Antagonistic effects of lesions of paramedian reticular nucleus on amphetamine-induced locomotion and striatal dopamine release in rats

Neuroscience Research, 16 (1993) 311-314 © 1993 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0168-0102/93/$06.00

311

NSR 00637

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Antagonistic effects of lesions of paramedian reticular nucleus on amphetamine-induced locomotion and striatal dopamine release in rats W.T. Chiu a, L.S. Lin a a n d M.T. Lin b a Department of Surgery, TaipeiMedical College, Taipei, Taiwan, ROC, b Department of Physiology, National Cheng Kung University Medical College, Tainan City, Taiwan, ROC (Received 15 March 1993; revised 3 April 1993; accepted 3 April 1993)

Key words: Locomotor activity; Paramedian reticular nucleus; Dopamine; Corpus striatum; Voltammetry

Summary Systemic administration of amphetamine (1.25 mg/kg) produced increases of locomotion (including horizontal motion, vertical motion, and total distance travelled), elevations of turnings (including both clockwise and anticlockwise) and inhibition of postural freezing in freely moving rats. All the afore-mentioned activity measures induced by amphetamine were suppressed following electrolytic lesions of the paramedian reticular nucleus (PRN) in rat medulla. In addition, the spontaneous level of either the locomotor activity, the direction of turnings, or the postural freezing were slightly but significantly affected by the PRN lesions. In vivo voltammetric data revealed that amphetamine administration greatly enhanced the striatal dopamine release. Furthermore, the enhanced dopamine release in corpus striatum produced by amphetamine were greatly attenuated by PRN lesions. The results indicate that there exists a PRN-striatal dopamine link in rat brain which mediates the amphetamine-induced increases of locomotion and turnings, as well as decreases of postural freezing.

Evidence has accumulated to suggest that he paramedian reticular nucleus (PRN) plays an important role in the regulation of somatosensory and somatomotor functions in rats. For example, stimulation of the PRN suppressed both the extensor reflex (Alexander, 1946) and the twitching induced by picrotoxin (Magoun and Rhines, 1946). Stroking or blowing on the integument of limbs and abdomen (Duggan and Game, 1975) or stimulation of the vestibular apparatus (Chelarducci et al., 1974) affected the majority of the neuronal activity in the PRN region. On the other hand, amphetamine is well known for its activity to enhance locomotor activity and to produce stereotyped behaviors in rats (Schiorring, 1971;

Correspondence to: Prof. Mao-Tsun Lin, Department of Physiology, National Cheng Kung University Medical College, Tainan city, Taiwan, Republic of China. Fax: (886)-6-2362780.

Segal, 1975). It is a well-known drug of abuse; it provides an animal model of schizophrenia, and it is widely used as an implicit model of dopamine activity in the brain. In the current experiments, in order to determine whether the PRN plays a role in the amphetamine-induced locomotion, we examined the activity measures as well as the dopamine release in the corpus striatum in both the sham-operated rats and the PRN-lesioned rats in response to systemic administration of amphetamine. Male Sprague-Dawley rats (250-300 g) were used in the present study. Upon receipt from the supplier (Animal Resource Center, National Cheng Kung University Medical College, Tainan City, Taiwan, ROC), the animals were housed in pairs in a temperature-regulated (22 + I°C) room on a 12/12-h light/dark cycle with food and water ad libitum. The animals were kept for at least 2 weeks before they were used. The light was turned on at 0600 h and turned off at 1800 h.

