Although the clinical use of deep brain stimulation (DBS) is increasing, its basic mechanisms of action are still poorly understood. Platinum/iridium electrodes were inserted into the subthalamic nucleus of rats with unilateral 6-OHDA-induced lesions of the medial forebrain bundle. Six behavioral parameters were compared with respect to their potential to detect DBS effects. Locomotor function was quantified by (i) apomorphine-induced rotation, (ii) initiation time, (iii) the number of adjusting steps in the stepping test, and (iv) the total migration distance in the open field test. Sensorimotor neglect and anxiety were quantified by (v) the retrieval bias in the corridor test and (vi) the ratio of migration distance in the center versus in the periphery in the open field test, respectively. In our setup, unipolar stimulation was found to be more efficient than bipolar stimulation for achieving beneficial long-term DBS effects. Performance in the apomorphine-induced rotation test showed no improvement after 6 weeks. DBS reduced the initiation time of the contralateral paw in the stepping test after 3 weeks of DBS followed by 3 weeks without DBS. Similarly, sensorimotor neglect was improved. The latter two parameters were found to be most appropriate for judging therapeutic DBS effects.
Electrical stimulation of the brain is an emerging area for the treatment of a growing number of neurological and psychiatric diseases. Deep brain stimulation (DBS) is well established for the treatment of movement disorders, such as Parkinson’s disease (PD) [
The STN is one of the most important target regions for high-frequency (approx. 130 Hz) DBS in patients, especially in patients in the advanced stages of PD who are refractory to conventional therapy [
Maesawa et al. [
Clearly, more information is needed to explore the full therapeutic potential of DBS. For example, optimum target regions are not always known, and the basic mechanisms by which DBS acts are still poorly understood [
In pioneering work on experimental DBS, stainless steel electrodes have been used to optimize the electrode position in the brain [
Here, we combined a revised version of our miniaturized constant-current-pulse generator [
Male Wistar Han rats (240 g–260 g; Crl:WI(Han) Rattus norvegicus: RRID:RGD_2308816) were obtained from Charles River Laboratory (Sulzfeld, Germany) and housed under temperature-controlled conditions in a 12-h light-dark cycle with conventional rodent chow and water provided ad libitum. The study was carried out in accordance with the European Community Council directive 86/609/EEC for the care of laboratory animals and was approved by the local animal care committee (LALLF M-V/TSEM/7221.3-1.2-019/10).
Two types of microelectrodes were custom-made from round Pt/Ir alloy (Pt90/Ir10) wires, which were insulated with polyesterimide but left bare at the tips (Figure
Photographs of tips (left), distal connections (center), and schematic drawings (right) of (a) the unipolar (200
The surgical procedures were performed using a stereotactic frame (Stoelting, Wood Dale, IL, USA). Rats were anesthetized by intraperitoneal injection of ketamine hydrochloride (10 mg per 100 g body weight, Ketanest S®, Pfizer, Karlsruhe, Germany) and xylazine (0.5 mg per 100 g body weight, Rompun®, Pfizer). During surgery, their eyes were protected from dehydration by Vidisic® (Bausch and Lomb, Berlin, Germany).
The skull was opened using a dental rose-head bur (Kaniedenta, Herford, Germany). To induce hemiparkinsonism, rats were lesioned with a unilateral injection of 6-OHDA into the right medial forebrain bundle. Twenty-four
Approximately 3 weeks after lesion induction, the electrodes were implanted with their stimulating tips localized in the STN. The tip coordinates, relative to bregma, were AP: −3.5 mm, ML: 2.4 mm, and DV: −7.6 mm ([
Schematic views of the unipolar DBS rat model. (a) Rat with stimulator in backpack; (b) sagittal view illustrating the locations of the implanted unipolar DBS electrode; (c) image of an explanted DBS mounting; (d) backpack vest with Velcro hooks; (e) stimulator in PMMA housing with pocket and current-pulse battery. 1: unipolar Pt/Ir electrode; 2: electrode cables; 3: gold-wire counter electrode; 4: biocompatible dental acrylic embedding all components; 5: anchor screw to fix the acrylic mounting to the skull; 6: electrode connector; 7: current-pulse battery connector.
