Psychopharmacology(1990) 101 : 137--141
Psychopharmacology © Springer-Verlag 1990
Control of food intake by kainate/quisqualate receptors in the median raphe nucleus David Wirtshafter and Joseph C. Krebs Department of Psychologyand Committee on Neuroscience, University of Illinois at Chicago, Box 4348, Chicago, I1 60680, USA ReceivedApril 6, 1989 / Final version November 29, 1989
Abstract. In previous studies we have reported that increases in food and water intake can be produced by microinjections of both N M D A selective and broad spectrum excitatory amino acid antagonists into the median raphe nucleus (MR). In the current experiments we examined the influence of kainate/quisqualate receptors in the M R on ingestive behavior. The consumption of food and water by deprived rats could be suppressed by intra-MR microinjections of the excitatory amino acid agonists kainic acid (5-10 ng in 0.5 gl vehicle) and quisqualic acid (125-500 ng). Conversely, intra-raphe injections of the kainate/quisqualate receptor antagonists pBB-PZDA (1.25-2.5 gg) and GAMS (10--20 pg) elicited feeding in nondeprived animals, pBB-PZDA was more potent in eliciting ingestive behavior than was the selective N M D A antagonist 2-amino-6-phosphonohexanoic acid, suggesting that the effects of pBB-PZDA were not mediated through the N M D A receptor. The current findings suggest that ingestive behaviors are tonically inhibited by excitatory amino acids acting at kainate/ quisqualate receptors in the vicinity of the MR. Key words: Food intake - Median raphe nucleus - Kaina t e - Quisqualate Receptors
Considerable evidence indicates that cells within the median raphe nucleus (MR), or its immediate vicinity, may exert a tonic, inhibitory influence on behavioral arousal. Electrolytic or excitotoxic lesions of the M R result in marked hyperactivity (Jacobs et al. 1974; Lorens 1978; Asin et al. 1979; Wirtshafter and Asin 1982; Asin and Fibiger 1983; Paris and Lorens 1988) and similar effects can be produced by intra-MR injections of the GABA-A agonist muscimol (Sainati and Lorens 1982; Paris and Lorens 1987; Wirtshafter et al. 1987, 1988). The locomoOffprint requests to: D. Wirtshafter
tor response to intra-MR injections of muscimol is much larger than that seen after injections into adjacent sites such as the dorsal raphe nucleus or the ventral tegmental area (Wirtshafter and Klitenick 1989) and this effect does not appear to be dependent on either serotonergic (Klitenick etal. 1985; Paris and Lorens 1987; Wirtsharer et al. 1987) or dopaminergic (Wirtshafter et al. 1988) mechanisms. Injections of muscimol into the M R also result in robust increases in food and water intake and, again, larger effects are seen after injections in the M R than after injections into nearby structures (Klitenick and Wirtshafter 1988, 1989). Recent anatomical data have suggested the existence of excitatory amino acid (EAA) containing afferents to the MR (Pritzel et al. 1985; Kalen et al. 1986) and we have reported that EAAs may act within the M R to influence activity levels and ingestive behavior (Wirtsharer and Trifunovic 1988; Wirtshafter et al. 1989). Available evidence suggests that there exist at least three different subtypes of EAA receptors, the N-methyl-Daspartate (NMDA) receptor, the kainate receptor and the quisqualate receptor (Watkins 1987). Feeding and drinking can be elicited in nondeprived rats by intra-MR injections of the broad spectrum EAA antagonist kynurenic acid and by injections of the specific N M D A antagonists 2-amino-5-phosphonovaleric acid (AP5) and CPP (Wirtshafter and Trifunovic 1988). These findings clearly implicate N M D A receptors within the M R in the control of ingestive behavior, but leave the question of the involvement of kainate/quisqualate receptors unanswered. The present study was designed to investigate whether manipulations of kainate/quisquatate receptors within the M R produce alterations in ingestive behavior similar to those seen after manipulations of GABA-A and N M D A receptors. We examined this question by studying deprivation-induced food intake following intra-MR injections of kainic and quisqualic acids and by examining food and water intake in nondeprived animals following injections of the relatively specific kainate/qu-
138 isquatate antagonists g a m m a - g t u t a m y l - a m i n o m e t h y l s u I fonic acid ( G A M S ) and p - b r o m o b e n z o y l - p i p e r a z i n e d i carboxylic acid ( p B B - P Z D A ) (Davies et at. 1984; Watkins 1987). In order to c o n t r o l for the possibility that the effects o f p B B - P Z D A m i g h t be exerted t h r o u g h the N M D A receptor, for which it has some affinity, we c o m pared the ingestive behaviors seen following i n t r a - M R injections o f equimolar doses o f p B B - P Z D A a n d o f the m o r e p o t e n t selective N M D A antagonist 2-amino-6p h o s p h o n o h e x a n o i c acid (AP6) (Watkins and O l v e r m a n 1987). The results o f these experiments suggest that kainate/quisqualate receptors in the vicinity o f the M R play a role in the c o n t r o l o f ingestive behavior.