312 In preparation for PRN lesions, the animals were placed in a Kopf stereotaxic instrument under general anesthesia (sodium pentobarbital 6 m g / 1 0 0 g, i.p.), and prepared by bilateral electrolytic destruction of the PRN (A1.31, L0.04 and V1.0 below dura) on the animal's left and right sides, according to the atlas of Paxinos and Watson (1982). Lesions were applied to these animals with a fine monopolar stimulating electrode by passage of a DC potential of 0.2 mA for 30 s from a Grass $48 stimulator. Sham operation had identical procedure but no current was applied to the PRN. One month after PRN lesions or sham operation, the animals were subjected to experimentation. All lesion placements were in region of the PRN as revealed by standard histological procedures. For histological verification of the lesioned sites in the PRN regions, animals were killed by decapitation at the end of all experiments and perfused with physiological saline followed by 10% formalin. Their brains were removed, sliced on a freezing microtome and stained with methylene blue (3 m g / m l ) for microscopic examination. The behavioral apparatus used were four activity chambers equipped with an infrared light matrix system as detailed elsewhere (Young et al., 1993). In this system, the first part was an IBM PC-AT compatible microcomputer which was used as the controller for data acquisition and as the analyzer for data location. The second part was the infrared light emitting and receiving circuits. The two kinds of devices adopted were the infrared light emitting diode (IRLED) and phototransistor, respectively. Twenty-four I R L E D or phototransistors were fixed on an acrylic bar with 1.6 cm interval between tow neighboring devices. For detecting the horizontal and vertical motions simultaneously, we installed the acrylic bar pair (both the emitting and receiving bars were precisely aligned at a distance of 45 cm) along the x-axis, y-axis and z-axis. The following locomotor activities were measured using this system: (1) horizontal fine motion time ( H F M T in s; time spent during horizontal displacements of less than 1.6 cm); (2) horizontal gross motion time ( H G M T in s; time spent for horizontal displacements of greater than 1.6 cm); (3) vertical motion time (VMT in s; time spent during vertical displacements in which one or more infrared light beams on the z-axis were blocked); (4) freezing time (FT in s; time elapsed when the animal did not have HFMT, H G M T or VMT); (5) total distance travelled (TDT in cm; the total sum of all horizontal gross motion); (6) clockwise turnings (CT in counts); and (7) anticlockwise turnings (ACT in counts). The animals were placed in the center of the open

field and were allowed to habituate to the open field for 90 min 1 week before testing. Each rat was tested only once. While the rat was being injected, the open field was washed. The rat was then returned to the open field for observation right after injection. All behavioral testings were prepared in the light phase of the diurnal cycle. A single carbon fiber (12 ~ m in diameter, AVCO, Lowell, MA) was inserted into the pulled glass micropipette (20-25 mm in length). The pipette tip was cut using fine scissors, so that the fiber fitted lightly inside glass tip, the carbon fiber was pushed out of the pipette tip. Electrical contact with the fiber was made using silver paste. The tip and blunt end of the pipette were sealed with cyanoacrylate adhesive (Super glue). The entire surface of a pyrolytic carbon fiber was 12 ~,m thick and 500 ~tm long. To improve the sensitivity and selectivity of carbon fiber electrodes for dopamine, the electrodes were electrically pretreated as detailed elsewhere (Gonon et al., 1984). This treatment consisted of a DC current applied in two stages, 2.2 V for 30 s in 0.1 M H2804, and 2.2 V for 30 s in 1 N HCI. The carbon fiber electrode was washed with distilled water. The tip of the carbon fiber electrodes were immersed into a nation drop (10 I*l of 15% solution, Aldrich Chemical Company, Inc., Milwaukee, WI, USA) 4 times. The nation-coated electrodes were then dried at 60°C for 20 s and used immediately for in vitro followed by in vivo experiments (Brazell et al., 1987; Crespi et al., 1988; Lin et al., 1992). Differential pulse amperometry was performed in vitro and in vivo with a Biopulse (Solea Tacussel Co., France) using the following scan parameters: imposed initial potential = - 2 2 0 mV; imposed final potential = - 7 0 mV; pulse cycle = 25; prepulse = 70 mV; measuring pulse = 40 ms; and measuring p o t e n t i a l - - 4 0 mV. The sensitivity of the nation-coated electrodes to dopamine in the concentration of 200-800 nM was determined using differential pulse amperometry in a temperature-controlled (37°C) water bath. Phosphate-buffered saline (0.1 M, pH 7.4) was used as blank and solvent for the test solution. To determine the sensitivity of the electrodes (resistance = 200 S2; capacitance=0.0056 /~F) for dopamine over DOPAC, a ratio of the sensitivity of d o p a m i n e / D O P A C was calculated. Our electrodes were 250 times more sensitive to dopamine than to DOPAC. The animals were anesthetized with sodium pentobarbital (6 m g / 1 0 0 g, i.p.), held in a stereotaxic frame and implanted unilaterally with a nation-coated electrode in the corpus striatum using the coordinates of Paxinos and Watson (1982). Auxiliary (silver wire) and

313 TABLE 1 E F F E C T S O F I.P. I N J E C T I O N O F N O R M A L S A L I N E O R A M P H E T A M I N E (1.25 m g / k g ) ON L O C O M O T O R ACTIVITIES IN I N T A C T RATS, IN S H A M - O P E R A T E D RATS, A N D IN P R N - L E S I O N E D R A T S Treatment Intact rats Normal saline Amphetamine Sham-operated rats Normal saline Amphetamine PRN-lesioned rats Normal saline Amphetamine

