Locomotor activity initiated by microinfusions of picrotoxin into the ventral tegmental area

Brain Research, 161 (1979) 311-319 © Elsevier/North-Holland Biomedical Press

311

L O C O M O T O R ACTIVITY I N I T I A T E D BY M I C R O I N F U S I O N S OF P I C R O T O X I N INTO T H E V E N T R A L T E G M E N T A L AREA

G. J. MOGENSON, M. WU and S. K. MANCHANDA* Department of Physiology, The University of Western Ontario, London, Ontario N6A 5C1 (Canada)

(Accepted May 18th, 1978)

SUMMARY Ambulatory activity in rats was increased in a dose-related manner by microinjections of picrotoxin, a GABA antagonist, bilaterally into the ventral tegmental area. Microinjections of strychnine, a glycine antagonist, had no effect on activity. The ambulatory activity induced by microinjections of picrotoxin into the ventral tegmental area was significantly attenuated when the nucleus accumbens was pretreated with spiroperidol, a dopamine antagonist. These findings provide additional evidence that dopaminergic (A10) neurons projecting from the ventral tegmental area to the nucleus accumbens, contribute to locomotor activity. It is suggested that picrotoxin disinhibits the A10 dopaminergic neurons projecting to the nucleus accumbens.

INTRODUCTION There is considerable evidence that dopaminergic neural projections to the nucleus accumbens contribute to ambulatory activity in rats. The microinjection of dopamine into the nucleus accumbens has been shown to increase ambulatory activity 1,7,1a,19,21 and, when the animals were pretreated with nialomide, a monoaminoxidase (MAO) inhibitor which reduces the breakdown of dopamine, the locomotor effects of the centrally administered dopamine were enhanced 21. Ambulatory activity was also increased by microinjections of amphetamine, which increases the release and blocks the reuptake of dopamine from nerve terminals into the nucleus accumbens 19. Following damage to dopamine nerve terminals by microinjections of 6hydroxydopamine to the nucleus accumbens, the increase in ambulatory activity following the systemic administration of amphetamine was not observed 14. It has been * Present address: Department of Physiology, All India Institute of Medical Sciences,Ansari Nagar, New Delhi-16, India.

312 concluded from such observations that stimulation of dopamine receptors in the nucleus accumbens enhances ambulatory activity. The origin of the dopamine terminals in the nucleus accumbens is the ventral tegmental area (VTA), the site of Alo dopamine neurons s. It has been suggested that these dopamine neurons have a GABA synaptic input believed to be inhibitorylO, 26,31. By blocking this inhibitory input using a GABA antagonist such as picrotoxin, the increased discharge of the Alo dopamine neurons should increase the release of dopamine from nerve terminals in the nucleus accumbens and increase ambulatory activity. This possibility was suggested by the observation of circling or rotational behavior after unilateral microinjections of picrotoxin into the region of A9 dopamine neurons of the substantia nigra, which have been shown to have a GABA-inhibitory input 29. The purpose of the present study was to investigate whether ambulatory activity could be enhanced by microinjections of picrotoxin into the ventral tegmental area. To obtain evidence that A10 dopamine neurons were involved in the locomotor effects of such microinjections of picrotoxin, presumably due to disinhibition of dopamine neurons, in some of the experiments the nucleus accumbens was pretreated with the dopamine antagonist, spiroperidol. METHODS Male Wistar rats weighing 225-250 g at the time of surgery were housed individually in wire mesh cages and received Purina Chow pellets and tap water ad libitum. For implantation of cannulae, rats were anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg, i.p.) and placed in a K o p f stereotaxic apparatus. Stainless steel guide cannulae were placed into the nucleus accumbens as well as the VTA. The stereotaxic coordinates for the VTA were 2.2 mm anterior to the interaural line, 0.75 mm lateral to the midline and 7.5 mm below the cortex, and for the nucleus accumbens were 9.4 mm anterior to the interaura[ line, 1.2-1.7 mm lateral to the midline and 6.0 mm below the cortex. Following surgery, the animals were allowed 5-7 days to recover. They were then handled and adapted to the behavioral test chamber for 2 days. Ambulatory activity was recorded in an open-field chamber (75 × 75 × 50 cm), illuminated by a 100 W bulb placed 100 cm above the floor. Light sources were mounted in two of the walls and photoelectric cells were placed on the opposite walls (Fig. IB). The floor was covered with white oilcloth marked off into 9 squares, 25 × 25 cm. Ambulatory activity was recorded by an electronic counter that detected the number of interruptions of light beams or by visually counting the number of squares the animal entered. A square was entered when head and shoulders crossed the boundary line of the square. Rotation, defined as turning 360 ° in one location in the apparatus, was also recorded. Intracerebral microinjections were made into unanesthetized animals using a 1 #1 Hamilton syringe connected by PE10 polyethylene tubing to a 30-gauge needle which extended 0.75 mm beyond the guide cannula. The volume was initially 0.5/A for microinjections of picrotoxin and strychnine sulphate into the VTA. Using this