Following electrode implantation, the cables of the stimulating and the counter electrode contacts were implanted subcutaneously with a central dorsal outlet port (Figure
(a) Details of the outlet port for the subcutaneous cables centered at the dorsum. (b) Rat with electrode connector one week after surgery. 1: suture clips; 2: dorsal cable outlet port; 3: crimped plug connector.
One week after surgery, a plug connector (M52-040023V0545, Harwin Plc, Hampshire, UK) was crimped to the electrode cables (Figure
The setup allowed for the completely free movement of the animals over long periods of time. The stimulator plate was protected from mechanical strain and moisture by a custom-made polymethyl-methacrylate box and was connected to the external current-pulse battery. At the start of the stimulation, the electrode connector was plugged into the stimulator (Figure
The jacket and cables were checked daily to ensure the long-term effectiveness of the device. Cables that were torn off by the animal in exceptional cases were replaced immediately. The jackets had to be replaced every week because of wear. The stimulator signal was checked with an oscilloscope at the same time as the jackets were replaced. There has never been a problem with the batteries or the stimulator hardware.
The stimulator provided rectangular monophasic current pulses. Different treatment groups were stimulated for 3 days, 3 weeks, or 6 weeks. In all experiments, the stimulators were adjusted to a pulse width of 60
The effects of lesion- and DBS-induced changes in the animals’ behavior were quantified using the drug-induced apomorphine-stimulated rotation test and three non-drug-induced tests (the stepping, corridor, and open field tests). Experiments were conducted at different times: (i) prior to lesion induction; (ii) 12–14 days after lesion or sham lesion induction; (iii) after 3 days of DBS or 3 days with the stimulator off (sham stimulation); (iv) after 3 weeks of DBS or 3 weeks with the stimulator off (sham stimulation); (v) ≥3 days after the cessation of DBS subsequent to 3 weeks of DBS; (vi) after 6 weeks of DBS; and (vii) 3 weeks after the cessation of DBS subsequent to 3 weeks of DBS. For details see Table
Experimental design. The number of rats refers to the group sizes at the time of the apomorphine-induced rotation tests.
Group name | 6-OHDA lesion | Electrode | DBS duration | Number of animals |
---|---|---|---|---|
Naive_3 d/3 w | − | — | — | 10 |
Naive_sham_3 w | − | Bipolar | — | 9 |
6-OHDA_sham_3 d/3 w | + | Bipolar | — | 7 |
Sham_bi_3 d | Sham | Bipolar | 3 days | 9 |
Sham_bi_3 w | Sham | Bipolar | 3 weeks | 10 |
Sham_uni_6 w | Sham | Unipolar | 6 weeks | 7 |
6-OHDA_bi_3 d | + | Bipolar | 3 days | 7 |
6-OHDA_bi_3 w | + | Bipolar | 3 weeks | 5 |
6-OHDA_uni_3 d | + | Unipolar | 3 days | 13 |
6-OHDA_uni_3 w/3 w + 3 d off | + | Unipolar | 3 weeks | 11 |
6-OHDA_uni_6 w | + | Unipolar | 6 weeks | 7 |
6-OHDA_uni_3 w + 3 w off | + | Unipolar | 3 weeks | 8 |
Schedules of the test procedures. RT, ST, OFT, and CT refer to the rotation, stepping, open field, and corridor tests, respectively.
For assessing drug-induced locomotor function, apomorphine (0.25 mg/kg body weight dissolved in saline) was injected subcutaneously. Rotation was quantified in a custom-made “rodent-rotometer” modified according to Ungerstedt and Arbuthnott [
The stepping test, which assesses forelimb akinesia, was essentially performed as described by Olsson et al. [
To assess sensorimotor neglect, we used the corridor test [
Spontaneous mobility and anxiety were evaluated by placing the rats in a square open field arena (46 cm × 45 cm) inside an isolation box. The animals were kept in the dark in the examination room 1 h before the start of the test. The open field was illuminated by a white photo bulb providing 200 to 250 Lux. During testing, rats were monitored by a video camera. The open field was divided into a center area (22 cm × 22 cm) and a peripheral zone using the tracking software Ethovision XT (Noldus Information Technology, Leesburg, VA, USA; RRID:SCR_000441). This allowed for the automatic recording of the rat’s movement in the two zones. Each rat was tested once for 10 minutes. After each session, the open field was cleaned to prevent odor from influencing the next animal’s behavior. The total migration distances were taken as a measure of spontaneous mobility and the ratio of the migration distance within the center area to the total distance moved was interpreted as a measure of anxiety.
