and (R)-ketamine (Cayman Chemical, Ann Arbor, MI, United States) were administered at a dose of 15 mg/kg (i.p.); (2R,6R)-HNK (NCATS Chemical Genomics Center, Bethesda, MD, United States) was administered at a dose of 30 mg/kg (i.p.). 0.9% saline was used as the vehicle for all experiments, and administered through the same route as drug treatment.

2.5. Fiber photometry apparatus

Ratiometric fiber photometry was conducted as previously described (Lerner et al., 2015) with a system comprised of a Tucker Davis (Alachua, Florida, United States) RZ5P processor controlled by the Synapse software suite. The RZ5P controlled excitation (473nm) and control signal (405nm) LEDs (Doric, Canada) modulated at 531 and 211hz respectively. LED power was adjusted to ensure the photodetector (Newport 2151,Irvine, Californa, United States) receiving light was not being saturated, and measured ~30uW at the fiber tip. Light was passed through a minicube (FMC6AE, Doric, Canada) that contained GFP excitation and emission filter sets. Animals were tethered to the system via fiber optic patch cord (400 uM core, 48NA, Doric, Quebec, Canada) connected to head mounted fiber optic cannula via ceramic sleeve.

2.6. Experimental design

For all experiments, viral infused mice were tethered to a patch cord, returned to their home cage and allowed a 30 minute recorded habituation period. Habituation not only served to allow the animals to normalize following tethering to the apparatus, but also allowed for rapid photobleaching observed early in some traces to abate prior to manipulation.

For the first experiment (n = 19, reported in 3.2), a racemic ketamine dose response, animals were injected with vehicle following habituation and activity was recorded for one hour. When the vehicle recording period was complete animals were injected with drug and again activity was recorded for one hour. After the recording period the patch cord was removed and animals were returned to their home cage. Animals first received doses of 3 or 10 mg/kg (i.p.) ketamine. For the higher doses animals that had received 10 mg/kg ketamine in a first recording session were subjected to a second recording session two weeks after the first, and received 30 or 100 mg/kg ketamine.

For the second experiment (n=26, reported in 3.3) we sought to compare the effects of (R,S)-ketamine with its (R)- and (S)-stereisomers and (2R,6R)-HNK metabolite, which are reported to have rapid antidepressant activity in rodents. For (S)- and (R)-ketamine we opted for doses (15 mg/kg, n=4/group) that were equivalent to the dose of (R,S)-ketamine (30 mg/kg, n=4) administered in the dose response experiment, and also tested (2R,6R)-HNK (n=5) at a dose of 30 mg/kg, which we have previously observed to produce antidepressant effects in rodent models (Fukumoto et al., 2019). Additionally, in this experiment a vehicle group was compared to treatment in a between subject fashion (n=4), and a second control, viral-GFP infused mice were also administered 30 mg/kg racemic ketamine (n=5). For this experiment animals were tethered to the fiber photometry apparatus and habituated as before. A single injection, drug or vehicle, followed the habituation period, and animals were recorded for one hour. Following the recording period the patch cord was removed and animals were returned to their home cage.

2.7. Fiber photometry data analysis

Using matlab scripts each signal was decimated to 381 hz and low pass filtered with a frequency cutoff of 5 Hz.The excitation signal was regressed against the control signal and ΔF/F was calculated with the excitation signal residuals (residuals/predicted). For each animal, the fifteen through twenty minute block of the habituation period was used for comparison to periods after vehicle or drug treatments. These periods were normalized by z-score to standardize signals across animals prior to analysis. Area under the curve (AUC) values were summated across signal time-bins. MAD was calculated for each habituation period and a threshold was set 2.91 deviations above the MAD (Calipari et al., 2016). MAD is a central tendency estimator robust against outliers, and in the normalized data set the 2.91 MADS-from-median threshold identifies data points highly distant from the median. This threshold was applied to the remainder of the signal. Areas with GCaMP signal above MAD threshold for >= 0.2 seconds (i.e. approximate decay rate for GCaMP6f) were identified as peaks. For analysis of samples above MAD threshold the sum of the number of samples above the MAD threshold was calculated for each time-bin.

