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Neuroscience Letters, 150 (1993) 183-186
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Motor activity induced by disinhibition of the primary motor cortex of the
rat is blocked by a non-NMDA glutamate receptor antagonist
Manuel A. Castro-Alamancos and Jos6 Borrell
Cajal Institute, Madrid (Spain)
(Received 16 September 1992; Revised version received 11 November 1992; Accepted 11 November 1992)
Key words: Neocortex; GABA; GABA A receptor; NMDA receptor; Glutamate
Application of a GABAA 0,-aminobutyric acid-A) receptor antagonist through a microdialysis probe into the forelimb primary motor cortex of
ketamine-anesthetized rats induced electromyographic activity in the contralateral forelimb. This activity consisted of spontaneous forelimb movements with a frequency of 0.8 +0.2 Hz. The motor activity induced by GABAA receptor blockade was suppressed by application through the dialysis
probe of a non-NMDA (N-methyl-D-aspartate) receptor antagonist, but not by an NMDA receptor antagonist. Glutamate eliminated the blocking
effect of the non-NMDA receptor antagonist upon GABAA receptor blockade mediated activity. In conclusion, the results show that an excitatory
input to the motor cortical output is mediated through a non-NMDA receptor, therefore the effects of cortical disinhibition may be controlled by
non-NMDA receptors.
The primary motor cortex (MI) can be mapped to consist of discrete areas from which movements are evoked
in corresponding parts of the body on the contralateral
side. This cortical map has been known for some time in
the rat [9, 10]. Recently, important advances have been
made in the understanding of the intracortical circuitry
of the motor cortex. During the pharmacological blockade of cortical inhibition of one part of the MI, stimulation of adjacent MI areas cause movements of neighboring representations [11]. These results have been interpreted as suggesting that intracortical connections form
a substrate for reorganization of cortical maps and that
inhibitory circuits are critically placed to maintain or readjust the form of cortical motor representations [11].
Thus, this mechanism has been hypothesized to mediate
the reorganization process which occurs within hours
after the transection of peripheral nerves in adult rats
[16] and possibly also to mediate use-dependent or learning-dependent changes in the organization of the motor
representations throughout life [3, 4, 14].
A model of intracortical connectivity in which the organization of the cortical motor map is regulated by inhibitory local circuit neurons has been proposed [11].
These neurons use the neurotransmitter ~'-aminobutyric
acid (GABA) and form the principal inhibitory cell class
Correspondence: M.A. Castro-Alamancos, Cajal Institute, Avenida
Dr. Arce 37, 28002-Madrid, Spain.
in the cortex. Pyramidal cells in the infragranular layers
of the motor cortex have been shown to have extensive
axon collaterals [7] which activate other pyramidal cells
up to 1 mm away [12]. The expression of these horizontal
connections in the cortex is normally weak because these
fibers simultaneously activate GABA neurons. These
neurons then project to nearby pyramidal cells and rapidly suppress any excitatory activity. Therefore, if the
local inhibitory circuitry is blocked, the spontaneous excitatory activity in the motor cortex is not suppressed
and spontaneous discharges of pyramidal cells activate
muscles in the periphery producing observable motor activity. Extensive evidence suggests that the neurotransmitter which mediates the excitatory input to the pyramidal cells is glutamate [6, 8]. Thus, the receptor mediating
the excitatory activity upon the pyramidal cells could be
of the N M D A (N-methyl-D-aspartate) type or of the
non-NMDA type [15]. In the present study, we addressed
this question by applying into the cortex an N M D A or a
non-NMDA receptor antagonist in conjunction with a
gabaergic receptor antagonist.
