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Abdallah'et'al.'2018'-'Neuropsychopharmacology!
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The Effects of Ketamine on Prefrontal Glutamate Neurotransmission in
Healthy and Depressed Subjects
Chadi G. Abdallah 1,2, Henk M. De Feyter 5, Lynnette A. Averill 1,2, Lihong Jiang 5, Christopher
L. Averill 1,2, Golam M.I. Chowdhury 2,5, Prerana Purohit 1,2, Robin A. de Graaf 5, Irina Esterlis
1,2, Christoph Juchem 3,4,5, Brian P. Pittman 2, John H. Krystal 1,2, Douglas L. Rothman 5,*, Gerard
Sanacora 1,2,*, Graeme F. Mason 2,5,*
1 National Center for PTSD – Clinical Neurosciences Division, US Department of Veterans Affairs, West Haven,
CT, USA
2 Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA
3 Departments of Biomedical Engineering and Radiology, Columbia University, New York, NY, USA
4 Department of Neurology, Yale University School of Medicine, New Haven, CT, USA
5 Yale Magnetic Resonance Research Center, Department of Radiology and Biomedical Imaging, Yale University
School of Medicine, CT, USA
* Equally contributed.
Corresponding Author: Chadi G. Abdallah, chadi.abdallah@yale.edu
Abstract
The ability of ketamine administration to activate prefrontal glutamate neurotransmission is thought
to be a key mechanism contributing to its transient psychotomimetic effects and its delayed and
sustained antidepressant effects. Rodent studies employing carbon-13 magnetic resonance
spectroscopy (13C MRS) methods have shown ketamine and other NMDA receptor antagonists to
transiently increase measures reflecting glutamate-glutamine cycling and glutamate neurotransmission
in the frontal cortex. However, there are not yet direct measures of glutamate neurotransmission in
vivo in humans to support these hypotheses. The current first level pilot study employed a novel
prefrontal 13C MRS approach similar to that used in the rodent studies for direct measurement of
ketamine effects on glutamate-glutamine cycling. Twenty-one participants (14 healthy and 7
depressed) completed two 13C MRS scans during infusion of normal saline or subanesthetic doses of
ketamine. Compared to placebo, ketamine increased prefrontal glutamate-glutamine cycling, as
indicated by a 13% increase in 13C glutamine enrichment (t = 2.4, p = 0.02). We found no evidence of
ketamine effects on oxidative energy production, as reflected by 13C glutamate enrichment. During
ketamine infusion, the ratio of 13C glutamate/glutamine enrichments, a putative measure of
neurotransmission strength, was correlated with the Clinician-Administered Dissociative States Scale
(r = –0.54, p = 0.048). These findings provide the most direct evidence in humans to date that ketamine
increases glutamate release in the prefrontal cortex, a mechanism previously linked to schizophrenia
pathophysiology and implicated in the induction of rapid antidepressant effects.
Introduction
The discovery [2] and replication [3] of the
antidepressant effects of ketamine, and the implication
of N-methyl-D-aspartate receptor (NMDAR)
hypofunction in the pathophysiology of schizophrenia
[4] have generated considerable excitement about the
potential of targeting glutamate neurotransmission to
develop safe, effective, and rapid acting
antidepressants (RAADs) [5], as well as to develop
glutamate-based “antipsychotics” to treat the often
resistant cognitive deficits and negative symptoms in
schizophrenia [6, 7].
However, to date not a single RAAD or glutamate-
based antipsychotic has been approved to treat
depression or schizophrenia, although two decades
have passed since the RAAD and psychotomimetic
effects of ketamine were first reported in the 1990s [4,
8]. A major obstacle in this field is the lack of
biomarkers that directly reflect synaptic glutamate
neurotransmission. Such markers would serve to 1)
test ketamine’s RAAD and psychotomimetic
mechanisms in humans and 2) permit expedited
screening and optimization of putative novel
glutamate-based RAAD and antipsychotic agents.
