ABSTRACT Ketamine is a psychotomimetic and antidepressant drug. Although antagonism of cell-surface NMDA receptors (NMDARs) may trigger ketamine’s psychoactive effects, ketamine or its major

metabolite norketamine could act intracellularly to produce some behavioral effects. To explore the viability of this latter hypothesis, we examined intracellular accumulation of novel

visualizable analogues of ketamine/norketamine. We introduced an alkyne “click” handle into norketamine (alkyne-norketamine, A-NK) at the key nitrogen atom. Ketamine, norketamine, and A-NK,

but not A-NK-amide, showed acute and persisting psychoactive effects in mice. This psychoactivity profile paralleled activity of the compounds as NMDAR channel blockers; A-NK-amide was

weak A-NK-amide labeling. Accumulation was not dependent on membrane potential, NMDAR expression, or NMDAR activity. Overall, the approach revealed a correlation among NMDAR activity,

intracellular accumulation/retention, and behavioral effects. Thus, we advance first generation chemical biology tools to aid in the identification of ketamine targets. SIMILAR CONTENT BEING

VIEWED BY OTHERS SUSTAINED ANTIDEPRESSANT EFFECT OF KETAMINE THROUGH NMDAR TRAPPING IN THE LHB Article Open access 18 October 2023 DART.2: BIDIRECTIONAL SYNAPTIC PHARMACOLOGY WITH

THOUSANDFOLD CELLULAR SPECIFICITY Article 14 June 2024 KETAMINE DECREASES NEURONALLY RELEASED GLUTAMATE VIA RETROGRADE STIMULATION OF PRESYNAPTIC ADENOSINE A1 RECEPTORS Article Open access

11 August 2021 INTRODUCTION The non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, ketamine, is a psychotomimetic, dissociative anesthetic, and fast acting antidepressant in

humans1,2,3,4,5 and has antidepressant-like actions in rodents6,7,8. Many questions remain about cellular effects underlying these actions. For instance, drugs with very similar actions on

NMDARs may not share ketamine’s behavioral effects9. One explanation is that metabolites of ketamine could have psychoactive effects10. Another non-exclusive possibility is that ketamine or

its metabolites could have undiscovered cellular targets relevant to their behavioral effects. For instance, as a weak base, the neutral species of ketamine may readily permeate cell

membranes and bind intracellular targets11,12,13. Targets other than NMDARs have recently been proposed to underlie antidepressant effects10. However, the nature and mechanisms of any

intracellular accumulation of ketamine are unclear. Ketamine is an open channel blocker of NMDARs, a major class of glutamate receptors governing excitation in the vertebrate CNS. It is

unusual among psychoactive NMDAR channel blockers in yielding a major metabolite, norketamine, which is also psychoactive. Although its plasma concentrations remain low relative to peak

ketamine levels, norketamine has a significantly longer half-life in humans than ketamine (11 h vs. 2.5 h)14,15. Norketamine blocks NMDARs, but its detailed pharmacodynamic properties have

not been explored16. It is also unclear whether norketamine possesses the acute psychoactive and antidepressant activity of the parent compound. Given that targets other than NMDARs may be

important for some behavioral effects of ketamine, cellular visualization of ketamine analogues could help reveal unanticipated targets relevant to drug effects. Here, we use “click”

chemistry, an approach that allows visualization of biologically active molecules by covalently linking them to their substrates and/or visual probes, to probe the possibility of

intracellular accumulation. To address competing ideas regarding ketamine’s actions, we tested ketamine, norketamine, and two novel analogues that can be visualized with click chemistry:

alkyne norketamine (A-NK) and alkyne norketamine amide (A-NK-amide), a non-protonatable analogue. We found that three of the four compounds exhibited strong psychoactive effects, including

antidepressant-like effects in the rodent forced swim test (FST). The fourth compound, A-NK-amide, was much weaker in behavioral assays. The three behaviorally active compounds exhibited

channel block at NMDARs in hippocampal neurons, while A-NK-amide exhibited much weaker activity at NMDARs. A-NK-amide, unlike the active analogue A-NK, failed to strongly accumulate in

intracellular compartments, retrospectively assessed using _in situ_ click chemistry on dissociated, fixed neurons. The subcellular compartments labeled by A-NK were diverse, suggesting the

possibility of multiple intracellular targets correlated with psychoactive effects. Thus, intracellular accumulation correlated with cellular and behavioral effects. Our work establishes

that the major metabolite of ketamine has psychoactive and antidepressant-like effects and demonstrates the importance of protonation for cell entry and/or retention. We introduce for the

first time a visualizable probe that retains electrophysiological and behavioral properties of ketamine/norketamine while enabling visualization of drug localization. The probe revealed

intracellular labeling potentially relevant to cellular actions of ketamine/norketamine. RESULTS NORKETAMINE AND A-NK EXHIBIT STRONG PSYCHOACTIVITY; A-NK-AMIDE IS WEAKER The compounds

evaluated in the present work are shown in Fig. 1. Recent structure-activity work on ketamine showed that the methyl group on the central nitrogen atom is expendable for anesthetic

activity17, so we performed chemical modifications with norketamine as precursor to the chemical biology analogues A-NK and A-NK-amide. Ketamine exhibits a range of psychoactive effects,

from acute (e.g., psychotomimetic, anesthetic at high doses) to delayed (e.g., antidepressant at low doses) behavioral changes. We first examined near-anesthetic doses of compounds to test

acute psychoactive effects18. We quantified locomotor activity and rearing behavior for 60 min following a single i.p. injection. Ketamine at 100 mg/kg (n = 8) significantly increased the

number of ambulations across the 60-min test session relative to vehicle controls (n = 7)[Treatment effect: F(1,13) = 11.88, p = 0.004], and the differences varied with time [Treatment x