The experiments were performed in male Wistar rats
(250-300 g). Rats were anesthetized with ketamine (100
mg/kg). Stainless steel insulated wires with 1-mm uninsulated tips were implanted into individual muscles of the
forepaw. The skin over the forepaw was incised and electrodes were inserted into the muscle about 2 mm apart,
and the skin was sutured closed. The correct placement
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of the electrodes in the muscle was verified by observing
the movement evoked by stimulating electrically through
the implanted wires (200 ps pulses, 0.5 mA). The electromyographic (EMG) signals were differentially amplified
(Grass P15) with the bandpass set at 300-3 kHz. The
animal was then placed in a stereotaxic apparatus and a
craniotomy extending from bregma to 2 mm anterior
and from 1 to 3 mm lateral of the midline was performed
in the hemisphere contralateral to the forelimb with the
implanted electrodes. The cerebral neocortex was exposed and a dialysis probe was inserted into the cortex.
The dialysis probe was of a concentric design [2] using a
cuprophan hollow dialysis membrane. The dialysis membrane extended 2 mm into the brain from the pia in order
to perfuse the cerebral neocortex. An electrode placed
adjacent to the dialysis membrane served to verify that
the area with the implanted dialysis probe was the forelimb primary motor cortex, by applying current trains
(100 ms of 300 Hz, 200-ps pulse duration monophasic
cathodal pulses of 30 pA) that elicited movements of the
contralateral forelimb. The dialysis probe was perfused
at a constant rate of 2 pl/min with Krebs-Ringer bicarbonate, and was left in place for at least 60 min prior to
the initiation of each experiment. After this period, each
rat was perfused with one of the following combinations
of test substances for 5 min: bicuculline methobromide
(2.5 mM), bicuculline methobromide (2.5 mM) + DL-2amino-5-phosphonovaleric acid (APV; 2.5 mM), bicuculline methobromide (2.5 mM) + 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 2.5 mM), bicuculline
methobromide (2.5 mM) + CNQX (2.5 mM) + glutamate (5 mM) or glutamate (5 mM). Bicuculline, APV
and CNQX were purchased from Cambridge Research
Biochemicals, and glutamate was purchased from Sigma.
During the period of perfusion and up to 60 min after
perfusion the EMG activity was monitored through an
oscilloscope and samples of 5-sec duration were recollected at a sample rate of 1 kHz through a computer. A
minimum of 5 samples were collected for each test substance combination. Each experiment was repeated at
least 3 times. A total of 15 rats were used in these experiments. In some cases the experiments were conducted in
such a way that a single animal received all the test substance combinations with an interval between combinations of 60 min. A minimum of 5 samples corresponding
to the effects of each substance combination were recorded.
The application of the GABA type A receptor antagonist bicuculline into the forelimb MI resulted in the occurrence of spontaneous bursts of EMG activity in the
contralateral forelimb (Fig. 1A) which reflected a clearly
observable forelimb movement. The forelimb movements observed were large and similar to those observed
after stimulation of the infragranular layers of the motor
cortex with low impedance electrodes which produce
large current spreads. The rate of the spontaneous EMG
activity was approximately of 0.8 + 0.2 Hz. Even though
the anesthesia used (ketamine) is a NMDA receptor antagonist we applied another NMDA receptor antagonist
(APV) in order to block more effectively and locally the
transmission through this receptor. Application of APV
did not affect the spontaneous EMG activity elicited by
bicuculline (Fig. 1B). Application of a non-NMDA receptor antagonist (CNQX), which blocks transmission
through the quisqualate and kainate receptors, completely abolished the spontaneous bursts of EMG activity elicited by bicuculline (Fig. 1C). Thus, after the joint
application of bicuculline and CNQX no movements
were observed in the contralateral forelimb. When bicuculline and CNQX were applied with a dose of glutamate the blocking effect of CNQX upon the bicucullineinduced EMG activity was abolished and forelimb movements were clearly visible (Fig. 1D). Therefore, the application of glutamate in combination with CNQX and bicuculline succeeded in restoring the motor cortical output. Also, the application of the same dose of glutamate
alone has no effect upon EMG activity.
The results show that disinhibition produced by blocking the GABAA receptors in the MI induces the appearance of spontaneous motor activity in the body part represented by this MI area. This effect is not observed
after the application of an excitatory neurotransmitter in
the same area. Thus, an excitatory neurotransmitter in
the absence of inhibition blockade does not excite effectively the pyramidal cells in the motor cortex, possibly
because this transmitter is also exciting the gabaergic
cells which inhibit more effectively the pyramidal cells.
