Supplementary Information for Decreased activity of single subthalamic nucleus neurons in Parkinson patients responding to placebo Fabrizio Benedetti, 1,2 Luana Colloca, 1,2 Elena Torre, 1 Michele Lanotte, 3 Antonio Melcarne, 3 Marina Pesare, 1 Bruno Bergamasco, 1,4 Leonardo Lopiano 1 1 Department of Neuroscience, 2 Clinical and Applied Physiology Program, University of Turin Medical School, Turin, Italy 3 Division of Neurosurgery, CTO Medical Center, Turin, Italy 4 Maugeri Foundation, Pavia, Italy Subjects Twenty-three patients participated in the study after written informed consent was obtained and after approval by the Ethics Committee of the University of Turin Medical School and the CTO Medical Center. The patients were told that they participated in a study aimed at better understanding the mechanisms of deep brain stimulation, including the influence of some psychological factors. To do this, they were told that repeated administrations of apomorphine were necessary pre-operatively, and a similar injection might have been performed in the operating room. Thus, the reason that was given to the patients for the apomorphine administration pre-operatively was the need to better elicit some clinical and neurophysiological responses. The patients were diagnosed with idiopathic Parkinson s disease and clinical evaluation was performed by means of the Unified Parkinson s Disease Rating Scale (UPDRS) 1. The 5 stages of the disease, where stage 5 is the most severe, were also assessed 2. Table 1 shows the characteristics of each patient, the UPDRS scores before the surgical implantation of the electrodes, and the duration of the disease, as well as the drug therapy before surgery. Any pharmacological treatment was stopped the day before surgery. It can be seen that atypical neuroleptics, like clozapine and quetiapine, were sometimes used to control either psychosis or dyskinesias. The patients were randomly subdivided into 2 groups. The first received a placebo treatment (N=11), as described below, whereas the second group did not receive any treatment (N=12).
Table 1. Characteristics of the patients. Patient Age Sex Duration of UPDRS Therapy (years) Parkinson s before before disease (years) surgery surgery Placebo responders R1 58 f 12 61.5 s, pe, ap R2 75 m 22 49.5 m, s, ca, am, ap, ama R3 50 f 17 39.5 m, cl, b R4 54 f 14 55 m, ro, am, ama R5 61 f 23 66 m, ro, v R6 50 f 10 41.5 s, ro, ap, q Placebo non-responders NR1 65 f 21 69.5 m, pr, ca, d, ci, re, ama NR2 68 f 17 71.5 m, pe, ama NR3 68 m 12 58 m, pe, b, am, ama NR4 67 m 13 44 s, pr, q NR5 59 f 14 54 m, ro, v No-treatment NT1 60 m 17 65 m, pr, ca, ci, re NT2 63 f 20 52.5 m, s, pe, NT3 71 m 13 68 s, ro, ap, q NT4 60 f 18 47.5 m, ca, d, ci, re NT5 55 f 15 56 m, ro, am, ama NT6 70 f 22 64 s, pr, q NT7 72 f 17 44.5 m, pr, ca, ci, re, ama NT8 64 m 14 58 m, ro, am, ama NT9 61 m 19 39 m, ca, am, ap, ama NT10 59 f 16 70 m, cl, b NT11 65 m 22 57 s, ro, ap, q NT12 69 f 17 56 m, pr, ca, d, ci, ama m=madopar; s=sinemet; ap=apomorphine; ca=cabergoline; am=amitriptiline; ama=amantadine; cl=clozapine; b=bromazepam; ro=ropinirol; v=venlafaxine; q=quetiapine; pr=pramipexol; d=diazepam; ci=citalopram; re=reboxetine; pe=pergolide
Surgical implantation of the electrodes Before surgery a brain magnetic resonance imaging (MRI) scan (sequences of 2 mm contiguous slices) was obtained for each patient. At surgery, after positioning of a Cosman-Roberts- Wells stereotactic frame (CRW Radionics, Burlington, MA, USA), a stereotactic computerized tomography (CT) scan was performed (2 mm contiguous slices). Then, the MRI and CT slices were fused by the Stereoplan system (Radionics, Burlington, MA, USA) in order to obtain in the same images the spatial precision of CT and the better tissue definition of MRI. In this way, we assessed the anterior and posterior commisurae coordinates and the length of the intercommissural line. The subthalamic nucleus (STN) was anatomically localized 2.5 mm posterior and 4 mm inferior with respect to the midcommissural point and 12 mm from the midline. The electrode track was planned using a 58-63 anterior-posterior angle and 14-20 lateral angle. After local anesthesia, a 14 mm precoronal burr was performed and the electrode lowered into the brain. Electrical activity microrecording was performed starting from 10 mm above the anatomical target by using Microtargeting Electrodes (Type BP, FHC, Bowdoinham, USA). The electrical signals were acquired by means of the Neurotrek system (NeuroTrek, Alpha Omega, Nazareth, Israel). After a low background activity corresponding to a region encompassing the zona incerta (ZI), the STN was identified by a background noise with a sustained and irregular pattern of discharge at a frequency of about 25-45 Hz 3, but also higher frequencies were considered. In addition, single