Carbon-13 magnetic resonance spectroscopy (13C
MRS) is a unique, noninvasive method to measure
neurotransmitter cycling and cell-specific
neuroenergetics [1, 9, 10]. In the current study, we
implemented a 13C MRS pharmacoimaging paradigm,
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using a novel method [11-13] to conduct quantitative
13C MRS acquisition in the human frontal lobe – a
region closely related to psychopathology that was not
initially accessible in 13C MRS studies. We aimed to
determine ketamine’s effect on glutamate-glutamine
cycling and to investigate the association between
changes in neurotransmission and the
psychotomimetic effects of ketamine.
13C MRS is presently the only method that provides
direct dynamic measurements of glutamate-glutamine
cycling in the human brain [1]. Briefly, labeled 13C-
glucose is infused intravenously over 120 minutes
during the MRS acquisition. The incorporation of the
13C in glutamate and glutamine generates unique
signals on the 13C spectrum (Fig. 1). Metabolites of
13C-glucose enter the tricarboxylic acid (TCA; also
known as Krebs) cycle, which in turn label glutamate.
Then, as 13C-glutamate is released into the synaptic
cleft, astrocytes take it up and convert it to 13C-
glutamine (Fig. 2). The glutamate and glutamine 13C
enrichments rise rapidly in the first 20-30 minutes of
13C-glucose infusion and reach a steady-state in the
last 40-60 minutes (Fig. S1). Accordingly, the
glutamate enrichment primarily reflects oxidative
energy production through the TCA cycle, while the
rate of glutamine enrichment primarily reflects
glutamate neurotransmitter cycling [1, 9].
A main limitation of previous methods is the lack of
distinction between presynaptic glutamate release and
postsynaptic activation, the latter has been related to
the RAAD effects of ketamine [14, 15]. While these
processes are highly coupled under normal conditions,
preclinical 13C MRS data suggest disproportionate
changes following ketamine administration [16, 17].
In particular, approximately 9 minutes after
intraperitoneal injection in rodents, ketamine induces
a rise in prefrontal 13C glutamine enrichment (~20%)
with no changes in 13C glutamate (~1%) [17],
suggesting a disproportionate change in
neuroenergetics (VTCA) vs. glutamate cycling (Vcycle) –
i.e., an alteration in neuronal energy-per-cycle (EPC)
ratio. By 18 minutes after the injection, both prefrontal
13C glutamate (~13%) and glutamine (~35%)
enrichment increased [17]. Under normal conditions,
neuronal energy largely supports postsynaptic
neuronal activation and functional increments are
consistently proportional to changes in glutamate-
glutamine cycling, resulting in a largely constant EPC
ratio across species and functional conditions [18, 19].
The alteration of EPC acutely post injection may
reflect a transient reduction in postsynaptic activation
[10] due to blockade of postsynaptic NMDAR by
ketamine and its metabolite, norketamine. Thus, an
alteration of EPC immediately post ketamine injection
(i.e., an increase in presynaptic release without
proportionate increase in postsynaptic activation)
would be consistent with the NMDAR hypofunction
model of schizophrenia, raising the question whether
EPC, as reflected by 13C glutamate/glutamine ratio,
would predict the psychotomimetic effects of
ketamine during infusion in humans. Consistent with
Figure 1. Prefrontal 13C magnetic resonance spectroscopy (MRS) acquisition and 13C spectrum. Sagittal (A)
and axial (B) view of the region of interest – based on the radius of the carbon coil – primarily rostral Brodmann
Area 10. (C) 13C magnetic resonance spectrum acquired at 4T from the prefrontal region of a study participant
during infusion of [U 13C]-glucose. Color code: blue = raw; red = fitted; green = residual. Abbreviations: GluC45
= 13C-Glutamate C45; GlnC45 = 13C-Glutamine C45; AspC34 = 13C-Aspartate C34.
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this hypothesis, the increase in 13C
glutamate/glutamine ratio in rodents is more evident
following the administration of low doses of ketamine
(3mg/kg & 10mg/kg), selective NMDAR subtype 2B
antagonist, and scopolamine – all of which are
expected to have lower psychotomimetic effects
compared to 30mg/kg of ketamine. This possibility, if
confirmed, would signify a paradigm shift suggesting
that the behavioral disturbances during ketamine
infusion may be driven by a loss of communication
fidelity between presynaptic neurotransmission
release and postsynaptic activation, rather than a
correlate only of increased glutamate
neurotransmission. Considering that the RAAD
effects are associated with postsynaptic glutamate
activation, the EPC biomarker will also provide a
mechanism through which novel drugs may induce
RAAD effects without psychotomimetic symptoms,
provided that these new agents equally increase
presynaptic release and postsynaptic activation.
