Ratio of Inhibited-to-Activated Pallidal Neurons Decreases Dramatically During Passive Limb Movement in the MPTP-Treated Monkey

T. Boraud, E. Bezard, B. Bioulac, and C. E. Gross

Basal Gang, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5543, Université Victor Segalen Bordeaux 2, 33076 Bordeaux Cedex, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Boraud, T., E. Bezard, B. Bioulac, and C. E. Gross. Ratio of Inhibited-to-Activated Pallidal Neurons Decreases Dramatically During Passive Limb Movement in the MPTP-Treated Monkey. J. Neurophysiol. 83: 1760-1763, 2000. Mink advanced the hypothesis in 1996 that the role of the basal ganglia (BG) is primarily one of focused selection; the encouragement of motor mechanisms inducing a desired movement and the inhibition of competing mechanisms. This would imply, in normal subjects, a ratio of inhibited-to-activated (I/A) movement-related globus pallidus pars internalis (GPi) neurons <1 and a drastic decrease of this ratio in the parkinsonian state. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication should therefore decrease the specificity of the response of this neuronal population. To test this working hypothesis we studied the activity of GPi neurons in response to passive limb movement in the normal and the parkinsonian monkey. Extracellular unit recordings monitored any correlation between passive limb movements and eventual modifications of the neuronal activity of the GPi in two calm, awake, and drug naive monkeys (Macaca fascicularis) before and after MPTP intoxication. In the normal animal, arm- and leg-related neurons were located in clusters in the medial part of the GPi. The I/A ratio was 0.22. Most GPi cells were linked to a single joint. In the MPTP-treated monkey, the number of movement-related neurons increased, the I/A ratio dropped significantly to 0.03, and most responding cells were linked to several joints. These data, which cannot be explained by the classic "box" model, endorse Mink's hypothesis.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The term basal ganglia (BG) comprises a certain number of nuclei in the forebrain, diencephalon, and midbrain thought to play a significant role in the control of posture and movement (Alexander and Crutcher 1990). It has been well documented that subjects with degenerative diseases of the BG such as Parkinson's disease (PD) can suffer from rigidity, abnormal posture, and slowness in movement, and eventually present involuntary movements induced by dopatherapy (Agid 1991). Several aspects of BG physiopathology can be explained by the "box" model of organization proposed in the late 1980s (Albin et al. 1989; Alexander and Crutcher 1990) but the brain mechanisms which actually underlie movement have not yet been elucidated. Recent advances in our knowledge of these structures have led Mink to postulate that the BG do not, in fact, instigate movement (Mink 1996). He suggests that when voluntary movement is generated the role of the BG is to regulate the competing motor mechanisms that come into action, inhibiting those that interfere with the desired movement and disinhibiting those contributing to it. In this way, the desired movement can proceed.

This hypothesis implies that the dopamine depletion observed in PD deprives the BG of the ability to disinhibit desired motor programs and completely inhibit competing motor programs; hence the appearance of major motor disorders, such as akinesia and rigidity. Mink has suggested that the validity of this theory could be tested by recording the extracellular unit activity of BG output neurons during movement in parkinsonian monkeys. This study has been designed to test the "Mink hypothesis" by comparing the influence of passive movement on neuronal activity in the globus pallidus pars internalis (GPi), the main BG output structure in primates, in both normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were carried out on two cynomolgus monkeys (Macaca fascicularis) weighing 3-4 kg. The recording chamber was stereotactically installed under general anesthesia (ketamine-hydrochloride 10-15 mg/kg im, Panpharma, France and xylazine 1.5-2.5 mg/kg im, Sigma) at an angle of 45° to the sagittal plane to facilitate the positioning of the microelectrodes which were then inserted parallel to the central axis of the chamber as previously described (Boraud et al. 1996, 1998). Monkeys were rendered severely parkinsonian by bilateral intracarotidian injection of an acute dose of MPTP (0.8 mg/kg each side) under arteriographic control as previously described (Benazzouz et al. 1993). Their behavior was then clinically assessed on a monkey parkinsonian rating scale (0-25) (see Bezard et al. 1997). Assessment showed that both monkeys developed a stable akineto-rigid parkinsonian syndrome rated respectively 15.2/25 and 16.3/25 without tremor. These ratings correspond approximately to stage IV on the Hoen and Yahr scale (Bezard et al. 1997).