Data analysis was conducted with the SAS software package, Version 9.4 for Windows (Copyright, SAS Institute Inc., Cary, NC, USA, RRID:SCR_008567). Descriptive statistics and tests for normality were calculated with the UNIVARIATE procedure using Base SAS software. Data that could be considered as approximately normal was analyzed by one-way repeated measurement ANOVA with the MIXED procedure of the SAS/STAT software module. The models for the investigated treatments contained the fixed factor “time” with different levels (prelesion, postlesion, 3 d, 3 w, 3 w + 3 d, and 6 w) for each treatment. Repeated measures on the same animal were taken into account in the REPEATED statement of the MIXED procedure using time as the repeated effect, the SUBJECT = animal option to define the blocks of the residual covariance matrix and the TYPE = CS option to define their covariance structure. Least-square means (LSM) and their standard errors (SE) were computed for each time level of each treatment and compared with the “postlesion”-LSM using the Dunnett-Hsu procedure (pairwise multiple comparisons with the control).
The investigated treatments for each time (prelesion, postlesion, 3 d, 3 w, 3 w + 3 d, and 6 w) were analyzed by one-way ANOVA with the MIXED procedure of the SAS/STAT software module. The models for the times contained the fixed factor treatment (see Table
The localization of electrode tips in the STN was evaluated by retrospective analyses of Nissl-stained cryosections of the STN of selected rats. It suggested a precise electrode placement in approximately 75% of the cases, analogous to the success rate of the lesion surgery (see below). A comprehensive histological evaluation is currently underway.
The success of lesion induction was evaluated based on the apomorphine-induced rotation test results 12–14 days after surgery. The success rate (rpm ≥ 2) was approximately 75%, and the mortality rate was less than 10%. In the apomorphine-induced rotation test, reduced rotation was detected with DBS after 3 days and after 3 weeks. In these cases, unipolar stimulation was more effective than bipolar stimulation (Figure
Short-term and long-term effects of DBS with uni- and bipolar electrodes on the apomorphine-induced rotation behavior of hemiparkinsonian rats. (a) and (b) refer to test groups and controls, respectively. Different column patterns indicate the different times of behavioral testing; black: 12–14 days after 6-OHDA or sham lesion; gray and hatched: after the durations of DBS or sham stimulation indicated in the group labels. For experimental details see Table
Spontaneous locomotor activity was assessed based on the total migration distance in the open field test. 6-OHDA lesions reduced the total migration distance, whereas naïve rats showed a marginal increase in total distance with each trial, which can be explained by habituation to the open field with repeated exposure. In contrast, DBS reduced the total migration distance, in most of the groups. The total migration distance increased at 3 days after the cessation of DBS subsequent to 3 weeks of DBS with unipolar electrodes (Figure
To assess the effects of the lesion-induced akinesia, the parameters “initiation time of paw movement” and “number of adjusting steps” were recorded in the stepping test. In rats receiving bipolar DBS, a significant reduction in the initiation time of contralateral forepaw stepping was observed after 3 days but not after 3 weeks of DBS (Figure
Short-term and long-term effects of DBS with uni- and bipolar electrodes on akinesia as measured by the initiation time of the first adjusting step of the contralateral forepaw in the stepping test. (a) and (b) refer to test groups and controls, respectively. The dashed line at 1 s allows for an easier comparison with Figure
Short-term and long-term effects of DBS with uni- and bipolar electrodes on the akinesia of hemiparkinsonian rats as measured by the initiation time of the first adjusting step of the ipsilateral forepaw in the stepping test. (a) and (b) refer to test groups and controls, respectively. The dashed line at 1 s allows for an easier comparison with Figure
Unexpectedly, we observed an increase in the initiation time of ipsilateral forepaw stepping after 6-OHDA lesioning in one group and no beneficial effect of DBS in any of the groups. Moreover, we found an aggravating effect of DBS effect after 3 weeks that vanished 3 days after the cessation of DBS (Figure
Impaired contralateral paw movement (contralateral bias) was determined based on the number of contralateral versus ipsilateral adjusting steps of the forepaws. A significant difference in the contralateral bias during forced sidestepping was found in only one case. The contralateral bias in the forehand direction worsened after 6 weeks of unipolar DBS (Figure