Methods Subjects. Subjects were 46 adult, male Sprague-Dawley derived rats obtained from a colony maintained by the University of Illinois at Chicago. Rats weighed between 270 and 320 g at the start of testing and were housed in individual wire mesh cages on a 12:12 h light:dark cycle with food and water available ad libitum, except as indicated below. Surgery. Under sodium pentobarbital anesthesia (50 mg/kg) rats were prepared With chronic 22 gauge stainless steel guide cannulae terminating 2 mm dorsal to the MR (AP: 1.2, H: 6.4, L: 0.0; Paxinos and Watson 1986). The cannulae were lowered in the sagittal plane following retraction of the superior sagittal sinus (Wirtsharer et aL 1979) and were attached to the skull with dental cement. A 28 gauge stainless steel obdurator which extended 2 mm beyond the end of the guide cannula was then inserted. Intracerebral injections. Rats were gently removed from their home cages and their obdurators removed and replaced by a 28 gauge stainless steel injector connected by a length of polyethylene tubing to a motor driven Hamilton microsyringe. The injector cannula was trimmed so as to extend 2 mm past the end of the guide. All injections were made in a volume of 0.5 gl at a rate of 0.25 gl/ rain. The injector was removed 30 s following the completion of the injection. The obdurator was then replaced and the subjects returned to their home cages. While the injections were being made, a weighed quantity of food (Wayne Lab Blox) was placed in the animals' cages and graduated drinking tubes attached to their outsides. Spillage was measured on sheets of paper placed under the cages. Water and food intakes were measured I h following injection. Drugs. Kainic and quisquatic acids and AP6 were purchased from the Sigma Chemical Company (St Louis, MO), GAMS from Cambridge Research Biochemicals (Valley Stream, NY) and pBBPZDA from Tocris Neuramin (Essex, England). All compounds were prepared in a vehicle of sterile normal saline with the pH adjusted to 7.2. Procedure. The effects of intra-MR injections of quisqualate and kainate on deprivation-induced feeding were studied in two separate groups consisting of eight and six animals, respectively. As pilot studies indicated that animals often eat less after their first exposure to deprivation than they do on subsequent trials, all subjects were given an initial run in which they were deprived of food for 24 h and then injected with saline into the MR, after which food was returned. Rats received subsequent tests of ingestive behavior, following 24 h food deprivation, at 4-day intervals. Rats in the quisqualate group were tested following injections of 0, 0.I25, 0.25 or 0.5 gg (0, 0.63, 1.26, 2.5t nmole) of quisqualate, and subjects in the kainate group received injections of 0, 5, or
10 ng (0, 23.4 or 46.9 pmole) of kainate. All injections were administered in a randomized order for individual animals. Three groups of seven to eight subjects were used to study the effects of EAA antagonists on ingestive behavior in nondeprived animals. All subjects received an initial "habituation" run in which they received injections of normal saline and were then allowed to feed for I h. After this, subjects in the GAMS group received intra-MR injections of 0, 5, 10 or 20 gg (0, 20.8, 41.6 or 83.2 nmole) of GAMS, subjects in the pBB-PZDA group received injections of 0, 1.25, or 2.5 gg (0, 3.6 or 7.1 nmole) of pBBPZDA, and subjects in the third group received injections of saline, 7.1 