,1 ,1 ,2 ,3
*Centre Hospitalier Universitaire (CHU) Clermont-Ferrand and Universit
e d’Auvergne,
Clermont-Ferrand, France
†Institut de Biologie du Developpement de Marseille (IBDM) UMR7288, Aix-Marseille Universite,
CNRS, Marseille, France
Abstract
The long-term effects and action mechanisms of subthalamic nucleus (STN) high-frequency stimulation (HFS) for Parkinson’s disease still remain poorly characterized, mainly due to the lack of experimental models relevant to clinical application. To address this issue, we performed a multilevel study in freely moving hemiparkinsonian rats undergoing 5-week chronic STN HFS, using a portable constant-current microstimulator.In vivo meta-bolic neuroimaging by 1H-magnetic resonance spectroscopy (11.7 T) showed that STN HFS normalized the tissue levels of the neurotransmission-related metabolites glutamate, glutamine and GABA in both the striatum and substantia nigra reticulata (SNr), which were significantly increased in hemiparkinsonian rats, but further decreased nigral GABA levels below control values; taurine levels, which were not affected in hemiparkinsonian rats, were significantly reduced. Slice electrophysiological recordings
revealed that STN HFS was, uniquely among antiparkinsonian treatments, able to restore both forms of corticostriatal synaptic plasticity, i.e. long-term depression and potentiation, which were impaired in hemiparkinsonian rats. Behavior analysis (staircase test) showed a progressive recovery of motor skill during the stimulation period. Altogether, these data show that chronic STN HFS efficiently counteracts metabolic and synaptic defects due to dopaminergic lesion in both the striatum and SNr. Comparison of chronic STN HFS with acute and subchronic treatment further suggests that the long-term benefits of this treatment rely both on the maintenance of acute effects and on delayed actions on the basal ganglia network.
Keywords: high-frequency stimulation, nuclear magnetic resonance, striatum, substantia nigra, subthalamic nucleus, synaptic plasticity.
J. Neurochem. (2016) 136, 1004–1016.
The motor symptoms of Parkinson’s disease (PD) are
primarily attributed to the dysfunction of the loop circuits
involving the cortex, basal ganglia (BG) and thalamus, mainly
due to the loss of dopaminergic innervation from the
substantia nigra pars compacta (SNc). Such dopaminergic
denervation leads to burst
firing of neurons of the subthalamic
Received August 24, 2015; revised manuscript received October 26,2015; accepted November 5, 2015.
Address correspondence and reprint requests to Paolo Gubellini, Institut de Biologie du Developpement de Marseille (IBDM), UMR7288 (Aix-Marseille Universite/CNRS), Case 907, Parc Scientifique de Luminy, 163 Avenue de Luminy, 13009 Marseille, France. E-mail: paolo.gubellini@univ-amu.fr
1These authors contributed equally to this work.
2Present address: Department of Applied Clinical and Biotechnological Sciences (DISCAB), University of L’Aquila, L’Aquila 67100, Italy. 3Present address: Departement de Psychiatrie et Neurosciences and Faculte de Medecine, Centre de Recherche du CHU de Quebec, Axe Neurosciences, Universite Laval, Quebec City, QC, Canada.
Abbreviations used: 1H-MRS, proton magnetic resonance spec-troscopy; 6-OHDA, 6-hydroxydopamine; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BG, basal ganglia; DBS, deep brain stimulation; EPSC, excitatory post-synaptic current; Gln, glutamine; Glu, glutamate; GPe/i, globus pallidus external/internal; HFS, high-frequency stimulation; LTD, long-term depression; LTP, long-term potentiation; MSN, medium spiny neuron; NMDA, N-methyl-D-aspartic acid; NMR, nuclear magnetic resonance; OD, optical density; PD, Parkinson’s disease; PRESS, point-resolved spectroscopy; Rm, input resistance; SNc/r, substantia nigra pars compacta/reticulata; STN, subthalamic nucleus; Tau, taurine; VOI, voxel of interest.
nucleus (STN) and causes hyperactivity of the
subthalam-opallidal, subthalamonigral, corticostriatal and thalamostriatal
pathways, resulting in hyperactivity of the BG output
structures, i.e. the internal globus pallidus (GPi or
entope-duncular nucleus, in rodents) and substantia nigra pars
reticulata (SNr) (DeLong 1990; Hollerman and Grace 1992;
Centonze et al. 1999; Blandini et al. 2000; Bevan et al.
2002; Aymerich et al. 2006). In animal models, dysfunctions
at the level of striatal glutamatergic neurotransmission and
synaptic plasticity, as well as GABAergic signaling, have
been implicated in PD pathophysiology (Lindefors and
Ungerstedt 1990; Centonze et al. 1999; Dehorter et al.
2009). For almost two decades, deep brain stimulation
(DBS) has been a successful and frequently practiced
neurosurgical approach for alleviating the motor symptoms
of PD, in particular high-frequency stimulation (HFS) of the
STN (Benabid et al. 2009; Karas et al. 2013). Despite some
discrepancies, literature data suggest that STN HFS
modu-lates glutamatergic and GABAergic neurotransmission in the
striatum, GPi/SNr and cortex (Bruet et al. 2003; Windels
et al. 2005; Boulet et al. 2006; Walker et al. 2009, 2012;
Favier et al. 2013; Melon et al. 2015). However, the
overwhelming majority of experimental studies deals with
short-term DBS (Gubellini et al. 2009; Spieles-Engemann
et al. 2010); thus, the impact of prolonged, chronic STN HFS
remains unknown and, most strikingly, the clinical use of
DBS is proceeding without a complete understanding of these
synaptic mechanisms (Rosa et al. 2012). This issue is crucial
not only for the sake of knowledge but also in light of recent
long-term clinical studies in PD patients undergoing STN
HFS, reporting a progressive worsening of both motor and
non-motor symptoms (Fasano et al. 2012; Rizzone et al.
