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Lithium: A switch from LTD- to LTP-like plasticity in human cortex
Neuropharmacology 63 (2012) 274e279
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Lithium: A switch from LTD- to LTP-like plasticity in human cortex Hanna Voytovych, Lucia Kriváneková, Ulf Ziemann* Department of Neurology, Goethe-University Frankfurt, Schleusenweg 2e16, D-60528 Frankfurt am Main, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2012 Received in revised form 5 March 2012 Accepted 23 March 2012
Lithium, a simple cation, is the mainstay treatment of bipolar disorder. Deficient synaptic plasticity is considered one important mechanism of this disease. Lithium inhibits glycogen synthase kinase-3beta (GSK-3b), which is involved in the regulation of synaptic plasticity. In animal preparations, inhibition of GSK-3b by lithium up-regulated long-term potentiation (LTP) of excitatory synapses but downregulated long-term depression (LTD). The effects of lithium on plasticity in the human brain are unexplored. We tested the effects of a single oral dose of 900 mg of lithium on LTP-/LTD-like plasticity in human motor cortex induced by established paired associative transcranial magnetic stimulation (PASLTP, PASLTD) protocols. We studied 10 healthy adults in a placebo-controlled double-blind randomized crossover design. PAS-induced plasticity was indexed by change in motor evoked potential amplitude recorded in a hand muscle. In the placebo session, subjects were stratified, according to the known variability of the PASLTP response, into PASLTP ‘LTP responders’ and PASLTP ‘LTD responders’ (n ¼ 5 each). Lithium did not affect the PASLTP-induced LTP-like plasticity in the ‘LTP responders’, but switched the PASLTP-induced LTD-like plasticity in the ‘LTD responders’ to LTP-like plasticity. In contrast, lithium had no effect on the PASLTD-induced LTD-like plasticity in the ‘LTD responders’. We provide first-time evidence that lithium significantly modulates brain stimulation induced plasticity in human cortex. The switch from LTD- to LTP-like plasticity is best explained by the inhibitory action of lithium on GSK3b. This conclusion is necessarily circumstantial because GSK-3b activity was not directly measured. We discuss that other important plasticity-related modes actions of lithium cannot explain our findings. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Lithium Transcranial magnetic stimulation Paired associative stimulation Long-term potentiation Long-term depression Motor cortex
1. Introduction Lithium is a monovalent cation that constitutes the mainstay treatment of bipolar disorder for the last 50 years (Geddes et al., 2004). How lithium exerts its therapeutic effects has still not been fully elucidated (Phiel and Klein, 2001). Lithium inhibits several enzymes, including the multifunctional serine/threonine kinase glycogen synthase kinase-3 (GSK-3) (Jope, 2003). The isoform GSK-3b is highly enriched in brain tissue (Woodgett, 1990) and shows high basal activity, which is primarily determined by its dephosphorylation status. Recent experiments have implicated a fundamental role of GSK-3b in the regulation of the direction and magnitude of N-methyl-D-aspartate receptor (NMDAR) dependent plasticity at excitatory synapses. In rat hippocampus, induction of NMDAR dependent long-term depression (LTD) by electrical stimulation resulted in further activation of GSK-3b through dephosphorylation (Peineau et al., 2007) while induction of long-term potentiation (LTP) resulted in deactivation of GSK-3b through
* Corresponding author. Tel.: þ49 69 6301 5739; fax: þ49 69 6301 4498. E-mail address: [email protected] (U. Ziemann). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.03.023
phosphorylation (Hooper et al., 2007; Peineau et al., 2007). GSK-3b inhibition by lithium prevented the induction of LTD (Peineau et al., 2007) and deficient LTP in GSK-3b overexpressing mice could be rescued by lithium (Hooper et al., 2007; Zhu et al., 2007). Therefore, the inhibitory action of lithium on GSK-3b implicates a modulating effect on NMDAR dependent bidirectional synaptic plasticity favoring LTP over LTD. In bipolar disorder and major depression, increased GSK-3b activity and associated dysregulation of synaptic plasticity have been proposed from animal disease models (Beaulieu et al., 2008; Carlson et al., 2006). Therefore, targeting hyperactive GSK-3brelated signaling pathways by lithium or other drugs has been advocated as a premier strategy for effective treatment of these disorders (Schloesser et al., 2011). Here we tested the effects of lithium for the first-time in a human experimental model of bidirectional LTD-/LTP-like plasticity. Paired associative stimulation (PAS) of motor cortex (Jung and Ziemann, 2009; Müller-Dahlhaus et al., 2010; Stefan et al., 2000; Wolters et al., 2003) was employed to induce long-term increase (PASLTP) or decrease (PASLTD) in motor evoked potential (MEP) amplitude, a marker of corticospinal excitability elicited by transcranial magnetic stimulation (TMS) (Hallett, 2007). The PAS-
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induced MEP changes are reminiscent of bidirectional spike timingdependent plasticity in neuronal cultures and slices (Bi and Poo, 2001). They have been termed LTP-like and LTD-like plasticity (Ziemann et al., 2004) because they show fundamental characteristics of LTP and LTD as identified at the cellular level in basic experiments, such as NMDAR dependency, duration > 30 min, associativity and cooperativity (Cooke and Bliss, 2006; MüllerDahlhaus et al., 2010). Inter-individual variability of the PASLTP response is well known, with a significant fraction of individuals showing a decrease in MEP amplitude (‘LTD responders’) rather than an increase (‘LTP responders’) (Müller-Dahlhaus et al., 2008). This variability may be determined by many factors, including gender, age, genetics or treatment with CNS active drugs (Ridding and Ziemann, 2010). Here we hypothesized that the PASLTP ‘LTD responders’ serve as natural model for high intrinsic GSK-3b activity, and that an LTPlike increase in MEP amplitude in the PASLTP ‘LTD responders’ could be rescued by pre-application with lithium. We provide firsttime evidence in humans in favor of these hypotheses: lithium switched the PASLTP-induced LTD-like response into an LTP-like response. The present findings are relevant for research of neuropsychiatric diseases such as bipolar disorder where excessive GSK3b activity is thought to play a pathogenetic role (Beaulieu et al., 2008). 2. Materials and methods 2.1. Subjects Ten healthy young adults participated in the PASLTP experiment (age range, 21e31 years; mean age, 24.3 2.8 years; 6 female). The groups of PASLTP ‘LTP responders’ (for definition, see below; n ¼ 5; mean age, 25.4 3.5; 3 female) and PASLTP ‘LTD responders’ (n ¼ 5; mean age, 23.2 1.6 years; 3 female) did not differ with respect to the demographic data. No subject had a history of neuropsychiatric disease or was on CNS active medication or recreational drugs. All subjects were right-handed, as indicated by the Edinburgh Handedness Inventory (Oldfield, 1971). The study was approved by the ethics committee of the medical faculty of Goethe University, Frankfurt am Main, Germany and by the Federal Institute for Drugs and Medical Devices of Germany (BfArM). The study conformed to the latest version of the Declaration of Helsinki. All volunteers gave their written informed consent prior to participation. 2.2. MEP measurements Focal TMS was performed using a flat figure-of-eight-shaped stimulating coil (outer diameter of each wing, 70 mm) connected to a Magstim 200 magnetic stimulator (Magstim Company, UK) with a monophasic current waveform. The coil