Summary of test results. The improvement and worsening of lesion-induced parkinsonian symptoms by DBS are marked by (+) and (−), respectively. Borderline changes
Group name | Rotation test | Stepping test |
Stepping test contralateral bias | Corridor test | Open-field test | ||
---|---|---|---|---|---|---|---|
Contralateral paw | Forehand | Backhand | Total distance | Distance |
|||
6-OHDA_bi_3 d | b+ | + | 0 | 0 | 0 | — | Up |
6-OHDA_bi_3 w | b+ | b+ | 0 | 0 | 0 | — | 0 |
6-OHDA_uni_ 3 d | + | b− | 0 | 0 | 0 | — | 0 |
6-OHDA_uni_ 3 w | + | 0 | 0 | 0 | + | 0 | Up |
6-OHDA_uni_ 3 w + 3 d off | 0 | 0 | 0 | 0 | n.d. | + | Up |
6-OHDA_uni_ 6 w | 0 | b+ | — | 0 | 0 | b− | Up |
6-OHDA_uni_ 3 w + 3 w off | 0 | + | 0 | 0 | b+ | 0 | 0 |
Short-term and long-term effects of DBS with uni- and bipolar electrodes on akinesia as measured by forced sidestepping of the forepaws in the forehand direction in the stepping tests. (a) and (b) refer to test groups and controls, respectively. Different column patterns indicate different times of behavioral testing; white: before 6-OHDA or sham lesion; black: 12–14 days after 6-OHDA or sham lesion; gray and hatched: after the durations of DBS or sham stimulation indicated in the group labels. For experimental details see Table
Short-term and long-term effects of DBS with uni- and bipolar electrodes on the akinesia of hemiparkinsonian rats as measured by forced sidestepping of the forepaws in the backhand direction in the stepping tests. (a) and (b) refer to test groups and controls, respectively. Different column patterns indicate different times of behavioral testing; white: before 6-OHDA or sham lesion; black: 12–14 days after 6-OHDA or sham lesion; gray and hatched: after the durations of DBS or sham stimulation indicated in the group labels. For experimental details see Table
In the corridor test, DBS reduced the amount of sensorimotor neglect when applied by unipolar electrodes for 3 weeks (Figure
Short-term and long-term effects of DBS with uni- and bipolar electrodes on the sensorimotor neglect of hemiparkinsonian rats as measured by the corridor test. (a) and (b) refer to test groups and controls, respectively. Different column patterns indicate different times of behavioral testing; white: before 6-OHDA or sham lesion; black: 12–14 days after 6-OHDA or sham lesion; gray and hatched: after the durations of DBS or sham stimulation indicated in the group labels. For experimental details see Table
Short-term and long-term effects of DBS with uni- and bipolar electrodes on the locomotor activity of hemiparkinsonian rats as measured by the total migration distance in the open field. (a) and (b) refer to test groups and controls, respectively. Different column patterns indicate different times of behavioral testing; white: before 6-OHDA or sham lesion; black: 12–14 days after 6-OHDA or sham lesion; gray and hatched: after the durations of DBS or sham stimulation indicated in the group labels. For experimental details see Table
The open field test provided information on locomotor activity and anxiety-like behavior. Although the total distance moved (Figure
Short-term and long-term effects of DBS with uni- and bipolar electrodes on the anxiety-like behavior (b) of hemiparkinsonian rats as measured by the ratio: migration distance in the center/total migration in the open field. (a) and (b) refer to test groups and controls, respectively. Different column patterns indicate different times of behavioral testing; white: before 6-OHDA or sham lesion; black: 12–14 days after 6-OHDA or sham lesion; gray and hatched: after the durations of DBS or sham stimulation indicated in the group labels. For experimental details see Table
Table
The 6-OHDA-induced hemiparkinsonian rat model has been established for the study of therapeutic approaches for treating PD [
Frequencies from 90 to 130 Hz are generally accepted as optimal to elicit the therapeutic effects of DBS in patients [
In PD patients, unipolar stimulation is the preferred mode of DBS. So et al. [
To test the success of lesioning and test initial locomotor function, the classical drug-induced rotation assay was used. Pathological rotation is measured in response to the administration of either the dopamine (DA) receptor agonist apomorphine or the DA-releasing drug amphetamine [
In partially lesioned animals, Hefti et al. [
Interestingly, we found a reduction in apomorphine-induced rotation if DBS was applied for 3 days or 3 weeks in either the bipolar or unipolar modes, with the latter being more effective (Figure
We believe that apomorphine-induced rotation is not an appropriate parameter for testing the beneficial effects of STN-DBS. Limitations of the apomorphine-induced rotation test have been previously demonstrated. Metz and Whishaw [