2014); such decline might be associated with the progression
of the disease, but long-term changes induced by chronic STN
HFS cannot be excluded and this needs to be elucidated. Until
now, a major limitation for studying the long-term action
mechanisms and outcome of DBS has been the absence of
functionally relevant models in rodents. Because of the
remarkable plasticity of the brain, acute and subchronic
effects of experimental DBS might thus not be representative
of its chronic action and application; indeed, adaptive
processes, including plasticity in brain connectivity (van
Hartevelt et al. 2014), might be involved in the long-term
maintenance
– or loss – of the beneficial effects of this
surgical treatment described thus far (Udupa and Chen 2015).
To address this issue, we have recently developed a portable
microstimulator allowing chronic continuous DBS at constant
current in freely moving rats (Forni et al. 2012), and we
adapted the stimulation procedures to be performed in the
magnetic environment for nuclear magnetic resonance
(NMR) (Melon et al. 2015). Here, we studied the effects of
5-week, chronic STN HFS in freely moving
hemiparkinso-nian rats, unilaterally lesioned with 6-hydroxydopamine
(6-OHDA) injection in the SNc. We utilized a multidisciplinary
approach associating in vivo 11.7 T proton magnetic
resonance spectroscopy (
1H-MRS), ex vivo (slice)
electro-physiology and behavioral tests to study the long-term impact
of DBS on metabolite levels in the striatum and the SNr,
corticostriatal synaptic plasticity and motor skills.
Materials and methods
Animals and experimental layout
All animal experiments have been carried out in accordance with EU Directive 2010/63/EU for animal experiments and conformed to the ethical guidelines of the French Ministry of Agriculture and Forests (Animal Health and Protection Veterinary Service), as well as to the local Ethical Committee (#04-03032011). All efforts were made to minimize the number and suffering of the animals. Male Wistar Han rats (Charles River, Saint-Germain-sur-l’Arbresle, France) aged 7 weeks (180–200 g) at their arrival were utilized, housed 5 per cage (Type IV cages, 1500 cm2) at 21 1°C in a light controlled environment (12-h light/dark cycle) with access to food and water ad libitum (except during deprivation for behavioral tests, see below). After surgery (see below), animals were kept individually in Type E cages (580 cm2). All animals received several surgeries: 6-OHDA or vehicle injection in the SNc, as well as electrode implantation. Experimental groups are defined as follows: sham (vehicle injection in the SNc); 6-OHDA (rats receiving 6-OHDA injection in the SNc) and DBS (rats receiving 6-OHDA injection in the SNc and undergoing 5 weeks STN HFS). For these groups, several time points (in weeks) are taken into account, where‘time 0’ is defined as the start of STN HFS. One subset of sham, 6-OHDA and DBS rats was used for 1H-MRS and was also tested for behavior. Another subset was used for electrophysiology. The experimental layout and time course are shown in Fig. 1.
Brain surgery and DBS procedures
All surgical procedures were performed under ketamine+xylazine anesthesia (100+ 10 mg/kg, i.p.).
6-OHDA and sham lesion
6-OHDA and DBS rats received a unilateral SNc injection of 12lg of 6-OHDA (Sigma-Aldrich, Saint-Quentin Fallavier, France) dissolved in 6lL of vehicle solution (0.9% sterile NaCl containing 0.1% ascorbic acid), at the rate of 1lL/min. Sham rats received only vehicle solution in the SNc. The stereotaxic coordinates of the injection site were (from the interaural) anteroposterior (AP) +2.2 mm, lateral (L) 2.0 mm and dorsoventral (DV) +3.3 mm, with the incisor bar at +5.0 mm above the interaural plane (De Groot 1959).
Implantation of DBS electrode and microstimulator
Two to 3 weeks after SNc surgery (6-OHDA or sham lesion), all rats were unilaterally implanted with a DBS electrode in the STN ipsilateral to the injected SNc. Home-made bipolar electrodes were used, consisting of two parallel platinum-iridium wires (distance between tips ~ 400 lm) insulated with Teflon and bared at the extremity for a length of 500lm (diameter of each wire: 110 lm insulated, 76lm bare). For NMR experiments, gold wires were used, based on pilot experiments showing their compatibility with
the magnetic environment and no tissue damage after several days of continuous DBS (Forni et al. 2012; Melon et al. 2015). The electrode was implanted so that the two wires were placed along the anteroposterior axis with the active zone covering the STN extent in depth. The stereotaxic coordinates for the electrode tips were calculated as the average of interaural (AP+5.2 mm, L 2.4 mm, DV +2.0 mm) and bregma (AP 3.8 mm taken at equidistance from the two wires, L 2.4 mm, DV 8.0 mm from dura) values (Paxinos and Watson 1998). The electrode and the microstimulator support were fixed on the skull with Super-Bond C&B dental cement (Sun Medical Co, Ltd, Moriyama City, Japan), with the upper side of the support being designed as a platform to‘plug in’ the microstim-ulator (Forni et al. 2012). In rats undergoing NMR, the electrode was welded to a non-magnetic connector and the microstimulator support was fixed to the skull by two plastic screws inserted in dental cement, in order to allow removing the support and the microstimulator when the animals entered the magnetic environment (in this case, rats were connected to an external stimulator, see below).
DBS procedures
In order to deliver chronic STN HFS in freely moving rats for a period of 5 weeks, we used a constant-current microstimulator that has been previously characterized (Forni et al. 2012). DBS param-eters (current intensity, voltage, pulse width and frequency) were tested and set using an oscilloscope and a 15-kΩ output reference resistance connected to the pins of the microstimulator. STN HFS was started after 5 days of post-surgical recovery. The current intensity was set at 80lA (below the threshold for dyskinesia), the frequency at 130 Hz and the pulse width at 80ls. During the whole 5-week STN HFS period, these parameters were checked every 1– 3 days.