2.8. Statistical analysis

Data were processed in Matlab r2018b (Mathworks, Natick, MA, United States) and analyzed using Prism 8 (GraphPad, San Deigo, CA, Untited States). Analysis consisted of two way ANOVA (treatment X time) implementing time, and where appropriate treatment, as a within-subject variable. The Geisser-Greenhouse correction was employed but had no bearing on results reporting so ANOVA results assuming sphericity are reported. Dunnet corrected posthoc tests are reported. Data are reported as mean ± SEM.

3. Results

3.1. Experimental approach and GCaMP6f viral expression in vmPFC

A GCaMP6f viral vector was infused into the vmPFC and then a fiber optic cannula was placed within an area covering the viral infused area (Fig. 1A). Following treatment and recording animals were sacrificed and cannula locations within the area of GCaMP6f were verified. Cannula locations across experiments were consistent in most animals, and fell within the target area delineated in Fig. 1A. Five animals showed off target viral infusions and/or low viral expression, resulting in low GCaMP6f activity peak when handling and injections took place. Data from the remaining 45 animals were z-normalized to signal obtained during the 15–20 min time bin of the habituation period prior to final analysis (for example, see Fig. 1B).

Fig. 1.

Fig. 1.

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Experimental methods and analysis. (a) Viral infusion and fiber optic cannula placement into vmPFC two to three weeks prior to treatment and recording. (b) Representative viral expression in vmPFC and area where cannula tips were observed (grey). (c) Example processed GCaMP6f signal with MAD threshold labeled. Scale bar = 100μm

3.2. (R,S)-Ketamine dose response effects on GCaMP6f activity in vmPFC

Visual inspection of the data demonstrated that ketamine administration altered calcium transients after dosing indicative of increased neuronal activity (Fig. 2A). To quantify this change, area under the curve was computed for individual signal traces in one minute time-bins across the 60 minute monitoring period. As animals were being compared to their own vehicle treatments (R,S)-ketamine doses were analyzed independently.

Fig. 2.

Fig. 2.

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GCaMP6f response following (R,S)-ketamine administration. (A) Minimally processed mean signal relative to prior vehicle treatment in 3mg/kg (n=7), 10mg/kg (n=6), 30mg/kg (n=5), and 100mg/kg (n=4). Top trace represents minutes 0–30 after treatment, bottom represents minutes 30–60. (B) 1 minute binned analysis of the AUC for GCaMP6f signal following ketamine treatment relative to vehicle treatment from the corresponding time period. (C) 1 minute binned analysis of the samples above MAD threshold relative to vehicle treatment from the corresponding time period. Data presented as mean ± sem.

3.2.1. 30 mg/kg (R,S)-Ketamine

Administration of 30 mg/kg (R,S)-ketamine (n=5) produced a large increase in AUC during the period immediately after ketamine that was substantiated by a significant interaction ((timeXtreatment, F(59,236)=3.84, p<0.0001, Fig. 2B). As it seems more likely that increased activity over a sustained period of time is important for influencing brain adaptive changes after (R,S)-ketamine treatment we further collapsed activity into 5 minute time bins for comparison between (R,S)-ketamine treatment and respective vehicle treatment periods. Analysis of 5 minute periods (timeXtreatment, F(11,44)=5.06, p=0.008, Fig. 3A) showed significant differences through the 20 minute period immediately following dosing when compared to the corresponding vehicle period (minutes 1–5, t(8)=6.99, p<0.0001; minutes 6–10, t(8)=6.02, p<0.0001; minutes 11–15, t(8)=4.55, p<0.0001; minutes 16–20, t(8)=2.45, p=0.018). Analysis of the samples above MAD threshold mirrored that of AUC (timeXtreatment F(59,236)=4.83, p<0.0001, Fig. 2C). GCaMP6f activity from four of the five animals remained above MAD threshold for approximately 10 minutes after injection limiting the utility of peak counting. However, the samples above MAD threshold following dosing were significantly different than vehicle (minutes 1–5, t(8)=7.77, p<0.0001; minutes 6–10, t(8)=5.94, p<0.0001; minutes 11–15, t(8)=5.72, p<0.0001; minutes 16–20, t(8)=2.91, p=0.006; Fig. 3B).