In the current study, we primarily aimed to implement
a novel, complex yet unique, pharmacoimaging
paradigm to translate the preclinical 13C MRS
ketamine findings into a biomarker to apply in humans
in vivo, investigating its potential utility in rational
drug development for mood and psychotic disorders,
as well as for a variety of other potential
neuropsychiatric indications. Additionally,
considering recent preclinical evidence, we tested the
hypothesis that ketamine in a cohort of healthy and
depressed subjects would induce a large increase in
prefrontal 13C glutamine enrichment, with a modest
increase in 13C glutamate enrichment comparable to
recent preclinical findings [17]. Increased 13C-
glutamine enrichment could be interpreted as a
ketamine-induced surge in glutamate-glutamine
cycling, and a rise in 13C glutamate enrichment would
suggest an acute increase in prefrontal oxidative
energy. Based on findings from glutamate inhibitor
studies [20-23], we predicted a positive association
between glutamate-glutamine cycling and the
behavioral psychotomimetic symptoms following
ketamine administration. We also examined whether
variability in 13C glutamate/glutamine ratio, a putative
indicator of EPC, would be associated with the
psychotomimetic effects of ketamine as suggested by
preclinical data.
Methods
Study Population
Twenty-four participants [healthy = 17; major
depressive disorder (MDD = 7)] between the ages of
21 and 65 were recruited for this study. Of the healthy
subjects, one subject did not complete the second scan,
and two subjects had unsuccessful scans. All study
procedures were approved by an institutional review
board (ClinicalTrials.gov NCT02037035). Subjects
completed an informed consent process and were
screened for study inclusion and exclusion criteria. For
all participants, exclusion criteria consisted of medical
conditions that contraindicate MRS or increase the
risks of ketamine administration. A negative drug test
and, for women a negative pregnancy test and use of a
medically accepted means of contraception were
required. Healthy participants were excluded if they
had a lifetime history of any psychiatric disorder, or if
they had a first-degree relative with mood, anxiety, or
psychotic disorder. MDD participants were included if
they met the following study criteria: 1) current major
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Figure 2. Carbon-13 (13C) labeling of glutamate
and glutamine via the TCA and glutamate-
glutamine cycle. Following glycolysis, 13C-Glucose
(13C-Glc) metabolites (i.e., Acetyl-CoA) enter the
mitochondrial tricarboxylic acid (TCA) cycle (also
known as Krebs cycle) and subsequently label
glutamate through exchange with α-ketoglutarate.
Next, 13C-glutamate is released into the synaptic cleft
and taken up by astrocytes, where the 13C-glutamate
is converted to 13C-glutamine and transferred to
neurons. Hence, the rate of 13C-glutamate enrichment
is primarily affected by neuroenergetics (the neuronal
TCA cycle) and the rate of 13C-glutamine enrichment
primarily reflects the rate of glutamate-glutamine
cycling. The development of this metabolic model
and potential impact of other metabolic pathways on
the measured labeling are discussed in reference [1].
The figure was adapted with permission from the
Emerge Research Program (emerge.care).
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depressive disorder as determined by a structured
interview, 2) Montgomery-Åsberg Depression Rating
Scale (MADRS) severity of 18 or more at baseline, 3)
no substance abuse or dependence in the previous 12
months, 4) no lifetime history of psychosis or bipolar
disorder, and 5) medication-free or on a stable (> 4
weeks) antidepressant treatment. The MDD group was
included in the current report to explore the possibility
of differences between the two groups and to test the
feasibility of conducting the 13C MRS
pharmacoimaging paradigm in this population. The
target sample for the MDD group is 18 subjects, which
will be reported in the future once enrollment is
completed.
Overview of Study Design
Two 13C MRS scans, separated by at least one week,
were performed using a single-blind, placebo-
controlled, fixed-order within-subject design (Fig.
