Deep brain stimulation of the medial forebrain bundle elevates striatal dopamine concentration without affecting spontaneous or reward-induced phasic release

Accepted Manuscript Deep brain stimulation of the medial forebrain bundle elevates striatal dopamine concentration without affecting spontaneous or reward-induced phasic release Marianne Klanker, Matthijs Feenstra, Ingo Willuhn, Damiaan Denys PII: DOI: Reference:

S0306-4522(17)30647-4 http://dx.doi.org/10.1016/j.neuroscience.2017.09.012 NSC 18020

To appear in:

Neuroscience

Received Date: Revised Date: Accepted Date:

24 March 2017 1 September 2017 6 September 2017

Please cite this article as: M. Klanker, M. Feenstra, I. Willuhn, D. Denys, Deep brain stimulation of the medial forebrain bundle elevates striatal dopamine concentration without affecting spontaneous or reward-induced phasic release, Neuroscience (2017), doi: http://dx.doi.org/10.1016/j.neuroscience.2017.09.012

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Title Page Title: Deep brain stimulation of the medial forebrain bundle elevates striatal dopamine concentration without affecting spontaneous or reward-induced phasic release Short Title: Deep brain stimulation of the medial forebrain bundle evokes striatal DA release Authors: Marianne Klanker1,2, Matthijs Feenstra1,2,*, Ingo Willuhn1,2,*, Damiaan Denys1,2*

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Netherlands Institute for Neuroscience Institute of the Royal Netherlands Academy of Arts and Sciences Meibergdreef 47 1105 BA Amsterdam The Netherlands Department of Psychiatry Academic Medical Center University of Amsterdam Postbus 22660 1100 DD Amsterdam The Netherlands These authors contributed equally to the work.

Corresponding author:

Marianne Klanker Netherlands Institute for Neuroscience (NIN) Meibergdreef 47 1105 BA Amsterdam The Netherlands Phone: +31 20 5665489 E-mail: [email protected]

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Abstract (max 250 words) Deep brain stimulation (DBS) of the medial forebrain bundle (MFB) induces rapid improvement of depressive symptoms in patients suffering from treatment-refractory major depressive disorder. It has been hypothesized that activation of the dopamine (DA) system contributes to this effect. To investigate whether DBS in the MFB affects DA release in the striatum, we combined DBS with fast-scan cyclic voltammetry (FSCV) in freely moving rats. Animals were implanted with a stimulating electrode at the border of the MFB and the ventral tegmental area, and a FSCV microelectrode in the ventromedial striatum to monitor extracellular DA during the acute onset of DBS and subsequent continued stimulation. DBS onset induced a significant increase in extracellular DA concentration in the ventromedial striatum that was sustained for at least 40 seconds. However, continued DBS did not affect amplitude or frequency of so-called spontaneous phasic DA transients, nor phasic DA release in response to the delivery of unexpected food pellets. These findings suggest that effects of DBS in the MFB are mediated by an acute change in extracellular DA concentration, but more research is needed to further explore the potentially sustained duration of this effect. Together, our results provide both support and refinement of the hypothesis that MFB DBS activates the DA system: DBS induces an increase in overall ambient concentration of DA, but spontaneous or rewardassociated more rapid, phasic DA dynamics are not enhanced. This knowledge improves our understanding of how DBS affects brain function and may help improve future therapies for depressive symptoms.

Keywords (max 6): Deep brain stimulation, dopamine, fast-scan cyclic voltammetry, medial forebrain bundle, ventral tegmental area, dopamine transients

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In the last decade, high-frequency deep brain stimulation (DBS) emerged as an innovative treatment for refractory psychiatric disorders, such as obsessive-compulsive disorder (OCD) and major depressive disorder (MDD). With significant responses in approximately 50% of patients, DBS offers last-resort treatment to otherwise therapy-resistant patients (Holtzheimer and Mayberg, 2011; Goodman and Alterman, 2012). Interestingly, successful remission can be achieved by stimulation in different target regions, the most widely used targets in depression being the nucleus accumbens (Schlaepfer et al., 2008; Bewernick et al., 2012), the anterior branch of the ventral capsule (Malone et al., 2009; Bergfeld et al., 2016) and the subcallosal cingulate (Mayberg et al., 2005; Puigdemont et al., 2012). However, a basic understanding of how stimulation of these different targets affects the activity of brain circuits and neurotransmission in general is still lacking.

A recent study investigated DBS to the supero-lateral branch of the medial forebrain bundle (MFB) and showed promising effects in a small patient group with MDD (Schlaepfer et al., 2013). It has been hypothesized that MFB DBS activates the “tonic” dopamine output from the VTA, thereby increasing the extracellular concentration of dopamine (DA) in the nucleus accumbens and prefrontal cortex (Schlaepfer et al., 2013, 2014). Dysregulation of dopaminergic pathways is thought to contribute to anhedonia and motivational dysfunction in depressive patients (Dunlop and Nemeroff, 2007; Drevets et al., 2008; Russo and Nestler, 2013) and has been linked to depression-like behavior in rodent models (Friedman et al., 2012; Chaudhury et al., 2013; Tye et al., 2013). However, the neurochemical effects of MFB DBS have not yet been directly studied and it is still unknown if activation of the DA system contributes to the therapeutic effect of MFB DBS.