nmole pBBpZDA, or 7.1 or 47.3 nmole AP6. All injections were separated from each other by at least 3 days and were administered in a randomized order for individual animals. A final group of eight subjects was used as a control for the tonicity of the infusates. These subjects received injections of either normal saline or of normal saline containing an additional 2.5 gg/0.5 gl sodium chloride. This tatter solution is approximately isotonic with the 20 gg/0.5 gl solution of GAMS, which was the most concentrated solution used in the present study. Histology. Following the completion of behavioral studies, rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with saline followed by 10% formalin. Brains were then removed and stored in formalin for at least 2 weeks at which time 64 g frozen sections were taken through the extent of the cannula tracks and stained with cresyl violet. Statistics. Data were analyzed either by repeated measures analyses of variance (ANOVAs) followed, where appropriate, by NewmanKeuls tests, or by planned comparison ANOVAs (Kirk 1968).
Results All cannulae terminated within the region o f the M R at sites c o m p a r a b l e to those we have illustrated in previous publications (Wirtshafter et aI. 1987, 1988; Wirtshafter a n d Trifunovic 1989). Feeding and drinking by deprived animals following i n t r a - M R injections o f quisqualate are shown in Fig. 1. Analysis o f these d a t a by means o f repeated measures A N O V A s d e m o n s t r a t e d a significant effect o f quisqualate injections on b o t h f o o d [ F ( 2 , 2 1 ) = 17.18, P < 0 . 0 1 ] and water [ F ( 2 , 2 1 ) = 9 . 8 4 , P < 0 . 0 1 ] intake. As can be seen in Fig. 2, i n t r a - M R injections o f kainate also produced a significant attenuation o f b o t h f o o d [ F ( 2 , 1 0 ) = 5.57, P < 0 . 0 5 ] and water [ F ( 2 , 1 0 ) = 7 . 3 9 , P < 0 . 0 5 ] intake. In contrast to the effects o f the agonists, i n t r a - M R injections o f the kainate/quisqualate antagonist G A M S p r o d u c e d significant feeding [ F ( 3 , 2 1 ) = 4 . 2 7 , P < 0 . 0 5 ] and drinking [F(3,21) =3.19, P < 0 . 0 5 ] in nondeprived rats (Fig. 3). Similar increases in f o o d intake were produced by p B B - P Z D A [ F ( 2 , 1 4 ) = 8.88, P < 0 . 0 1 ] (Fig. 4) a n d this d r u g also tended to p r o d u c e an increase in water intake, b u t this effect fell short o f the usual criteria o f statistical significance [F(2,14) = 3.48, P < 0.08]. Figure 5 displays the c o m p a r a t i v e effects o f pBBP Z D A and A P 6 on feeding and drinking. Analysis o f these d a t a by means o f planned c o m p a r i s o n A N O V A s d e m o n s t r a t e d that 2.5 gg (7.1 nmole) o f p B B - P Z D A p r o d u c e d a significant increase in b o t h f o o d [ F ( 1 , 9 ) = 18.75, P < 0 . 0 1 ] and water [ F ( 1 , 9 ) = 8 . 3 1 , P < 0 . 0 2 ] in-
1t
'o 1 g4 8 v t.,1J
v
z Ci 0 0
oi
L=
7 6 5 4
3 2 I
@
o
0 0
.125
.25
QUISOUALKrE
DOSE
.5
5
o
lO
20
CAMS DOSE (ug)
(ug) 14
1211-
12 11 10
10v
E
t,t
z bJ
87gS-
O
ml
N
nbJ
4-
21
0
.125
.25
0
.5
QUISQUALATE DOSE (ug)
Fig. 1. Effects of intra-median raphe injections of quisqualic acid on food and water intake following 24 h food deprivation. Vertical bars indicate SEMs and stars indicate P < 0 . 0 5 versus vehicle injections
10
5
2O
GAMS DOSE ( u g )
Fig. 3. Effects of intra-median raphe injections of G A M S on food and water intake in nondeprived rats. Vertical bars indicate SEMs and stars indicate P < 0.05 versus vehicle injections
a-
10. g m
v
7.