HFMT (s)

HGMT (s)

VMT (s)

FT (s)

CT (counts)

ACT (counts)

TDT (cm)

2+ 1 267+38 a

3+ 2 1895:28 a

16+ 3 3565:61 a

3492+77 24685:99 a

5+2 62_+4 a

6+2 63+5 a

191 5 : 2 3 5770+581 a

35:1 2445:35 a

25:1 1735:24 a

20 5 : 3 3375:58 a

3485+69 2675+88 a

45:2 585:5 a

55:2 575:6 a

217 5 : 2 8 5368_+614 a

45 5: 4 b 136 5 : 2 a,b

2804+69b 3474 5:75 a,b

115:2b 35 5:7 a,b

12 5 : l b 15 +_ 3 a,b

350 5: 3 5 b 1769 5 : 6 4 a,b

85: l b 267 5:38 a,b

95: 2 b 22 +_ 4 a.b

Values are expressed as m e a n s + SEM of 8 rats for each group. The activity measures were recorded for 60 min. a Significantly different form corresponding control values (saline groups), P < 0.05 (one-way ANOVA). b Significantly different form corresponding control values (saline group of intact rats), P < 0.05 (one-way ANOVA).

reference (Ag/AgC1) electrodes were placed on the dura surface of the parietal skull. Differential voltammograms were then recorded every 2 s. Experiments were carried out to assess the effects of amphetamine on striatal dopamine release in both sham-operated rats and in PRN-lesioned rats 3-4 days after electrode implantation. At the end of each experiment, an electrolytic lesion was performed by applying a DC current to the carbon fiber electrode. In order to verify correct placement of the electrodes, the region of the brain stem containing the site of recording was taken from each rat and was fixed by immersion in Faglu (0.5% glutaraldehyde and 4% formaldehyde in phosphate buffer) for subsequent histological analysis. Forty-eight rats were randomly assigned to six equal groups. The animals were acclimatized to the behavioral apparatus for 30 min before an i.p. dose of normal saline or amphetamine (1.25 mg/kg). The locomotor activity responses of these groups of animals were recorded for a 60-min period after injection. The effects of the PRN lesions on the amphetamine-induced locomotor activities are summarized in Table 1. As revealed in Table 1, amphetamine injection had significant effects on the following activity measures: increases in HFMT, HGMT, VMT, TDT, CT or ACT, as well as decreases in FT (P < 0.05; one-way ANOVA). The table also showed that all the aforementioned activity measures induced by amphetamine were significantly suppressed by lesions of the PRN in rats ( P < 0.05; one-way ANOVA). However, it was found that PRN lesions slightly elevated the spontaneous levels of both the locomotor activities and the

direction of turnings, but depressed the spontaneous levels of the postural freezing in rats. Another 16 rats were randomly assigned to two equal groups. The effects of amphetamine (1.25 mg/kg, i.p.) on striatal dopamine release in both the sham-operated rats and the PRN-lesioned rats are summarized in Table 2. Lesions of the PRN produced a slight increase in the spontaneous levels of the striatal dopamine release. Also, the amphetamine-induced increases of striatal dopamine release were attenuated following the PRN lesions. The present results provide new evidence that electrolytic destruction of the PRN area in rat medulla increased the spontaneous levels of striatal dopamine release. The present results also showed that PRN lesions produced increases of locomotion (including

TABLE 2 E F F E C T S O F A M P H E T A M I N E (1.25 m g / k g , I.P.) O R N O R M A L SALINE I N J E C T I O N O N T H E S T R I A T A L D O P A M I N E REL E A S E IN S H A M - O P E R A T E D R A T S A N D IN P R N - L E S I O N E D RATS Groups of animals

Sham operation P R N lesions

Striatal dopamine (nM) Before injection

After injection

Difference

158 + 23 245+21 a

477 + 34 336+ 17 a

209 + 18 91 + 8 a

Values are expressed as m e a n s + SEM of 8 rats for each group. a Significantly different form corresponding control values (saline groups), P < 0.05 (one-way A N O V A ) .