Assessment of 6-OHDA lesion and of electrode location in the STN Dopaminergic denervation was quantified by analyzing [3 H]-mazindol binding to dopamine uptake sites, as previously described (Salin et al. 2002; Cuomo et al. 2009). Briefly, brain coronal tissue sections (10lm thick) were cut at 20°C with a cryostat (Leica CM3050; Wetzlar, Germany) at AP interaural coordinates 8.6–
9.2 mm (Paxinos and Watson 1998) corresponding to the striatum. The sections were then mounted on glass slides and incubated for 40 min with 15 nM [3H]-mazindol (NEN DuPont; specific activity, 17 Ci/mM). [3H]-sensitive Hyperfilm photographic films (Kodak BioMax MR Film; Sigma, Munich, Germany) were then exposed to the slides in X-ray cassettes at 23-25°C for 30 days. Autoradiogram films were then digitized with Densirag analysis system (BIOCOM Explora Nova, La Rochelle, France) to measure the levels of [3 H]-mazindol labeling as gray levels, which were then converted to optical density using external standards (calibrated density step tablet; Kodak, Rochester, NY, USA). The background signal was measured on each section by scanning an area of the corpus callosum, which is known to lack dopaminergic terminals. The mean optical density value was then determined from at least four sections per animal after subtracting the background signal. The loss of [3H]-mazindol labeling was calculated as the ratio (expressed as %) between the 6-OHDA and DBS groups versus sham, for both the ipsilateral and the contralateral side. Both 6-OHDA and DBS rats showed a dramatic loss of [3H]-mazindol binding in the ipsilateral striatum compared with sham ( 92.6 and 89.8%, respectively) and no significant change in the contralateral side (Fig. 2). The location of the DBS electrode was verified on cryostat sections covering the anteroposterior extent of the STN. No major tissue damage was observed after the 5 weeks of continuous stimulation, and only animals showing a correct placement of the two poles of the electrode within the STN were selected for further studies.
In vivo1H-MRS
1H-MRS scans were performed at 11.7 T on a Bruker BioSpec 117/ 16 Ultra Shielded Refrigerated system at the end of the experiment time course (Fig. 1) as previously described (Melon et al. 2015). During1H-MRS acquisitions, the portable microstimulator and its support were removed, and an external home-made non-magnetic stimulator was plugged directly into the electrode via the connector using identical stimulation parameters. Throughout the experiment, animals were kept anesthetized with 1–2.4% isoflurane combined with a mixture of air and O2(70–30%, respectively; 300 mL/min). They were secured with two earpieces and a bite bar in a holder. A circular polarized 1H rat brain radiofrequency coil used for
6-OHDA
DBS Sham
Vehicle inj. Electrode impl.
DBS 6-OHDA inj.
6-OHDA inj. Staircase test
Week –4 –3 –2 –1 0 1 2 3 4 5 Histology 1 H-MRS Electrophysiology –5
Fig. 1 Experimental layout and time course. Three groups of rats were used, all unilaterally injected in the SNc at week 4 (white arrows) with either 6-hydroxydopamine (6-OHDA) (‘deep brain stimulation (DBS)’ and‘6-OHDA’ groups) or vehicle (‘sham’ group), and implanted with the stimulating electrode in the subthalamic nucleus (STN) at week 1 (black arrows). DBS rats received chronic 5-week STN high-frequency
stimulation starting at week 0 (gray arrow). The staircase test was performed at three time points, i.e. at week 2, 2 and 5 (as shown by the orange rectangles). At the end of week 5, animals were used for eitherin vivo proton magnetic resonance spectroscopy or killed for ex vivo (slice) electrophysiology, and their brains were used for assessing 6-OHDA lesion.
excitation and signal reception wasfixed on the holder, and then all the system was placed at the isocenter of the magnet. Breathing and temperature parameters were monitored along the session, and temperature was maintained at 37.8°C by warm air system. Anesthesia was adjusted according to the respiratory rate. For positioning of the spectroscopic voxels in the striatum and the SNr, T2-weighted multislice images were acquired using a fast spin echo technique with afield of view of 35 9 35 mm2; 2569 256 data matrix; 1-mm slice thickness; effective TE= 15.72 ms; TR= 2500 ms and number of averages = 2. Localized1H-MRS spectra were acquired in a 8-lL (2 9 2 9 2 mm) voxel of interest (VOI) from the striatum and in a 2.9-lL (1.8 9 1.0 9 1.6 mm) VOI from the SNr (Fig. 3). The standard point-resolved spec-troscopy sequence was used for spectra acquisition with an echo time of 8.8 ms, a repetition time of 4000 ms and a spectral width of 5000 Hz, combined with VAPOR water suppression module consisting of variable-power RF pulses with optimized relaxation delays (Tkac et al. 2004). The middle of the striatum voxel was positioned 0.36 mm posterior and 3.0 mm left from the bregma and 5.5 mm from the skull surface according to the Paxinos and Watson atlas (Paxinos and Watson 1998). In the same manner, the middle of the SNr voxel was positioned 4.92 mm posterior and 2.5 mm left from the bregma and 8.5 mm from the skull surface. The VOI localized on the SNr was selected as the smallest volume around the SNr, and its location was positioned in the most reproducible way, using anatomical landmarks on the T2-weighted images: the aspect of the corpus callosum and the volume of the ventricles. Further-more, higher gray level allowed delineating the SNr from the SNc. Magnetic field homogeneity was adjusted manually using 1st-and 2nd-order shims. Each spectrum corresponded to 512 averaging scans for the dorsal striatum and 1664 averaging scans for the SNr. Absolute quantification was performed using the water signal as an
internal reference. Water spectra were acquired in the same conditions in the striatum and the SNr without water suppression and with 256 averaging scans. Experimental spectra werefitted with jMRUI software using the time-domain semiparametric algorithm QUEST, based on signals for a basis set of simulated metabolites (version 5.0, http://www.mrui.uab.es/mrui). The metabolite basis set included lactate (Lac), N-acetylaspartate (NAA), total creatine (creatine and phosphocreatine; tCr), total choline (glycerophospho-choline and phospho(glycerophospho-choline; tCho), glutamate (Glu), glutamine (Gln),c-aminobutyric acid (GABA), taurine (Tau) and myo-inositol (Myo-Ins). It also included a simulated signal of macromolecules and lipids (MM). Concentrations were not corrected for T1 and T2 relaxation times effects because, at long TR (≥ 4000 ms) and short TE (≤ 30 ms), moderate variations in T1 and T2 times have minor impact. The reliability of metabolite quantification was assessed from the average Cramer-Rao lower bounds (CRLB) calculated by jMRUI. Only results with a CRLB≤ 25% were included.