S2). On day 1, participants received normal saline and
uniformly 13C-labeled glucose infused over 120
minutes as described previously [24]. Concurrently,
13C MRS of the frontal brain region was used to
observe glutamate and glutamine 13C enrichments
during the placebo infusion. On day 8+, healthy
participants received a subanesthetic dose that has
been commonly used in studies examining the
psychotomimetic effects of the drug (0.23 mg/kg bolus
followed by 0.58 mg/kg infusion over approximately
75 minutes). This dose was administered to ensure
rapid and robust psychotomimetic effect [25, 26],
which we believed would enhance statistical power to
ascertain the relationship between glutamate cycling
and the psychotomimetic symptoms during ketamine
infusion. MDD patients received the dose most
commonly used in depression clinical trials (0.5 mg/kg
infused over 40 minutes) [2, 3], concurrently with
otherwise identical 13C-glucose infusion and 13C MRS
scans. The psychotomimetic effects of ketamine were
assessed using the Clinician-Administered
Dissociative States Scale (CADSS) and Brief
Psychiatric Rating Scale (BPRS) (Fig. S2). The
behavioral assessments in the MDD participants will
be investigated in future reports once a larger cohort is
enrolled.
13C MRS Acquisition & Processing
MRS data were acquired on a 4.0 T whole-body
magnet interfaced to a Bruker AVANCE spectrometer
(Bruker Instruments, Billerica, MA, USA). Subjects
were placed supine in the magnet, with their head
immobilized with foam. An RF probe consisting of
one circular 13C coil (8.5 cm Æ) and two circular,
quadrature driven 1H RF coils (12.5 cm Æ) were used
for acquisition of 13C MR spectra from the frontal lobe
(Fig. 1A & 1B). Following tuning and acquisition of
scout images, second-order shimming of the region of
interest (ROI) was performed using phase mapping
provided by Bruker.
13C MR spectra were acquired with a pulse-acquire
sequence using an adiabatic 90˚ excitation pulse and
optimized repetition time. Nuclear Overhauser
enhancement (nOe) was achieved by applying 1H
block pulses before the 13C excitation pulse. 1H
decoupling during acquisition consists of pseudo noise
decoupling as described by Li et al. [12], to decouple
the long-range 1H-13C coupling of the carboxylic
carbon positions. The pseudo noise decoupling pulse
has a constant amplitude and the phase of each 1.2-ms
unit pulse is randomly assigned to either 0° or 180°.
Following the start of [U 13C]-glucose infusion, 6.5-
minute blocks of MR spectra were acquired for 120
minutes (Fig. 1C & S1). In the last 60 minutes of
acquisition, we alternated 6.5-minute blocks of
acquisition using our standard parameters with
acquisition without nOe and a 30 second delay in order
to obtain a correction for nOe efficiency and saturation
effects.
Spectral data were analyzed with –2Hz/6Hz
Lorentzian-to-Gaussian conversion and 16-fold zero-
filing followed by Fourier transformation. An LC
model approach was used to fit the peak areas of the
labeled carbon positions of glutamate C45 and
glutamine C45 (Fig. 1C), which overlapped with
aspartate C34. Due to the overlap, we analyzed the
combined aspartate and glutamate peaks. However, it
is unlikely that the surge in glutamine enrichment was
affected by changes in aspartate during ketamine
considering that the kinetics of aspartate labeling
closely track that of its glutamate precursor
[27], which was not shown to be affected by ketamine
infusion. The amplitude of 13C-labeling over early
time points (approximately 20 minutes) was averaged
and normalized by labeling over the steady-state
amplitude of the 13C enrichment time course (post-90
minutes). This procedure provided a normalized
measure of 13C glutamate and 13C glutamine
enrichment, which reflects the enrichment rate of these
metabolites early during the infusion of placebo and
ketamine. Cramer-Rao Lower Bounds were used to
estimate the quality of the individual measurements,
averaging 3% for glutamate and 11% for glutamine.
To ensure that the observed 13C enrichment values are
not affected by variability in input glucose enrichment,
plasma fractional enrichment (FE) of 13C glucose was
determined during placebo (early FE = 51.2%; steady
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state FE = 61.1%) and ketamine (early FE = 51.6%;
steady state FE = 61.8%), which did not differ between
sessions (p > 0.2). To provide an estimate of the
variability of the 13C MRS measures between sessions,
we computed the ratio of the mean over the standard
deviation of each of the measures across all 21 subjects
during the placebo scans. We found relatively low
variability, with ratios of 7.3 for 13C glutamine
enrichment and 11 for 13C glutamate enrichment. The
ratios were comparable across healthy (7 for 13C
glutamine and 11 for 13C glutamate) and depressed
subjects (8 for 13C glutamine and 10 for 13C
glutamate).