314 h o r i z o n t a l motion, vertical motion, a n d total d i s t a n c e travelled), e l e v a t i o n s of t u r n i n g s (including b o t h clockwise a n d anticlockwise), and inhibition o f p o s t u r a l f r e e z i n g in rats. Brain d o p a m i n e system has long b e e n i m p l i c a t e d in the expression of l o c o m o t o r b e h a v i o r s ( R a n d r u p a n d M u n k v a d , 1970). T h e s e o b s e r v a t i o n s p r o m p t e d us to t h i n k that t h e r e exists a P R N - s t r i a t a l d o p a m i n e link in rat b r a i n which m e d i a t e s the s p o n t a n e o u s levels o f l o c o m o t o r activities. A m p h e t a m i n e is well known for its ability to enh a n c e l o c o m o t o r activity a n d to p r o d u c e s t e r e o t y p e d b e h a v i o r s in rats. C e r t a i n dose o f a m p h e t a m i n e p r o d u c e d a t h r e e - p h a s e b e h a v i o r a l r e s p o n s e in rats consisting of e n h a n c e d l o c o m o t o r activity a n d rears, stereotypy, a n d an a f t e r p h a s e of e n h a n c e d l o c o m o t i o n s (Schiorring, 1971; Segal, 1975). T h e s t e r e o t y p y p h a s e is c h a r a c t e r i z e d by a b s e n c e of l o c o m o t i o n and by c h a r a c teristic r e p e t i t i v e h e a d m o v e m e n t s a n d i n t e n s e sniffing, licking, or biting of a highly r e s t r i c t e d area. Several lines of e v i d e n c e i n d i c a t e that " s t e r e o t y p i c sniffing" is m e d i a t e d by the nucleus a c c u m b e n s a n d t h a t the r e p e t itive h e a d m o v e m e n t s , licking a n d biting a r e m e d i a t e d by the c o r p u s s t r i a t u m (Kelly et al., 1975). In the c u r r e n t results, using a m o d u l a r i z e d i n f r a r e d light matrix system for m e a s u r i n g a n i m a l ' s b e h a v i o r s ( Y o u n g et al., 1993), a m p h e t a m i n e at a dose o f 1.25 m g / k g p r o d u c e d i n c r e a s e s o f l o c o m o t i o n (including h o r i z o n t a l motion, vertical m o t i o n , a n d total d i s t a n c e travelled), elevations o f turnings (including b o t h clockwise a n d anticlockwise), a n d inhibition o f p o s t u r a l freezing in rats. F u r t h e r m o r e , the p r e s e n t results p r o v i d e a n o t h e r new e v i d e n c e t h a t the a m p h e t a m i n e - i n d u c e d i n c r e a s e s o f l o c o m o t i o n , e l e v a t i o n s o f turnings, as well as inhibition of p o s t u r a l freezing w e r e all s u p p r e s s e d following P R N lesions in rats. In vivo v o l t a m m e t r i c d a t a also r e v e a l e d t h a t P R N lesions a t t e n u a t e d t h e amp h e t a m i n e - i n d u c e d increases in the striatal d o p a m i n e r e l e a s e in rats. T h e s e results i n d i c a t e t h a t P R N lesions s u p p r e s s the a m p h e t a m i n e - i n d u c e d i n c r e a s e s in the striatal d o p a m i n e r e l e a s e and result in a r e d u c t i o n in the a m p h e t a m i n e - i n d u c e d l o c o m o t o r activities in rats. In fact, m o n o a m i n e r g i c a n d p e p t i d e r g i c m e c h a n i s m s have b e e n i m p l i c a t e d in the actions on psychology a n d b e h a v i o r ( R a n d r u p a n d M u n k v a d , 1970). A l t h o u g h t h e p r e s e n t results can b e e x p l a i n e d in c o n n e c t i o n with d o p a m i n e r g i c m e c h a n i s m s , involvement of s e r o t o n e r g i c m e c h a n i s m s m a y not b e excluded, since t h e lesions w e r e p l a c e d n e a r the r a p h e nucleus c o n t a i n i n g serot o n e r g i c cells ( H a r v e y et al., 1975; Fuxe, 1965). T h e s e s e r o t o n i n e r g i c n e u r o n s w e r e shown to be involved in a variety of b e h a v i o r a l r e s p o n s e s ( H a r v e y et al., 1975).

A c k n o w l e d g e m e n t s T h e w o r k r e p o r t e d h e r e was supp o r t e d by g r a n t s from the N a t i o n a l Science Council of R e p u b l i c of China. T h e a u t h o r s wish to t h a n k Miss Y.Y. C h a n for h e r excellent technical assistance.

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