Ex vivo electrophysiology
At the end of the experiment time course (Fig. 1), coronal corticos-triatal slices (250lm) were prepared from brains of sham, 6-OHDA and DBS rats (aged 4 months) by a vibratome (Leica VT1000 S) in oxygenated ice-cold solution containing (in mM): 110 choline, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, 7 glucose, 300–310 mOsm, pH 7.4. For DBS and 6-OHDA rats, only slices ipsilateral to 6-OHDA injection were used. Slices were then stored in oxygenated artificial cerebrospinal fluid (ACSF) at 23-25°C, which composition was (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 glucose, and 25 NaHCO3, 300–310 mOsm, pH 7.4, and also containing 250lM kynurenic acid and 1 mM sodium pyruvate. Whole-cell patch-clamp recordings were performed in oxygenated ACSF (without kynurenic acid and sodium pyruvate) at 35°C, containing 50 lM picrotoxin (Tocris Bioscience, Bristol, UK) andflowing at ~ 2.5 mL/min. Striatal medium spiny neurons (MSNs) were recorded by borosilicate micropipettes (5–6 MΩ) filled with (in mM): 125 K-gluconate, 10 NaCl, 1 CaCl2, 2 MgCl2, 0.5 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid, 19 4-(2-hydro-xyethyl)piperazine-1-ethanesulfonic acid (HEPES), 0.3 guanosine triphosphate, 1 Mg-adenosine triphosphate (Mg-ATP), pH 7.3 and 290–300 mOsm. MSNs of the dorsolateral striatum were identified visually (by infrared videomicroscopy) and by their electrophysio-logical properties (Jiang and North 1991). Glutamatergic excitatory post-synaptic currents (EPSCs) were evoked by a stimulating bipolar electrode placed on the corpus callosum between the cortex and the dorsal striatum as previously described (Cuomo et al. 2009), using a S88 stimulator and a SIU5 constant-voltage isolation unit (Grass Instruments, Warwick, RI, USA). Electrophysiological data were recorded by pClamp 10.2 software and an AxoPatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) at a sampling frequency of 10 kHz. MSNs were voltage-clamped at 80 mV, and resting membrane potential was determined by finding the membrane potential at which injected current was null. Series and input resistance were continuously monitored by sending 5 mV pulses, and MSNs showing> 20% change in series resistance and/or unstable EPSCs were discarded. Data were analyzed offline by pClamp 10.2. EPSC amplitude for monitoring long-term depression (LTD) and long-term potentiation (LTP) was measured on averaged EPSCs (6/ min) to obtain time-course plots and to compare this parameter before Fig. 2 Assessment of 6-hydroxydopamine (6-OHDA) lesion. The graph
shows the optical density (OD) of mazindol staining in the striatum of sham, 6-OHDA and deep brain stimulation (DBS) rats (expressed as % of sham value), ipsilateral and contralateral to the injected site (data are presented as mean SEM; *p < 0.05, **p < 0.01, Mann–Whitney test; n = 4–6 rats). The inset depicts a coronal section of the brain from a 6-OHDA rat showing the loss of mazindol staining in the striatum ipsilateral to the 6-OHDA-injected side (red asterisk).
and after the induction protocols. LTD induction protocol consisted in three stimulation trains (100 Hz) of 3-s duration separated by 20-s intervals. LTP induction protocol was identical but, during each train, MSNs were depolarized to 10 mV in order to allow N-methyl-D -aspartate receptor activation (Calabresi et al. 1992a,b, 2000a; Partridge et al. 2000; Paille et al. 2010).
Staircase test
This test is conceived for measuring the independent use of forepaws in skilled reaching and grasping food pellets, and it also takes advantage of a motivational cue due to food deprivation. STN HFS was maintained also during this test, notably thanks to the new implantable microstimulator allowing the rats performing in the narrow staircase apparatus (Campden Instruments, Ltd., Loughborough, Leics., UK). The stairs of both sides were loaded with two pellets in each of the seven wells, i.e. 14 pellets per side (Montoya et al. 1991). We measured the number of pellets eaten [14 - (pellets remained+ pellets missed)] on the side ipsilateral and contralateral to vehicle or 6-OHDA injection, where pellets remained are those remained in the wells and pellets missed are those fallen out of the wells, similarly to previous reports (Kloth et al. 2006; Klein et al. 2007). During the whole experiment time
course, rats underwent the staircase test on three sessions lasting 5 min each (Fig. 1). The first session was held at the 2 weeks time point for the three groups (sham, 6-OHDA and DBS): animals were trained once a day for four consecutive days in the staircase apparatus, and only the 5th day was considered for measurements. The following sessions were performed at 2 and 5 weeks: each consisted in three training days followed by measurements on the 4th day. Rats were food deprived (10 g/day of food per animal) for 4 days before each session.