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was placed over hand area of the left primary motor cortex (M1), at the site giving rise to the largest motor evoked potentials (MEP) in the abductor pollicis brevis (APB) muscle of the right hand. The coil was held tangentially to the skull with the handle pointing backward and laterally at a 45 angle to the sagittal plane. Thus, the induced current in the brain flowed from lateral-posterior to medial-anterior. This current direction is optimal for trans-synaptic excitation of the corticospinal neurons (Di Lazzaro et al., 2008). MEP recordings were obtained by surface electromyography (EMG), using AgeAgCl cup electrodes in a bellyetendon montage. The EMG raw signal was amplified and filtered (0.02e2 kHz; D360 amplifier; Digitimer Ltd., UK), digitized (analogedigital rate, 5 kHz; CED Micro 1401Ò; Cambridge Electronic Design, UK), and fed into a laboratory computer for online visual display and offline analysis. All measurements were obtained during muscle rest, which was continuously monitored by high-gain EMG audioevisual feedback. 2.3. Paired associative stimulation (PAS) PAS consisted of 225 pairs (rate, 0.25 Hz) of electrical stimulation of the right median nerve at the wrist followed by focal TMS of the APB representation of left M1. Electrical stimulation was applied through a bipolar electrode (cathode proximal), using constant-current square-wave pulses (duration, 1 ms) at an intensity of three times the perceptual sensory threshold. TMS intensity was adjusted to elicit MEP of, on average, 1 mV peak-to-peak amplitude (MEP1mV) in the right APB when given without the preceding median nerve stimulus. In the first experiment (all 10 subjects participated), the interstimulus interval between the median nerve stimulus and TMS was set to the individual N20-latency of standard median nerve somatosensory-evoked potential recordings plus 2 ms (N20 þ 2 ms). At this interval, PAS produced an LTP-like increase in MEP amplitude in the majority of subjects in previous studies (PASLTP), with a fraction of subjects however showing a LTD-like MEP decrease (Fratello et al., 2006; Müller-Dahlhaus et al., 2008). In the present study, these PASLTP ‘LTD responders’ (i.e. MEPP1eP6/MEPB1 1.0) were subjected to another PAS protocol with a slightly shorter interstimulus interval of N20e5 ms (PASLTD), which in previous studies consistently resulted in a LTD-like decrease of MEP amplitude (Müller et al., 2007; Ziemann et al., 2004). Because attention may have considerable effects on the magnitude of a PAS effect (Stefan et al., 2004), subjects were instructed to continuously watch the stimulated hand, to count randomly occurring flashes of an LED attached to the dorsum of this hand, and to report the number at the end of the PAS intervention. 2.4. Lithium serum concentration Blood samples were taken immediately before and 2 h after drug intake (Fig. 1) for measurements of the plasma concentration of lithium. 2.5. Experimental design The study was performed in a double-blind, randomized, placebo-controlled crossover design. In the PASLTP and in the PASLTD experiment, subject participated in two sessions each (Fig. 1): in one session a single oral dose of 900 mg of lithium carbonate, equivalent to 24.4 mmol of lithium (QuilonumÒ, GlaxoSmithCline), and in the other session placebo (PBO) was applied. The dose of 900 mg of lithium was chosen because (1) it is equivalent to a medium daily dose in the treatment of patients with bipolar disorder to reach therapeutic blood concentrations of
10 Subjects
B0
Blood Sample
Lithium Placebo
Blood Sample
B1
PASLTP
P1…P6 0 - 30 min
5 Subjects: PASLTP LTD Responder
B0
Blood Sample
Lithium Placebo
Blood Sample
B1
PASLTD
P1…P6 0 - 30 min
Fig. 1. Timeline of experiments. B0, baseline MEP measurements (before drug intake); B1, MEP measurements 2 h after drug intake and immediately prior to PAS; P1eP6, six blocks of MEP measurements at 5, 10, 15, 20, 25 and 30 min post-PAS. MEP measurements consisted of 20 trials per time point. Ten subjects participated in the PASLTP experiment, and the five PASLTP LTD responders were tested a few weeks later in the PASLTD experiment. Both experiments consisted of two pseudo-randomized double-blinded sessions (lithium versus placebo, separated by at least 7 days).
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0.5e1.2 mmol/L (summary of product characteristics of QuilonumÒ); (2) previous unpublished data from our group showed that a single oral dose of 900 mg of lithium, in contrast to lower and higher doses, had no significant effect on corticospinal excitability as measured by MEP amplitude. This limited the possibility that effects of lithium on PAS-induced plasticity as measured by changes in MEP amplitude would be contaminated by direct effects of lithium on corticospinal excitability. The PBO and lithium sessions within one PAS experiment were at least 1 week apart to rule out carry over effects. The PASLTD experiment in the PASLTP ‘LTD responders’ followed the PASLTP experiment by several weeks. To test the effects of drug (lithium versus PBO) on corticospinal excitability and PAS-induced plasticity, MEP amplitudes were measured at eight time points (Fig. 1): baseline (time point B0, prior to drug intake), 2 h post-dose and immediately prior to PAS (time point B1), and 6 times post-PAS in steps of 5 min, covering 30 min after PAS (time points P1eP6). At each time point, 20 trials were recorded with a random intertrial interval (range, 8e12 s) to limit anticipation of the next trial. TMS intensity at B0 was set to MEP1mV. The experimental protocol prescribed to adjust stimulus intensity at B1, if the MEP amplitude at B1 deviated by >20% from the one at B0, in order to re-install MEP1mV prior to PAS. However, this was necessary only once in one subject each in the PASLTP and PASLTD experiments, indicating that lithium and PBO had no significant effect on corticospinal excitability. The timing of B1 2 h after drug intake was chosen because plasma levels of lithium after a single oral dose peak 2e4 h after intake (summary of product characteristics of QuilonumÒ). 2.6. Statistical analysis The software package StatsView for Windows (SAS Institute Inc., Version 5.0.1) was used for statistical analysis. Peak-to-peak MEP amplitudes were analyzed in the single trials and then averaged for each subject, time point and drug condition. Possible differences in baseline MEP amplitudes (time point B0) or 2 h after drug intake (time point B1) between drug (lithium versus PBO) were checked by twosided t-tests for paired samples, and MEP differences between responder status (PASLTP ‘LTP responders’ versus PASLTP ‘LTD responders’; see below for definition) were tested by two-sided t-tests for unpaired samples. In addition, MEP amplitudes were compared between time points B0 and B1 within drug and responder status conditions by two-sided t-tests for paired samples. For analysis of the PAS effects, MEP amplitudes at time points P1eP6 were normalized to those at time point B1. In the PASLTP experiment, subjects were defined as PASLTP ‘LTP responders’, if in the PBO condition MEPP1eP6/MEPB1 > 1.0, and as PASLTP ‘LTD responders’, if in the PBO condition MEPP1eP6/MEPB1 1.0. The effects of PAS on MEP amplitude in the PASLTP experiment were analyzed in a three-way mixed analysis of variance (ANOVA) with the within-subject effects of time (seven levels: B1, P1eP6) and drug (two levels: lithium and PBO), and the between-subject effect of responder status (two levels: LTP responder versus LTD responder). According to a significant responder status * drug interaction (see Results) two subsequent two-way repeated measures ANOVAs (rmANOVAs) explored the effects of time and drug for the PASLTP ‘LTP responders’ and PASLTP ‘LTD responders’ separately. Similarly, in the PASLTD experiment (only PASLTP ‘LTD responders’ included), the effects of PAS on MEP amplitude were tested in a two-way rmANOVA with the within-subject effects of time (seven levels: B1, P1eP6) and drug (two levels: lithium and PBO). In case of significant main effects or interactions, Fisher’s LSD test was applied for post hoc analysis. All results are expressed as means SEM. P values < 0.05 were considered statistically significant.
3. Results No adverse events were observed during the experiments except for one subject that reported self-limiting diarrhea a few hours after lithium intake. 3.1. MEP amplitude at baseline (B0) and 2 h after drug intake (B1) MEP amplitudes at baseline (time point B0) and 2 h after drug intake (time point B1) did not differ between responder status or drug conditions (all P > 0.25), and were always close to the targeted amplitude of 1 mV (Table 1). In addition, there was no effect of time on MEP amplitude (comparison of time points B1 and B0) within any of the drug or responder status conditions (all P > 0.25). These nil findings indicate that there were no baseline differences in motor excitability or significant drug effects on motor excitability that could account for the effects of drug on PAS-induced plasticity (see below).
Table 1 MEP amplitude (in mV) and lithium (Li) plasma concentration (in mmol/L) at time points B0 and B1. Group
Drug
MEP (B0)
PASLTP ‘LTP responder’ PASLTP ‘LTD responder’ PASLTD
PBO Lithium PBO Lithium PBO Lithium
0.95 1.16 1.07 1.03 1.14 1.07
0.09 0.05 0.10 0.05 0.03 0.03
MEP (B1) 1.03 0.99 0.96 1.00 1.05 1.07
0.09 0.08 0.06 0.03 0.12 0.11
Li (B0) 0.04 0.04 0.05 0.03 0.03 0.03
Li (B1) 0.01 0.02 0.02 0.02 0.01 0.01
0.05 0.41 0.04 0.48 0.03 0.35
0.01 0.07 0.02 0.11 0.01 0.14
All data are means 1 SEM.