Fig. 3.

Fig. 3.

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(R,S)-ketamine dose response. GCaMP6f signal relative to prior vehicle treatment in 3mg/kg (n=7), 10mg/kg (n=6), 30mg/kg (n=5), and 100mg/kg (n=4) (R,S)-ketamine treated mice. (A) 5 minute binned analysis of the AUC for GCaMP6f signal following ketamine treatment relative to vehicle treatment from the corresponding time period. (B) 5 minute binned analysis of the samples above MAD threshold relatiave to vehicle treatment from the corresponding time period. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. Data presented as mean ± sem.

3.2.2. 10 mg/kg (R,S)-ketamine

Analysis of AUC following 10 mg/kg (R,S)-ketamine treatment (n=6) produced a significant interaction (timeXtreatment, F(59,295)=2.10, p<0.0001, Fig. 2B), although in this case treatment effects appear during the period immediately after treatment, as well as in the final minutes of the recording period. The dynamic response is evident in analysis of five minute time periods (timeXtreatment, F(11,55)=2.66, p=0.008, Fig. 3A). In the 5 minutes following treatment a significant increase in AUC is observed (t(10)=3.49, p=0.001). 10 mg/kg (R,S)-ketamine also produced elevated AUC effects in the later sampling periods (minutes 51–55, t(10)=2.39, p=0.021; minutes 55–60, t(10)=2.13, p=0.038). Analysis of the samples above MAD threshold showed a significant interaction (timeXtreatment, F(59, 295)=1.54, p=0.012, Fig. 2C), and significant post-hoc effects in the first five minute period (t(10)=4.28, p<0.0001) and during the 51–55 minute period (t(10)=2.10, p=0.04, Fig. 3B). We did not observe a change in peak event number after 10 mg/kg ketamine treatment.

3.2.3. 3 mg/kg (R,S)-Ketamine

Analysis of GCaMP6f activity following 3 mg/kg (R,S)-ketamine (n=7) produced a significant interaction (timeXtreatment, F(59,354)=2.04, p<0.0001, Fig. 2B) with between treatment effects evident during the last 30 minutes of the recording period. A significant interaction was observed in the 5 minute analysis (timeXtreatment, F(11,66)=2.91, p=0.004, Fig. 3A) with significantly elevated signal appearing after 30 minutes and persisting through the remainder of the recording period (minutes 31–35, t(12)=3.06, p=0.003; minutes 36–40, t(12)=2.44, p=0.018; minutes 41–45, t(12)=2.02, p=0.047; minutes 46–50, t(12)=3.31, p=0.002; minutes 51–55, t(12)=1.93, p=0.059; minutes 55–60, t(12)=2.48, p=0.016). Analysis of the samples above MAD threshold in one minute bins produced a significant effect of treatment (F(1, 6)=6.70, p=0.041, Fig. 2C), similary evident in analysis of 5 minute periods, though this attained post-hoc significance only during the 31–35 minute period (t(12)=2.34, p=0.023, Fig. 3B). These findings indicate increased activity after (R,S)-ketamine, that does not persist above MAD threshold for extended periods of time. Consistent with this, we observed a greater number of peak events following 3 mg/kg (R,S)-ketamine treatment in the 30–60 minute time period (Wilcoxon matched pairs W=28, n=7, p=0.016).

3.2.4. 100 mg/kg (R,S)-ketamine

Analysis of AUC following 100 mg/kg (R,S)-ketamine dosing (n=4) produced a significant interaction (timeXtreatment, F(59,177)=1.54, p=0.016, Fig. 2B) with changes appearing to be restricted to the period immediately after (R,S)-ketamine injection. However, analysis of 5 minute periods did not produce an interaction (timeXtreatment, F(11,33)=1.47, p=0.188, Fig. 3A) or main effects. When analyzing the samples above MAD threshold a significant interaction was observed over 1 minute periods (timeXtreatment, F(59,177)=2.35, p<0.0001, Fig. 2C), and 5 minute periods (timeXtreatment, F(11,33)=2.49, p=0.021, Fig. 3B) with samples above MAD threshold significantly elevated during the first five minute period (t(6)=4.32, p=0.0001; Fig. 3B).