Statistical analysis
The size of the samples (n) indicated in the Results andfigures refers to number of rats, except for electrophysiology where it indicates the number of recorded MSNs. Statistical analysis was performed by Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). For analyzing two data sets, we used the Mann–Whitney test. For analyzing≥ 3 data sets (either ‘within group’ or ‘between groups’), we usedANOVAor Kruskal–Wallis tests, followed, respectively, by Bonferroni or Dunn’s post-test. The p value for significance was set at 0.05. Data are expressed as mean SEM. The statistics tests used and significance values are reported in the Results and in figure legends.
Fig. 3 In vivo proton magnetic resonance spectroscopy acquisitions. Typical spectra from the dorsal striatum and from the SNr. Coronal T2-weighted image shows the voxel of interest (VOI) (in red) centered in the dorsal striatum (a) and in the SNr (b). VOIs’ volumes are, respectively, 29 2 9 2 mm and 1.8 9 1 9 1.6 mm. Spectra shown are obtained in a sham, a 6-hydroxydopamine (6-OHDA) and a deep brain stimulation (DBS) rat, in the striatum (a) and the SNr (b), with a PRESS localization sequence (TE= 8.8 ms; TR = 4 s; NS = 512 for acquisitions in the striatum and NS= 1024 for the SNr; line broadening= 10 Hz). Metabolites are assigned as macromolecules
(MM) at 0.92 ppm; lactate (Lac) at 1.33 ppm; N-acetylaspartate (NAA) at 2.008 ppm (signal of CH3) and at 2.49 ppm (signal of bCH2); glutamate (Glu)+ glutamine (Gln) at 2.12 ppm (signal of bCH2) and 3.75 ppm (signal ofaCH); c-aminobutyric acid (GABA) at 2.27 ppm; glutamate (Glu) at 2.34 ppm (signal ofcCH2); glutamine (Gln) at 2.42 ppm (signal ofcCH2); total creatine (tCr) at 3.03 (signal of CH3) and 3.94 ppm (signal of CH2), total choline (tCho) at 3.22 ppm; taurine (Tau) at 3.24–3.42 ppm (respectively for NCH2and SCH2), myo-inositol (Myo-Ins) at 3.48–3.52 ppm for the [1,3] CH and the [4,6] CH.
Results
Measurement of absolute concentration of metabolites by
in vivo
1H-MRS
Typical in vivo
1H-MRS spectra obtained in the striatum are
depicted in Fig. 3(a). After shimming, a half-height
line-width of the water signal of 9–12 Hz was achieved in the
dorsal striatum. A neurochemical pro
file of the SNr was also
achieved in 2.9-
lL volumes (Fig. 3b). The half-height
linewidth of the water resonance was 13
–15 Hz. The
majority of the metabolites had average CRLBs lower than
14% in the striatum, with the exception of GABA (15–20%)
and Gln (15–18%), and lower than 25% in the SNr.
In both the striatum (Fig. 4a) and the SNr (Fig. 4b) of
6-OHDA rats, compared with sham, Tau levels were not
affected but the levels of Glu, Gln and GABA were
significantly increased. Interestingly, in DBS rats, such
increases were reversed in both striatum and SNr, and Tau
levels were significantly decreased below sham and 6-OHDA
values (Fig. 4a and b).
In 6-OHDA rats, compared with sham, striatal levels of
Glu, Gln and GABA increased in average by 66.9%
(from 9.73
0.36 to 16.25 1.57 mM), 104.3% (from
4.23
0.74 to 8.46 0.50 mM) and 93.7% (from
1.58
0.21 to 2.64 0.29 mM), while in the SNr by
48.4% (from 8.59
0.69 to 11.50 1.28 mM), 87.9%
(from 3.13
0.52 to 5.15 0.46 mM) and 34.0% (from
3.15
0.29 to 4.68 0.39 mM), respectively. In DBS rats,
compared with 6-OHDA, striatal levels of Glu, Gln and
GABA decreased in average by 78.5% (9.10
1.15 mM),
102.8% (4.26
1.06 mM) and 55.3% (1.97 0.19 mM)
and in the SNr by 36.7% (9.33
1.15 mM), 79.3%
(3.28
0.40 mM) and 108.9% (2.02 0.26 mM),
respec-tively. It is worth noting that nigral GABA levels in the DBS
group were even lower than sham value, although this effect
did not reach significance. The levels of Tau in DBS rats
compared with 6-OHDA were reduced by 61.9% (from
14.40
1.07 to 8.89 0.54 mM) in the striatum and by
89.4% (from 4.28
0.19 to 2.26 0.17 mM) in the SNr,
while in sham they were, respectively, 12.98
0.97 and
4.21
0.17 mM. The levels of the other metabolites
measured were not significantly affected by either 6-OHDA
lesion or 5-week STN HFS (not shown).