Statistical Analyses
Before conducting each analysis, the distributions of
outcome measure were examined. Estimates of
variation are provided as standard error of mean
(SEM). Considering that this is a first-in-human study,
this study should be considered a first-level pilot study
implementing a novel technique, rather than a
confirmatory study. Effect sizes are provided as
Cohen’s d. All tests are 2-tailed with significance set
at p ≤ 0.05.
The percent change of 13C glutamate and 13C
glutamine enrichment during ketamine compared to
placebo was computed and represented the primary
outcome measures. One sample t-tests examined
whether the percent changes of each metabolite were
statistically significant. Follow-up analysis used
independent t-tests to determine whether the percent
changes of each metabolite differed between the study
groups. Spearman’s correlations examined the
relationship between the metabolites enrichment and
psychotomimetic effects of ketamine as determined by
CADSS and BPRS in healthy participants.
Results
A total of 21 participants (healthy = 14; MDD = 7)
successfully completed both study sessions [mean
(SEM) age = 30 (1.2) years; 15 males]. In the MDD
group, the baseline MADRS was 27 ± 1.8 (mean ±
SEM), and 3 subjects were on a stable antidepressant
treatment. As described in the Methods, the full
assessment of the behavioral
(antidepressant/psychotomimetic) effects of ketamine
in the MDD group, and their association with 13C
enrichment, will be investigated in future reports with
a larger cohort currently being recruited and studied.
Ketamine Effects on Glutamine and Glutamate
Enrichment
To assess the presence of a glutamate surge, we
compared early time points of the enrichments of
glutamate and glutamine between the placebo and
ketamine groups. This period was comparable to that
studied previously by 13C MRS in rodent models [16,
17]. In the current study, the 13C glutamine
enrichment was 13% greater during ketamine infusion
than with placebo (t = 2.4, df = 20, p = 0.02; Fig. 3),
but no significant effect of ketamine on 13C glutamate
enrichment was seen (t = 0.2, df = 20, p = 0.81; Fig.
3). To examine whether the cycling surge differed in
MDD, we conducted an exploratory analysis which
showed a comparable effect size in healthy (d = 0.48)
and MDD (d = 0.78), with no statistically significant
differences between groups (p = 0.85; Fig. 4).
Exploratory Analyses: Psychotomimetic Effects of
Ketamine
Compared to placebo, ketamine in the healthy group
induced significant increases in CADSS [mean
difference (SEM) = 28 (4.1), t = 6.9, df = 13, p < 0.001]
and BPRS Positive symptoms [mean difference (SEM)
= 10 (1.8), t = 5.5, df = 13, p < 0.001], but not BPRS
Negative symptoms [mean difference (SEM) = 1 (0.6),
t = 1.7, df = 13, p = 0.10].
During the ketamine infusion, the 13C
glutamate/glutamine enrichment ratio was negatively
correlated with CADSS (r = –0.54, n = 14, p = 0.048;
Cohen’s d = 1.3; Fig. 5A), but not with BPRS Positive
(r = –0.43, n = 14, p = 0.13; Cohen’s d = 1.0; Fig. 5B)
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Figure 3. Effects of ketamine on prefrontal
glutamate-glutamine cycling and
neuroenergetics. In the first 20 minutes post
infusion, ketamine induced a rapid increase in 13C-
glutamine enrichment, indicating an acute surge in
prefrontal glutamate-glutamine cycling. The
enrichment of 13C-glutamate remained stable,
suggesting no changes in oxidative energy
production early during ketamine infusion.
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or BPRS Negative symptoms (r = 0.02, n = 14, p =
0.96; Cohen’s d = 0.03). Otherwise, there were no
significant correlations between 13C glutamine or 13C
glutamate enrichment and any of CADSS, BPRS
Positive, or BPRS Negative scores (p > 0.1). During
placebo infusion, there were no significant
correlations between any of the metabolites and
psychotomimetic measures (p > 0.1).