Time Blocks interaction: F(5,65) = 5.26, p = 0.001] (not shown). Further, ketamine decreased behavioral rearing frequency [Treatment effect: F(1,13) = 46.47, p < 0.00005], which also

varied with time [Treatment x Time Blocks interaction: F(5,65) = 8.16, p = 0.0002] (not shown). Subsequent pair-wise comparisons confirmed that ketamine reduced rearing beyond Bonferroni

multiple comparisons correction (p < 0.005) across the first 50 min of the test session. Differences were significant for ambulations during only the first 20 min (blocks 1, 2; p < 

0.003). These results confirm observations on ambulatory activity and rearing by others18. Based on these results, we focused on rearing behavior as a sensitive indicator of psychoactive

effects of ketamine-like compounds in the C57Bl/6 J mice studied here. In an interleaved cohort of animals, ambulatory activity following ketamine (50 mg/kg), norketamine, A-NK, and

A-NK-amide (100 mg/kg each) did not differ among drugs or against vehicle control (Fig. 2a; n = 5 for each group), suggesting no anesthetic effects of any of the drugs. Doses were chosen

based on relatively potencies from previous work16. There was, however, a drug by post-injection time interaction effect on ambulation (Fig. 2a), hinting at differences in psychoactivity

sometime during the test session. In the more sensitive measure of rearing frequency, ketamine, norketamine, and A-NK had strong effects (Fig. 2b), but A-NK-amide was weaker than the other

compounds; rearing frequency was significantly reduced in the A-NK-amide treated mice only early in the session (Fig. 2b). Rearing duration showed similar effects (Fig. 2c). Collectively

these results indicate that high, subanesthetic doses of ketamine, norketamine, and A-NK robustly depress rearing behavior, while A-NK-amide induces weaker psychoactive effects compared with

ketamine and the other two analogues. To test effects in the FST, a screen for antidepressant drugs, mice were dosed i.p. with 3 or 10 mg/kg of ketamine or with vehicle (n = 20 per

condition) and then tested 3 h later on the FST6,8,9,19. An ANOVA conducted on these data (Fig. 2d1) revealed a significant effect of Treatment, [F(2,57) = 5.89, p = 0.005] on immobility

time, with subsequent pair-wise comparisons showing that both the 3 and 10 mg/kg doses significantly reduced immobility, (p = 0.021 and 0.002, respectively) compared to vehicle-treated mice.

Another independent cohort of naive mice was injected with 3 or 10 mg/kg of norketamine or vehicle and then assessed on the FST to test the hypothesis that the primary metabolite of

ketamine (i.e., norketamine), may also have antidepressant-like properties. An ANOVA of these data yielded a significant effect of Treatment, [F(2,46) = 5.31, p = 0.008], on immobility time

(n = 16–17 mice per condition). Follow-up comparisons indicated that the 10 mg/kg dose of norketamine resulted in a significant (beyond Bonferroni correction: p = 0.025) reduction in

immobility time (p = 0.003) while the 3 mg/kg dose also decreased immobility (p = 0.039), relative to vehicle controls (data not shown). We replicated the norketamine (10 mg/kg) finding

alongside a test of A-NK (10 mg/kg), a synthetic analogue of norketamine in a separate cohort of animals (Fig. 2e1). An ANOVA of these data (n = 32–34 mice per condition) indicated a

significant Treatment effect, [F(2,97) = 6.73, p = 0.002], on immobility and pair-wise comparisons showed that both norketamine and A-NK significantly reduced immobility relative to vehicle

control injections (p = 0.0005 and 0.019, respectively). In contrast, injection of the other synthetic analogue, A-NK-amide, failed to affect immobility time in the FST compared to vehicle

control injections (Fig. 2f1) in additional independent groups of naive mice (n = 25 per condition). The FST was tested with a 3 hr delay and lower dose compared with initial tests of acute

psychoactivity (Fig. 2a–c), so acute psychotomimetic or anesthetic effects should not have affected performance during the FST. Nevertheless, we examined the effects of ketamine,

norketamine, A-NK, and A-NK-amide on general ambulatory activity and vertical rearing using the same dosing and post-injection delay used for FST testing. Ketamine (3 mg/kg and 10 mg/kg; n =

 13 per condition) failed to show significant effects of either dose on ambulatory activity (total ambulations), and the same was true for norketamine (n = 17), A-NK (n = 18), and A-NK-amide

(10 mg/kg, n = 12; Fig. 2d2). In addition, none of the drug treatments had any significant effects on vertical rearing frequency at this later time point and lower dose (Fig. 2d3,e3,f3).

These data suggest that it is unlikely that ketamine, norketamine or A-NK altered motor activity to cloud interpretation of the antidepressant-like effects of the drugs during the FST.

VOLTAGE DEPENDENCE AND KINETIC STUDIES OF ANALOGUES There have been few evaluations of the effect of norketamine on NMDAR function16,20. No work has assessed activity of chemical biology

analogues of ketamine/norketamine at NMDARs. In dissociated cultures of rat hippocampal neurons, we explored properties of the analogues on NMDAR function. All three NMDAR-active compounds

exhibited voltage dependence at equimolar concentration (10 μM; Fig. 3), suggesting similar mechanism of action. Norketamine and A-NK exhibited indistinguishable block at −70 mV but

significantly different block at +50 mV, suggesting that A-NK exhibits slightly weaker voltage dependence (Fig. 3e,f). Interestingly, the amide derivative was completely inert at 10 μM (Fig.