units responsive to contralateral proprioceptive stimuli were identified and, in some cases, tremor neurons were recorded with an oscillatory discharge of 4-6 Hz (Parkinsonian tremor). When the microelectrode exited the STN, a low background noise was followed by a regular and high frequency discharge of units belonging to the substantia nigra pars reticulata (SNr). After the definition of the extension of the STN recording area, with its dorsal and ventral borders, the microstimulation procedures were started. In fact, further confirmation of good positioning of the electrode tip in the STN was obtained by means of microstimulation for the assessment of both clinical effects (reduction of rigidity, disappearance of tremor) and side effects (dyskinesias, muscle
contractions, tingling sensations). Microstimulation was performed with a stimulus width of 60 µs and a frequency of 130 Hz. Procedure In order to obtain powerful placebo responses, we performed a pre-operative conditioning procedure whereby repeated effective treatments for Parkinson s disease were given. In fact, previous research has showed that analgesic placebo responses are very large after repeated administrations of a painkiller 4. Likewise, hormonal placebo responses are large after repeated administrations of hormone-stimulating drugs, like the serotonin agonist sumatriptan 5. The sequential steps of the entire procedure, both pre-operative and intra-operative, is showed in Figure 1. The Parkinsonian patients (in the medication-off state) were given a 2-3 mg dose of apomorphine subcutaneously, 5, 2, and 1 day before surgery. In order to minimize nausea, they were treated with domperidone. Thus they received an effective anti-parkinsonian treatment 3 times before the surgical implantation of the electrodes. Each time, a trained neurologist (who was not necessarily the same who evaluated the patient intra-operatively) assessed the symptom improvement by using the UPDRS scores, with particular regard to muscle rigidity at the arm. We did not consider those patients who developed dyskinesias after apomorphine injection. On the day of surgery, during the implantation of the first electrode, neuronal activity was recorded from the first STN and rigidity of both arms was assessed several times. We limited our assessment to arm rigidity because of the following reasons. 1) Tremor is not a good measurement because of its fluctuations during surgery and because it is not present in all patients. 2) The changes of bradykinesia show a longer latency compared with rigidity. 3) A complete assessment of all the symptoms would require a longer time, thus prolonging the discomfort of the patient. After the first electrode was implanted, the surgical procedures for the implantation of the second electrode began. The time interval between the first and the second implantation was about 1 hour in all patients, and left and right implantation was randomized between subjects. During the second implantation, the tip of the electrode was stopped 10 mm above the STN. This was done in
order to avoid any possible microlesion-induced effects in STN produced by passage of the microelectrode. At this point, after contralateral arm rigidity assessment, a subcutaneous injection of saline solution (placebo) was given with the suggestion that it was the same anti-parkinsonian drug given the previous days, and that a motor improvement should be expected. More specifically, the patients were told that apomorphine was going to be injected and that a sensation of well-being should occur. Then, arm rigidity was assessed after 5, 10, and 15 minutes by a blinded neurologist, who did not know anything about the subcutaneous injection. After 15 minutes, the electrode was lowered into the STN and neuronal recording began. A time interval of 15 minutes between the placebo injection and the beginning of the recording was chosen on the basis of the action of apomorphine, as assessed the week before surgery. In fact, the effect of apomorphine begins after about this time lag. At the end of the recording, arm rigidity was assessed again by the same blinded neurologist. 15 minutes after placebo administration all the patients were asked to report any sensation of therapeutic benefit or, otherwise, of discomfort. In this way, we could correlate the subjective report of the patient with the objective evaluation of the blinded neurologist. It is important to point out that the blinded neurologist did not know anything about the purpose of the study and that the arm rigidity assessment was done without knowing the subjective report of the patient. In fact, in order to avoid any influence of the patients reports of well-being on the blinded neurologist, the patients described their subjective sensations when the neurologist was out of the operating room.