Corticostriatal synaptic plasticity
Electrophysiological recordings from striatal MSNs (n
= 53)
of sham, 6-OHDA and DBS rats showed similar intrinsic
membrane properties (respectively, resting membrane
poten-tial
80.3
2.4, 79.8 2.1 and 78.6 3.2 mV; input
resistance 52.4
5.3, 55.6 4.4 and 53.7 3.2 MΩ;
n
= 13, 16 and 24). In the striatum of sham rats, both LTD
and LTP were induced and showed a time course and
amplitude comparable to those previously described in the
literature (Calabresi et al. 1992a,b; Gubellini et al. 2004;
Paille et al. 2010). For LTD (Fig. 5a), average EPSC
amplitude
after
the
induction
protocol
decreased
to
50.48
12.26% of baseline, while for LTP (Fig. 5b), it
increased to 158.50
18.76%. These two forms of synaptic
plasticity were lost in 6-OHDA rats, in agreement with
previous reports (Centonze et al. 2001). In this experimental
group, after LTD induction protocol, the average EPSC
amplitude was 83.57
10.21% of baseline (Fig. 5a), and
after LTP induction protocol, it was 85.39
17.04%
(Fig. 5b). Interestingly, in DBS rats, both LTD and LTP
were restored. For LTD, average EPSC amplitude was
reduced to 70.36
8.23% of baseline. However, while in
average signi
ficant, the time course of EPSC amplitude
reduction in DBS rats was delayed compared to sham, and
full depression was generally observed only after 10
–15 min
Fig. 4 Absolute concentrations of Tau, glutamate (Glu), glutamine (Gln)and GABA in the striatum and SNr are modulated by 6-hydroxydopa-mine (6-OHDA) lesion and/or 5-week subthalamic nucleus high-frequency stimulation. In the striatum (a), Tau concentration was unaffected in 6-OHDA rats (n = 4) but was significantly reduced in deep brain stimulation (DBS) rats (n = 6) compared with sham (n = 5), while Glu, Gln and GABA concentrations were all significantly increased in 6-OHDA rats and normalized in DBS. In the SNr (b), Tau concentration was unaffected in 6-OHDA rats but significantly reduced in DBS, while Glu, Gln and GABA concentrations were significantly increased in 6-OHDA rats and normalized in DBS. Data are presented as mean SEM. °p < 0.05, °°p < 0.01; °°°°p < 0.0001, 1-wayANOVAand Bonferroni post-test;n = 4–6 rats.ANOVAresults for striatal Tau, Glu, Gln and GABA are, respectively: F(2, 12) = 12.44, p < 0.01; F(2, 12)= 11.61, p < 0.01; F(2, 11) = 7.67, p < 0.01; F(2, 12) = 5.13, p < 0.05. ANOVA results for nigral Tau, Glu, Gln and GABA are, respectively:F(2, 12) = 46.46, p < 0.0001; F(2, 10) = 4.27, p < 0.05; F(2, 10) = 5.86, p < 0.05; F(2, 13) = 18.57, p < 0.001.
from the induction protocol (Fig. 5a). For LTP, average
EPSC amplitude was increased to 166.00
18.51% of
baseline, and this form of synaptic plasticity was also
restored in terms of time course (Fig. 5b). Between-groups
statistical analysis revealed that LTD and LTP obtained in
sham and DBS rats were also significantly different from the
results obtained in 6-OHDA (Fig. 5a and b).
Reaching and grasping skill
The staircase test showed that the number of pellets eaten by
sham rats remained relatively constant during the three
sessions. On the ipsilateral side (Fig. 6a), these scores were
6.2
1.4, 8.8 1.2 and 8.8 1.1 at 2, 2 and 5 weeks,
respectively,
and
contralateral
values
(Fig. 6b)
were
8.2
1.3, 9.8 0.8 and 11.2 0.8. In 6-OHDA animals,
the number of pellets eaten on both sides was signi
ficantly
lower compared with sham and remained stable: ipsilateral
scores at
2, 2 and 5 weeks were, respectively, 1.6
0.8,
3.1
1.1 and 3.1 1.4 (Fig. 6a), and contralateral scores
were 2.4
1.0, 3.1 1.1 and 3.4 1.4 (Fig. 6b). In DBS
rats (Fig. 6a and b), the number of pellets eaten at
2 weeks
was similar to 6-OHDA at the same time point, namely
2.4
0.6 and 2.8 0.8 for the ipsilateral and contralateral
side, respectively. Interestingly, these scores increased
significantly during the 5-week STN HFS period: at 2 weeks
they reached, respectively, 8.0
1.0 and 5.3 0.9, and at
5 weeks 10.4
1.1 and 8.1 1.1 (Fig. 6a and b). Overall,
these data show that STN HFS could ameliorate
progres-sively the de
ficit in reaching and grasping skill of
hemi-parkinsonian rats, with a complete recovery at 5 weeks.
Discussion
In this multidisciplinary study, we characterized the
meta-bolic, neurophysiological and behavioral effects of chronic
5-week STN HFS in a rat model of PD, in order to help
Fig. 5 Corticostriatal long-term depression (LTD) and long-term potentiation (LTP) are lost after 6-hydroxydopamine (6-OHDA) lesion and restored by 5 weeks subthalamic nucleus high-frequency stimu-lation. (a) The graph above shows the time course for LTD in sham, 6-OHDA and deep brain stimulation (DBS) rats (data are presented as mean SEM for each time point; the black arrow represents the induction protocol). Excitatory post-synaptic current (EPSC) amplitude is normalized as % of baseline, i.e. the period before the induction protocol. Note that LTD was lost in 6-OHDA rats and, while restored in the DBS group, its time course was delayed and differed from sham. The traces show samples of EPSCs recorded before (black) and after (gray) the LTD induction protocol in the three experimental groups. The histogram below shows the average EPSC amplitude of the whole period after the induction protocol, normalized to baseline (data are presented as mean SEM). (b) The time-course plot, traces and histogram (see above for description) show that LTP was lost in 6-OHDA rats and fully restored in the DBS group. **p < 0.01, ***p < 0.001 versus baseline, Mann–Whitney test; °p < 0.05, °°°p < 0.001, Kruskal–Wallis and Dunn’s post-test; n = 6–12 striatal medium spiny neurons.elucidating the long-term mechanisms by which this
treat-ment impacts parkinsonian deficits.
Here, we show that the levels of Glu, Gln, and GABA
measured by
1H-MRS were all significantly increased in
6-OHDA rats compared with sham, in both the striatum and
SNr. Such increases are consistent with the view that
glutamatergic and GABAergic systems become hyperactive
in these BG structures in response to dopamine depletion
(Lindefors and Ungerstedt 1990; Centonze et al. 1999;
Blandini et al. 2000; Gubellini et al. 2002, 2006; Obeso
et al. 2008; Dehorter et al. 2009) and are in line with
previous NMR studies from our groups and others (Chassain
et al. 2008; Bagga et al. 2013; Melon et al. 2015), although
another article showed increase of GABA but a decrease of
Glu (Coune et al. 2013). These discrepancies might be due
to different PD models (6-OHDA lesion of the medial
forebrain bundle vs. the SNc) and/or different post-lesion
time when metabolite levels were measured (3 vs. 9 weeks).