3d). At −70 mV both norketamine and A-NK displayed significantly slower onset kinetics than ketamine, with A-NK exhibiting especially slow onset and offset kinetics during continuous

agonist exposure (Fig. 3g). In summary, both norketamine and A-NK retained voltage dependence similar to ketamine, with subtle differences in kinetics, suggesting that both mimic the actions

of the parent compound. To compare relative potencies of the active analogues, we challenged cells with increasing concentrations of antagonist in the prolonged presence of 10 μM NMDA as

agonist (Fig. 4). We found that norketamine was approximately 4-fold less potent than ketamine (IC50 ketamine: 0.4 μM, IC50 norketamine: 2.0 μM, n = 9–10, Fig. 4a,b,d) and A-NK was

equipotent to norketamine (IC50: 1.8 μM, n = 9, Fig. 4c,d). Figures 3 and 4 suggest that A-NK has slower kinetics than the parent compounds when applied during continuous agonist

presentation. The most direct comparison is between A-NK and norketamine, because these two compounds proved equipotent at −70 mV. We and others have previously shown that kinetics of

trapping NMDAR channel blockers, including ketamine, is rate limited by low channel open probability21,22. The additional slowness of A-NK could result from intrinsically slow

pharmacodynamics (drug binding and dissociation), or it could represent slow access to the NMDAR through routes other than aqueous diffusion. For instance, previous work has shown that

ketamine can reach at least one site via a lipophilic pathway23. Given the enhanced lipophilicity that the hydrocarbon chain imparts to the analogue (cLogP A-NK: 3.86 ± 0.54 vs. ketamine:

2.75 ± 0.33 vs. norketamine: 2.32 ± 0.33, see Methods), we hypothesized that A-NK may need to reach a _non-aqueous_ binding site, causing kinetics to be rate limited by membrane

partitioning, as seen with steroid modulators of GABAA receptors24. This is in contrast to local anesthetics acting at voltage-gated sodium channels, where hydrophobicity and a membranous

access route speed drug actions25,26. If cellular accumulation or compartmentalization explains the slow kinetics of A-NK, then A-NK pre-application should speed the block observed upon

ensuing agonist/A-NK co-application. To test this, we pre-applied A-NK for 60 s, followed by co-application with agonist. We compared kinetics of block onset with kinetics observed in a

co-application-only protocol. The rate of A-NK block upon NMDA co-application was unaffected by A-NK pre-application (Fig. 5). This suggests that A-NK pharmacodynamics accounts for the slow

block during continuous agonist presentation, perhaps because of steric-hindrance from the bulky side chain. These data also support the idea that A-NK, like ketamine, is a use-dependent

blocker because in the pre-application protocol, there was no evidence that A-NK interacted with the receptor/channel before agonist was presented27. Results from Figs 3, 4 and 5 show that

the psychoactive compound A-NK inhibits responses to sustained agonist application. However, does the slow kinetics of A-NK hinder its ability to depress synaptic responses, which are

generated in response to very brief agonist presentation? To address this question we applied norketamine and A-NK prior to evoking NMDAR-mediated EPSCs. Both drugs significantly reduced the

peak of the EPSC (norketamine: 22.6 +/− 3.0% reduction from baseline, n = 18, p < 0.05 paired t-test; A-NK: 30.5 ± 2.9% reduction from baseline, n = 20, p < 0.05). A-NK’s effect on

the EPSC decay time course was only trend level (p = 0.045) compared with a stronger effect of norketamine (Fig. 6a–c). Nevertheless, upon repetitive stimulation at 0.033 Hz, both

norketamine and A-NK behaved nearly indistinguishably (Fig. 6d–g). Both had strong, cumulative effects on EPSC peak and total charge that slowly returned toward baseline following compound

removal (Fig. 6f,g). These results are consistent with the idea that the primary mode of norketamine and A-NK inhibition of NMDA receptor function is through activation-dependent trapping

block, similar to ketamine28,29,30. We conclude that the slower kinetics of A-NK do not prevent it from interacting with synaptic neurotransmission. A-NK INTRACELLULAR ACCUMULATION One

recent hypothesis suggests that ketamine may trigger antidepressant downstream signaling pathways by binding nascent intracellular NMDA receptors resident in the endoplasmic reticulum12.

A-NK and ANK-amide allowed us to incubate live cells with the unlabeled analogue, then visualize the localization of analogue following cell fixation and _in situ_ fluorescence click

chemistry with a fluorescent dye. After bath incubation with unlabeled A-NK or A-NK-amide in live cells, we washed away extracellular analogue, fixed cells and “clicked” azide-Alexa Fluor

488 to residual compound (see Methods). Because localization occurred in fixed cells when excess extracellular alkyne was no longer present, we posit that this protocol should reveal sites

of localization of the alkyne analogues at the time of fixation. We observed intracellular labeling in neurons, with accumulation of A-NK most evident. A-NK-amide accumulation was barely

detectable above background (though statistically significant, Fig. 7a,b). Prolonged wash following fixation only modestly altered intensity and pattern of A-NK accumulation, suggesting that

the analogue was indeed fixed in place by the protocol (Fig. 7c; n = 40 cells in 4 experiments, 64 ± 3% vs. 52 ± 3% increase in fluorescence with a 1 h wash following fixation, vs. DMSO

control). We tested whether A-NK accumulated in a specific cellular compartment by co-labeling with antibodies against proteins resident in specific organelles. Antibody co-labeling of A-NK

with anti-PDI, an endoplasmic reticulum marker, failed to reveal specific overlap (Fig. 8a). Although overlap appeared higher with the Golgi marker giantin and the mitochondrial marker COXIV