An important first step in understanding the mechanisms of action of MFB DBS is to study the neurochemical and physiological effects of electrical brain stimulation itself. Preclinical studies have proven useful tools for the investigation of DBS effects on DA release in the brain (Bruet et al., 2001; Hamani et al., 2010; Shon et al., 2010; Van Dijk et al., 2012; Paek et al., 2013; Bregman et al., 2015; Jiménez-Sánchez et al., 2016). However, these studies mainly used microdialysis measurements (Bruet et 3

al., 2001; Hamani et al., 2010; Van Dijk et al., 2012; Bregman et al., 2015; Jiménez-Sánchez et al., 2016), which detect slow changes in DA release. Fast-scan cyclic voltammetry (FSCV) is the method of choice to detect rapid (sub second) changes in DA release (e.g. spontaneously occurring transients or transients timelocked to events such as reward-delivery (Wightman et al., 2007) in freely moving animals. Studies using FSCV to investigate DBS effects on DA release have been limited to the detection of DBS-induced effects on electrically stimulated DA release in anesthetized preparations (Shon et al., 2010; Paek et al., 2013). Here, we demonstrate the feasibility of combining FSCV and DBS of the MFB (bordering the VTA, similar to Furlanetti et al (2015 a,b) in freely moving rodents. The combination of these techniques allows us to investigate if MFB DBS affects DA release patterns in the ventromedial striatum. To investigate the MFB as a relatively new target area for DBS and in order to get a better understanding of the effects of MFB DBS on brain function in general, we used healthy animals in this study. The use of healthy animals can provide important insight into the physiological effects of DBS and may reveal possible side effects of stimulation in new target regions (Feenstra and Denys, 2012). Our findings suggest that MFB DBS with clinically relevant stimulation parameters may elevate overall extracellular concentrations of DA in the ventromedial striatum, but does not modify spontaneous or reward-induced rapid DA dynamics. These findings support the hypothesis that rapid effects of MFB DBS may be mediated by the release of neurotransmitters.

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Experimental procedures All experiments were approved by the Animal Experimentation Committee of the Royal Netherlands Academy of Arts and Sciences and were carried out in agreement with Dutch law (Wet op de Dierproeven, 1996) and European regulations (Guideline 86/609/EEC).

Animals Male Wistar rats (Charles River, DBS group n=16; control group n=8) were socially housed (per 2) under a reversed day-night schedule (lights on from 7 p.m. to 7 a.m.). Unlimited access to food and water was available the day before and 3-4 days following surgery. During behavioral training and experiments, rats were food-restricted (16 grams/animal/day) with unlimited access to water. After surgery, rats were housed individually.

Surgery Rats (weighing ~300 grams) were anesthetized with Isoflurane (induction: 3%, maintenance: 1.8-2.5%), received subcutaneous Metacam® (Meloxicam, 1 mg/kg, Boehringer-Ingelheim, Germany) as analgesic, and were then placed in a stereotactic frame. Body temperature was maintained by a temperature controller and heating pad. A custom made guide cannula (made in house) was implanted above the right nucleus accumbens (anterior posterior +1.3 mm, lateral +1.3 mm relative to bregma (Paxinos and Watson, 2007)). An Ag/AgCl reference electrode was implanted in the contralateral hemisphere. For the DBS group, a bipolar stimulation electrode (Plastics one, Roanoke, VA, USA) was inserted above the MFB (targeted at AP -4.6 mm, ML 1.0mm, DV -7 mm from bregma (Paxinos and Watson, 2007)). A micromanipulator holding a glass-enclosed carbon fiber electrode (electrode tip: diameter 7 μm, length 100-150 μm) was placed into the guide cannula and the electrode was lowered into the ventromedial striatum. A head-mounted amplifier for FSCV recordings was attached to the reference and working electrode. A resting potential of -0.4 V was applied to the carbon fiber electrode (vs the reference electrode). Every 100 ms, the potential was driven to 1.3 V and back in a triangular waveform (lasting 8.5 ms). At specific applied potentials in the waveform, changes in current can be measured due to redox 5