In a recent work from our group (Melon et al. 2015), we
showed that acute STN HFS could normalize the levels of
Glu, Gln and GABA in the striatum but not in the SNr. In the
same article, we reported that subchronic (7 days) STN HFS
maintained this normalizing effect in the striatum for Glu and
GABA but not for Gln, while in the SNr only GABA was
normalized. Here, we show that 5-week STN HFS could
normalize the levels of Glu, Gln and GABA in the striatum,
similarly to acute stimulation, and, interestingly, it could also
normalize them in the SNr, with a supranormalization of
GABA. This demonstrates that STN HFS has rapid beneficial
effects on glutamatergic and GABAergic
metabolism/trans-mission in the striatum that are maintained in the long term,
whereas chronic stimulation is required to restore these
parameters also in the SNr. In addition, such kinetics of DBS
action provides a mechanistic substrate that can explain its
progressive bene
ficial symptomatic effects, as shown here
with the staircase test. Such different outcomes between
chronic and subchronic/acute STN HFS highlight the view
that short-term effects of DBS might not be representative of
the changes induced by longer, and more clinically relevant,
stimulation periods, during which progressive responses to
this treatment can occur in the brain. Moreover, these data
suggest that, while acute STN HFS might have an immediate
and
– possibly – direct impact on striatal neurotransmitters/
metabolites, longer stimulation periods are required for
progressively achieving a full and stable effect in both the
main input and output structures of the BG. Concerning the
levels of Tau, we previously showed that they were not
affected by 6-OHDA lesion and they were decreased after
subchronic, but not acute, STN HFS in both the striatum and
SNr (Melon et al. 2015). Here, we show that such reduction
of Tau levels in both these structures is conserved after
long-term chronic STN HFS, illustrating again the progression and
maintenance of DBS-induced plasticity. Given the multiple
roles played by Tau, in particular as a neuromodulator and as
a neuroprotectant (Wu and Prentice 2010; Kumari et al.
2013), the mechanisms underlying this proper effect of DBS,
which is independent from the dopaminergic lesion, and its
functional consequences, deserve further studies.
Concerning synaptic plasticity, here we report for the
first
time the effects of STN HFS on corticostriatal LTD and LTP.
These are classically described as a persistent decrease
(LTD) or increase (LTP) in the amplitude of evoked
corticostriatal glutamatergic events, occurring after specific
induction protocols and receptor stimulation (Calabresi et al.
1992b,a; Lovinger et al. 1993; Partridge et al. 2000; Fino
et al. 2005; Pawlak and Kerr 2008; Lovinger 2010; Paille
et al. 2010; Bagetta et al. 2011). Here, we show that both
Fig. 6 Chronic 5-week subthalamic nucleus (STN) high-frequencystimulation (HFS) improves motor behavior. Forepaw skill in reaching and grasping food pellets (number of pellets eaten) remained stable in sham rats (n = 7) during the whole experiment, both on the ipsilateral (a) and contralateral (b) side. The number of pellets eaten by 6-hydroxydopamine (6-OHDA) rats (n = 11) was significantly reduced bilaterally (a, b) compared to sham and remained low during the whole monitoring period. In deep brain stimulation (DBS) rats (n = 15), a progressive and bilateral (a, b) increase in the number of pellets eaten was observed during chronic STN HFS. Such improvement was significant (compared to 2 weeks) for the ipsilateral side at 2 weeks and for both sides at 5 weeks. Data are presented as mean SEM; $p < 0.05,$$p < 0.01 versus sham, *p < 0.05, ***p < 0.001 versus 6-OHDA, Kruskal–Wallis and Dunn’s post-test; °°p < 0.01, °°°p < 0.001, Kruskal–Wallis and Dunn’s post-test; n = 7–15.
LTD and LTP were lost after 6-OHDA lesion, as previously
well
demonstrated,
since
their
induction
mechanisms
strongly depend, inter alia, on the stimulation of dopamine
receptors (Calabresi et al. 2000b; Centonze et al. 2001;
Bagetta et al. 2010). Interestingly, 5-week STN HFS
restored both forms of synaptic plasticity, suggesting that
such treatment might act on the function of the corticostriatal
pathway through a mechanism bypassing the dopaminergic
system. This might be linked to the restoration of glutamate
levels in the striatum shown by the present
1H-MRS data,
presumably reflecting a normalization of glutamatergic
transmission, and hence of the activation of the glutamate
receptors involved in the induction of LTD and LTP, i.e.
a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid
(AMPA), N-methyl-
D-aspartic acid and group I metabotropic
glutamate receptors (Gubellini et al. 2004; Lovinger 2010).
In addition to local action at striatal level, which might
involve direct subthalamo-striatal afferents (Kita and Kitai
1987), STN HFS could also impact the function of the
corticostriatal pathway via its complex action on brain
circuitry within and beyond the BG, notably the antidromic
stimulation of the cortex, in particular the motor areas
projecting to the striatum (Li et al. 2007; Gradinaru et al.
2009; Degos et al. 2013; de Hemptinne et al. 2015): this
could result in normalizing cortical neuron activity and
counteracting corticostriatal hyperactivity, leading to the
restoration of corticostriatal synapse function. Concerning
the delayed LTD induction observed here in DBS rats,
several alternative or concomitant mechanisms could be
implicated. For example, we have previously shown that the
gene expression pro
file is modified in the striatum of
hemiparkinsonian rats undergoing STN HFS (Lortet et al.