Mean firing frequency and firing pattern were determined both in the normal and the MPTP situation in the GPi. The electrophysiological signal was displayed and stored in a computer (PowerPc 6400, Apple) via the first channel of a MacLab interface (MacLab/4S V1.0.6/1.7.2/0, AD Instruments) using Chart software (Chart V3.5.6, AD Instruments), allowing both online and offline analysis. We used mechanograms, obtained using a manipulandum connected to a potentiometer (16S6-MCB, PK) and recorded via the second channel of the MacLab interface to correlate changes in firing frequency to movements. The manipulandum could be adapted to each joint to record the passive movements we imposed. After carrying out a 10-s control recording of basal neuronal activity, we initiated a sequence of 15 pseudosinusoidal flexion-extension (45°) movements for each joint. Each movement, followed by a short rest interval, was performed bilaterally at 0.2 Hz (under visual control) at a regular rhythm imposed by a metronome. Mechanograms obtained before and after MPTP treatment were compared with the use of analysis of variance (ANOVA) and those which were significantly different (P < 0.05) were rejected. The threshold of significance was defined as follows: a neuron was considered to be joint related (Fig. 1A) if the mean activity provoked by 15 flexion-extensions was at least three times greater than the standard deviation from mean activity at rest as described elsewhere (Mushiake and Strick 1995).



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1. A: example of the activation of globus pallidus pars internalis (GPi) neuronal firing frequency induced by passive limb movement in normal animal. Top: mean mechanogram track of 15 movements. Middle: unit activity. Bottom: mean instant frequency of 15 movements (horizontal line, threshold of significance; vertical line, onset of movement). B: penetration track on a serial frontal slice of the GP of monkey Q-297 before (left) and after (right) MPTP treatment. CA, commissura anterior. Vertical line, sagittal stereotactic plane; horizontal line, horizontal plane passing through the CA-CP line. Circles, neurons whose activity was modified during passive movement of a contralateral limb joint. Squares, neurons whose activity was modified during passive movement of an ipsilateral limb joint. Diamonds, neurons whose activity was modified during passive movement of either an ipsi- or a contralateral limb joint. Shading shows the limb concerned. Top shading, upper limbs; bottom shading, lower limbs.

We analyzed the following parameters: effect on firing frequency (activation or inhibition), side(s) linked (ipsilateral, contralateral, or bilateral), and limb(s) and joint(s) linked. Distribution histograms were built for each parameter and the normal and MPTP situations were compared using the frequency of occurence method (chi 2, df = delta  - 1, delta  = number of categories for each parameter studied, if delta  = 1, then P < 0.05 when chi 2 > 3.841 and if delta  = 2, then P < 0.05 when chi 2 > 5.991).

Marking lesions were made by passing DC (30 µA for 10 s) through the recording electrode at selected points. These were then used, together with electrophysiological landmarks and the dark lines of gliosis indicating recording tracks in the pallidal complex, to retrace the anatomic path of electrode penetration (Fig. 1B shows an axonometric reconstruction). Tyrosine hydroxylase (TH) immunohistochemistry was used to assess the degree of midbrain dopaminergic cell loss (see Bezard et al. 1997 for methods). This showed a dramatic reduction in TH-immunoreactive cell body density in the substantia nigra of the two MPTP-treated monkeys; 83.7% and 77.2% neuronal loss compared with control values obtained in four normal animals.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In GPi, both mean firing frequency and the number of bursting neurons increased significantly after MPTP treatment. These results agreed with previously published results obtained in the same conditions (Boraud et al. 1996, 1998).

In normal animals, passive movement modified the firing frequency of 82/168 GPi neurons (Fig. 2A). After MPTP-treatment this proportion increased, albeit nonsignificantly (97/171) (Fig. 2A). In the normal situation, mean firing frequency significantly decreased during passive movement in 18.3% (15/82) neurons and increased in 81.7% (67/82) (P < 0.05, Fig. 2A, left), giving an inhibited-to activated cells (I/A) ratio of 0.22. In the MPTP situation this ratio dropped to 0.03, with only 3.1% (3/97) neurons decreasing and 96.9% (94/97) increasing their frequency of discharge (P < 0.05, Fig. 2A).