reactions of DA molecules (~0.6 V oxidation, ~-0.2 V reduction) close to the electrode surface (Phillips et al., 2003). To ensure placement of the stimulating electrode in a region were DA could be evoked, we gradually lowered the stimulating electrode into the MFB with 0.2 mm increments. Whenever the stimulating electrode was lowered, a biphasic stimulation (24/60 pulses, 60 Hz frequency, 2 ms pulse width per phase, 125 μA intensity) was applied. The stimulating electrode was lowered until evoked DA release could be measured at the working electrode in the ventromedial striatum. There has been inconsistent targeting of specific subregions of the MFB in preclinical experiments, with some studies stimulating the MFB close to the lateral hypothalamus (Bregman et al., 2015; Edemann-Callesen et al., 2015) and others more posterior, close to the VTA (Furlanetti et al., 2015a, 2015b, 2016). Our placement is located at the border of the MFB and VTA and corresponds to the placements of Furnaletti and colleagues (Furlanetti, et al., 2015a.b). Then, the working electrode was removed and the stimulating electrode was fixed with dental cement. Only animals that showed evoked DA release during surgery proceeded to the experiments described below. For the control group, a bipolar stimulating electrode was implanted in the VTA (AP 5.2 mm, ML 1.0 mm, DV ~8-9 mm from skull) and no stimulations were applied during surgery. A removable stylet was used to close the cannula after surgery.

Apparatus Experiments were performed in a custom made operant chamber (40 x 40 x 40 cm, mechanical workshop of the institute) with MED Associates parts (Med Associates, Sandown Scientific, Hampton, UK). A house light was located in the middle of one of the walls close to the chamber ceiling. A food dispenser was placed in the middle of the opposite wall. Nose pokes in the food dispenser to retrieve sucrose pellets (Dustless precision pellets®, 45 mg, Bio-Serv) were detected by an infrared sensor.

Experimental Procedure Before the start of the experiment, rats were habituated to the operant chamber and sucrose pellets. On the experimental day, rats were placed into the operant chamber and electrode implants attached to a head stage 6

to allow DBS and FSCV recordings. For FSCV recordings (Phillips et al., 2003), a micromanipulator holding a fresh glass-enclosed carbon fiber electrode was placed into the cannula and connected to a headmounted amplifier. The electrode was gradually lowered into the ventromedial striatum. The electrode was placed at a depth where spontaneous DA transients and DA release to unexpected food pellets were observed. For DBS, the head stage was connected to an isolator (DLS 100, World Precision Instruments (WPI), Sarasota, FL, USA) and stimulator (DS 8000, WPI, Sarasota, FL, USA). Fig 1 shows the experimental timeline. During the first 90 minutes, the animals could habituate. In the subsequent 10 minutes (T1), spontaneous DA transients were recorded, but rats were not presented with any other stimulus. During the following 20 minutes, spontaneous transients were collected and rats received food pellets (10-16 per session) delivered with a variable inter-trial interval (30/60/90/120/150 s; total session time: 20 minutes) throughout this period. After this period, DBS was applied (120Hz, 80 μs pulse width, 200 μA) to animals in the DBS group. To prevent artefacts from the DBS obscuring FSCV measurements, DBS stimulation was only applied during the inter-scan intervals in between voltammetry scans. Thus, instead of continuous stimulation, 1 pulse that would overlap with the FSCV waveform was omitted and 11 pulses were applied during each inter-scan interval. When DBS was switched on, the same procedure was applied: first 10 minutes of spontaneous transients were recorded (T2), followed by 20 minutes in which the rats received sucrose pellets at unexpected time points. Then DBS was switched off and spontaneous transients were recorded for a final 10 minutes (T3). After the experiment, electrical stimulations with parameters known to evoke DA release were applied to evoke DA release at the recording site (biphasic, 8/10/12/16/30 pulses, 30Hz frequency, 125 μA or 200 μA intensity, 2 ms pulse width per phase). A second cohort of animals (control group) underwent a similar experimental procedure, but did not receive DBS: These between-animal controls were placed into the operant chamber, attached to the headstage for voltammetry recordings, and a carbon fiber electrode was lowered into the ventromedial striatum. After habituation, spontaneous transients were recorded for 10 minutes, then rats received food pellets for 20 minutes delivered with a variable inter-trial interval. This procedure was then repeated: first 10 minutes of spontaneous transient recordings, followed by 20 minutes in which 7

the rats received sucrose pellets delivered at unexpected time points. Thus, between-animal controls never received DBS, but otherwise underwent the same experimental procedure and sequence of treatments as the DBS group. After the final experiment, a stainless steel electrode was lowered to the recording location and a direct current (100 μA) was passed to mark placement of recording and stimulating electrodes. Rats were euthanized with an overdose of pentobarbital (Erasmus MC, Rotterdam, the Netherlands) and decapitated. Brains were removed and frozen. Histological sections were cut on a cryostat and stained with cresyl violet to reveal electrode positions.

Data Analysis Chemometric analysis was performed on all FSCV recordings to separate DA-related currents from those of other electro-active species (Heien et al., 2004; Keithley et al., 2009). In vivo Training sets (for DA and pH) were prepared from electrically evoked DA release in a representative group of animals in our lab that were implanted with carbon fiber electrodes of the same material, construction design, and length (Clark et al., 2009; Willuhn et al., 2012; Howe et al., 2013; Collins et al., 2016). All data was smoothed using a 5-point moving average.