3.3. (R,S)-ketamine effects on GCaMP6f activity in vmPFC, comparison with (R)- and (S)- ketamine and (2R,6R)-HNK

A clear increase in signal was observed after injection of (R,S)-ketamine (30mg/kg) and (S)-ketamine (15mg/kg, Fig. 4A). For AUC, main effects of treatment (F(5, 20)=6.99, p<0.001) and time (F(59, 1180)=6.54, p<0.0001) were observed, and these were substantiated by a significant interaction (timeXtreatment, F(295, 1180)=2.78, p<0.0001, Fig. 4B). Consistent with our prior experiment, (R,S)-ketamine produced an elevation in GCaMP6f signal in the period immediately after the injection until approximately 20 minutes post-injection. Notably, (S)-ketamine produced an increase in signal along the same time-scale. To assess this, we again compiled data into five minute time periods after treatment. We observed significant main effects (time F(11, 220)=6.80, p<0.0001, treatment F(5, 20)=7.04, p<0.001 ) as well as a significant interaction (timeXtreatment, F(55, 220)=3.61, p=0.0006; Fig. 5A). Post-hoc comparisons were made to the vehicle control group (n=4). As in our prior experiment (see section 3.2.1), increased signal was observed in the (R,S)-ketamine treated animals during the 20 minutes after treatment (minutes 1–5, t(6)=3.53, p=0.002; minutes 6–10, t(6)=3.70, p=0.001; minutes 11–15, t(6)=3.93, p=0.001; minutes 16–20, t(6)=2.94, p=0.015). Elevated signal was also observed following (S)-ketamine administration during minutes 1–5 (t(6)=7.07, p<0.0001), 6–10 (t(6)=5.65, p<0.0001), and 11–15 (t(6)=3.80, p=0.001). Increased signal was not observed following treatment with (R)-ketamine (15mg/kg) or (2R,6R)-HNK (30mg/kg) during any of the 5 minute periods analyzed (all p>0.9). The results obtained with (R,S)-ketamine and (S)-ketamine were recapitulated when treatment groups were compared to the GFP expressing control condition (treated with 30mg/kg (R,S)-ketamine).

Fig. 4.

Fig. 4.

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GCaMP6f response following rapid acting antidepressant treatment. (A) Minimally processed mean signal relative to prior vehicle treatment (n=4) in (R,S)-ketamine (30mg/kg, n=4), (R)-ketamine (15mg/kg, n=4), (S)-ketamine (15mg/kg, n=4), and (2R,6R)-HNK (n=5) treated mice as well as 30mg/kg (R,S)-ketamine treated GFP control (n=5). Top trace represents minutes 0–30 after treatment, bottom represents minutes 30–60. (B) 1 minute binned analysis of the AUC for GCaMP6f signal following treatment relative to vehicle treatment from the corresponding time period. (C) 1 minute binned analysis of the samples above MAD threshold relatiave to vehicle treatment from the corresponding time period. Data presented as mean ± sem.

Fig. 5.

Fig. 5.

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5 minute binned analysis of GCaMP6f signal relative to vehicle (n=4) treatment in (R,S)-ketamine (30mg/kg, n=4), (R)-ketamine (15mg/kg, n=4), (S)-ketamine (15mg/kg, n=4), and (2R,6R)-HNK (n=5) treated mice as well as 30mg/kg (R,S)-ketamine treated GFP control (n=5). (A) Analysis of the AUC following treatment relative to vehicle treatment from the corresponding time period. (B) Analysis of the samples above MAD threshold relatiave to vehicle treatment from the corresponding time period. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. Data presented as mean ± sem.