(Fig. 8b,c), the co-labeling revealed no selective accumulation in any one compartment. We conclude that robust intracellular labeling characterizes an active ketamine/norketamine analogue,

but there appears to be no selective or specific compartmentalization. Some studies suggest that ketamine may preferentially inhibit interneurons8 and/or may alter postsynaptic (dendritic)

glutamate signaling6,8. To test whether A-NK compartmentalization can help explain these observations, we co-labeled neurons with an antibody directed against GABA, a broad marker of

hippocampal interneurons, and MAP2, a marker of dendrites (Fig. 8d, left). We examined A-NK accumulation on somas and dendrites of GABA and non-GABA cells (Fig. 8d, right) and found no

evidence for preferential compartmentalization in dendrites or in interneurons (Fig. 8e). To address whether intracellular accumulation is associated with nascent or recycled NMDARs, we used

HEK cells, which do not express endogenous NMDARs. We transfected cells with DsRed and NMDAR subunits or DsRed alone as a control and imaged A-NK accumulation. We found that A-NK

accumulated in both NMDAR-transfected cells and in control cells at similar levels (Fig. 9a,b), suggesting that intracellular accumulation is not accounted for by interaction with

intracellular NMDARs. This result also suggests that permeation of A-NK through surface NMDARs down its electrochemical gradient is not an important mechanism by which A-NK accumulates. To

test this directly, we challenged neurons with A-NK while co-treating with 50 μM D-APV to prevent channel opening or while co-treating with agonist (10 μM NMDA and 10 μM glycine) to open

channels and provide opportunity for A-NK entry. Subsequent visualization revealed that A-NK accumulation was not increased and in fact was slightly reduced by agonist co-treatment relative

to D-APV treatment (Fig. 9c,d). Finally, we addressed whether the difference in accumulation between positively charged A-NK and electroneutral A-NK-amide might result from electrostatic

attraction of protonated A-NK to the negative membrane potential of the intracellular compartment. This experiment further tests the validity of the assumption that the uncharged form of

ketamine is the only major permeant species. To test this, we incubated cells in A-NK with 120 mM KCl (replacement for NaCl) to reduce the membrane potential. If cell permeation was aided by

negative membrane potential we would expect that intracellular staining would be reduced in cells treated with KCl. Instead, A-NK accumulation was slightly altered in the direction opposite

of our prediction (Fig. 9e,f). Although we cannot fully exclude the possibility that membrane potential disruption caused by fixation influenced retention, the difference in A-NK and A-NK

amide accumulation does not appear to be caused by electrostatic attraction. DISCUSSION Ketamine is a multifaceted psychoactive drug whose mechanisms of action are still under exploration.

This paper addressed two questions regarding its actions. First, we examined the properties of norketamine, the major primary metabolite of ketamine, on psychoactivity, including performance

during the FST, the most extensively used test for assessing the behavioral effects of antidepressant drugs in rodents31,32, and we also evaluated norketamine’s activity at NMDARs. Our work

indicates that norketamine is subtly different than ketamine in its effects on NMDARs but could participate in the psychoactive actions of ketamine. Second, we introduce a novel chemical

biology probe that has actions similar to ketamine and norketamine. Because of its similar effects, it and subsequent generations of analogues can be used to visualize and/or biochemically

label novel targets of ketamine’s actions in neurons. Previous work examining norketamine at NMDARs confirmed that it is neuroactive, but the work did little to characterize its mechanism of

action. Here, we provide evidence that norketamine and the analogue A-NK both mimic ketamine’s actions at NMDARs and in tests of ketamine-like psychoactivity. Both norketamine and A-NK

displayed characteristics consistent with voltage-dependent, open- channel blockers. The alkyne chain considerably slowed A-NK’s rate of block during exogenous agonist application (Figs 3

and 4). In contrast to steroid modulators of GABAA receptors, for instance33, this slowing was not attributable to differences in the rate of access to the receptor. Instead, receptor

interactions of A-NK are slow relative to norketamine, perhaps resulting from steric effects imparted by the alkyne side-chain. We note that all of our _in vitro_ recordings were performed

in the absence of physiological Mg2+, and Mg2+ influences the interaction of ketamine-like compounds with the NMDAR channel9,34. We therefore cannot exclude the possibility that Mg2+ might

change the details of the various analogues’ interactions with NMDARs. In this regard, it is encouraging that our _in vivo_ results (Fig. 1) largely parallel the _in vitro_ findings.