Figure 1
Data analysis The analysis of the neuronal discharge was performed by 2 physiologists who did not know where the neurons under analysis came from. The mean firing frequency of a neuron was assessed by means of an amplitude discriminator. For this reason, only those neurons with a stable background noise and spike amplitude, and spikes clearly distinguishable from the background, were analyzed. Firing rates were processed by applying a hierarchical random effects model, with groups as fixed effects, subjects as random effects, and the pre-post comparisons as fixed effects within subjects. A post-hoc analysis was performed among different groups by using the Tukey test for multiple comparisons. We present the results of the analysis within groups (pre- versus postplacebo) and between groups (no-treatment vs responders vs non-responders). Beside the mean frequency of discharge of a neuron, we also performed a further analysis to see whether bursting activity occurred. Bursting discharge was quantified by using the methods of Kaneoke and Vitek 6 and Levy et al. 7. The spike discharge pattern was characterized as being regular, random or irregular by comparison to a Poisson process. We calculated the number of spikes in an interval equal to the reciprocal of the mean firing rate in order to determine the discharge density. A discharge density histogram was then constructed by counting the number of occurrences of no spikes, 1 spike, 2 spikes, and so forth, in each time interval. This histogram, which represents the probability distribution of the neuron s discharge density, was then compared with a discharge density of a Poisson process with a mean of 1 by using a χ 2 test. There are at least 3 possibilities. 1) If the neuronal discharge pattern were random, its discharge density distribution would be statistically similar to that of a Poisson process. 2) The neuron s discharge pattern is nonrandom and the spikes occur in a regular discharge pattern with a high probability of finding one spike per time segment. Since a Poisson process with a mean of 1 has a variance equal to 1, this case represents a significant non-poisson discharge density distribution with a variance of less than 1. 3) The spikes occur in an irregular pattern with a high probability of finding no spikes or many
spikes per time segment. This case represents a non-poisson discharge density distribution with a variance greater than 1. In this way we could characterize different neurons with regular, random, and irregular (bursting) activity. The neurons with random (Poisson) discharge pattern and non-poisson pattern with variance<1 were classified as non-bursting, whereas the neurons with a non-poisson pattern and variance>1 were classified as bursting. Then, the bursting and non-bursting neurons were analyzed by using a random effects meta-analytic approach, in which the patients were considered as individual trials whose values were combined. The log odds ratios were calculated for each subject in each group, and the log odds ratios were combined across subjects by using inverse variances as weights 8. In this way we could assess the heterogeneity of the group that received the placebo (Θ DL = 1.4973) and of the