The activity induced by GABAA receptor blockade is not
suppressed by application of an NMDA antagonist,
while the application of a non-NMDA receptor antagonist completely blocks the motor activity. Finally, the
results also show that glutamate is able to eliminate the
blocking effect of the non-NMDA receptor antagonist
upon the spontaneous motor activity induced by
GABAA receptor blockade. The spontaneous EMG activity observed after GABAA receptor blockade in MI is
probably the consequence of the typical paroxysmal discharges which are observed in the spontaneous firing
rate of cortical cells after GABAA receptor blockade [1].
Thus, these discharges are most likely also eliminated by
blockade of the non-NMDA receptor. Furthermore, our
results are in accordance with electrophysiological recordings from the visual cortex where it was shown that
vertical connections from layer II III cells over the pyramidal cells in layer V are mostly mediated through
non-NMDA receptors and not by NMDA receptors [13].
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,a,
part. Since the i n t r a c o r t i c a l o r g a n i z a t i o n o f m o s t p a r t s o f
the c o r t e x is similar, o u r results can be generalized to
o t h e r cortical a r e a s [13].
In conclusion, the effect o f d i s i n h i b i t i o n in the M I
u p o n E M G activity is b l o c k e d b y a n o n - N M D A r e c e p t o r
a n t a g o n i s t . This result indicates t h a t the o u t p u t o f the
m o t o r c o r t e x is m e d i a t e d t h r o u g h n o n - N M D A r e c e p t o r
c o n t a i n i n g neurons. F u r t h e r , local c h a n g e s in G A B A A
r e c e p t o r s a n d n o n - N M D A r e c e p t o r s in the M I m i g h t
m e d i a t e the r e o r g a n i z a t i o n o b s e r v e d after p e r i p h e r a l
nerve injury [16], r e c o v e r y o f f u n c t i o n after b r a i n d a m age [5] a n d the activity o r l e a r n i n g - d e p e n d e n t c h a n g e s o f
the m o t o r r e p r e s e n t a t i o n s which occur t h r o u g h o u t life
m
B
[3, 4, 141 .
This w o r k was s u p p o r t e d b y g r a n t s f r o m C A M a n d
D G I C Y T . We greatly a p p r e c i a t e the technical assistance
o f C. G a r c i a .
C
,.~i~d,..~ . i , a , , . . ~ .ma.L k,~ . ~ J1~. J . . ~ - ~1. h ~ ida . . . . . .
, ,..=~~ , ...L J.g ,h ~ . . L l , l , c . J m . , ,
. g , J. A ~L J . ,~.~....1
Fig. 1. Typical electromyographic recordings showing (A) the effect of
application of a GABAAreceptor antagonist (bicuculline; 2.5 mM) into
the forelimb primary motor cortex upon forelimb muscle activity, (B)
the lack of effect of application of an NMDA receptor antagonist
(APV; 2.5 raM) upon bicuculline-induced motor activity, (C) the total
blockade of bicuculline-induced motor activity by a non-NMDA receptor antagonist (CNQX; 2.5 mM), and (D) the suppression of the effect
of CNQX upon bicuculline-induced motor activity by glutamate
(5 mM). Recording time per trace: 5 s.
This is p r o b a b l y n o t o n l y the case for vertical c o n n e c tions in the c o r t e x b u t also for h o r i z o n t a l connections.
Thus, after d i s i n h i b i t i o n in the cortex, p y r a m i d a l cells
receive e x c i t a t o r y i n p u t s f r o m b o t h vertical a n d h o r i z o n tal c o n n e c t i o n s w h i c h w o u l d n o r m a l l y be s u p p r e s s e d b y
the g a b a e r g i c input. A s a consequence, the p y r a m i d a l
cells t r a n s m i t this i n p u t t h r o u g h the c o r t i c o s p i n a l p a t h w a y a n d a m o v e m e n t is o b s e r v e d in the r e p r e s e n t e d b o d y
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