ILl
e.
Psychopharmacology © Springer-Verlag 1990
Control of food intake by kainate/quisqualate receptors in the median raphe nucleus David Wirtshafter and Joseph C. Krebs Department of Psychologyand Committee on Neuroscience, University of Illinois at Chicago, Box 4348, Chicago, I1 60680, USA ReceivedApril 6, 1989 / Final version November 29, 1989
Abstract. In previous studies we have reported that increases in food and water intake can be produced by microinjections of both N M D A selective and broad spectrum excitatory amino acid antagonists into the median raphe nucleus (MR). In the current experiments we examined the influence of kainate/quisqualate receptors in the M R on ingestive behavior. The consumption of food and water by deprived rats could be suppressed by intra-MR microinjections of the excitatory amino acid agonists kainic acid (5-10 ng in 0.5 gl vehicle) and quisqualic acid (125-500 ng). Conversely, intra-raphe injections of the kainate/quisqualate receptor antagonists pBB-PZDA (1.25-2.5 gg) and GAMS (10--20 pg) elicited feeding in nondeprived animals, pBB-PZDA was more potent in eliciting ingestive behavior than was the selective N M D A antagonist 2-amino-6-phosphonohexanoic acid, suggesting that the effects of pBB-PZDA were not mediated through the N M D A receptor. The current findings suggest that ingestive behaviors are tonically inhibited by excitatory amino acids acting at kainate/ quisqualate receptors in the vicinity of the MR. Key words: Food intake - Median raphe nucleus - Kaina t e - Quisqualate Receptors
Considerable evidence indicates that cells within the median raphe nucleus (MR), or its immediate vicinity, may exert a tonic, inhibitory influence on behavioral arousal. Electrolytic or excitotoxic lesions of the M R result in marked hyperactivity (Jacobs et al. 1974; Lorens 1978; Asin et al. 1979; Wirtshafter and Asin 1982; Asin and Fibiger 1983; Paris and Lorens 1988) and similar effects can be produced by intra-MR injections of the GABA-A agonist muscimol (Sainati and Lorens 1982; Paris and Lorens 1987; Wirtshafter et al. 1987, 1988). The locomoOffprint requests to: D. Wirtshafter
tor response to intra-MR injections of muscimol is much larger than that seen after injections into adjacent sites such as the dorsal raphe nucleus or the ventral tegmental area (Wirtshafter and Klitenick 1989) and this effect does not appear to be dependent on either serotonergic (Klitenick etal. 1985; Paris and Lorens 1987; Wirtsharer et al. 1987) or dopaminergic (Wirtshafter et al. 1988) mechanisms. Injections of muscimol into the M R also result in robust increases in food and water intake and, again, larger effects are seen after injections in the M R than after injections into nearby structures (Klitenick and Wirtshafter 1988, 1989). Recent anatomical data have suggested the existence of excitatory amino acid (EAA) containing afferents to the MR (Pritzel et al. 1985; Kalen et al. 1986) and we have reported that EAAs may act within the M R to influence activity levels and ingestive behavior (Wirtsharer and Trifunovic 1988; Wirtshafter et al. 1989). Available evidence suggests that there exist at least three different subtypes of EAA receptors, the N-methyl-Daspartate (NMDA) receptor, the kainate receptor and the quisqualate receptor (Watkins 1987). Feeding and drinking can be elicited in nondeprived rats by intra-MR injections of the broad spectrum EAA antagonist kynurenic acid and by injections of the specific N M D A antagonists 2-amino-5-phosphonovaleric acid (AP5) and CPP (Wirtshafter and Trifunovic 1988). These findings clearly implicate N M D A receptors within the M R in the control of ingestive behavior, but leave the question of the involvement of kainate/quisqualate receptors unanswered. The present study was designed to investigate whether manipulations of kainate/quisquatate receptors within the M R produce alterations in ingestive behavior similar to those seen after manipulations of GABA-A and N M D A receptors. We examined this question by studying deprivation-induced food intake following intra-MR injections of kainic and quisqualic acids and by examining food and water intake in nondeprived animals following injections of the relatively specific kainate/qu-