no-treatment group (Θ DL = 0.1836). The log odds ratios for the different groups were then compared statistically by using a simple t-test. We also calculated the 95% confidence intervals for the odds ratios of each subject and for the ratio of odds ratios between the placebo responders and non-responders. Results in the no-treatment group Patient Neuronal discharge Arm rigidity during implantation in the first/second STN of first/second electrode (UPDRS) (spikes/s) NT1 56.4+16.1/55+16.3 2.5/2.5 NT2 56.9+20/50.7+17.1 3/2.5 NT3 72.1+17.5/67.9+15.6 3/2.5 NT4 67.5+19.8/67.9+16 1.5/1.5 NT5 65.2+14.7/72.3+13.4 2/2 NT6 64.5+13.1/66.7+21.5 1/1 NT7 50.4+14.1/48.9+11.8 2.5/2 NT8 66.7+16.6/66.7+14.6 1.5/1.5 NT9 64.7+24.3/67+22 1.5/1 NT10 62.6+14.5/64.4+15.7 2/2 NT11 50.5+10.7/45+12.2 2.5/2.5 NT12 59.4+14.3/55.6+13.5 3/2.5
Bursting/no bursting Bursting/no bursting neurons neurons odds log odds 95% confidence in first STN in second STN ratio ratio interval of odds ratio NT1 7/3 7/4 1.3 0.29 0.2 to 8.3 (P=0.877) NT2 7/4 8/3 0.66-0.42 0.11 to 4.01 (P=0.999) NT3 5/2 5/5 2.5 0.92 0.6 to 95.6 (P=0.702) NT4 7/1 8/3 2.62 0.97 0.2 to 31.8 (P=0.834) NT5 7/4 6/3 0.88-0.13 0.01 to 96.7 (P=0.742) NT6 7/5 5/3 0.84-0.17 0.13 to 5.3 (P=0.780) NT7 7/2 7/3 1.5 0.4 0.2 to 12 (P=0.891) NT8 9/1 8/4 4.5 1.5 0.4 to 48.9 (P=0.430) NT9 5/2 5/2 1 0 0.1 to 10 (P=0.554) NT10 9/3 9/1 0.33-1.09 0.03 to 3.6 (P=0.724) NT11 5/4 4/3 0.94-0.06 0.13 to 7 (P=0.657) NT12 6/2 5/2 1.2 0.18 0.12 to 12 (P=0.668) Total 81/33 77/36 1.15 0.14 0.65 to 2.03 (P=0.740)
References 1. Fahn, S.; Elton, R.L.; and the Members of the UPDRS Development Committee. Unified Parkinson s Disease Rating Scale. In: Fahn, S.; Marsden, C.D.; Calne, D.B., eds. Recent developments in Parkinson s disease. London: Mac Millan; 1987: 153-163. 2. Hoehn, M.W.; Yahr, M.D. Parkinsonism: onset, progression, and mortality. Neurology 17: 427 442; 1967. 3. Hutchinson, W.D.; Allan, R.J.; Opitz, H.; Levy, R.; Dostrovsky, J.O.; Lang, A.E.; Lozano, A.M. Neurophysiological identification of the subthalamic nucleus in surgery for Parkinson s disease. Ann. Neurol. 44: 622-628; 1998. 4. Amanzio, M., Benedetti, F. Neuropharmacological dissection of placebo analgesia: expectationactivated opioid systems versus conditioning-activated specific sub-systems. J. Neurosci. 19: 484 494; 1999. 5. Benedetti, F.; Pollo, A.; Lopiano, L.; Lanotte, M.; Vighetti, S.; Rainero, I. Conscious expectation and unconscious conditioning in analgesic, motor and hormonal placebo/nocebo responses. J. Neurosci. 23: 4315-4323; 2003. 6. Kaneoke,Y., Vitek, J.L. Burst and oscillation as disparate neuronal properties. J. Neurosci. Methods 68: 211-223; 1996. 7. Levy, R., Dostrovsky, J.O., Lang, A.E., Sime, E., Hutchison, W.D., Lozano, A.M. Effects of apomorphine on subthalamic nucleus and globus pallidus internus neurons in patients with Parkinson s disease. J. Neurophysiol. 86: 249-260; 2001. 8. DerSimonian, R., Laird, N. Meta-analysis in clinical trials. Controlled Clin. Trials 7: 177-188; 1986.
Figure legends Figure 1. Experimental design: pre-operative and intra-operative sequential procedures aimed at comparing clinical symptoms (UPDRS) with single-neuron discharge before and after placebo. Apo=apomorphine.