313 A

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Fig. 1. A: parasagittal view of the rat brain showing cannula positioned into the midbrain to microinject picrotoxin into the region of the Alo dopamine neurons (VTA, ventral tegmental area) which project to the nucleus accumbens (N. Acc.) in the basal forebrain. In some experiments cannulae were also placed into the nucleus accumbens to investigate the effects of spiroperidol, a dopamine antagonist, on locomotor activity initiated by administering picrotoxin to the VTA. B: schematic diagram of an open-field apparatus (75 x 75 cm) equipped with light sources (L) and photoreceptor relays (P) used to measure ambulatory activity. C: locomotor activity recorded during a 30 min test period in 9 rats. Sal: following bilateral microinjections of isotonic saline 0.5 btl into the VTA; Pic. following bilateral microinjections of picrotoxin (0.10/~g in 0.5 #1) into the VTA; Stry" following bilateral microinjections of strychnine sulphate (0.10/~g in 0.5/~1) into the VTA. volume it is estimated that diffusion takes place into a sphere of tissue approximately 1 m m in diameter 4,es. In later experiments, to reduce the spread of the injected substances, the volume was reduced to 0.2 #1, estimated to spread to a region 0.5--0.6 m m in diameter. The cannulae were positioned 0.24).3 m m more medially into the VTA, in later experiments, as an additional measure to reduce the possibility of spread of picrotoxin to the substantia nigra. For microinjections of spiroperidol into the nucleus accumbens the volume was 1.0/A, which, according to estimates from our previous experiments, should not spread beyond the boundary of the nucleus accumbens 4. Within each experiment drug treatments were given into a randomized sequence. At least two days were allowed between treatment. The following drugs were used: picrotoxin (Sigma), strychnine sulphate (Sigma) dissolved in isotonic saline, spiroperidol (Janssen) dissolved in tartaric or lactic acid, the p H adjusted with 1 N N a O H . At the completion of the experiments, the loci of cannulae were verified histologically to be in the nucleus accumbens and VTA. In earlier experiments (Figs. 1 and 2 and Table I) several of the cannulae were in the lateral VTA near the border of the substantia nigra. In later experiments (Fig. 3 and Table II), the cannulae were in the medial VTA 0.8-1.2 m m from the midline. RESULTS

Bilateral microinjections of picrotoxin (0.10 #g in 0.5 bd) into the VTA significantly increased (P < 0.01) ambulatory activity (Fig. 1C). Microinjections of strych-

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Fig. 2. Bilateral microinjections of picrotoxin in to the ventral tegmental area resulted in a doserelated increase in locomotor activity during 30 min test periods. Results for 0 (isotonic saline), 0.05, 0.10 and 0.20/~g were obtained from a series of 5 rats (injection volume 0.5/~1). The results for the 0.15 #g dose have been included from Table I (n -- 8; injection volume 0.2 #1). nine sulphate (0.10 #g in 0.5 #1) did not increase ambulatory activity in comparison to control microinjections of 0.9 ~ NaCl solutions (P > 0.10). Bilateral microinjections of picrotoxin significantly (P < 0.01) increased ambulatory activity in a dose-related manner in the range of 0.10~0.20 #g (Fig. 2). For subsequent experiments 0.15/~g was selected as the standard dose of picrotoxin and the volume was reduced to 0.2/~1. The effects of unilateral and bilateral microinjections ofpicrotoxin were compared to the effects of similar microinjections of strychnine sulphate and to sham injections. Ambulatory activity was increased significantly by unilateral as well as by bilateral microinjections of picrotoxin (Table I). In addition it was noted that the increase in ambulatory activity was significantly greater (P < 0.01) following bilateral microinjections than following either of the unilateral microinjections. Significant increases in rotational behavior were observed following unilateral microinjections of picrotoxin (P < 0.01). To determine whether or not rotation was the result of picrotoxin diffusing to the substantia nigra, 3 rats were prepared with chronic bilateral cannulae positioned more medially in the VTA. Following unilateral microinjections of picrotoxin (0.15/tl in 0.2/A), no rotational behavior was observed after microinjections into 5 of the sites and only 5 rotations during the 30 min observation period were observed in the sixth site. Ambulatory activity following these unilateral microinjections of picrotoxin increased from a mean of 24 ± 7 for the 6 cannulae to a mean of 102 ± 17 (P < 0.01). In animals prepared with chronic bilateral cannulae into the nucleus accumbens and with chronic bilateral cannulae into the medial VTA, spiroperidol was infused into the nucleus accumbens to see whether the effects of microinfusing picrotoxin into the VTA on ambulatory activity could be blocked. The results for 5 rats are shown in Table II. Bilateral microinfusions of spiroperidol significantly reduced ambulatory activity induced by bilateral microinfusions of picrotoxin (P < 0.01). Unilateral