Analysis of samples above the MAD threshold produced results similar to AUC analysis. We observed a significant effect of time (F(59, 1180)=12.74, p<0.001) and treatment (F(5,20)=13.44, p<0.001), as well as a significant interaction (timeXtreatment, F(295, 1180)=3.47, p<0.001) when analyzing the data in one minute time periods (Figure 4C). Analysis of 5 minute time periods following drug treatment also produced significant main effects (time F(11, 220)=20.43, p<0.0001; treatment F(5, 20)=13.43, p<0.0001), and a significant interaction (timeXtreatment, F(55, 220)=5.55, p<0.0001; Fig. 5B). Within the first five minute period after injection we observed significant increase in samples above the MAD threshold in (R,S)-ketamine (t(6)=7.36, p<0.0001) and (S)-ketamine (t(6)=7.77, p<0.0001) injected animals in comparison to vehicle injected mice. This increase continued into the 6–10 minute period ((R,S)-ketamine, t(6)=6.83, p<0.0001; (S)-ketamine, t(6)=6.51, p<0.0001), and 11–15 minute period ((R,S)-ketamine, t(6)=6.98, p<0.0001; (S)-ketamine, t(6)=4.95, p<0.0001). During the 16–20 minute time period an increase in points above MAD threshold was only observed in the (R,S)-ketamine condition (t(6)=4.05, p=0.0003). We observed no change in points above MAD threshold following (R)-ketamine or (2R,6R)-HNK injection. (R,S)-ketamine and (S)-ketamine signal was also found to be significantly different from that obtained from GFP expressing controls injected with (R,S)-ketamine. Additionally, we observed no change in the number of peaks recorded during sampling after administration of (R)-ketamine or (2R,6R)-HNK when compared to vehicle treated animals.

4. Discussion

This study utilized indwelling fiber-optic cannula and ratiometric fiber photometry with the genetically encoded calcium indicator GCaMP6f to assess the activity of Camk2α expressing neurons in the vmPFC after treatment with (R,S)-ketamine, its stereoisomers (R)- and (S)- ketamine, and metabolite (2R,6R)-HNK. Infusion of (R,S)-ketamine (30 mg/kg) produced a rapid increase in GCaMP6f signal shortly after treatment that persisted for approximately 25 minutes. A similar increase in signal was observed following treatment with (S)-ketamine (15 mg/kg), but not (R)-ketamine (15 mg/kg). These data indicate that the higher NMDA receptor antagonist activity of the (S)-stereoisomer accounts for the increased activity of racemic ketamine. These data are also consistent with the lack of effect of the (R)-stereoisomer and (2R,6R)-HNK on GCaMP6f signal as both have low affinity for the NMDA receptor. These findings are consistent with reports demonstrating transiently increased glutamate following (R,S)-ketamine administration (Moghaddam et al., 1997), as well as reports suggesting that (R)-ketamine (Chang et al., 2019; Fukumoto et al., 2017) and (2R,6R)-HNK (Lumsden et al., 2019; Zanos et al., 2016) initiate their antidepressant effects through mechanisms distinct from racemic ketamine or (S)-ketamine. To our knowledge, irrespective of sampling technique, this work represents the first direct comparison of neuronal activity following application of these compounds in rats or mice. Notably, this work demonstrates the utility of genetically encoded calcium indicators for studying pharmacological responses in a region and cell population specific fashion, and has clear application for identifying pharmacological responses in a projection specific fashion.

Racemic ketamine has a polypharmacologic profile that produces a multi-system effect following administration. Pre-clinical work shows that the vmPFC is a critical region for the response to (R,S)-ketamine as inhibition and pharmacological blockade of this area blocks its antidepressant actions (Fuchikami et al., 2015; Fukumoto et al., 2014, 2016; Hare et al., 2019; Lepack et al., 2014). Studies using microdialysis in rats have reported increased levels of extracellular glutamate in the mPFC after ketamine and increased pyramidal cell activity following NMDA antagonist administration (Homayoun and Moghaddam, 2007; Jackson et al., 2004; Moghaddam et al., 1997). This work demonstrated an inverted U dose response whereby low, subanesthetic doses (10 and 30 mg/kg) of (R,S)-ketamine produce an increase in extracellular glutamate that persists for a longer period of time (80 to 100 min), and total lack of effect at higher doses (50 and 200 mg/kg). We observed an increase in GCaMP6f AUC after 30 mg/kg (R,S)-ketamine in two separate sets of experiments. The time course showed a rapid onset, faster than the increase in extracellular glutamate (Moghaddam et al., 1997), but similar to the timecourse for activation observed by extracellular single unit recording immediately after NMDA antagonist administration (Homayoun and Moghaddam, 2007); the GCaMP6f signal then decayed to baseline levels over approximately 25 minutes. The high, anesthetic dose tested produced a brief increase in signal for the first 5 min bin, followed by a rapid reduction in activity coinciding with sedation. This differs from studies of extracellular glutamate, which reported no effects even during the first time period tested, which could be due analysis of a larger time bin (20 min) (Moghaddam et al., 1997). Our assessment of points above MAD offers insight into the degree of activation relative to baseline. Increases in number of points above MAD following (R,S)-ketamine and (S)-ketamine indicate sustained, simultaneous activity that is well outside of the range of typical vmPFC activity that we observed during the baseline period.