Additionally, our results showing that a reduction of the initiation time of the contralateral forepaw was induced by DBS were not consistent with the results of the rotation test with unipolar stimulation. The shortest initiation times of the contralateral paw were observed 3 weeks after the cessation of DBS subsequent to 3 weeks of continuous DBS (Figure
Locomotor activity changes detected in the open field test should be interpreted with caution as they may be influenced by various modifying factors, including habituation, the need for exploration, and anxiety effects. Indeed, we observed a habituation to the open field in naïve rats in both the total migration distance and in the anxiety parameter distance ratio. Lesions induced a reduction in locomotor activity, as measured by total migration distance. DBS induced a further decrease, even in sham-lesioned rats (Figure
In PD patients, anxiety may result from not only the impairment of motor function but also dysfunction of the STN. Experiments with bilaterally STN-lesioned rats in the elevated plus maze test also suggest such a connection [
The corridor test was originally established to detect lateralized sensorimotor integration [
These findings raise questions about whether different mechanisms are responsible for the observed effects of acute and chronic DBS, as well as about the persistent effects on locomotor and sensorimotor functions. One possible reason for these differences may be the development of an insensitivity toward DBS, reflecting changes in the basal ganglia network [
Here, we propose that tests of spontaneous locomotion, such as the stepping test, are more relevant for detecting the beneficial effects of DBS and provide different information than the apomorphine-induced rotation test. However, this conclusion does not necessarily apply to the amphetamine-induced rotation test because of the different mechanisms of these two rotation tests (see Appendix).
Our results suggest that persistent DBS effects in 6-OHDA-lesioned neuronal networks may be the result of the protection or regeneration of part of the physiological function of these networks in relation to locomotor activity in the absence of dopaminergic neurons. Alternatively, persistent effects of DBS could arise from DBS-induced effects that mimic a permanent lesion of the STN, for example, by space-consuming effects of the developing adventitia. Such mechanisms may explain our findings of persistent DBS effects on the initiation time of the contralateral forepaw in the stepping test (Figure
The different brain states and resulting behavioral effects are considered in Appendix. Figure
Simplified scheme of the lesioned hemisphere describing the effects of the lesion (red), DBS (blue), and apomorphine administration (light gray) on neurotransmitter release and on the activity (colored arrows) of different brain areas. Induced alterations in receptor numbers or sensitivities are not depicted; for explanation see Appendix. The brain areas are given in the boxes designated by SNc, GP, STN, and EP/SNr. The neurotransmitters glutamate (Glu), DA, and GABA are designated by triangular boxes pointing toward the affected brain areas that may be either excited (direct line input) or downregulated (input with circle). D1 and D2 stand for the dopaminergic receptors in the striatum, which are excited or inhibited by DA or apomorphine, respectively. Rectangular text balloons mark the input sites of lesioning, DBS, and apomorphine. The colored “+” and “−“ signs in the neurotransmitter triangles designate the effects of lesioning (red) and DBS (blue) on transmitter release. Color-coding was not attempted for the effects of apomorphine on transmitter release.
Based on previous studies, apparently contradictory results have been obtained at both the molecular and receptor levels. The DBS-related decreases in the levels of extracellular DA and its metabolites in the dorsal part of the striatum described by Walker et al. [
At the level of the receptors, missing neurotransmitter inputs are believed to induce a compensatory upregulation of receptor numbers or sensitivity. According to this view, a lesion-induced reduction in glutamate release to the cortex and the striatum resulting from alterations in activity along the striatum-D1 receptor-EP/SNr-thalamus pathway will result in an upregulation in glutamate receptors in the cortex and striatum. However, DBS after lesioning was found to reverse the increased striatal glutamate receptor numbers [
Most biochemical studies have been conducted under acute or subchronic (up to 7 days) STN-DBS. However, a deeper insight into the DBS-mechanisms and its long-term or persistent effects (≥6 weeks) require animal models that are suitable for combining biochemical, electrophysiological, optical microscopy, and other imaging methods, along with behavioral testing.