Discussion
The study results provide the most direct evidence yet
in humans of a ketamine-induced increase in
prefrontal glutamate release, based on the detection of
increased 13C labeling of glutamine. The rapid initial
labeling of glutamine is consistent with previous
findings in preclinical models. Comparable to
preclinical findings with 13C MRS [17], the changes in
13C-glutamine (13%) were more prominent than 13C-
glutamate (1%). Given the exponential decrease in
ketamine levels after injection, ketamine and
norketamine brain levels during infusions in humans
are likely to resemble the immediate time period post-
injection in rodents, in which there was no significant
increase in 13C glutamate enrichment (also ~1%). The
previous 13C MRS work with rodents also examined a
set of animals in which 13C administration was begun
with an 18-minute delay after ketamine
administration, and in that condition the increase in
13C glutamate was more apparent, though still less than
glutamine.
The ketamine-induced acute glutamate surge also
plays an important role in the psychotomimetic effects
of the drug, behavioral alterations that are believed to
capture core phenomenology of the pathophysiology
of schizophrenia [28]. Lacking a direct measure of
glutamate neurotransmission in vivo, investigators
over the past two decades have employed several
elegant approaches to infer the presence of a ketamine-
induced glutamate surge in humans and to relate this
surge to its psychotomimetic effects. Early studies
have shown that inhibitors of glutamate release
attenuate the ketamine-induced psychotomimetic
symptoms [20-23]. Other studies have employed
pharmacoimaging paradigms to demonstrate a
relationship between ketamine-induced prefrontal
activation/connectivity and its transient behavioral
disturbances [29-36], or to block prefrontal
activation/connectivity using glutamate inhibitors [22,
37]. More recently, a metabotropic glutamatergic
receptor tracer was also used to explore the acute
effects of ketamine [38-40]. 1H MRS, a method
capable of estimating total – but not dynamic cycling
of – prefrontal glutamate and glutamine levels show
changes in tissue content of glutamate and/or
glutamine during ketamine infusion [[36, 41-43] but
also see [44]]. Together, the data provided indirect
evidence supporting the presence of a ketamine-
induced surge in glutamate neurotransmission in
humans and that this surge may be associated with the
psychotomimetic effects of the drug.
While underscoring the pilot nature of the correlation
analyses in the current report, it is intriguing that
CADSS scores significantly correlated with
glutamate/glutamine ratio (r = –0.54, n = 14, p =
0.048; Cohen’s d = 1.3), but not with glutamine
enrichment (r = –0.38, n = 14, p = 0.19; Cohen’s d =
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Figure 4. Separate effects of ketamine on prefrontal glutamate-glutamine cycling and neuroenergetics in
healthy and major depressive disorder (MDD) subjects. (A) Mean and SEM of 13C-glutamate and 13C-glutamine
during placebo (Plc) and during ketamine (Ket). (B) Individual values, as well as mean and SEM, of percent change
in enrichment of 13C-glutamine during ketamine compared to placebo in each group. The pattern of changes in 13C-
glutamine and 13C-glutamate enrichments was comparable between healthy and depressed subjects, with no
statistically significant differences between the two groups. The similarities indicate that while the impacts of
ketamine on glutamate-glutamine cycling were significant, both groups were affected to about the same extent.
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0.8). A possible explanation is that the low
glutamate/glutamine enrichment ratio reflects low
EPC. In this model, healthy individuals with high
sensitivity to ketamine (i.e., NMDAR hypofunction)
would have higher blockade of synaptic NMDAR,
leading to reduced postsynaptic activation per
glutamate cycle, and subsequent increase in
psychotomimetic symptoms. This hypothesis could be
assessed in future 13C MRS studies enrolling larger
sample of healthy individuals with variable sensitivity
to ketamine (e.g., with/without family history of
alcoholism [45, 46]), as well as by testing the ability
of NMDAR agonists to affect the 13C
glutamate/glutamine ratio. The approach parallels
extent preclinical data [16, 17], which facilitates the
translatability of the biomarker between species.