Interestingly, we found that the A-NK-amide structure abolishes activity at NMDARs, has decreased behavioral effects, and reduces intracellular retention/labeling. We did detect significant

short-term psychoactive effects (Fig. 2b,c) and significant intracellular fluorescence labeling above background levels, confirming the success of the click reaction (Fig. 7b). The weak

A-NK-amide labeling could reflect poor permeation of the plasma membrane or poor intracellular retention. Either way, A-NK-amide seems to differ from the other analogues in its potential for

interaction with intracellular targets. Permeation and retention could be governed by drug physicochemical properties. Estimated pKa values for the compounds in this study are as follows:

ketamine (7.18), norketamine (6.78), A-NK (6.88), and A-NK-amide (−4.1). Calculated cLogP values are ketamine (2.75), norketamine (2.32), A-NK (3.86), and A-NK-amide (3.26). Thus A-NK-amide

has high lipophilicity and is not protonated. It is therefore unclear what would reduce its permeation and retention; simple lipophilicity is often not sufficient to predict membrane

permeability35. One possibility is that permeation of the positively charged analogue A-NK is facilitated by an active process that shuttles A-NK across the membrane. This process does not

appear to involve permeation of the NMDAR channel itself or more general electro-attraction (Fig. 9). Another possibility is that by virtue of its status as a weak base, A-NK is better

retained than A-NK-amide, perhaps in part by being trapped in its protonated state within acidic organelles13 and subsequently fixed in place. Binding to intracellular NMDARs does not appear

to constitute a major mechanism of intracellular retention of A-NK (Fig. 9a,b). Although A-NK exhibited more intracellular uptake/retention compared with A-NK-amide, A-NK did not exhibit

clear selective partitioning into a single organelle sub-compartment within neurons. We consider two caveats to the conclusion that A-NK yields a realistic picture of ketamine/norketamine

intracellular distribution. First, A-NK was not anchored before imaging, so diffusion beyond initial preferential sites of accumulation could have occurred during processing. Wash following

fixation did not notably alter the accumulation pattern (Fig. 7c), suggesting that the compound is ultimately fixed in place, but we cannot exclude rapid redistribution during fixation. This

issue has been dealt with in other contexts by introducing a photolabel to anchor the analogue _in situ_ prior to fixation36,37. On the other hand, non-covalently anchored optical probes

retain specific organelle distribution in other situations38,39,40. A second caveat is that the higher lipophilicity of A-NK over norketamine and ketamine may lead to a different

distribution than the parent compounds. Because A-NK-amide, which shares high lipophilicity with A-NK, exhibits poor intracellular accumulation, we argue that lipophilicity is unlikely to

lead to different intracellular accumulation properties compared with ketamine and norketamine. Rather, the intracellular accumulation of A-NK suggests that intracellular targets could

participate in the unique actions of ketamine11,12. The broad intracellular distribution of the analogues (and presumably ketamine/norketamine) also raises the question of specificity of any

intracellular actions. Previous reviews of intracellular ketamine actions have suggested that ketamine may have specific intracellular actions11,12. How could such non-selective

distribution of drug be associated with specific, compartmentalized functional effects? One possibility is that not all intracellular binding sites for ketamine and its analogues are

functionally relevant11. Norketamine, ketamine, and A-NK each induced strong acute psychotomimetic effects and reduced immobility time in the FST assay. Although limiting compound quantities

precluded full time course studies, the FST was performed with a 3 h delay, to help ensure that other acute psychoactive effects did not contribute to FST results. Although NMDAR activity

is one commonality among these compounds that could trigger behavioral effects, our data suggest that other intracellular targets might be important for behavioral and perhaps synaptic

effects of ketamine analogues, some of which take several hours to manifest in both humans and rodents6,9,41,42. This could explain why the pharmacologically similar compound memantine does

not share ketamine’s antidepressant-like effects9. Memantine, unlike A-NK, ketamine, and norketamine, is nearly fully protonated at physiological pH (pKa 10.3, 99% protonated at

physiological pH20), which may prevent it from entering cells. Another possibility is that a common downstream metabolite of the ketamine core structure mediates the antidepressant-like

effect10. A common metabolite for both ketamine and norketamine is 6-hydroxynorketamine10, a neuroactive compound but weak NMDAR antagonist43. The cyclohexane ring, which is modified in both

ketamine and norketamine to form these hydroxyketamine and hydroxynorketamines, can also be modified in A-NK. Although ketamine is rapidly metabolized to produce norketamine through

demethylation, our observed differences as well as those reported previously17 suggest that longer alkyl chains such as those in A-NK and A-NK-amide are not lost through metabolic pathways,

leaving the terminal alkyne intact. It therefore seems unlikely that a common downstream metabolite of ketamine, norketamine and A-NK can explain the behavioral effects, although it is

possible that a common metabolite helps explain the acute behavioral effect of A-NK-amide observed at high concentration (Fig. 2b,c). Furthermore, cellular esterase/amidases are unlikely to

explain the lack of cellular labeling or weak behavioral activity of A-NK-amide; precedent examples from the literature on local anesthetics show that amidation is correlated with increased

_in vivo_ stability (procainamide, lidocaine) over that of procaine44,45,46. METHODS MICE AND DRUG TREATMENTS The study was carried out in strict accordance with the recommendations in the

Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The protocol(s) was/were approved by the Washington University Animal Studies committee. All efforts

were made to minimize animal discomfort by use of anesthesia/analgesia. Male C57BL/6 J mice (Jackson Labs) that were 2–2.5 months old were administered ketamine (3–100 mg/kg), norketamine

(3–100 mg/kg), A-NK (10–100 mg/kg), A-NK-amide (10–100 mg/kg), or vehicle via intraperitoneal injection. Vehicle was matched across all conditions for each experiment and included up to 30%

DMSO or 7% Cremophor-EL in 0.9% saline. Drug effects on behavioral performance were evaluated immediately (acute psychoactivity studies) or three hours (forced swim test, FST) post-

injection. FORCED SWIM TEST (FST) Mice were transported from a housing room to the test room where they remained for at least 1 h before testing commenced. The FST procedure involved testing

drug- and vehicle-treated mice by placing a mouse inside one of two 2000 ml glass beakers (144 mm diam.) that contained 1600 ml of water at 24 ± 1 °C where each remained for a 6-min trial.