315 TABLE i Ambulatory activity and rotation during a 30 min test period following bilateral and unilateral microinjections of picrotoxin or strychnine sulphate into the ventral tegmental area Two weeks later this series of 8 rats were tested in activity wheels (Wahmann, Model LC-34) and the effects of bilateral microinjections of picrotoxin and strychnine sulphate (0.15/~g in 0.2/~1) into the VTA were compared. During a 30 min test period a mean of 79 wheel revolutions was recorded after microinjections of picrotoxin compared to a mean of 22 wheel revolutions after microinjections of strychnine sulphate (P < 0.01). Variable

Site of microinjeetion Bilateral

Right side

Left side

Locomotion Picrotoxin Strychnine Control

339 4- 53** 60 4- 14 53 ± 13

187 4- 54* 74 4- 23 43 ± 14

152 4- 45* 82 d_ 11 49 4- 18

Rotation Picrotoxin Strychnine Control

5 4- 3 0 0

27 4- 8** 0 0

29 ± 13" 0 0

* P < 0.05, ** P < 0.01, picrotoxin (0.15/zg in 0.2 #1) compared to same dose of strychnine sulphate and control (sham injection) (n = 8). TABLE II Ambulatory activity following bilateral and unilateral microinjections of picrotoxin into the ventral tegmental area with and without pretreatment of nucleus accumbens with spiroperidol n - 5 (30 min test period) ; 0.15/zg in 0.20 #1 of picrotoxin microinjected into VTA, 1.0/~g in 1/tl of spiroperidol into nucleus accumbens. At bottom spiroperidol microinjected into nucleus accumbens compared to control sham injection. Drug

Picrotoxin Spiroperidol 4- picrotoxin Spiroperidol Control

Site of drug microinjection Bilateral

Right side

Left side

415 86 14 37

221 48 18 52

132 61 17 33

4- 24 4- 30* -4- 4 4- 14

4444-

73 11"* 5 14

~ 42 4- 17"* 4- 5 4- 11

* P < 0.01; * * P > 0.05 < 0.10.

m i c r o i n f u s i o n s o f s p i r o p e r i d o l also r e d u c e d a m b u l a t o r y a c t i v i t y i n d u c e d b y i p s i l a t e r a l m i c r o i n j e c t i o n s o f p i c r o t o x i n ( P < 0.01). F o l l o w i n g m i c r o i n j e c t i o n s o f s p i r o p e r i d o l i n t o t h e n u c l e u s a c c u m b e n s a m b u l a t o r y a c t i v i t y was a t t e n u a t e d in c o m p a r i s o n to c o n t r o l tests, b u t the differences w e r e n o t s t a t i s t i c a l l y significant. T h e s p i r o p e r i d o l b l o c k i n g e x p e r i m e n t s w e r e r e p e a t e d in a s e c o n d series o f rats, in w h i c h p i c r o t o x i n was m i c r o i n j e c t e d u n i l a t e r a l l y i n t o t h e V T A a n d s p i r o p e r i d o l was m i c r o i n j e c t e d i n t o i p s i l a t e r a l n u c l e u s a c c u m b e n s (Fig. 3). A s a c o n t r o l , spiro-

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Fig. 3. Increased locomotor activity resulting from unilateral microinjection of picrotoxin (Pic) into the ventral tegmental area (VTA) is significantly attenuated by the ipsilateral (ipsi), but not by contralateral (contra) microinjection of spiroperidol (Spi) into the nucleus accumbens (N.Acc). Bars from left to right: Sal: saline into the VTA; Pic: picrotoxin (0.15/~g in 0.2 pl) into the VTA; Veh ÷ Pic: vehicle (lactic acid) into N.Acc followed by Pic into i psilateral VTA; Spi ÷ Pic: spiroperidol (1.0 #g in 1.0 #1) into N.Acc followed by Pic into ipsilateral VTA ; Con ÷ Con: sham microinjections into N.Acc and VTA; Spi ÷ Con: spiroperidol into N.Acc and sham microinjection ipsilateral VTA; Spi -- Pic; picrotoxin microinjected into VTA and spiroperidol (1.0/~g in 1.0/al) microinjected into contralateral nucleus accumbens (n -- 9). In a subsequent test ambulatory activity following bilateral microinjections of amphetamine sulphate (1 /~g in 1.0/~1) was 504 ± 106 compared to 39 ÷ 7 for a control test with microinjections of isotonic saline (P < 0.01), confirming the observations of previous investigators 19. peridol was also microinjected into the contralateral nucleus accumbens. Unilateral microinjections of picrotoxin (0.15 # g in 0.2 #1) into the V T A increased ambulatory activity from 26 4- 8 to 192 4- 30 ( P < 0.01) (Fig. 3, open bars). The effects of microinjections of spiroperidol (1 # g in 1 /zl) into the ipsilateral nucleus accumbens on ambulatory activity produced by microinjections o f picrotoxin into the V T A were compared to the effects of unilateral microinjections of the vehicle into the nucleus accumbens. A m b u l a t o r y activity was reduced to 44 :]_ 10 by spiroperidol, compared to 178 4- 30 for the vehicle ( P < 0.01). A m b u l a t o r y activity after unilateral microinjection of spiroperidol into nucleus accumbens was reduced, but compared to sham injections (Con) the difference was not statistically significant. Microinjections o f spiroperidol into the contralateral nucleus accumbens did not significantly reduce ambulatory activity elicited by microinjections o f picrotoxin into the V T A ( P ) 0.10) (Fig. 3, bar at extreme right). DISCUSSION Microinjections of picrotoxin, a G A B A antagonist, into the V T A were shown to increase ambulatory activity in an open-field apparatus in a dose-related manner. Rats tested in activity wheels also were more active following the administration o f