Limitations: Thus far, this study provided first-level
pilot evidence about the potential utility of the 13C
MRS pharmacoimaging paradigm in human models of
schizophrenia, demonstrating the long-hypothesized
ketamine-induced glutamate surge and presenting
pilot evidence associating the psychotomimetic effects
of ketamine with reduced EPC, as estimated by the 13C
glutamate/glutamine ratio. However, considering the
pilot nature of the association findings, and the lack of
direct modulation implicating NMDAR hypofunction
in the variability of 13C glutamate/glutamine ratio,
future replications and laboratory testing are required.
To assess the psychotomimetic effects in the healthy
group, we administered a larger dose along with a
bolus. Future studies aiming to compare
neurotransmission changes between healthy and
depressed groups would require administering the
same doses in both populations. Other limitations
include the small sample size which requires
replication in larger studies, and the fixed order
acquisition, which requires future test-retest reliability
data or randomized parallel groups design. Moreover,
the overlap between glutamine and aspartate peaks
prevented the individual fitting of aspartate and
glutamine. Finally, due to limitations that may include
biological variability and low spatial resolution of 13C
MRS, the application of the 13C-glutamate/glutamine
ratio may be limited to alterations in glutamate
neurotransmission in groups of individuals or within
subjects. However, new higher spatial resolution 1H-
13C MRS methods that can measure glutamate and
glutamine, without aspartate, may allow application to
evaluate individual subject responses in the future
[47].
Preclinical studies have found that the NMDAR
antagonist ketamine blocks glutamate
neurotransmission, but only at high anesthetic doses
[48]. At subanesthetic doses, ketamine induces a
paradoxical increase in glutamate neurotransmission
[15, 49]. The acute surge in glutamate transmission
increases prefrontal synaptic connectivity, through
enhanced activity-dependent brain-derived
neurotrophic factor (BDNF) and related downstream
signaling [15]. Other putative RAADs (e.g.,
scopolamine and (2R,6R)-Hydroxynorketamine)
show a comparable acute surge in prefrontal glutamate
[17, 50-53] and enhanced BDNF signaling [54, 55].
Together, the preclinical evidence to date underscores
the critical role of acute prefrontal glutamate
neurotransmission in the mechanism of action of
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Figure 5. Association between glutamate neurotransmission and the psychotomimetic effects of ketamine in
healthy participants. (A) Reduced neuroenergetics relative to neurotransmitter cycling, as measured by the 13C-
glutamate/13C-glutamine (13C Glu/Gln) ratio, is associated with the psychotomimetic effects of ketamine, as measured
by the Clinician-Administered Dissociative States Scale (CADSS). (B) The association with the Brief Psychiatric
Rating Scale (BPRS) positive symptoms was comparable in effect size, but did not reach statistical significance in
this sample of 14 subjects.
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various RAADs. It also highlights the role of
prefrontal synaptic plasticity – i.e., the increased
density and connectivity evident at 24 hours post
treatment and lasting for a week [5, 56].
In humans, functional global brain connectivity was
previously used to provide a well-replicated evidence
of reduced prefrontal connectivity in individuals with
MDD [37, 57-59], a dysconnectivity that is normalized
24 hours post-ketamine treatment [37, 57]. The
ketamine-induced subacute increases in prefrontal
connectivity were positively associated with the
antidepressant response [57]. In healthy subjects, a
direct link between prefrontal connectivity and
glutamate neurotransmission was demonstrated, while
showing an acute ketamine-induced surge in
prefrontal connectivity which is attenuated by a
glutamate inhibitor [37]. Together, the human
evidence is consistent with the findings from
preclinical models, with particular focus on the
essential role of acute and subacute prefrontal synaptic
neurotransmission and connectivity in the mechanism
of ketamine and perhaps other RAADs.
In this context, the 13C MRS pharmacoimaging
paradigm and findings offer the following evidence
and possibilities: (1) It is consistent with the
occurrence of an acute ketamine-induced glutamate
surge in humans, using a paradigm that is readily
translated between animals and humans. (2) The
similar time frames between preclinical observations
with human connectivity and neurotransmitter cycling
data raises the question whether acute increases in
glutamate-glutamine cycling would predict the
antidepressant effects of ketamine, comparable to
findings from preclinical and connectivity data. (3)
The findings, taken together with published
observations, suggest the possibility of a positive
relationship between prefrontal glutamate cycling and
global connectivity. Such data would not only inform
us about the mechanisms of RAADs, but also provide
important information about the interplay between
molecular neurotransmission and large-scale intrinsic
networks in the human brain. (4) It may be that cycling
and connectivity measures could facilitate the
optimization of antipsychotics and RAADs doses prior
to testing in costly clinical trials. Taken together, these
points raise the possibility of application as rigorous
and reproducible biomarkers of treatment target
engagement and validation to improve the
development of novel antipsychotics and RAADs.