It is notable that the observed increase in GCaMP6f signaling immediately following injection of 30 mg/kg (R,S)-ketamine or 15 mg/kg of (S)-ketamine is shorter in duration than the increase in glutamate release observed with microdialysis (Moghaddam et al., 1997). This may be due to a difference in the temporal resolution of the techniques, but may also indicate that the increase in glutamate captured in perfusate over extended time periods (30–100 minutes) does not produce a robust increase in neuronal activity that we observe immediately after injection and lasting up to 25 min. In vivo single unit recording following NMDA antagonist treatment demonstrates an increase in firing, but the firing occurs in randomly distributed single spikes. This type of activation may not be sufficient to produce the changes in bulk GCaMP signal necessary to capture an activity increase by fiber photometry. Indeed, this may also provide the most parsimonious explanation for the lack of an increase in signal peaks observed in the 30–60 minute period following (R,S)-ketamine administration. Interestingly however, we do observe increased indices of activity within this period in animals injected with lower doses of (R,S)-ketamine. It is unclear how these different timescales of vmPFC activation following NMDA antagonist administration relate to the antidepressant outcome, but it is clear that some level of vmPFC activation is evident across the doses examined.

The effects of (S)-ketamine on GCaMP6f activity largely mirrored that of (R,S)-ketamine. In contrast to (S)-ketamine, there was no significant effect of (R)-ketamine or (2R,6R)-HNK on GCaMP6f activity, including AUC, signal peaks, or samples above MAD threshold. (R)-ketamine is reported to produce rapid antidepressant actions in mice at the same doses utilized in the current study (Fukumoto et al., 2017; Yang et al., 2015). Similarly, the dose of (2R,6R)-HNK used in the current study produces rapid antidepressant effects in pre-clinical models (Fukumoto et al., 2019). (R)-ketamine has approximately 4-fold lower affinity for the NMDA receptor compared with (S)-ketamine (White et al., 1985). At the dose used here (R)-ketamine does not produce the side effect profile in rodents observed with (S)-ketamine, but it may at higher doses (Zanos et al., 2019). (2R,6R)-HNK has 70–100-fold lower activity for the NMDA receptor compared to (S)-ketamine (Lumsden et al., 2019; Zanos et al., 2016) but see (Kavalali and Monteggia, 2018). Togther, these findings indicate that the rapid and sustained effects of (R,S)- or (S)-ketamine on the GCaMP signal is mediated by NMDA antagonistic properties that are significantly lower in (R)-ketamine and (2R,6R)-HNK.

A recent study has reported that (2R,6R)-HNK as well as (R,S)-ketamine result in elevated levels of extracellular glutamate 24 hours after dosing (Pham et al., 2018); extracellular glutamate was not analyzed immediately after (2R,6R)-HNK or (R,S)-ketamine dosing in this study. This delayed increase in glutamate transmission in response to (2R,6R)-HNK administration presumably is mediated via a different initial mechanism compared to (R,S)-ketamine. Both (2R,6R)-HNK and (R,S)-ketamine produce a delayed increase in AMPA receptor levels in the mPFC (Shaffer et al., 2019; Zanos et al., 2016), which could contribute to the delayed increase in glutamate transmission (e.g., increased AMPA receptor stimulated firing of pyramidal neurons). It would be interesting to test if pretreatment with an AMPA receptor antagonist, which blocks the rapid antidepressant behavioral actions of these agents, also blocks the delayed increase in extracellular glutamate observed with (2R,6R)-HNK and (R,S)-ketamine. Nevertheless, these findings indicate that (2R,6R)-HNK, as well as (R)-ketamine engage different initial targets.