DBS onset For the effect of DBS onset on DA release, we analyzed a 50 s period during which DBS was switched ON. Data from this file was binned in three time bins: a 10 s baseline bin and two 20 s bins in which DBS was on. Using three bins allowed us to see whether any effect of DBS would sustain over this period. We compared the first 40 s of DBS (T2) to two control periods: 1) the first 40 s period of “no DBS” (T1) (within-animal comparison; animals are their own controls), and 2) the first 40 s period of T2 recorded in a different cohort of animals that underwent the exact same procedures (see Fig 1), but were not implanted with DBS electrodes (time-matched between-animal comparison). Data were analyzed statistically using repeated measures ANOVA. Because FSCV is based on background subtraction (measuring deviations from baseline concentration, not baseline concentration itself), FSCV is not well suited to track slow changes in concentration over prolonged time periods in which the background may 8

shift (Robinson et al., 2003). Thus, we limited our analysis to a period of 40 s, which could reliably be analyzed in all animals.

Spontaneous transients Spontaneous fluctuations in DA concentration commonly called ’DA transients’ can be observed in striatal regions (Robinson et al., 2003). Only transients greater than 0.3 nA were included in the analysis (calculated from 20 nM cutoff used in Vander Weele et al., 2014 using standard calibration factor). Transient amplitude and frequency were quantified using MiniAnalysis (Synaptosoft). Amplitude and frequency of spontaneous transients were analyzed during three blocks of 10 minutes (T1,T2,T3; repeated measures ANOVA) as well as during the two blocks in which animals received unexpected food rewards (paired t-test; see below section unexpected food rewards). General activity and number of nose pokes made in the food receptacle were recorded throughout these three blocks (T1, T2, T3). Behavioral data was analyzed with a repeated measures ANOVA.

Unexpected food rewards Rats were presented with unexpected food rewards during two blocks of 20 minutes (see Fig1). Throughout the 20-minute period, the following behavioral parameters were recorded: general activity, total number of nose pokes, and latency between pellet delivery and the first nose poke in the food receptacle to retrieve the pellet. Behavioral data were analyzed with paired t-tests. For the DA response to unexpected food pellets, all DA traces were aligned to the time of pellet delivery in the food receptacle. The peak DA response to pellet delivery was obtained by taking the maximum value in a two-second window that followed pellet delivery. For each rat, peak DA values to pellet delivery were averaged across the entire session. Peak DA responses during DBS OFF and DBS periods were compared with paired t-tests.

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All statistical analyses were performed with SPSS Statistics 21 (IBM, Amonk, NY). Statistical significance was set to p<0.05. All data was normally distributed. Greenhouse-Geisser corrections were applied when the assumption of sphericity was violated.

Results Of the animals originally implanted with DBS electrodes, eight were excluded from the experiment: n=4 rats did not show stimulated DA release during surgery, n=1 needed significantly higher stimulation parameters during surgery to achieve DA release and also did not show stimulated DA release (with standard parameters) after the experiment, n=1 died after surgery, and n=2 were not recorded from due to technical problems. Final group size for the DBS group was n=8. For the between-animal control group, n=3 animals were excluded from analysis because recordings contained too much noise (final group size n=5).

Histology Fig 2 shows the placement of recording (left) and stimulating electrodes (right) in the ventromedial striatum and MFB, respectively. In three animals, histological verification of stimulating electrode was not possible due to errors with histological processing. However, these three animals did show electrically stimulated dopamine release during surgery, and thus were included in the analysis. Depth of recording electrodes was estimated based on depth of cannula during surgery and how deep the electrode was lowered during the experiment.

Onset of MFB DBS induces DA release in the striatum For this experiment, one animal was excluded from data analysis due to a loss of baseline recordings. Fig 3 shows the effect of DBS onset on striatal DA release for the 7 remaining animals. Data was separated into three time bins: a 10-s baseline bin and two 20-s DBS bins. We compared the DA trace during the first 40 s of transient recording period T1 (NO DBS) and T2 (DBS) to see if onset of DBS would influence DA release (Fig 3B,C). A repeated measures ANOVA showed a DBS*time-bin interaction effect F(2,12)=5.676, p=0.018 (main effect DBS F(1,6)=2.541, p=0.162; main effect time-bin F(1.103,6.616)=3.656, p=0.098), indicating that the DA trace over three bins differed depending on stimulation condition (NO DBS vs DBS). 10