3.2. Lithium blood concentrations Lithium blood concentration was 0.03e0.05 mmol/L at baseline (time point B0) and in the PBO condition 2 h later (time point B1) but increased significantly 2 h after lithium intake to 0.35e0.48 mmol/L (Table 1). Importantly, there was no difference between lithium blood concentrations at time point B1 between responder status in the PASLTP experiment (P ¼ 0.60) or between PASLTP and PASLTD experiment in the PASLTP ‘LTD responders’ (P ¼ 0.47) that could have explained the differential effects of lithium on PASLTP-induced plasticity in PASLTP ‘LTP responders’ versus PASLTP ‘LTD responders’ or on PASLTP-induced versus PASLTDinduced plasticity in the PASLTP ‘LTD responders’ (see next paragraphs). 3.3. Effects of lithium on PASLTP-induced plasticity In the PBO condition, there were 5 PASLTP LTP responders (i.e. MEPP1eP6/MEPB1 > 1.0, mean MEPP1eP6/MEPB1 ¼ 1.33 0.04) and 5 PASLTP LTD responders (i.e. MEPP1eP6/MEPB1 1.0, mean MEPP1eP6/ MEPB1 ¼ 0.75 0.08). The mixed ANOVA revealed a significant effect of responder status (F1, 8 ¼ 12.76, P ¼ 0.007), time (F6, 48 ¼ 3.09, P ¼ 0.012), and of the interaction between responder status * drug (F1, 8 ¼ 10.46, P ¼ 0.012) (Fig. 2AeB), while the effect of drug and any of the other interactions were not significant. A subsequent two-way rmANOVA showed that within the group of PASLTP ‘LTP responders’ time had a significant effect on normalized MEP amplitude (F6, 24 ¼ 2.82, P ¼ 0.032) while the effect of drug and of the interaction of drug * time had no effect (Fig. 2A). In contrast, the two-way rmANOVA within the group of PASLTP ‘LTD responders’ showed a significant effect of drug (F1, 4 ¼ 19.04, P ¼ 0.012) but no effect of time or the interaction of drug * time (Fig. 2B). Post hoc comparisons revealed that MEP amplitude normalized to B1 was larger under lithium than PBO at time points P2 and P5 after PASLTP (P < 0.05, Fig. 2B). 3.4. Effects of lithium on PASLTD-induced plasticity in PASLTP ‘LTD responders’ In the PBO condition, PASLTD in the PASLTP ‘LTD responders’ resulted in an LTD-like effect similar to the PASLTP experiment (mean MEPP1eP6/MEPB1 ¼ 0.71 0.05, P > 0.7) (Fig. 2C). The rmANOVA revealed a significant effect of time (F6, 24 ¼ 3.79, P ¼ 0.0085) but no effects of drug or of the interaction drug * time (Fig. 2C). A three-way rmANOVA in the PASLTP ‘LTD responders’ with the main effects drug (lithium versus PBO), PAS experiment (PASLTP versus PASLTD) and time (B1, P1eP6) demonstrated a significant effect of drug (F1, 4 ¼ 9.68, P ¼ 0.036), and of the interactions PAS experiment * drug (F1,4 ¼ 37.05, P ¼ 0.0037) and PAS experiment * time (F6,24 ¼ 2.57, P ¼ 0.046), while the other effects and interactions were not significant (cf. Fig. 2BeC).
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1.8 1.6
PBO Lithium
1.4 1.2 1 .8 .6 .4
B1
P1
P2
P3
P4
P5
P6
PASLTP : LTD responder 1.8
PBO Lithium
1.6
1.4
1.4
1.2
1.2
1 .8 .6 .4
PASLTD (PASLTP LTD responder)
1.8
PBO Lithium
1.6
MEP amplitude normalized to B1
C
B PASLTP : LTP responder
MEP amplitude normalized to B1
MEP amplitude normalized to B1
A
277
B1
P1
Time
P2
P3
Time
P4
P5
P6
1 .8 .6 .4
B1
P1
P2
P3
P4
P5
P6
Time
Fig. 2. A. MEP amplitude after PASLTP (time points P1eP6) normalized to baseline prior to PASLTP (time point B1) in PASLTP LTP responders (n ¼ 5) under placebo (PBO, black circles) versus lithium (white circles). B. Same conventions and arrangements as in A for the PASLTP LTD responders (n ¼ 5) in the PASLTP experiments, and C. in the PASLTD experiment. All data are means 1 SEM. Asterisks denote significant difference between drug conditions (post hoc paired t-tests, P < 0.05).
4. Discussion The main novel finding of this study is that a single oral dose of lithium switched individuals with a PASLTP-induced LTD-like MEP decrease under placebo conditions to a LTP-like MEP increase. We replicated earlier findings that, without CNS active drugs, PASLTP typically results in a LTP-like increase in MEP amplitude in healthy young adults but, in a substantial fraction of subjects, also in a LTD-like MEP decrease (Fratello et al., 2006; Müller-Dahlhaus et al., 2008). The reasons for this substantial inter-individual variability to a standardized non-invasive brain stimulation protocol are not fully understood, but a multitude of determinants, such as gender, age, preceding motor activity, genetic polymorphisms of important regulatory molecules such as brain-derived neurotrophic factor, and pharmacological modulation may influence the direction and magnitude of brains stimulation induced MEP change (Ridding and Ziemann, 2010). Here we were interested in the effects of lithium, a simple cation that has been shown recently in animal experiments in vitro and in vivo to modulate LTD-/LTP through inhibition of GSK-3b. Before we will enter a detailed discussion that inhibition of GSK-3b by lithium is the most likely candidate mechanism to explain the present findings, two caveats should be emphasized: First, any conclusion toward the proposed link between modulation of PAS-induced LTP-/LTD-like plasticity and inhibition of GSK3b by lithium is necessarily circumstantial because GSK-3b activity was not directly measured. Second, lithium has several other identified modes of action related to regulation of synaptic plasticity, which may have contributed to the current findings (Phiel and Klein, 2001). Lithium depletes inositol-1,4,5 trisphosphate (IP3) through direct inhibition of inositol monophosphatase. IP3 mediates release of intracellular Ca2þ from internal stores. Consequently, IP3 receptor antagonists led to significant attenuation of hippocampal LTP (Gärtner et al., 2006). Thus, this mode of action would not explain the lithium-induced switch from PASLTP-induced LTD- to LTP-like plasticity in the present experiments. In addition, lithium enhances glutamatergic neurotransmission by increasing the open probability of AMPA receptors in rat hippocampus (Gebhardt and Cull-Candy, 2010) resulting in enhanced excitatory postsynaptic potentials (Colino et al., 1998). However, an increase in MEP amplitude that should be expected by enhancement of ionotropic glutamatergic neurotransmission (Di Lazzaro et al., 2003) was not observed (Table 1). Therefore, while a contribution by those mechanisms cannot be
entirely excluded they would lead to results that are inconsistent with the present findings. In contrast, our findings are in excellent agreement with previous studies on the effects of lithium on regulation of LTP/LTD through GSK-3b inhibition. Transgenic mice with an overexpression of GSK-3b showed significantly less LTP at Schaffer commissuralpyramidal cell synapses in the CA1 region of the hippocampus compared to wild-type controls (Hooper et al., 2007). This LTP deficiency in the transgenic mice was rescued to the level of the wild-type controls if the transgenic mice were chronically pretreated with lithium (Hooper et al., 2007). Also, pharmacological overactivation of GSK-3b by wortmannin resulted in LTP deficiency in rat hippocampus which could be prevented by simultaneous inhibition of GSK-3b by lithium (Zhu et al., 2007). It is very suggestive that these data directly translate to the present experiments where a fraction of healthy subjects exhibited an LTD-like response to the PASLTP protocol under placebo conditions. This LTD-like response was switched to an LTP-like response by a single oral pre-application of 900 mg of lithium (Fig. 2B). The mean LTPlike MEP increase (MEPP1eP6/MEPB1 ¼ 1.23 0.08) was similar as for the PASLTP ‘LTP responders’ under placebo conditions (MEPP1eP6/MEPB1 ¼ 1.33 0.04, P > 0.25). Together, these findings suggest that the PASLTP ‘LTD responders’ have intrinsically high GSK-3b activity, but that a LTP-like response can be induced if GSK3b activity is inhibited at the time of PASLTP by lithium. Importantly, lithium had no effect on corticospinal excitability per se as indicated by the absence of change in MEP1mV amplitude at time point B1 2 h after lithium intake compared to baseline at time point B0 (Table 1). This largely excludes the possibility that the observed lithiuminduced switch from PASLTP LTD- to LTP-like plasticity was caused by e.g. homeostatic mechanisms that would be based on a decrease of corticospinal excitability prior to an LTP-like plasticity inducing protocol (Lang et al., 2004). In our experiments, lithium had no significant effect on the LTPlike response in the PASLTP ‘LTP responders’ (Fig. 2A). At first sight, this was surprising because the inhibitory action of lithium on GSK3b activity should facilitate LTP. Why this was not observed may be explained by the following reasons: (1) the intrinsic activity of GSK3b in PASLTP ‘LTP responders’ is probably low and further suppressed by an LTP-inducing stimulation protocol (Hooper et al., 2007). Therefore, a floor effect may have prevented lithium from further down-regulating GSK-3b activity; (2) data reported from LTP experiments in rat hippocampus have been inconsistent: lithium suppressed LTP via blockade of postsynaptic guanine nucleotide-binding proteins (Ballyk and Goh, 1993), or enhanced