An opaque partition was placed between the beakers, and the water was changed for each pair of subjects. Testing of drug- and vehicle-treated mice was counterbalanced across the two beakers

with typically one drug- and one vehicle-injected mouse being tested at the same time. A video camera positioned above the beakers was used to record an overhead view of the mice, which was

used to evaluate their performance. Immobility, defined as a lack of extraneous movement except for that required to keep the head above water, was quantified (seconds) during the last 4 min

of the trial by an observer who viewed the video recording and was unaware of the treatment status of individual mice. LOCOMOTOR ACTIVITY To examine the effects of the drug treatments on

general activity/exploratory behavior levels, mice were evaluated over a 1-h period in transparent polystyrene enclosures measuring 47.6 × 25.4 × 20.6 cm high, according to our previously

published procedures47. Each enclosure was surrounded by two frames containing pairs of photocells. Beam breaks were recorded by a computer, and measured parameters were quantified by

standard algorithms (MotorMonitor, Kinder Scientific LLC, Poway, CA). Dependent variables that were analyzed included total ambulations (whole body movements), which were derived from a

lower frame used to measure horizontal movement, and vertical rearing frequency, which was computed using a second frame that was raised slightly above the first. The effect of high doses of

ketamine (50–100 mg/kg) and analogues (100 mg/kg) were assessed using the locomotor instrumentation described above and a modified procedure previously utilized with rats to evaluate the

effects of high doses of ketamine and metabolites48. Relevant dependent variables were ambulations and vertical rearing, which served as measures of psychoactivity (see Results). The acute,

high-dose effects of ketamine on these behaviors have been interpreted as psychotomimetic-like effects18. Animals dosed with vehicle, ketamine, norketamine, A-NK, and A-NK-amide were all

evaluated during the same session, in an interleaved design, immediately following i.p. injection. The same instrumentation and general procedures described above were used to assess

possible activity-related drug effects in the FST studies by evaluating the same doses with the former methods.These control studies involved mice that had served as vehicle controls in the

FST. Locomotor assessments were performed 1–2 weeks later on the 1-h locomotor activity test following drug (10 mg/kg) or vehicle injections using the same post-injection delay interval (3 

h) that was utilized in the FST studies. HIPPOCAMPAL CULTURES Hippocampal cultures were prepared as either mass cultures or microcultures (as indicated in figure legends) from postnatal day

1–3 female rat pups anesthetized with isoflurane, under protocols consistent with NIH guidelines and approved by the Washington University Animal Studies Committee. Methods were adapted from

earlier descriptions49,50,51,52. Hippocampal slices (500 μm thickness) were digested with 1 mg ml−1 papain in oxygenated Leibovitz L-15 medium (Life Technologies, Gaithersburg, MD, USA).

Tissue was mechanically triturated in modified Eagle’s medium (Life Technologies) containing 5% horse serum, 5% fetal calf serum, 17 mM D-glucose, 400 μM glutamine, 50 U ml−1 penicillin and

50 μg ml−1 streptomycin. Cells were seeded in modified Eagle’s medium at a density of ~650 cells mm−2 as mass cultures (onto 25 mm cover glasses coated with 5 mg ml−1 collagen or 0.1 mg ml−1

poly-D-lysine with 1 mg ml−1 laminin) or 100 cells mm−2 as microcultures (onto 35 mm plastic culture dishes coated with collagen microdroplets on a layer of 0.15% agarose). Cultures were

incubated at 37 °C in a humidified chamber with 5%CO2/95% air. Cytosine arabinoside (6.7 μM) was added 3–4 days after plating to inhibit glial proliferation. The following day, half of the

culture medium was replaced with Neurobasal medium plus B27 supplement (Life Technologies). ELECTROPHYSIOLOGY Whole-cell recordings were performed at room temperature from neurons cultured

for 4–7 days (exogenous applications of agonist) or for 10–12 days (EPSCs) using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) and Digidata 1440 A converter with

Clampex 10.1 software. Young cells were favored for biophysical experiments with exogenous applications to minimize spatial voltage-clamp errors and to keep current amplitudes small. For

recordings, cells were transferred to an extracellular (bath) solution containing (in mM): 138 NaCl, 4 KCl, 2 CaCl2, 10 glucose, 0.01 glycine and 10 HEPES, pH 7.25 adjusted with NaOH.

Solutions were nominally Mg2+ free to promote NMDAR activation. D-(-)−2-Amino-5-phosphonopentanoic acid (D-APV, 25–50 μM) was included until seal formation to prevent excitotoxicity.

Solutions with adjusted composition described below were perfused using a gravity-driven local perfusion system from a common tip. The estimated solution exchange times were <100 ms

(10–90% rise), estimated from junction current rises at the tip of an open patch pipette. For synaptic recordings, these solutions contained (in mM) 0.001

2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (NBQX) and 0.025 bicuculline methobromide or 0.01 gabazine. EPSCs were elicited from single-cell microcultures by brief depolarization to

0 mV to elicit a breakaway axonal action potential49. For exogenous NMDA application, 0.25 mM CaCl2 was used (in APV-free perfusion solutions) to minimize Ca2+-dependent NMDAR

desensitization53,54. This concentration balanced stability of NMDA responses with cell/membrane seal stability. Tetrodotoxin (250 nM) was added to prevent network activity when evoked

synaptic activation was not required. Unless otherwise noted, exogenous NMDA concentration was 30 μM. The open-tip resistance of patch pipettes was 3–6 MΩ when filled with an internal

solution containing (in mM): 130 cesium methanesulfonate, 0.5 CaCl2, 5 EGTA, 4 NaCl, and 10 HEPES at pH 7.25, adjusted with KOH. Holding voltage was typically −70 mV unless otherwise noted.