Separate post-hoc ANOVA’s for DBS and NO DBS showed that when DBS was switched on (T2 period), DA increased above baseline (significant effect time-bin F(1.022,6.134)=14.489, p=0.008, simple contrasts showed significant differences between time-bin1 and time-bin2 F(1,6)=15.205, p=0.008 and between timebin1 and time-bin3 F(1,6)=14.319, p=0.009). During the control period recorded in these same animals (T1, NO DBS), DA was not different across bins (F(1.144,6.865)=0.477, p=0.537). When excluding animals with recording electrodes placed outside of the nucleus accumbens (black and red dot in Fig 2), effects look similar. In addition, to verify this result, we compared the DBS onset trace to a similar time period in a between-animal control group (first 30 s of T2, importantly these animals underwent the same experimental procedure as the DBS group, thus they also received a 20 min session during which they received unexpected food pellets before transient recording T2). Similar to the findings described above, DA release across the three time bins was different between the DBS group and control group (significant Bin*Group interaction effect F(1.097,10.975)=7.924, p=0.015 (main effect Bin F(1.097,10.975)=8.483, p=0.013; Main effect Group F(1,10)=3.000, p=0.114). In the between-animal control group, DA did not change from baseline in the time period analyzed (T2; Main effect Bin F(2,8)=0.529, p=0.608). Together, these findings indicate that MFB DBS induced a rapid increase of striatal DA above baseline, which persisted for (at least) 40 seconds, whereas in a control period, DA did not change from baseline. We also checked whether switching the DBS off would affect DA release. Data was separated into three time-bins: a 10 s baseline bin during which DBS was still switched on, and two 20 s bins during which DBS was switched off. (Baseline: 0.02 ± 0.08 nA, time-bin 1: -0.10 ± 0.22 nA, time-bin 2: -0.11 ± 0.22 nA; data not shown). A repeated measures ANOVA did not show any effect of time-bin on DA release (F(2,12)=0.525, p=0.604), suggesting that switching off DBS did not affect DA release.

Continuous MFB stimulation does not affect spontaneous fluctuations in striatal DA We recorded spontaneous dopamine transients during three 10-minute periods: T1: NO DBS, T2: DBS and T3: DBS OFF (see Fig 1 for timeline experimental procedure). General activity and number of nose pokes made in the food receptacle were similar for all three 10-minute periods of transient recordings (data not 11

shown), suggesting that general motor behavior was not affected by stimulation. DBS did not influence amplitude or frequency of spontaneously occurring DA transients during the three 10-minute periods of spontaneous transient measurements (T1,T2,T3; amplitude F(2,14)=0.548, p=0.590; frequency F(2,14)=0.149, p=0.863, Fig 4A,B). We separately analyzed transient frequency in the first minute of T1 and T2, to see whether onset of DBS would affect the occurrence of spontaneous transients. Transient frequency did not differ between first minute of T1 and first minute of T2 period (t(6)=-1.457, p=0.195), suggesting that onset of DBS did not influence transient frequency. Thus, it is likely that the increased extracellular DA level following DBS onset is not maintained by an increase in phasic dopamine events or presumably dopamine neuron burst firing. We also quantified spontaneous transients during the two 20-minute periods in which rats received unexpected food rewards (excluding the DA responses to food pellet delivery), and found no effect of DBS (frequency – period 1: 5.61 ± 0.98, period 2: 5.75 ± 1.00, t(7)=-0.329, p=0.752; amplitude – period 1: 0.45 ± 0.02 nA, period 2: 0.46 ± 0.20 nA, t(7)=-1.133, p=0.295). Together, this shows that continuous DBS in the MFB does not influence the occurrence or amplitude of spontaneous dopamine transients.

Reward-induced DA is not altered by MFB DBS Rats were presented with unexpected food rewards during two 20-minute sessions. Rats received 11.5 ± 0.4 food pellets during the first session (NO DBS) and 13.1 ± 0.7 pellets during the second session (DBS). DBS did not influence general activity or nose poke behavior: latency until first nose poke after pellet delivery (session 1: 5.6 ±2.8 s; session 2: 2.3 ± 0.2 s) and number of nose pokes (session 1: 232.9±31.7, session 2: 279.6±29.8) made into the food dispenser were similar in both sessions. Fig 4C shows the DA response to reward delivery in presence (red line) and absence (black line) of DBS averaged across all animals. Rewardinduced DA release was not influenced by DBS (peak DA to reward delivery –NO DBS: 0.93 ± 0.12 nA, DBS ON: 1.10 ± 0.16 nA; t(7)=-2.180, p=0.066, Fig 4D).

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Discussion We investigated the effects of MFB DBS on different aspects of dopamine release in the ventromedial striatum. To our knowledge, this is the first study reporting effects of MFB DBS on basal and reward-related DA release in awake, freely moving rodents. Acute onset of DBS in the MFB induced an increase in DA release over baseline in the ventromedial striatum that lasted for at least 40 seconds. Continuation of MFB DBS beyond 40 seconds did not affect the frequency and amplitude of spontaneously occurring DA fluctuations (‘transients’), nor did it affect DA release in response to unexpected food rewards. These findings show that DBS onset induces an increase in DA output from the VTA, an activation of the DA system that may be of relevance for the rapid effects of MFB DBS in depressive patients (Schlaepfer et al., 2014).