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LTP, probably through activation of intracellular signaling pathways such as the cAMP response element binding protein (CREP) controlled by GSK-3b (Son et al., 2003). These divergent results are likely explained by differences in experimentation such as induction of LTP in the CA1 region (Ballyk and Goh, 1993) versus dentate gyrus (Son et al., 2003), implying that predictions from already heterogeneous animal models to human studies are difficult. Finally, the PASLTP ‘LTD responders’ showed a similar amount of LTD-like plasticity after PASLTD which, however, was not modulated by lithium (Fig. 2C). This is a particularly important finding because it demonstrates a crucial difference in the physiology of the LTDlike responses induced by PASLTP versus PASLTD in these individuals: the LTD-like MEP decrease after PASLTP was fragile and could be explained by the proposed intrinsically high activity of GSK-3b which was not overcome to a sufficient extent by the proposed PASLTP-induced inhibition of GSK-3b. Therefore, under placebo conditions, the net effect would still favor a LTD-like response, but the inhibitory action of lithium on GSK-3b activity would tip the balance toward an LTP-like response. In contrast, PASLTD should result in GSK-3b activation in these subjects with an already intrinsically high GSK-3b activity so that the addition of lithium was no longer sufficient to attenuate this PASLTD-induced LTD-like plasticity. Our findings are in apparent contrast to experiments in slices of rat hippocampus where bath application of lithium resulted in a blockade of LTD induction (Peineau et al., 2007). However, that effect was dose-dependent and fully expressed only at a lithium concentration of 20 mmol/L (Peineau et al., 2007), which is w57 times higher than the plasma concentration in our subjects at the time of PASLTD (Table 1). Similarly, an acute bath application of 10 mmol/L lithium to rat corticostriatal slices did not interfere with initially full expression of LTD and reduced LTD only at later stages (Calabresi et al., 1993). These data strongly suggest that the lithium dose in our experiments was too low to attenuate PASLTD-induced LTD-like plasticity. However, significantly higher doses were not feasible due to the risk of lithium-induced neurotoxicity at plasma levels >1.5 mmol/L (Sheean, 1991). A few issues might limit the conclusions from the current findings: (1) the number of included subjects was relatively small and it would certainly be of interest to replicate the data in a larger cohort. We did not plan to include more subjects because previous pharmacological PAS studies with significant results used equal or even smaller numbers of participants (Heidegger et al., 2010; Korchounov and Ziemann, 2011; Kuo et al., 2008); (2) GSK-3b activators would be of interest to test the specificity of the current findings further. It would be predicted that pharmacological activation of GSK-3b should increase the probability of induction of LTD-like plasticity and, therefore, should switch PASLTD ‘LTP responders’ (should they exist) to a LTD-like response. However, to the best of our knowledge, no specific GSK-3b activating drug is currently available for use in humans. In the mouse striatum, enhancement of dopaminergic neurotransmission resulted in inhibition of protein kinase B (also referred to as Akt), which in turn activates GSK-3b by release of the inhibitory action of Akt on GSK3b, an effect that could be blocked by D2 receptor blockers (Beaulieu et al., 2004). In accord, several studies demonstrated a non-linearly dose-dependent attenuation of PASLTP-induced LTPlike plasticity or even switch to LTD-like plasticity by D2 receptor agonists or the dopamine precursor L-dopa (Monte-Silva et al., 2009; Thirugnanasambandam et al., 2011) while the D2 receptor antagonist sulpiride resulted in a trend toward enhancement of PASLTP-induced LTP-like plasticity (Nitsche et al., 2009). However, relation of these data to GSK-3b modulation has to be interpreted with caution because D2 receptor agonists/antagonists may affect neuroplasticity through a multitude of alternative mechanisms (Gu, 2002).
5. Conclusion We provided first-time evidence for a modulating role of lithium on non-invasive brain stimulation induced plasticity in human cortex. The switch from PASLTP-induced LTD- to LTP-like plasticity is best explained by the inhibitory action of lithium on GSK-3b activity and the assumption that PASLTP ‘LTD responders’ provide a natural model of high intrinsic GSK-3b activity. Findings are relevant for research of neuropsychiatric diseases such as bipolar disorder where excessive GSK-3b activity is thought to play a pathogenetic role. Acknowledgments This study was supported by a grant of the German Research Foundation (DFG grant ZI 542/5-1). We thank Drs. Annemarie Hübers and Tonio Heidegger for support of this study. Conflicts of interest All authors declare that they have no conflicts of interest. References Ballyk, B.A., Goh, J.W., 1993. A postsynaptic G-protein in hippocampal long-term potentiation. Brain Res. 611, 81e86. Beaulieu, J.M., Sotnikova, T.D., Yao, W.D., Kockeritz, L., Woodgett, J.R., Gainetdinov, R.R., Caron, M.G., 2004. Lithium antagonizes dopamine-dependent behaviors mediated by an AKT/glycogen synthase kinase 3 signaling cascade. Proc. Natl. Acad. Sci. Unit. States Am. 101, 5099e5104. Beaulieu, J.M., Zhang, X., Rodriguiz, R.M., Sotnikova, T.D., Cools, M.J., Wetsel, W.C., Gainetdinov, R.R., Caron, M.G., 2008. Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency. Proc. Natl. Acad. Sci. Unit. States Am. 105, 1333e1338. Bi, G., Poo, M., 2001. Synaptic modification by correlated activity: Hebb’s postulate revisited. Annu. Rev. Neurosci. 24, 139e166. Calabresi, P., Pisani, A., Mercuri, N.B., Bernardi, G., 1993. Lithium treatment blocks long-term synaptic depression in the striatum. Neuron 10, 955e962. Carlson, P.J., Singh, J.B., Zarate Jr., C.A., Drevets, W.C., Manji, H.K., 2006. Neural circuitry and neuroplasticity in mood disorders: insights for novel therapeutic targets. NeuroRx 3, 22e41. Colino, A., Garcia-Seoane, J.J., Valentin, A., 1998. Action potential broadening induced by lithium may cause a presynaptic enhancement of excitatory synaptic transmission in neonatal rat hippocampus. Eur. J. Neurosci. 10, 2433e2443. Cooke, S.F., Bliss, T.V., 2006. Plasticity in the human central nervous system. Brain 129, 1659e1673. Di Lazzaro, V., Oliviero, A., Profice, P., Pennisi, M.A., Pilato, F., Zito, G., Dileone, M., Nicoletti, R., Pasqualetti, P., Tonali, P.A., 2003. Ketamine increases motor cortex excitability to transcranial magnetic stimulation. J. Physiol. 547, 485e496. Di Lazzaro, V., Ziemann, U., Lemon, R.N., 2008. State of the art: physiology of transcranial motor cortex stimulation. Brain Stimulation 1, 345e362. Fratello, F., Veniero, D., Curcio, G., Ferrara, M., Marzano, C., Moroni, F., Pellicciari, M.C., Bertini, M., Rossini, P.M., De Gennaro, L., 2006. Modulation of corticospinal excitability by paired associative stimulation: reproducibility of effects and intraindividual reliability. Clin. Neurophysiol. 117, 2667e2674. Gärtner, A., Polnau, D.G., Staiger, V., Sciarretta, C., Minichiello, L., Thoenen, H., Bonhoeffer, T., Korte, M., 2006. Hippocampal long-term potentiation is supported by presynaptic and postsynaptic tyrosine receptor kinase B-mediated phospholipase C gamma signaling. J. Neurosci. 26, 3496e3504. Gebhardt, C., Cull-Candy, S.G., 2010. Lithium acts as a potentiator of AMPAR currents in hippocampal CA1 cells by selectively increasing channel open probability. J. Physiol. 588, 3933e3941. Geddes, J.R., Burgess, S., Hawton, K., Jamison, K., Goodwin, G.M., 2004. Long-term lithium therapy for bipolar disorder: systematic review and meta-analysis of randomized controlled trials. Am. J. Psychiatr. 161, 217e222. Gu, Q., 2002. Neuromodulatory transmitter systems in the cortex and their role in cortical plasticity. Neuroscience 111, 815e835. Hallett, M., 2007. Transcranial magnetic stimulation: a primer. Neuron 55, 187e199. Heidegger, T., Krakow, K., Ziemann, U., 2010. Effects of antiepileptic drugs on associative LTP-like plasticity in human motor cortex. Eur. J. Neurosci. 32, 1215e1222. Hooper, C., Markevich, V., Plattner, F., Killick, R., Schofield, E., Engel, T., Hernandez, F., Anderton, B., Rosenblum, K., Bliss, T., Cooke, S.F., Avila, J., Lucas, J.J., Giese, K.P., Stephenson, J., Lovestone, S., 2007. Glycogen synthase kinase-3 inhibition is integral to long-term potentiation. Eur. J. Neurosci. 25, 81e86.