Access resistance for all recordings was 8–10 MΩ and was compensated for current amplitudes >2 nA. HEK CELLS AND TRANSFECTION Procedures were essentially similar to those previously

published by our group55,56. Briefly, HEK 293 cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% fetal bovine serum and 1 mM glutamine. Cells

on a 35 mm culture plate were transfected with wild-type GluN1a subunit DNA (pRc/CMV vector; 0.3 μg), GluN2B subunit DNA (pcDNA1; 1 μg), and DsRed fluorescent protein DNA

(pDsRed2–1;Clontech, Mountain View, CA 0.05 μg). The separate plasmids were co-transfected using Lipofectamine2000 (Life Technologies) according to the manufacturer’s protocol. Using this

method, DsRed fluorescence predicts functional NMDAR expression with more than 80% accuracy55,56. CONFOCAL IMAGING For evaluating labeling of A-NK and A-NK-amide, stocks of 10 mM compound

were prepared in DMSO and diluted to 10 μM in culture medium for 1 h at 37 °C. The drugs were then washed from the cells three times with PBS. Cells were then fixed with 4% paraformaldehyde

and 0.05% glutaraldehyde for 10 min. Cells were washed with PBS and then exposed to click reaction labeling for 1 h in the dark using azide-conjugated Alexa Fluor 488 (1 μM). The click

buffers contained 100 μM Tris[(1-benzyl-1_H_-1,2,3-triazol-4-yl)methyl]amine (TBTA), 2 mM (+)- Sodium L-ascorbate in distilled water, 1 μM azide-Alexa Fluor 488 in DMSO, 1 mM CuSO4. Pilot

experiments demonstrated that additional membrane permeabilization beyond cell fixation was not required for azide-Alexa Fluor 488 entry. Experiments examining _in situ_ click labeling

compared A-NK and A-NK-amide click labeling with control conditions in which all click reagents were present except pre-incubation in A-NK or A-NK. As expected, the control fluorophore

incubation yielded some background fluorescence above that obtained with no fluorophore (46.2 ± 4.0 fluorescence units to 105.2 ± 8.4 fluorescence units from 20 fields in four independent

experiments). All reported fluorescence values in text and figures represent labeling above the background labeling performed with all click reagents (including fluorophore) present, but

without pre-incubation in unlabeled analogue. For antibody co-labeling experiments, we incubated in 10 μM A-NK for 1 h at 37 °C then fixed cells with 4% paraformaldehyde in phosphate

buffered saline for 10 min. After washing, we incubated in primary antibody (anti-PDI, Giantin, or COX IV at 1:2000) in 4% normal goat serum and 0.04% Triton X-100 in phosphate buffered

saline for 2 h. Cells were subsequently incubated in secondary antibody (Alexa Fluor 647, 1:500) for 1 h, then clicked (azide-Alexa Fluor 488) for another hour in the above conditions.

CALCULATED LOGP AND PKA DERIVATIONS Calculated LogP (cLogP) and pKa values were calculated using software available on the virtual computational chemistry laboratory, www.vcclab.org57. cLogP

values are represented as the average ± SD of the output by 6 different algorithms consulted for the calculation. pKa values were calculated using SPARC online software58 or were obtained

from the literature59. DRUGS AND REAGENTS Anti-PDI (Abcam Cat# ab2792, RRID:AB_303304), Anti-COXIV (Abcam Cat# ab16056, RRID:AB_443304) and Anti-Giantin (Covance Research Products Inc Cat#

PRB-114C-200, RRID:AB_10063713) antibodies were all chosen for established specificity60,61,62. (R,S)-Ketamine and (R,S)- norketamine were obtained from Tocris as racemic mixtures. Racemic

(R,S)-A-NK and (R,S)-A-NK-amide were synthesized as described below, starting from racemic norketamine. Norketamine was synthesized using previously published methods17; and the alkyne

(A-NK) or amide (A-NK-amide) were synthesized by alkylation or amidation of norketamine. There was no attempt to resolve diasteromers, and all subsequent transformations likely resulted in

racemic products. 1H NMR and 13C NMR spectrometry was performed on an I400 Varian Inova NMR instrument (Agilent, Palo Alto, CA, 400 MHz for 1H NMR and 100.5 MHz for 13C NMR). High resolution

positive ion electrospray mass spectra were obtained on a Bruker MaXis 4 G Q-TOF mass spectrometer. ALKYNE-NORKETAMINE, A-NK (1) Norketamine (13 mg, 0.058 mmol) and finely ground potassium

carbonate (16 mg, 0.12 mmol) were dissolved in DMF (N, N-dimethylformamide) in a 5-ml reaction vessel. 5-Iodo-1-pentyne (33.8 mg, 0.17 mmol) was added to the solution. The vessel was sealed,

and the reaction solution was heated to 100 °C for 24 h. After the reaction solution was cooled to room temperature, the solution was dissolved in dichloromethane (5 ml), and washed with

10% sodium thiosulfate (1 ml × 2), and brine (1 ml × 2). The organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered under vacuum and concentrated to

viscous liquid. The crude product was purified by silica gel column chromatography eluting with hexane: ethyl acetate (3:2) to afford a light yellow viscous liquid (16 mg, 95.2%). 1H NMR