2013). Interestingly, among the genes affected, some code
for proteins involved in corticostriatal LTD, namely protein
kinase C (Calabresi et al. 1994) as well as CB1 receptor
(Gerdeman et al. 2002) and
a7 nicotinic ACh receptor
subunit (Partridge et al. 2002); their altered expression might
thus play a role in the slower time course of this form of
synaptic plasticity. Another possibility could be that the
dramatic decrease of AMPA receptor-mediated corticostriatal
glutamatergic transmission induced by STN HFS (Gubellini
et al. 2006) might partially occlude or delay the induction of
LTD. Finally, it is worth noting that previous works in rodent
PD models showed that corticostriatal LTD can be restored
by treatments using phosphodiesterase inhibitors (Picconi
et al. 2011), D2 receptor agonists or inhibition of
endo-Neuronal activity markers in the SNr/EP1–4 Corticostriatal synaptic transmission5 Corticostriatal LTD/LTP6
Glu and GABA levels in the striatum6,7 Glu and GABA levels in the SNr6,7 Motor behavior3,5–8 Adult neurogenesis9 Chronic Subchronic Acute 6-OHDA Naïve
Hours Days Weeks
6-OHDA lesion
start STN HFS
Fig. 7 Simplified scheme of the multiple experimental parameters studied at different time points (acute, subchronic and chronic) during subthalamic nucleus (STN) high-frequency stimulation (HFS) in hemi-parkinsonian rats. Data are issued from this article and other articles of our laboratory (indicated with numbers) using similar deep brain stimulation (DBS) conditions. All parameters refer to the side ipsilateral to 6-hydroxydopamine (6-OHDA) lesion, except motor behavior that refers to the contralateral side (bilateral effects, when present, are not mentioned). Lines represent the outcome of 6-OHDA lesion and STN HFS for each parameter (middle level= physiological conditions, also indicated by the white dotted line; up= increase; down = decrease or impairment). Neuronal activity markers in the SNr/EP (green) are intended as the mRNA levels of activity- or neurotransmitter-related markers (cytochrome oxidase subunit I and GAD67, respectively). Corticostriatal synaptic transmission (black) regroups spontaneous
excitatory post-synaptic currents as well as the sensitivity of AMPA and N-methyl-D-aspartic acid receptors studied by patch-clamp electro-physiology in brain slices. Corticostriatal long-term depression (LTD)/ long-term potentiation (LTP) (purple) indicate the possibility (or not) to induce these two forms of synaptic plasticity. Glutamate and GABA levels in the striatum (red) and SNr (pink) refer to the tissue levels of these neurotransmitters, as measured by in vivo proton magnetic resonance spectroscopy. Motor behavior (blue) was measured by the cylinder and the staircase test. Adult neurogenesis (orange) refers specifically to newborn cell survival in the rostral migratory stream and olfactory bulb. Numbers indicate the articles in which these effects are described:1Salinet al. (2002); 2Bacci et al. (2004);3Oueslatiet al. (2007); 4Lacombe et al. (2009) 5Gubellini et al. (2006); 6(Present paper);7Melon et al. (2015);8Forniet al. (2012);9Khaindravaet al. (2011).
cannabinoid degradation (Kreitzer and Malenka 2007), while
LTP by dopaminergic cells grafts (Rylander et al. 2013) or
L-DOPA administration (Picconi et al. 2003). Thus, this is
the
first article reporting that a single antiparkinsonian
treatment, i.e. chronic STN HFS, is capable of restoring both
corticostriatal LTD and LTP in a rodent model of PD.
Corticostriatal synaptic plasticity is supposed to underlie
motor skills, cognitive performances and reward mechanisms
and, as mentioned above, the loss of striatal LTD and LTP in
PD animal models is paralleled by behavioral de
ficits
relevant to this pathology (Calabresi et al. 1996, 2007;
Wickens et al. 2003; Mahon et al. 2004; Pisani et al. 2005).
Here, we report that the unilateral 6-OHDA lesion indeed
impaired performance in the staircase test, but, surprisingly,
it affected both forepaws. A possible cause could be that
performance in the staircase test also depends on a correct
postural balance for which both paws are essential.
More-over, a ipsilateral deficit, although milder, has already been
reported in hemiparkinsonian rats with a modified version of
the original staircase test (Klein et al. 2007). Interestingly, in
our rats, the deficit was progressively relieved by STN HFS,
with the ipsilateral side recovering more quickly than the
contralateral one, thus revealing that an asymmetry bias was
present anyhow. Such bilateral effects are consistent with the
interhemispheric functional interactions between the two
STN (Brown et al. 2001; Levy et al. 2002; Williams et al.
2003; Walker et al. 2011; Darvas and Hebb 2014) and might
be due, at least in part, to enhanced motivation for food and/
or increased perseverative behavior, which is a known
outcome of STN HFS (Baunez et al. 2007; Baunez and
Gubellini 2010).
Conclusion
The present work shows that chronic continuous STN HFS
applied for 5 weeks in hemiparkinsonian rats is able to
normalize neurotransmitter levels in both the input and
output structures of the BG and to rescue synaptic plasticity,
thus contributing to the restoration of BG function, which, in
turn, might underlie the improvement of motor skills.
Moreover, the time-course analysis of acute, subchronic
and chronic STN HFS effects integrating results from this
study and previous ones from our laboratory (Fig. 7) reveals
that the long-term benefits of this treatment may rely on the
maintenance of acute effects as well as on delayed actions
within and outside the BG network. In addition, STN HFS
also has proper effects that are independent from the
dopaminergic lesion and are maintained in the long term,
as exemplified here by the decrease in Tau levels, whose
functional consequences need to be elucidated. Finally, our
data further support the need for using experimental
proto-cols relevant to clinical conditions for studying the impact
and action mechanisms of STN HFS for PD therapy.
Acknowledgments and conflict of interest
disclosure
This work was supported by St. Jude Medical, Inc., the Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Universite, Universite d’Auvergne and Fondation de l’Avenir pour la Recherche Medicale Appliquee (grant ET3-702). 1H-MRS experiments were performed on the NMR platform ‘AgroReso-nance’ of INRA-Centre Auvergne-Rh^one-Alpes.
All experiments were conducted in compliance with the ARRIVE guidelines.
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