H. Voytovych et al. / Neuropharmacology 63 (2012) 274e279 Jope, R.S., 2003. Lithium and GSK-3: one inhibitor, two inhibitory actions, multiple outcomes. Trends Pharmacol. Sci. 24, 441e443. Jung, P., Ziemann, U., 2009. Homeostatic and non-homeostatic modulation of learning in human motor cortex. J. Neurosci. 29, 5597e5604. Korchounov, A., Ziemann, U., 2011. Neuromodulatory neurotransmitters influence LTP-like plasticity in human cortex: a pharmaco-TMS study. Neuropsychopharmacology 36, 1894e1902. Kuo, M.-F., Paulus, W., Nitsche, M.A., 2008. Boosting focally-induced brain plasticity by dopamine. Cereb. Cortex 18, 648e651. Lang, N., Siebner, H.R., Ernst, D., Nitsche, M.A., Paulus, W., Lemon, R.N., Rothwell, J.C., 2004. Preconditioning with transcranial direct current stimulation sensitizes the motor cortex to rapid-rate transcranial magnetic stimulation and controls the direction of after-effects. Biol. Psychiatry 56, 634e639. Monte-Silva, K., Kuo, M.-F., Thirugnanasambandam, N., Liebetanz, D., Paulus, W., Nitsche, M., 2009. Dose-dependent inverted U-shaped effect of dopamine (D2like) receptor activation on focal and nonfocal plasticity in humans. J. Neurosci. 29, 6124e6131. Müller-Dahlhaus, F., Ziemann, U., Classen, J., 2010. Plasticity resembling spiketiming dependent synaptic plasticity: the evidence in human cortex. Front. Syn Neurosci. 2, 1e11. Müller-Dahlhaus, J.F., Orekhov, Y., Liu, Y., Ziemann, U., 2008. Interindividual variability and age-dependency of motor cortical plasticity induced by paired associative stimulation. Exp. Brain Res. 187, 467e475. Müller, J.F.M., Orekhov, Y., Liu, Y., Ziemann, U., 2007. Homeostatic plasticity in human motor cortex demonstrated by two consecutive sessions of paired associative stimulation. Eur. J. Neurosci. 25, 3461e3468. Nitsche, M.A., Kuo, M.F., Grosch, J., Bergner, C., Monte-Silva, K., Paulus, W., 2009. D1receptor impact on neuroplasticity in humans. J. Neurosci. 29, 2648e2653. Oldfield, R.C., 1971. The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9, 97e113. Peineau, S., Taghibiglou, C., Bradley, C., Wong, T.P., Liu, L., Lu, J., Lo, E., Wu, D., Saule, E., Bouschet, T., Matthews, P., Isaac, J.T., Bortolotto, Z.A., Wang, Y.T.,
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Lithium: A switch from LTD- to LTP-like plasticity in human cortex Hanna Voytovych, Lucia Kriváneková, Ulf Ziemann* Department of Neurology, Goethe-University Frankfurt, Schleusenweg 2e16, D-60528 Frankfurt am Main, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2012 Received in revised form 5 March 2012 Accepted 23 March 2012
Lithium, a simple cation, is the mainstay treatment of bipolar disorder. Deficient synaptic plasticity is considered one important mechanism of this disease. Lithium inhibits glycogen synthase kinase-3beta (GSK-3b), which is involved in the regulation of synaptic plasticity. In animal preparations, inhibition of GSK-3b by lithium up-regulated long-term potentiation (LTP) of excitatory synapses but downregulated long-term depression (LTD). The effects of lithium on plasticity in the human brain are unexplored. We tested the effects of a single oral dose of 900 mg of lithium on LTP-/LTD-like plasticity in human motor cortex induced by established paired associative transcranial magnetic stimulation (PASLTP, PASLTD) protocols. We studied 10 healthy adults in a placebo-controlled double-blind randomized crossover design. PAS-induced plasticity was indexed by change in motor evoked potential amplitude recorded in a hand muscle. In the placebo session, subjects were stratified, according to the known variability of the PASLTP response, into PASLTP ‘LTP responders’ and PASLTP ‘LTD responders’ (n ¼ 5 each). Lithium did not affect the PASLTP-induced LTP-like plasticity in the ‘LTP responders’, but switched the PASLTP-induced LTD-like plasticity in the ‘LTD responders’ to LTP-like plasticity. In contrast, lithium had no effect on the PASLTD-induced LTD-like plasticity in the ‘LTD responders’. We provide first-time evidence that lithium significantly modulates brain stimulation induced plasticity in human cortex. The switch from LTD- to LTP-like plasticity is best explained by the inhibitory action of lithium on GSK3b. This conclusion is necessarily circumstantial because GSK-3b activity was not directly measured. We discuss that other important plasticity-related modes actions of lithium cannot explain our findings. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Lithium Transcranial magnetic stimulation Paired associative stimulation Long-term potentiation Long-term depression Motor cortex
1. Introduction Lithium is a monovalent cation that constitutes the mainstay treatment of bipolar disorder for the last 50 years (Geddes et al., 2004). How lithium exerts its therapeutic effects has still not been fully elucidated (Phiel and Klein, 2001). Lithium inhibits several enzymes, including the multifunctional serine/threonine kinase glycogen synthase kinase-3 (GSK-3) (Jope, 2003). The isoform GSK-3b is highly enriched in brain tissue (Woodgett, 1990) and shows high basal activity, which is primarily determined by its dephosphorylation status. Recent experiments have implicated a fundamental role of GSK-3b in the regulation of the direction and magnitude of N-methyl-D-aspartate receptor (NMDAR) dependent plasticity at excitatory synapses. In rat hippocampus, induction of NMDAR dependent long-term depression (LTD) by electrical stimulation resulted in further activation of GSK-3b through dephosphorylation (Peineau et al., 2007) while induction of long-term potentiation (LTP) resulted in deactivation of GSK-3b through
* Corresponding author. Tel.: þ49 69 6301 5739; fax: þ49 69 6301 4498. E-mail address: [email protected] (U. Ziemann). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.03.023
phosphorylation (Hooper et al., 2007; Peineau et al., 2007). GSK-3b inhibition by lithium prevented the induction of LTD (Peineau et al., 2007) and deficient LTP in GSK-3b overexpressing mice could be rescued by lithium (Hooper et al., 2007; Zhu et al., 2007). Therefore, the inhibitory action of lithium on GSK-3b implicates a modulating effect on NMDAR dependent bidirectional synaptic plasticity favoring LTP over LTD. In bipolar disorder and major depression, increased GSK-3b activity and associated dysregulation of synaptic plasticity have been proposed from animal disease models (Beaulieu et al., 2008; Carlson et al., 2006). Therefore, targeting hyperactive GSK-3brelated signaling pathways by lithium or other drugs has been advocated as a premier strategy for effective treatment of these disorders (Schloesser et al., 2011). Here we tested the effects of lithium for the first-time in a human experimental model of bidirectional LTD-/LTP-like plasticity. Paired associative stimulation (PAS) of motor cortex (Jung and Ziemann, 2009; Müller-Dahlhaus et al., 2010; Stefan et al., 2000; Wolters et al., 2003) was employed to induce long-term increase (PASLTP) or decrease (PASLTD) in motor evoked potential (MEP) amplitude, a marker of corticospinal excitability elicited by transcranial magnetic stimulation (TMS) (Hallett, 2007). The PAS-