(400 MHz, CDCl3): δ 7.54 (dd, _J_ = 7.6 Hz, 1 H), 7.35 (dd, _J_ = 8.0 Hz, 1 H), 7.30 (t, _J_ = 7.6 Hz, 1 H), 7.22 (t, _J_ = 7.6 Hz, 1 H), 2.78–2.74 (m, 1 H), 2.51–2.40 (m, 3 H), 2.23–2.20

(m, 2 H), 2.16–2.13 (m, 1 H), 2.07 (s, 1 H), 2.02–1.98 (m, 1 H), 1.88 (t, _J_ = 2.6 Hz, 2 H), 1.80–1.76 (m, 3 H), 1.66–1.61 (m, 2 H). 13C NMR (100.5 MHz, CDCl3): 209.31, 138.71, 134.03,

131.49, 129.52, 128.92, 126.94, 84.35, 70.15, 68.84, 41.40, 39.76, 39.44, 29.58, 28.22, 22.20, 16.48. HRMS (ESI): calculated for C17H20ClNO [M+H]+: 290.1306; found 290.1302.

ALKYNE-NORKETAMINE AMIDE, A-NK-AMIDE (2) 4-Pentynoic acid (10.8 mg, 0.11 mmol) and HBTU (43.7 mg, 0.12 mmol) were dissolved in DMF (200 μl) in a 1 ml vial. N, N-diisopropylethylamine (60 μl,

0.34 mmol) was added to the solution, and the reaction solution was stirred for 15 min. Then a solution of norketamine (19 mg, 0.085 mmol) in DMF (65 μl) was added to the solution, and the

reaction was stirred at room temperature overnight. The crude product was purified on a short silica gel column with an elution of hexane: ethyl acetate (3:2) to afford a yellow viscous

liquid (17 mg, 65.7%). 1H NMR (400 MHz, CDCl3): δ 7.92 (d, _J_ = 8.0 Hz, 1 H), 7.58 (br, 1 H), 7.38–7.22 (m, 3 H), 3.96 (dd, _J_ = 14.4 Hz, 1 H), 2.43–2.32 (m, 6 H), 2.08–2.06 (m, 1 H), 1.92

(s, 1 H), 1.83–1.72 (m, 3 H), 1.63 (t, _J_ = 14.8 Hz, 1 H). 13C NMR (100.5 MHz, CDCl3): 209.63, 169.57, 134.74, 133.83, 132.37, 131.15, 129.80, 126.52, 83.03, 69.42, 68.23, 39.45, 38.55,

35.88, 31.24, 22.54, 14.88. HRMS (ESI): calculated for C17H18ClNO2 [M+Na]+: 326.0918; found 326.0938. STATISTICAL ANALYSES Behavioral data were analyzed using one-way or repeated measures

(rm) analysis of variance (ANOVA) models. These models included one between-subjects variable (Treatment) and the rmANOVAs also included one within-subjects variable (Time Blocks) for

analyzing the general ambulatory and vertical rearing data. The Huynh-Feldt adjustment of alpha levels was utilized for all within-subjects effects containing more than two levels to protect

against violations of sphericity/compound symmetry assumptions underlying rmANOVA models. Pairwise comparisons were conducted following relevant, significant overall ANOVA effects, which

were subjected to Bonferroni correction when appropriate. Upaired _t_ tests, Bonferroni corrected for multiple comparisons where appropriate, were used to analyze electrophysiological and

cyto-fluorescence results. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Emnett, C. _et al_. A Clickable Analogue of Ketamine Retains NMDA Receptor Activity, Psychoactivity, and

Accumulates in Neurons. _Sci. Rep._ 6, 38808; doi: 10.1038/srep38808 (2016). PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and

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Cell 4, 679–693 (1993). CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank members of the Taylor Institute for Innovative Psychiatric

Research for discussion and Amanda Taylor for technical help with cultures. We acknowledge support from the Bantly Foundation and from NIH grants R01AA017413, R01MH077791, R01MH078823,

R01MH101874, and R21MH104506. Collection of mass spectral data were supported by grants from the National Institute of General Medical Sciences (8 P41 GM103422–35). AUTHOR INFORMATION Author

notes * Emnett Christine and Li Hairong contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Psychiatry, Washington University School of Medicine, USA Christine

Emnett, Xiaoping Jiang, Ann Benz, Joseph Boggiano, Sara Conyers, David F. Wozniak, Charles F. Zorumski & Steven Mennerick * Division of Biology and Biomedical Sciences, Graduate Program

in Neurosciences, Washington University School of Medicine, USA Christine Emnett * Department of Radiology, Washington University School of Medicine, USA Hairong Li & David E. Reichert *

Taylor Family Institute for Innovative Psychiatric Research, Washington University School of Medicine, USA David F. Wozniak, Charles F. Zorumski, David E. Reichert & Steven Mennerick *

Department of Neuroscience, Washington University School of Medicine, USA Charles F. Zorumski & Steven Mennerick Authors * Christine Emnett View author publications You can also search

for this author inPubMed Google Scholar * Hairong Li View author publications You can also search for this author inPubMed Google Scholar * Xiaoping Jiang View author publications You can

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inPubMed Google Scholar CONTRIBUTIONS C.E., D.E.R., S.M. and D.W. conceived experiments. C.E., H.L., X.J., A.B., J.B., S.C. performed experiments. All authors wrote, edited, and reviewed the

manuscript. ETHICS DECLARATIONS COMPETING INTERESTS CFZ is a member of the Scientific Advisory Board for Sage Therapeutics. SM holds a sponsored research agreement from Sage Therapeutics.

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