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2. Distribution histograms indicating the influence of passive limb movement on the firing activity of GPi neurons. A: GPi neurons responding to passive movement in normal (left) and MPTP-treated monkeys (right) are distinguished according to the type of response (activated in black and inhibited in white). * P < 0.05. B: distribution histograms showing the selectivity of joint movement-related neurons in response to passive movement: left, side selectivity (ispilateral, contralateral, or bilateral); middle, limb selectivity (upper, lower, or both); right, joint selectivity (single or multi-joint). Decrease in selectivity was observed for each of these parameters in the MPTP situation. * P < 0.05.

In the normal monkey, contralateral limb movement modified the firing frequency of 77/82 neurons, ipsilateral limb movement modified the firing frequency of 2/82 neurons, and either ipsi- or contralateral movement modified the firing frequency of 3/82 neurons (Fig. 2B, left). These proportions changed significantly in the MPTP-treated animal (P < 0.05, Fig. 1B, left). The number of neurons responding to either ipsi- or contralateral (13/97) and to ipsilateral movement (8/97) increased considerably, whereas only 78.4% (76/97) of neurons continued to respond to contralateral movement only.

MPTP intoxication also increased significantly the number of neurons responding to both superior and inferior limb movement (P < 0.05; normal, 1/82; MPTP, 21/97; Fig. 2B, middle), and the number of cells responding to multi-joint movement (P < 0.05; normal, 5/82; MPTP, 64/97; Fig. 2B, right). At the same time, the number of neurons responding to single joint movement decreased from 77/82 to 33/97.

Both before and after MPTP treatment, neurons were located in clusters with no obvious somatotopic organization (Fig. 1B).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that after MPTP treatment the percentage of GPi neurons inhibited by passive movement decreases and the number of movement-related GPi neurons responding less specifically than in the normal situation to more than one joint, to both upper and lower limbs and bilaterally, increases.

Mink has advanced the postulate that, in the normal situation, inhibition of GPi neurons disinhibits desired motor programs and inhibits competing programs (Fig. 3A) (Mink 1996). This would imply that in the normal animal the I/A ratio should be <1. Our results in normal monkeys show an I/A ratio of 0.22 which corroborates this hypothesis and is consistent with other studies (Turner and Anderson 1997). Mink has also suggested that dopamine depletion impairs this selection process and brings the I/A ratio down close to 0 (Fig. 3B) (Mink 1996). In our study MPTP treatment reduced the I/A ratio significantly to 0.03. This result also concords with Mink's model.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. Relationship of GPi activity to inputs from striatum. A: normal situation. During movement, inhibitory striatopallidal neurons inhibit the functional center, resulting in a focused output pattern. Pallidal activity changes are conveyed to the targets in thalamus (VLo) and midbrain extrapyramidal area (MEA) causing disinhibition of neurons involved in the desired motor program and inhibition of surrounding neurons involved in competing motor programs. Inhibited-to-activated (I/A) ratio is <1. B: parkinson situation. Dopamine depletion impairs the selection process and brings the I/A ratio down close to 0 (loosely adapted from Mink 1996).

A decrease in the specificity of neuronal response has already been reported by Filion et al. (1988) but these results were based on subjective evaluation and the type of modification induced by passive movement (inhibition or activation of the firing frequency) was not investigated in this study. Bergman has suggested that dopamine regulates the coupling between the different subcircuits of the BG (Bergman et al. 1998) and Mink has suggested that dopamine depletion should therefore reduce and eventually lead to the disappearance of this selective process (Mink 1996). Our results seem to concord with this hypothesis. However, we do not resolve the debate as to whether this loss of specificity is the primary mechanism for the genesis of rigidity in PD or a consequence of it.

The decrease in the I/A ratio and the sharp decrease in the specificity of the GPi neuronal response to passive limb movement that we observed supports Mink's hypothetical model of BG function, although his hypothesis refered to active movements. These results therefore need to be confirmed in a comparable study of the influence of active limb movement on GPi neuronal activity.


    ACKNOWLEDGMENTS

We thank C. Imbert and S. Dovero for technical assistance.

This study was supported by the Centre National de la Recherche Scientifique, the Institut Fédératif de Neurosciences (Institut National de la Santé et de la Recherche Médicale No. 8 and Centre National de la Recherche Scientifique No. 13), and the University Hospital of Bordeaux.


    FOOTNOTES

Address for reprint requests: T. Boraud, Basal Gang, CNRS UMR 5543, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 August 1999; accepted in final form 29 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society