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induced MEP changes are reminiscent of bidirectional spike timingdependent plasticity in neuronal cultures and slices (Bi and Poo, 2001). They have been termed LTP-like and LTD-like plasticity (Ziemann et al., 2004) because they show fundamental characteristics of LTP and LTD as identified at the cellular level in basic experiments, such as NMDAR dependency, duration > 30 min, associativity and cooperativity (Cooke and Bliss, 2006; MüllerDahlhaus et al., 2010). Inter-individual variability of the PASLTP response is well known, with a significant fraction of individuals showing a decrease in MEP amplitude (‘LTD responders’) rather than an increase (‘LTP responders’) (Müller-Dahlhaus et al., 2008). This variability may be determined by many factors, including gender, age, genetics or treatment with CNS active drugs (Ridding and Ziemann, 2010). Here we hypothesized that the PASLTP ‘LTD responders’ serve as natural model for high intrinsic GSK-3b activity, and that an LTPlike increase in MEP amplitude in the PASLTP ‘LTD responders’ could be rescued by pre-application with lithium. We provide firsttime evidence in humans in favor of these hypotheses: lithium switched the PASLTP-induced LTD-like response into an LTP-like response. The present findings are relevant for research of neuropsychiatric diseases such as bipolar disorder where excessive GSK3b activity is thought to play a pathogenetic role (Beaulieu et al., 2008). 2. Materials and methods 2.1. Subjects Ten healthy young adults participated in the PASLTP experiment (age range, 21e31 years; mean age, 24.3 2.8 years; 6 female). The groups of PASLTP ‘LTP responders’ (for definition, see below; n ¼ 5; mean age, 25.4 3.5; 3 female) and PASLTP ‘LTD responders’ (n ¼ 5; mean age, 23.2 1.6 years; 3 female) did not differ with respect to the demographic data. No subject had a history of neuropsychiatric disease or was on CNS active medication or recreational drugs. All subjects were right-handed, as indicated by the Edinburgh Handedness Inventory (Oldfield, 1971). The study was approved by the ethics committee of the medical faculty of Goethe University, Frankfurt am Main, Germany and by the Federal Institute for Drugs and Medical Devices of Germany (BfArM). The study conformed to the latest version of the Declaration of Helsinki. All volunteers gave their written informed consent prior to participation. 2.2. MEP measurements Focal TMS was performed using a flat figure-of-eight-shaped stimulating coil (outer diameter of each wing, 70 mm) connected to a Magstim 200 magnetic stimulator (Magstim Company, UK) with a monophasic current waveform. The coil
275
was placed over hand area of the left primary motor cortex (M1), at the site giving rise to the largest motor evoked potentials (MEP) in the abductor pollicis brevis (APB) muscle of the right hand. The coil was held tangentially to the skull with the handle pointing backward and laterally at a 45 angle to the sagittal plane. Thus, the induced current in the brain flowed from lateral-posterior to medial-anterior. This current direction is optimal for trans-synaptic excitation of the corticospinal neurons (Di Lazzaro et al., 2008). MEP recordings were obtained by surface electromyography (EMG), using AgeAgCl cup electrodes in a bellyetendon montage. The EMG raw signal was amplified and filtered (0.02e2 kHz; D360 amplifier; Digitimer Ltd., UK), digitized (analogedigital rate, 5 kHz; CED Micro 1401Ò; Cambridge Electronic Design, UK), and fed into a laboratory computer for online visual display and offline analysis. All measurements were obtained during muscle rest, which was continuously monitored by high-gain EMG audioevisual feedback. 2.3. Paired associative stimulation (PAS) PAS consisted of 225 pairs (rate, 0.25 Hz) of electrical stimulation of the right median nerve at the wrist followed by focal TMS of the APB representation of left M1. Electrical stimulation was applied through a bipolar electrode (cathode proximal), using constant-current square-wave pulses (duration, 1 ms) at an intensity of three times the perceptual sensory threshold. TMS intensity was adjusted to elicit MEP of, on average, 1 mV peak-to-peak amplitude (MEP1mV) in the right APB when given without the preceding median nerve stimulus. In the first experiment (all 10 subjects participated), the interstimulus interval between the median nerve stimulus and TMS was set to the individual N20-latency of standard median nerve somatosensory-evoked potential recordings plus 2 ms (N20 þ 2 ms). At this interval, PAS produced an LTP-like increase in MEP amplitude in the majority of subjects in previous studies (PASLTP), with a fraction of subjects however showing a LTD-like MEP decrease (Fratello et al., 2006; Müller-Dahlhaus et al., 2008). In the present study, these PASLTP ‘LTD responders’ (i.e. MEPP1eP6/MEPB1 1.0) were subjected to another PAS protocol with a slightly shorter interstimulus interval of N20e5 ms (PASLTD), which in previous studies consistently resulted in a LTD-like decrease of MEP amplitude (Müller et al., 2007; Ziemann et al., 2004). Because attention may have considerable effects on the magnitude of a PAS effect (Stefan et al., 2004), subjects were instructed to continuously watch the stimulated hand, to count randomly occurring flashes of an LED attached to the dorsum of this hand, and to report the number at the end of the PAS intervention. 2.4. Lithium serum concentration Blood samples were taken immediately before and 2 h after drug intake (Fig. 1) for measurements of the plasma concentration of lithium. 2.5. Experimental design The study was performed in a double-blind, randomized, placebo-controlled crossover design. In the PASLTP and in the PASLTD experiment, subject participated in two sessions each (Fig. 1): in one session a single oral dose of 900 mg of lithium carbonate, equivalent to 24.4 mmol of lithium (QuilonumÒ, GlaxoSmithCline), and in the other session placebo (PBO) was applied. The dose of 900 mg of lithium was chosen because (1) it is equivalent to a medium daily dose in the treatment of patients with bipolar disorder to reach therapeutic blood concentrations of
10 Subjects
B0
Blood Sample
Lithium Placebo
Blood Sample
B1
PASLTP
P1…P6 0 - 30 min
5 Subjects: PASLTP LTD Responder
B0
Blood Sample
Lithium Placebo
Blood Sample
B1
PASLTD
P1…P6 0 - 30 min
Fig. 1. Timeline of experiments. B0, baseline MEP measurements (before drug intake); B1, MEP measurements 2 h after drug intake and immediately prior to PAS; P1eP6, six blocks of MEP measurements at 5, 10, 15, 20, 25 and 30 min post-PAS. MEP measurements consisted of 20 trials per time point. Ten subjects participated in the PASLTP experiment, and the five PASLTP LTD responders were tested a few weeks later in the PASLTD experiment. Both experiments consisted of two pseudo-randomized double-blinded sessions (lithium versus placebo, separated by at least 7 days).