Onset of stimulation induces striatal DA release The MFB was recently proposed as a new DBS target for MDD (Schlaepfer et al., 2013). With connections to all previously described target sites, the MFB is well positioned to influence activity throughout corticalstriatal networks (Coenen et al., 2011; Döbrössy et al., 2015). One of the ways by which DBS may modulate network activity is by influencing the release of neurotransmitters/neuromodulators (Hamani et al., 2010; Anderson et al., 2012). As it has previously been hypothesized that MFB DBS may influence DA neurotransmission (Schlaepfer et al., 2013, 2014), we set up an experiment to directly test the effect of MFB DBS on DA release in the striatum. We observed increased DA release in the ventromedial striatum following DBS onset. Although the increase in striatal DA upon DBS onset was only moderate, it was reliably detectable compared to both a within and between animal control group. The MFB DBS-induced increase in striatal DA release over baseline was immediately apparent upon DBS onset and was sustained over a prolonged period (at least 40 seconds). This suggests that the effect of DBS onset may not be limited to a single evoked release event immediately upon onset, but could persist over a period that extends beyond the sampled period of time. Because of its differential nature (based on background subtraction: FSCV measures deviations from baseline concentration, not baseline concentration itself), FSCV is not well-suited to track neurotransmitter 13

concentration over periods of several minutes to hours (Robinson et al., 2003). Thus, we did not determine if the initial increase in DA concentration to onset of stimulation was sustained throughout the entire period of DBS. What speaks against a permanent increase of DA by DBS is that we did not see an immediate effect on DA release when DBS was switched off (data not shown). There may be two explanations for this: 1) the DA elevation induced by DBS onset may not reflect a sustained elevation, but instead DA levels may gradually return to baseline during DBS; or 2) DA is chronically elevated during DBS, and this elevation persists for some time even after stimulation terminates. Future studies using experimental techniques that can measure neurotransmitter fluctuations across minutes (microdialysis with high sampling rates or fastscan adsorption voltammetry (Atcherley et al., 2015)), will be able to shed more light on such slower DA dynamics. Bregman et al (2015) previously used microdialysis but could not detect effects of MFB DBS on DA release. However, their use of anesthetized rats and 30 minute dialysate samples may not have created optimal conditions to detect changes in DA release. Other studies that used FSCV to report the effects of continuous, high-frequency MFB stimulation on striatal DA release have been limited by the use of full anesthesia (Shon et al., 2010; Paek et al., 2013), which can affect both basal and induced DA release (Ståhle et al., 1990; Adachi et al., 2005, 2008). A second limitation of using FSCV in an anesthetized preparation is that only effects of DBS on electrically evoked DA release can be studied, ignoring possible effects on spontaneously occurring or stimulus-induced DA transients. In contrast to continuous, high-frequency DBS, the effects of short bouts of lower-frequency MFB stimulation on striatal DA release have been studied extensively, for example, in intracranial selfstimulation (ICSS) experiments or to study striatal DA dynamics. Indeed, this work showed that stimulation with various parameters can induce DA release in the striatum (Gratton et al., 1988; Miliaressis et al., 1991; Garris et al., 1999; Kilpatrick et al., 2000; You et al., 2001; Hernandez et al., 2006; Cossette et al., 2016; Rodeberg et al., 2016). However, voltammetry experiments generally used isolated stimulus trains with stimulus parameters (50/60 Hz frequency, 2-ms pulse width) that are optimized to induce DA release, rather than DBS parameters (120/130 Hz frequency, ~0.1 ms pulse width) (Stamford et al., 1987; Garris et al., 1997). In addition, there are more fundamental differences between stimulation in ICSS and DBS experiments: ICSS is self-initiated and consists of a relatively short sequence of stimulation pulses (stimulus 14