There are several issues and limitations to be considered. First, the results of the current study do not address the initial trigger underlying the increase in GCaMP signal in response to (R,S)-ketamine and (S)-ketamine. A recent report from our laboratory has demonstrated that cell specific NMDA-GluN2B knockdown on GABA interneurons, but not pyramidal cells in the medial PFC blocks the antidepressant response to ketamine (Gerhard et al., 2019). We have also reported that neuronal silencing or optogenetic stimulation of GABAergic interneurons in the mPFC blocks the antidepressant actions of (R,S)-ketamine (Fuchikami et al., 2015; Ghosal et al., 2019). These findings indicate that the increased GCaMP signal is due to blockade of NMDA receptors on GABAergic interneurons, leading to disinhibition of glutamate and stimulation of pyramidal cell firing. These effects are thought to underlie activity dependent increases in synaptic number and function that are associated with the rapid and sustained antidepressant actions of (R,S)-ketamine. Second, it is difficult to dissociate the role of GCaMP/neuronal firing in the synaptic/behavioral actions vs. side effects of (R,S)-ketamine. In patients, the dissociative side effects of racemic ketamine predict the sustained antidepressant response (Luckenbaugh et al., 2014), suggesting a causal link of both the antidepressant and side effects of (R,S)-ketamine with the GCaMP/neuronal activity. Third, while the doses of (R)-ketamine and (2R,6R)-HNK were chosen based on behavioral studies demonstrating an antidepressant response, we cannot rule out the possibility that other doses would increase GCaMP signaling. In particular, (R)-ketamine is reported to produce side effects in rodent models at doses that are higher than needed for antidepressant responses, and we would predict that these higher doses would increase GCaMP signaling (Zanos et al., 2019).

These findings add support to the growing literature indicating different initial mechanisms for (R,S)-ketamine, (R)-ketamine, and the metabolite (2R,6R)-HNK. Notably, downstream of the activating mechanism, BDNF release, increased synaptic levels of AMPA receptors, and mPFC neuroplasticity are necessary for the rapid and sustained antidepressant response of of these agents (Fukumoto et al., 2019; Lepack et al., 2016; Lepack et al., 2014; Li et al., 2010; Li et al., 2011; Moda-Sava et al., 2019; Zanos et al., 2016). One advantage of virally encoded tools is to characterize cell population and projection specific targets. Recent studies demonstrate that distinct populations of vmPFC projection neurons are disproportionately involved in the response to ketamine (Hare et al., 2019). GCaMP fiber photometry of projection defined targeting neurons will provide a more refined understanding of the cellular impact of (R,S)-ketamine, and provide more well-defined targets for novel rapid and efficacious antidepressant agents with fewer off-target effects.

Highlights.

R,S)-ketamine and (S)-stereoisomer administration increase vmPFC GCaMP signal.

(R,S)-ketamine causes a a dose dependent fashion increase in vmPFC GCaMP.

(R)-ketamine and (2R,6R)-HNK do not immediately increase the GCaMP signal in vmPFC

8. Acknowledgements

We thank Colin Bond for discussion and advice surrounding fiber-photometry analysis. This work was supported by the NIMH (R01 MH105910–04 and RO1 MH093897–06A1 to R.S.D), and a P&S Fund donation to the Brain and Behavior Research Foundation (NARSAD to B.D.H)

Abbreviations

PFC

prefrontal cortex

vmPFC

ventral medial prefrontal cortex

NMDA

N-methyl-D-aspartate

(2R,6R)-HNK

(2R,6R)-hydroxynorketamine

TRD

treatment resistant depression

GABA

Gamma aminobutyric acid

CAMK2a

Calmodulin dependent protein kinase 2a

MAD

Median absolute deviation

AUC

Area under the curve

Footnotes

7.

Declaration of Interest

We declare that Dr. Duman has consulted and/or received research support from Naurex, Aptynx, Lilly, Relmada, Navitor, Johnson & Johnson, Taisho, and Sunovion. The remaining authors have no competing financial interests.

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