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0.5e1.2 mmol/L (summary of product characteristics of QuilonumÒ); (2) previous unpublished data from our group showed that a single oral dose of 900 mg of lithium, in contrast to lower and higher doses, had no significant effect on corticospinal excitability as measured by MEP amplitude. This limited the possibility that effects of lithium on PAS-induced plasticity as measured by changes in MEP amplitude would be contaminated by direct effects of lithium on corticospinal excitability. The PBO and lithium sessions within one PAS experiment were at least 1 week apart to rule out carry over effects. The PASLTD experiment in the PASLTP ‘LTD responders’ followed the PASLTP experiment by several weeks. To test the effects of drug (lithium versus PBO) on corticospinal excitability and PAS-induced plasticity, MEP amplitudes were measured at eight time points (Fig. 1): baseline (time point B0, prior to drug intake), 2 h post-dose and immediately prior to PAS (time point B1), and 6 times post-PAS in steps of 5 min, covering 30 min after PAS (time points P1eP6). At each time point, 20 trials were recorded with a random intertrial interval (range, 8e12 s) to limit anticipation of the next trial. TMS intensity at B0 was set to MEP1mV. The experimental protocol prescribed to adjust stimulus intensity at B1, if the MEP amplitude at B1 deviated by >20% from the one at B0, in order to re-install MEP1mV prior to PAS. However, this was necessary only once in one subject each in the PASLTP and PASLTD experiments, indicating that lithium and PBO had no significant effect on corticospinal excitability. The timing of B1 2 h after drug intake was chosen because plasma levels of lithium after a single oral dose peak 2e4 h after intake (summary of product characteristics of QuilonumÒ). 2.6. Statistical analysis The software package StatsView for Windows (SAS Institute Inc., Version 5.0.1) was used for statistical analysis. Peak-to-peak MEP amplitudes were analyzed in the single trials and then averaged for each subject, time point and drug condition. Possible differences in baseline MEP amplitudes (time point B0) or 2 h after drug intake (time point B1) between drug (lithium versus PBO) were checked by twosided t-tests for paired samples, and MEP differences between responder status (PASLTP ‘LTP responders’ versus PASLTP ‘LTD responders’; see below for definition) were tested by two-sided t-tests for unpaired samples. In addition, MEP amplitudes were compared between time points B0 and B1 within drug and responder status conditions by two-sided t-tests for paired samples. For analysis of the PAS effects, MEP amplitudes at time points P1eP6 were normalized to those at time point B1. In the PASLTP experiment, subjects were defined as PASLTP ‘LTP responders’, if in the PBO condition MEPP1eP6/MEPB1 > 1.0, and as PASLTP ‘LTD responders’, if in the PBO condition MEPP1eP6/MEPB1 1.0. The effects of PAS on MEP amplitude in the PASLTP experiment were analyzed in a three-way mixed analysis of variance (ANOVA) with the within-subject effects of time (seven levels: B1, P1eP6) and drug (two levels: lithium and PBO), and the between-subject effect of responder status (two levels: LTP responder versus LTD responder). According to a significant responder status * drug interaction (see Results) two subsequent two-way repeated measures ANOVAs (rmANOVAs) explored the effects of time and drug for the PASLTP ‘LTP responders’ and PASLTP ‘LTD responders’ separately. Similarly, in the PASLTD experiment (only PASLTP ‘LTD responders’ included), the effects of PAS on MEP amplitude were tested in a two-way rmANOVA with the within-subject effects of time (seven levels: B1, P1eP6) and drug (two levels: lithium and PBO). In case of significant main effects or interactions, Fisher’s LSD test was applied for post hoc analysis. All results are expressed as means SEM. P values < 0.05 were considered statistically significant.
3. Results No adverse events were observed during the experiments except for one subject that reported self-limiting diarrhea a few hours after lithium intake. 3.1. MEP amplitude at baseline (B0) and 2 h after drug intake (B1) MEP amplitudes at baseline (time point B0) and 2 h after drug intake (time point B1) did not differ between responder status or drug conditions (all P > 0.25), and were always close to the targeted amplitude of 1 mV (Table 1). In addition, there was no effect of time on MEP amplitude (comparison of time points B1 and B0) within any of the drug or responder status conditions (all P > 0.25). These nil findings indicate that there were no baseline differences in motor excitability or significant drug effects on motor excitability that could account for the effects of drug on PAS-induced plasticity (see below).
Table 1 MEP amplitude (in mV) and lithium (Li) plasma concentration (in mmol/L) at time points B0 and B1. Group
Drug
MEP (B0)
PASLTP ‘LTP responder’ PASLTP ‘LTD responder’ PASLTD
PBO Lithium PBO Lithium PBO Lithium
0.95 1.16 1.07 1.03 1.14 1.07
0.09 0.05 0.10 0.05 0.03 0.03
MEP (B1) 1.03 0.99 0.96 1.00 1.05 1.07
0.09 0.08 0.06 0.03 0.12 0.11
Li (B0) 0.04 0.04 0.05 0.03 0.03 0.03
Li (B1) 0.01 0.02 0.02 0.02 0.01 0.01
0.05 0.41 0.04 0.48 0.03 0.35
0.01 0.07 0.02 0.11 0.01 0.14
All data are means 1 SEM.
3.2. Lithium blood concentrations Lithium blood concentration was 0.03e0.05 mmol/L at baseline (time point B0) and in the PBO condition 2 h later (time point B1) but increased significantly 2 h after lithium intake to 0.35e0.48 mmol/L (Table 1). Importantly, there was no difference between lithium blood concentrations at time point B1 between responder status in the PASLTP experiment (P ¼ 0.60) or between PASLTP and PASLTD experiment in the PASLTP ‘LTD responders’ (P ¼ 0.47) that could have explained the differential effects of lithium on PASLTP-induced plasticity in PASLTP ‘LTP responders’ versus PASLTP ‘LTD responders’ or on PASLTP-induced versus PASLTDinduced plasticity in the PASLTP ‘LTD responders’ (see next paragraphs). 3.3. Effects of lithium on PASLTP-induced plasticity In the PBO condition, there were 5 PASLTP LTP responders (i.e. MEPP1eP6/MEPB1 > 1.0, mean MEPP1eP6/MEPB1 ¼ 1.33 0.04) and 5 PASLTP LTD responders (i.e. MEPP1eP6/MEPB1 1.0, mean MEPP1eP6/ MEPB1 ¼ 0.75 0.08). The mixed ANOVA revealed a significant effect of responder status (F1, 8 ¼ 12.76, P ¼ 0.007), time (F6, 48 ¼ 3.09, P ¼ 0.012), and of the interaction between responder status * drug (F1, 8 ¼ 10.46, P ¼ 0.012) (Fig. 2AeB), while the effect of drug and any of the other interactions were not significant. A subsequent two-way rmANOVA showed that within the group of PASLTP ‘LTP responders’ time had a significant effect on normalized MEP amplitude (F6, 24 ¼ 2.82, P ¼ 0.032) while the effect of drug and of the interaction of drug * time had no effect (Fig. 2A). In contrast, the two-way rmANOVA within the group of PASLTP ‘LTD responders’ showed a significant effect of drug (F1, 4 ¼ 19.04, P ¼ 0.012) but no effect of time or the interaction of drug * time (Fig. 2B). Post hoc comparisons revealed that MEP amplitude normalized to B1 was larger under lithium than PBO at time points P2 and P5 after PASLTP (P < 0.05, Fig. 2B). 3.4. Effects of lithium on PASLTD-induced plasticity in PASLTP ‘LTD responders’ In the PBO condition, PASLTD in the PASLTP ‘LTD responders’ resulted in an LTD-like effect similar to the PASLTP experiment (mean MEPP1eP6/MEPB1 ¼ 0.71 0.05, P > 0.7) (Fig. 2C). The rmANOVA revealed a significant effect of time (F6, 24 ¼ 3.79, P ¼ 0.0085) but no effects of drug or of the interaction drug * time (Fig. 2C). A three-way rmANOVA in the PASLTP ‘LTD responders’ with the main effects drug (lithium versus PBO), PAS experiment (PASLTP versus PASLTD) and time (B1, P1eP6) demonstrated a significant effect of drug (F1, 4 ¼ 9.68, P ¼ 0.036), and of the interactions PAS experiment * drug (F1,4 ¼ 37.05, P ¼ 0.0037) and PAS experiment * time (F6,24 ¼ 2.57, P ¼ 0.046), while the other effects and interactions were not significant (cf. Fig. 2BeC).
H. Voytovych et al. / Neuropharmacology 63 (2012) 274e279
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Fig. 2. A. MEP amplitude after PASLTP (time points P1eP6) normalized to baseline prior to PASLTP (time point B1) in PASLTP LTP responders (n ¼ 5) under placebo (PBO, black circles) versus lithium (white circles). B. Same conventions and arrangements as in A for the PASLTP LTD responders (n ¼ 5) in the PASLTP experiments, and C. in the PASLTD experiment. All data are means 1 SEM. Asterisks denote significant difference between drug conditions (post hoc paired t-tests, P < 0.05).