Funding and Disclosure
Funding and research support were provided by NIMH
(K23MH101498), NIAAA (R01AA021984), the VA National
Center for PTSD, Brain & Behavior Foundation (NARSAD),
Patterson Trust Award, American Psychiatric Foundation, Clinical
Neuroscience Research Unit (CNRU) at Connecticut Mental Health
Center, and Yale Center for Clinical Investigation (YCCI UL1
RR024139), an NIH Clinical and Translational Science Award
(CTSA). The content is solely the responsibility of the authors and
does not necessarily represent the official views of the sponsors, the
Department of Veterans Affairs, NIH, or the U.S. Government.
CGA has served as a consultant and/or on advisory boards for
Genentech and Janssen, and editor of Chronic Stress for Sage
Publications, Inc.; JHK is a consultant for AbbVie, Inc., Amgen,
Astellas Pharma Global Development, Inc., AstraZeneca
Pharmaceuticals, Biomedisyn Corporation, Bristol-Myers Squibb,
Eli Lilly and Company, Euthymics Bioscience, Inc., Neurovance,
Inc., FORUM Pharmaceuticals, Janssen Research & Development,
Lundbeck Research USA, Novartis Pharma AG, Otsuka America
Pharmaceutical, Inc., Sage Therapeutics, Inc., Sunovion
Pharmaceuticals, Inc., and Takeda Industries; is on the Scientific
Advisory Board for Lohocla Research Corporation, Mnemosyne
Pharmaceuticals, Inc., Naurex, Inc., and Pfizer; is a stockholder in
Biohaven Pharmaceuticals; holds stock options in Mnemosyne
Pharmaceuticals, Inc.; holds patents for Dopamine and
Noradrenergic Reuptake Inhibitors in Treatment of Schizophrenia,
U.S. Patent No. 5,447,948 (issued Sep 5, 1995), and Glutamate
Modulating Agents in the Treatment of Mental Disorders, U.S.
Patent No. 8,778,979 (issued Jul 15, 2014); and filed a patent for
Intranasal Administration of Ketamine to Treat Depression. U.S.
Application No. 14/197,767 (filed on Mar 5, 2014); U.S. application
or Patent Cooperation Treaty international application No.
14/306,382 (filed on Jun 17, 2014); GS reports personal consulting
fees from Alkermes, Allergan, Biohaven Pharmaceuticals, Eli Lilly
and Co., Genetech, Intra-Cellular Therapies, Janssen
Pharmaceuticals, Lundbeck Research USA, Merck & Co., Naurex,
Navitor Pharmaceuticals, Noven Pharmaceuticals, Teva
Pharmaceuticals Industries, Taisho Pharmaceutical Co., Takeda
Pharmaceutical Co, Sage Pharmaceuticals Inc., Sevier, Valeant
Pharmaceuticals, and Vistagen Therapeutics Inc.; grants and
research contracts from Eli Lilly and Co., Janssen Pharmaceuticals,
Merck & Co., and Sevier and support from Sanofi-Aventis, in the
form of free medication for an NIH sponsored study over the last 36
mos. In addition, Dr. Sanacora is a stockholder and holds stock
options in Biohaven Pharmaceuticals; and has a patent for
Glutamate Modulating Agents in the Treatment of Mental
Disorders, U.S. Patent No. 8,778,979 (issued Jul 15, 2014) with
royalties paid from Biohaven Pharmaceuticals; GFM is a consultant
for Sumitomo Dainippon Pharma Co. Ltd and UCB Pharma SA, and
serves on the Scientific Advisory Board of Elucidata Inc.; All other
authors report no competing interests.
Acknowledgments
The authors would like to thank the individuals who participated in
these studies for their invaluable contribution.
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