trains), whereas the onset of DBS is not determined by the subject, and the stimulation is applied continuously after onset of stimulation. Importantly, here we show that onset of MFB DBS with clinically relevant stimulation parameters induces DA release in the striatum in awake, freely moving animals. Onset of DBS in other target regions can also acutely induce neurotransmitter release (Hamani et al., 2010; Van Dijk et al., 2012; Paek et al., 2013; Jiménez-Sánchez et al., 2016). Interestingly, clinical studies report rapid effects of DBS onset on mood and anxiety (Mayberg et al., 2005; Greenberg et al., 2008; Malone et al., 2009; Denys et al., 2010) that occur within seconds to minutes after onset of stimulation (Greenberg et al., 2008; Denys et al., 2010). Thus, one possibility is that elevation of neurotransmitters following DBS onset contributes to these rapid effects of DBS onset on mood and anxiety reported in clinical settings. The next question is how DBS of the MFB induces increased DA release in striatal regions. With the stimulus parameters commonly used for DBS (high frequency, low pulse width, and a relatively large electrode surface), direct activation of DA axons is unlikely (Yeomans et al., 1988; Yeomans, 1989). Although DA axons are capable of following high-frequency stimulation for a limited time period (1-3 s), sustained stimulation at high frequencies is not accompanied by steady DA release, probably due to adjustment of DA neurons (Stamford et al., 1987). This suggests that although the DBS onset effect on striatal DA release may result from a brief activation of DA neurons, any sustained effect on DA release may be indirect via recruitment of one of the many fiber components present in the MFB (Nieuwenhuys et al., 1982; Veening et al., 1982). This has also been suggested by Schlaepfer et al (2013;2014), who hypothesized that MFB DBS recruits glutamatergic fibers that originate in the PFC and project to DA neurons in the VTA, thereby modulating the activity of the DA system. MFB DBS affects activity in prelimbic regions (Bregman et al., 2015) and it was recently shown that selective activation of prefrontal terminals in the VTA can induce striatal DA release (Kim et al., 2015). However, it remains to be determined whether modulation of PFC activity during MFB DBS is indeed responsible for the increase in DA release to DBS onset.

Continuous DBS does not affect spontaneous or reward-induced DA release 15

Apart from an effect on baseline concentration, DBS might influence other DA release parameters such as spontaneously occurring transients or DA release that is associated with reward processing. A brief increase in the firing rate of DA neurons (burst firing) is thought to result in a brief increase in the extracellular DA concentration in projection regions (DA transient). These DA transients can occur spontaneously (or at least in the absence of any discernable external or environmental factor), or can be evoked by external salient events, such as the presentation of an unexpected food reward or alerting stimulus (Robinson et al., 2002; Cheer et al., 2004). We did not observe any changes in the number or amplitude of such spontaneously occurring transients: Frequency or amplitude changed neither in the seconds/minutes immediately following DBS onset, nor over a longer time period (30 minutes – 10 minute transient recordings and 20 minutes in between unexpected rewards). These findings suggest that although striatal DA release can be induced by the onset of MFB DBS, phasic DA release dynamics are not altered by continuous stimulation. This also suggests that the longer-term striatal DA release we observed following onset of stimulation cannot be explained by an increase in the frequency of DA transients. Thus, onset of DBS may increase basal DA levels in the striatum, without influencing the occurrence of spontaneous release events. Apart from the spontaneously occurring transients, we therefore specifically looked at DA transients induced by presentation of unexpected food rewards, a robust phenomenon that has been replicated numerous times and can be used to probe physiological and behavioral function of dopamine. We did not see an effect of MFB DBS on the latency to pick up food rewards from the food receptacle. In addition, MFB DBS did not change the amount of phasic DA release following presentation of an unexpected food reward. A previous study failed to find an effect of MFB DBS on reward-seeking behavior in a selfstimulation paradigm (Edemann-Callesen et al., 2015). Together, these findings suggest that sensitivity to rewards in normal, healthy animals is not altered by MFB DBS.

Future directions The ability to study the effects of DBS on DA release is not just relevant for the mechanisms of action of DBS in depression. OCD is also associated with dysfunctions of the DA system (Denys et al., 2004a, 2004b, 2013; Perani et al., 2008; Schneier et al., 2008). Moreover, OCD patients also show altered activity in the 16

ventral striatum during reward-related learning (Figee et al., 2011; Jung et al., 2011; Marsh et al., 2015), considered an indirect indicator of altered DA reward-processing. DBS in the nucleus accumbens/anterior limb of the internal capsule induces changes in D2/3 receptor binding potential, suggesting that DBS may increase striatal DA release (Figee et al., 2014). In addition, a recent study describes successful MFB DBS in two OCD patients, suggesting the MFB as a potential new target region for this disorder (Coenen et al., 2016) Our study has methodological value: The combination of techniques we described here can be used to directly study the effects of DBS in different target regions on DA release and may provide insight into potential different effects of stimulation between regions relevant for treatment of both MDD and OCD. Moreover, the possibility of using extended stimulation periods and the option of measuring DA release in different regions provide an interesting opportunity to enhance our understanding of the physiological effects of DBS. Future studies may also use the combination of FSCV and DBS during reward-learning paradigms in freely moving animals to further investigate whether DBS can influence DA responses during reward-related learning.

Since we used healthy animals, we cannot exclude that stimulation of a compromised brain system may induce different results than stimulation in a healthy subject. However, a first step in the direction of optimizing DBS treatment is to understand the basic mechanisms of it. Here, we collected data that improves our understanding of how DBS affects dopamine function, which may help improve future therapies for depressive symptoms. Furthermore, it is important to realize that the relation between clinical symptoms of MDD and their underlying neurobiological disturbances is not completely understood (Lujan et al., 2008; Anderson et al., 2012), which makes it difficult to model such symptoms in animals. Therefore investigating the effects of DBS in healthy rats is required as well, as it may provide insight into physiological and cognitive effects of DBS and can hint towards possible side effects of stimulation in new target areas (Feenstra and Denys, 2012). This study provides a first step in the characterization of DBS effects on DA release in freely moving animals. Future studies may use this combination of techniques to investigate the

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effects of DBS in different target regions on phasic DA release as well as combine DA measurements with DBS in animal models of depression.