4. Discussion The main novel finding of this study is that a single oral dose of lithium switched individuals with a PASLTP-induced LTD-like MEP decrease under placebo conditions to a LTP-like MEP increase. We replicated earlier findings that, without CNS active drugs, PASLTP typically results in a LTP-like increase in MEP amplitude in healthy young adults but, in a substantial fraction of subjects, also in a LTD-like MEP decrease (Fratello et al., 2006; Müller-Dahlhaus et al., 2008). The reasons for this substantial inter-individual variability to a standardized non-invasive brain stimulation protocol are not fully understood, but a multitude of determinants, such as gender, age, preceding motor activity, genetic polymorphisms of important regulatory molecules such as brain-derived neurotrophic factor, and pharmacological modulation may influence the direction and magnitude of brains stimulation induced MEP change (Ridding and Ziemann, 2010). Here we were interested in the effects of lithium, a simple cation that has been shown recently in animal experiments in vitro and in vivo to modulate LTD-/LTP through inhibition of GSK-3b. Before we will enter a detailed discussion that inhibition of GSK-3b by lithium is the most likely candidate mechanism to explain the present findings, two caveats should be emphasized: First, any conclusion toward the proposed link between modulation of PAS-induced LTP-/LTD-like plasticity and inhibition of GSK3b by lithium is necessarily circumstantial because GSK-3b activity was not directly measured. Second, lithium has several other identified modes of action related to regulation of synaptic plasticity, which may have contributed to the current findings (Phiel and Klein, 2001). Lithium depletes inositol-1,4,5 trisphosphate (IP3) through direct inhibition of inositol monophosphatase. IP3 mediates release of intracellular Ca2þ from internal stores. Consequently, IP3 receptor antagonists led to significant attenuation of hippocampal LTP (Gärtner et al., 2006). Thus, this mode of action would not explain the lithium-induced switch from PASLTP-induced LTD- to LTP-like plasticity in the present experiments. In addition, lithium enhances glutamatergic neurotransmission by increasing the open probability of AMPA receptors in rat hippocampus (Gebhardt and Cull-Candy, 2010) resulting in enhanced excitatory postsynaptic potentials (Colino et al., 1998). However, an increase in MEP amplitude that should be expected by enhancement of ionotropic glutamatergic neurotransmission (Di Lazzaro et al., 2003) was not observed (Table 1). Therefore, while a contribution by those mechanisms cannot be
entirely excluded they would lead to results that are inconsistent with the present findings. In contrast, our findings are in excellent agreement with previous studies on the effects of lithium on regulation of LTP/LTD through GSK-3b inhibition. Transgenic mice with an overexpression of GSK-3b showed significantly less LTP at Schaffer commissuralpyramidal cell synapses in the CA1 region of the hippocampus compared to wild-type controls (Hooper et al., 2007). This LTP deficiency in the transgenic mice was rescued to the level of the wild-type controls if the transgenic mice were chronically pretreated with lithium (Hooper et al., 2007). Also, pharmacological overactivation of GSK-3b by wortmannin resulted in LTP deficiency in rat hippocampus which could be prevented by simultaneous inhibition of GSK-3b by lithium (Zhu et al., 2007). It is very suggestive that these data directly translate to the present experiments where a fraction of healthy subjects exhibited an LTD-like response to the PASLTP protocol under placebo conditions. This LTD-like response was switched to an LTP-like response by a single oral pre-application of 900 mg of lithium (Fig. 2B). The mean LTPlike MEP increase (MEPP1eP6/MEPB1 ¼ 1.23 0.08) was similar as for the PASLTP ‘LTP responders’ under placebo conditions (MEPP1eP6/MEPB1 ¼ 1.33 0.04, P > 0.25). Together, these findings suggest that the PASLTP ‘LTD responders’ have intrinsically high GSK-3b activity, but that a LTP-like response can be induced if GSK3b activity is inhibited at the time of PASLTP by lithium. Importantly, lithium had no effect on corticospinal excitability per se as indicated by the absence of change in MEP1mV amplitude at time point B1 2 h after lithium intake compared to baseline at time point B0 (Table 1). This largely excludes the possibility that the observed lithiuminduced switch from PASLTP LTD- to LTP-like plasticity was caused by e.g. homeostatic mechanisms that would be based on a decrease of corticospinal excitability prior to an LTP-like plasticity inducing protocol (Lang et al., 2004). In our experiments, lithium had no significant effect on the LTPlike response in the PASLTP ‘LTP responders’ (Fig. 2A). At first sight, this was surprising because the inhibitory action of lithium on GSK3b activity should facilitate LTP. Why this was not observed may be explained by the following reasons: (1) the intrinsic activity of GSK3b in PASLTP ‘LTP responders’ is probably low and further suppressed by an LTP-inducing stimulation protocol (Hooper et al., 2007). Therefore, a floor effect may have prevented lithium from further down-regulating GSK-3b activity; (2) data reported from LTP experiments in rat hippocampus have been inconsistent: lithium suppressed LTP via blockade of postsynaptic guanine nucleotide-binding proteins (Ballyk and Goh, 1993), or enhanced
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LTP, probably through activation of intracellular signaling pathways such as the cAMP response element binding protein (CREP) controlled by GSK-3b (Son et al., 2003). These divergent results are likely explained by differences in experimentation such as induction of LTP in the CA1 region (Ballyk and Goh, 1993) versus dentate gyrus (Son et al., 2003), implying that predictions from already heterogeneous animal models to human studies are difficult. Finally, the PASLTP ‘LTD responders’ showed a similar amount of LTD-like plasticity after PASLTD which, however, was not modulated by lithium (Fig. 2C). This is a particularly important finding because it demonstrates a crucial difference in the physiology of the LTDlike responses induced by PASLTP versus PASLTD in these individuals: the LTD-like MEP decrease after PASLTP was fragile and could be explained by the proposed intrinsically high activity of GSK-3b which was not overcome to a sufficient extent by the proposed PASLTP-induced inhibition of GSK-3b. Therefore, under placebo conditions, the net effect would still favor a LTD-like response, but the inhibitory action of lithium on GSK-3b activity would tip the balance toward an LTP-like response. In contrast, PASLTD should result in GSK-3b activation in these subjects with an already intrinsically high GSK-3b activity so that the addition of lithium was no longer sufficient to attenuate this PASLTD-induced LTD-like plasticity. Our findings are in apparent contrast to experiments in slices of rat hippocampus where bath application of lithium resulted in a blockade of LTD induction (Peineau et al., 2007). However, that effect was dose-dependent and fully expressed only at a lithium concentration of 20 mmol/L (Peineau et al., 2007), which is w57 times higher than the plasma concentration in our subjects at the time of PASLTD (Table 1). Similarly, an acute bath application of 10 mmol/L lithium to rat corticostriatal slices did not interfere with initially full expression of LTD and reduced LTD only at later stages (Calabresi et al., 1993). These data strongly suggest that the lithium dose in our experiments was too low to attenuate PASLTD-induced LTD-like plasticity. However, significantly higher doses were not feasible due to the risk of lithium-induced neurotoxicity at plasma levels >1.5 mmol/L (Sheean, 1991). A few issues might limit the conclusions from the current findings: (1) the number of included subjects was relatively small and it would certainly be of interest to replicate the data in a larger cohort. We did not plan to include more subjects because previous pharmacological PAS studies with significant results used equal or even smaller numbers of participants (Heidegger et al., 2010; Korchounov and Ziemann, 2011; Kuo et al., 2008); (2) GSK-3b activators would be of interest to test the specificity of the current findings further. It would be predicted that pharmacological activation of GSK-3b should increase the probability of induction of LTD-like plasticity and, therefore, should switch PASLTD ‘LTP responders’ (should they exist) to a LTD-like response. However, to the best of our knowledge, no specific GSK-3b activating drug is currently available for use in humans. In the mouse striatum, enhancement of dopaminergic neurotransmission resulted in inhibition of protein kinase B (also referred to as Akt), which in turn activates GSK-3b by release of the inhibitory action of Akt on GSK3b, an effect that could be blocked by D2 receptor blockers (Beaulieu et al., 2004). In accord, several studies demonstrated a non-linearly dose-dependent attenuation of PASLTP-induced LTPlike plasticity or even switch to LTD-like plasticity by D2 receptor agonists or the dopamine precursor L-dopa (Monte-Silva et al., 2009; Thirugnanasambandam et al., 2011) while the D2 receptor antagonist sulpiride resulted in a trend toward enhancement of PASLTP-induced LTP-like plasticity (Nitsche et al., 2009). However, relation of these data to GSK-3b modulation has to be interpreted with caution because D2 receptor agonists/antagonists may affect neuroplasticity through a multitude of alternative mechanisms (Gu, 2002).
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