317 picrotoxin to the VTA. Microinjections of strychnine, which is a glycine antagonist but not a GABA antagonist, did not increase ambulatory activity. Pretreating the nucleus accumbens with microinjections of spiroperidol, a dopamine antagonist, significantly attenuated the ambulatory activity initiated by microinjecting picrotoxin into the VTA; these results implicate mlo dopaminergic neurons, which project from the VTA, in the observed enhancement of ambulatory activity. The ventral midbrain region contains GABA 24, and recent reports indicate that GABA inhibitory terminals synapse on A9 dopamine neurons in the substantia nigra 12,16 and on Alo dopamine neurons in the VTA 1°,26,31. Rotational behavior, associated with the unilateral release of dopamine from nigrostriatal neurons 2,80, has been observed after microinjections of picrotoxin into the substantia nigra29. The release of dopamine in the striatum has been attributed to the disinhibition of A9 dopaminergic neurons from the blocking action of picrotoxin on the GABA synaptic inputs. Similarly, microinjections of picrotoxin into the VTA might also disinhibit Alo dopaminergic neurons and result in the release of dopamine from axon terminals in the nucleus accumbens. This suggestion is supported by the recent report that neurons in the VTA reduce their rate of discharge following the iontophoretic application of baclofen17. It is also supported by our observation of a significant attenuation of the increased ambulatory activity initiated by administering picrotoxin to the VTA when the nucleus accumbens had been pretreated with spiroperidol, a dopamine antagonist. The Alo dopamine system was previously implicated in ambulatory activity in experiments in which microinjections of dopamine or amphetamine markedly increased ambulatory activity1,~3,~9-21. Whether the dopaminergic neurons projecting from the VTA to the nucleus accumbens synapse on interneurons (possibly Ach or GABA interneurons) or on output neurons is not known 1s,25. However, the activity of these dopaminergic neurons apparently influence the output of the nucleus accumbens, thereby modulating ambulatory activity. There has been evidence for some time of a projection from the nucleus accumbens to the substantia nigra which sends efferents to the striatum; the striatum projects to the globus pallidus, which has an output to the motor cortex, via the thalamus, and to the lower brain stem 15. Another possibility suggested by recent histological evidence is that the nucleus accumbens sends signals directly to the globus pallidus6,23,27. In any case, the nucleus accumbens is strategically located at the interface of the limbic system and motor system, and may have an important role in translating the intention to respond into behaviodL The nucleus accumbens may be an important integrative link between neural systems concerned with motivation and those concerned with the motor control of behavior22. Rotational behavior following the microinjection of picrotoxin into the VTA was observed in some animals. In initial experiments with bilateral microinjections, rotation was observed when the cannulae were asymmetrically placed, one cannula near the substantia nigra. Because rotation is associated with release of dopamine in the caudate nucleus2 and in particular following injections of picrotoxin into the substantia nigra 29, it seemed that the rotation observed after microinjections of picrotoxin to the VTA might be due to diffusion of picrotoxin to the substantia nigra.

318 Accordingly, in later experiments the c a n n u l a e were symmetrical a n d placed more medially at a greater distance from the s u b s t a n t i a nigra, a n d r o t a t i o n was i n f r e q u e n t or did n o t occur, even after unilateral microinjections of picrotoxin. ACKNOWLEDGEMENTS The authors acknowledge the assistance of Blanche Box, Becky Woodside, Vince Nicol a n d Professor F. R. Calaresu. Dr. Albert Wauquier, Janssen Pharmaceutica, Belgium kindly supplied the spiroperidol used in these experiments. This research was supported by the Medical Research Council of Canada. S. K. M a n c h a n d a was a visiting scientist from the All I n d i a Institute of Medical Sciences.

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