To our knowledge, we present the first behavioral investigation in freely moving rats with chronic instrumentation for up to 6 weeks, which allowed the animals to adapt to the instrumentation and allowed us to conduct comparative behavioral tests at different times under acute DBS conditions and after the cessation of DBS. In our setup, we found unipolar stimulation to be more efficient for achieving several beneficial long-term DBS effects. In our tests of behavioral changes, the stepping and corridor tests were the most appropriate for the evaluation of DBS-induced locomotor and sensorimotor improvements. When DBS was stopped after 3 weeks, some effects persisted for at least 3 more weeks, such as the reduction of initiation time of the contralateral paw in the stepping test and the slight reduction of the contralateral bias in the corridor test. In contrast, performance in the apomorphine-induced rotation test showed no improvement after 6 weeks. Our findings may indicate a regeneration of neuronal circuits in the absence of dopaminergic neurons. This would make apomorphine-induced rotation a suitable test to determine the long-term success of 6-OHDA lesioning but not a very informative test for determining the beneficial effects of DBS. In interpreting anxiety-like behaviors, researchers must consider habituation effects in relation to the durations between test repetitions in both sham and experimental animals.
The determination of very fast reaction times was difficult. To improve the statistical power of these tests, a larger sample size should be combined with video detection of reaction times. Our model can be considered a versatile platform that allows for the independent testing of separate elements, such as electrodes and counter-electrodes. Relatively simple modifications to our model will allow for the testing of unexplored target regions in other neurodegenerative disorders.
In addition, various electrical parameters can be tested, such as stimulation frequency and signal shape. To our knowledge, no systematic investigations have been conducted on whether the frequencies applied to humans are suitable for use with much smaller animals. We believe that this topic needs further investigation, taking into account allometric effects for organisms with various brain sizes. Our results may help in developing a reduced set of test parameters to facilitate this research.
Major problem remains to be elucidated about the mechanism by which DBS acts. Although 6-OHDA lesioning induces PD-like symptoms, the long-term DBS effects in our model may be a result of the emergence of new or a strengthening of existing neuronal circuits that compensate for the absence of dopamine in the brains of young rats. This outcome may suggest that the DBS-related locomotor and sensorimotor improvements, with no detectable improvements in the results of the rotation test, indicate DBS effects in the activation of neuronal substitute circuits. If this hypothesis is supported by future research, investigations of the effects of stimulation may be helpful in other areas, such as stroke research.
Figure
The depicted effects will, in principle, also apply when one of the successive treatments is omitted. Thus, the scheme allows for predicting multiple scenarios, such as the effects of DBS without a lesion or apomorphine administration without DBS. Nevertheless, the actual magnitudes of the combined effects on the activity of the various brain areas may vary significantly, leading to different individual responses. Short summaries of the different states illustrated by Figure
These considerations suggest a higher relevance of amphetamine-induced rotation tests for assessing therapeutic DBS effects, although apomorphine-induced rotation tests are useful for determining the success and degree of lesion induction.
The present address for Immo Weber is Department of Neurology, University Hospital Giessen and Marburg, Marburg, Germany.
The authors declare that they have no conflicts of interest.
The authors wish to thank their keeper Gerda Brüsch for taking care of the animals and assistance with the behavioral tests. The authors are grateful to Professor Reiner Benecke for his encouraging support and for providing the facilities for the experiments and to Professor Andreas Wree and Karl Nowak who helped establish the surgical procedures and chronic instrumentation setup. The authors are indebted to Dr. Robert Arndt (Rückmann & Arndt, Berlin, Germany) and the Steinbeis-Zentrum STZ 1050 (Rostock, Germany) for a fruitful cooperation in the development of the stimulator systems. The authors are deeply indebted to Dr. Eilhard Mix for his help in designing the animal experiments. He was a source of constant guidance for Kathrin Badstuebner throughout the whole study. The authors regret that he refrained from a coauthorship for personal reasons after his retirement. The German Research Foundation (DFG, Research Training Group 1505/1,2 “welisa”) financed a scholarship to Kathrin Badstuebner as well as small equipment and consumables for the experiments.