Conclusion Here, we demonstrate the feasibility of combining DBS with measurements of phasic DA release in awake, freely moving animals. Importantly, the stimulation parameters that were used in the current experiment (high frequency, low pulse width) are in line with DBS parameters that have been shown to successfully reduce depressive symptoms in clinical (Schlaepfer et al., 2013) and preclinical studies (Bregman et al., 2015; Edemann-Callesen et al., 2015; Furlanetti et al., 2015a, 2015b, 2016). Patients with MDD show dysregulation of DAergic pathways and impairments in reward-related learning (Kapur and John Mann, 1992; Dunlop and Nemeroff, 2007; Kumar et al., 2008; Gradin et al., 2011; Russo and Nestler, 2013; Vrieze et al., 2013). Our results show that MFB DBS with clinically relevant stimulation parameters does not modify reward-related DA release, but that DBS onset can elevate the extracellular concentration of dopamine in the striatum. These findings support the hypothesis that increased DA may be involved in the rapid effects of MFB DBS.

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Figure 1 Approximate timeline of experimental procedures. During the first ~90 minutes, the animals could habituate. In the subsequent 10 minutes (T1), spontaneous DA transients were recorded, but rats were not presented with any stimuli. During the following 20 minutes, spontaneous transients were collected and rats received food pellets unexpectedly. After this period, DBS was applied (120Hz, 80 μs pulse width, 200 μA) to animals in the DBS group. Control animals never received DBS. When DBS was switched on, first spontaneous transients were assessed for 10 minutes (T2), followed by 20 minutes in which rats received sucrose pellets at unexpected time points. Then DBS was switched off and spontaneous transients were recorded for a final 10 minutes (T3). Figure 2 Illustrations of coronal brain sections indicating placement of recording (left panel) and stimulating (right panel) electrodes. Each colored dot represents an electrode position for a single animal (n=8). In three animals, histological verification of stimulating electrode was not possible due to errors with histological processing. However, these three animals did show good dopamine release during surgery, and thus were included in the analysis. Each colored circle represents one animal. Numbers indicate distance from Bregma in millimeters (anterior to Bregma for FSCV electrodes, posterior to Bregma for stimulating electrodes). Adapted from (Paxinos and Watson, 2007). Figure 3 DBS onset induces an increase in striatal dopamine (DA) release. (A) Example traces of DA release following DBS onset from an individual animal. Right panel shows DA release following onset of DBS (first 40 s T2), left panel shows DA release during a similar time period without DBS (first 40 s T1). Color plots show current changes (z-axis, color) at the recording electrode plotted against applied voltage (y-axis) and time (x-axis) (B) Average trace showing DA release to DBS onset across all animals. Left panel – DBS onset induces DA release in the striatum. Red line represents DBS, black line represents NO DBS in the same animals. Right panel – DBS onset trace (red line) compared to a “NO DBS” time period (black line) inthe between-animal control group. (C) Quantification of DA response to DBS onset. Left panel compares first 50 s of T2 to first 50 s of T1 (within-animal control), right panel shows first 50 s of T2 to first 50 s of T2 of control group (between-animal control). When DBS is switched on (red bars), DA significantly increases above baseline. Without DBS (black bars), DA remains stable over time bins. Asterisks indicate significant difference from baseline (p<0.01). Figure 4 Continuous DBS does not affect striatal DA dynamics. MFB DBS does not affect amplitude (A) or frequency (B) of spontaneously occurring DA transients. Grey bars indicate group average, colored dots correspond to individual animals. (C) Unexpected food-reward presentation induces DA release in the ventromedial striatum. DBS does not affect the DA response to unexpected food reward. Black line – control period (NO DBS), Red line – DBS, lines reflect mean, shaded region reflects SEM. (D) Quantification of peak DA response to reward delivery. DBS does not influence peak DA response to reward delivery. Grey bars indicate group average, each colored dot corresponds to an individual animal.

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Acknowledgements: We wish to thank Ralph Hamelink, Sjak Verschuren (NIN mechatronics department), and Scott Ng-Evans (University of Washington, Seattle) for technical assistance.

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Figure1

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Highlights (3-5, max 125 characters including spaces) -

The rapid antidepressant effect of MFB DBS may be due to increased striatal dopamine release. We found that onset of MFB DBS acutely increased dopamine release in the striatum of freely moving rats. Continuous MFB DBS did not affect spontaneous phasic dopamine release or reward-related changes in dopamine release. The acute dopamine increase after DBS onset may be involved in the rapid effects of MFB DBS in depressed patients.

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