138 isquatate antagonists g a m m a - g t u t a m y l - a m i n o m e t h y l s u I fonic acid ( G A M S ) and p - b r o m o b e n z o y l - p i p e r a z i n e d i carboxylic acid ( p B B - P Z D A ) (Davies et at. 1984; Watkins 1987). In order to c o n t r o l for the possibility that the effects o f p B B - P Z D A m i g h t be exerted t h r o u g h the N M D A receptor, for which it has some affinity, we c o m pared the ingestive behaviors seen following i n t r a - M R injections o f equimolar doses o f p B B - P Z D A a n d o f the m o r e p o t e n t selective N M D A antagonist 2-amino-6p h o s p h o n o h e x a n o i c acid (AP6) (Watkins and O l v e r m a n 1987). The results o f these experiments suggest that kainate/quisqualate receptors in the vicinity o f the M R play a role in the c o n t r o l o f ingestive behavior.
Methods Subjects. Subjects were 46 adult, male Sprague-Dawley derived rats obtained from a colony maintained by the University of Illinois at Chicago. Rats weighed between 270 and 320 g at the start of testing and were housed in individual wire mesh cages on a 12:12 h light:dark cycle with food and water available ad libitum, except as indicated below. Surgery. Under sodium pentobarbital anesthesia (50 mg/kg) rats were prepared With chronic 22 gauge stainless steel guide cannulae terminating 2 mm dorsal to the MR (AP: 1.2, H: 6.4, L: 0.0; Paxinos and Watson 1986). The cannulae were lowered in the sagittal plane following retraction of the superior sagittal sinus (Wirtsharer et aL 1979) and were attached to the skull with dental cement. A 28 gauge stainless steel obdurator which extended 2 mm beyond the end of the guide cannula was then inserted. Intracerebral injections. Rats were gently removed from their home cages and their obdurators removed and replaced by a 28 gauge stainless steel injector connected by a length of polyethylene tubing to a motor driven Hamilton microsyringe. The injector cannula was trimmed so as to extend 2 mm past the end of the guide. All injections were made in a volume of 0.5 gl at a rate of 0.25 gl/ rain. The injector was removed 30 s following the completion of the injection. The obdurator was then replaced and the subjects returned to their home cages. While the injections were being made, a weighed quantity of food (Wayne Lab Blox) was placed in the animals' cages and graduated drinking tubes attached to their outsides. Spillage was measured on sheets of paper placed under the cages. Water and food intakes were measured I h following injection. Drugs. Kainic and quisquatic acids and AP6 were purchased from the Sigma Chemical Company (St Louis, MO), GAMS from Cambridge Research Biochemicals (Valley Stream, NY) and pBBPZDA from Tocris Neuramin (Essex, England). All compounds were prepared in a vehicle of sterile normal saline with the pH adjusted to 7.2. Procedure. The effects of intra-MR injections of quisqualate and kainate on deprivation-induced feeding were studied in two separate groups consisting of eight and six animals, respectively. As pilot studies indicated that animals often eat less after their first exposure to deprivation than they do on subsequent trials, all subjects were given an initial run in which they were deprived of food for 24 h and then injected with saline into the MR, after which food was returned. Rats received subsequent tests of ingestive behavior, following 24 h food deprivation, at 4-day intervals. Rats in the quisqualate group were tested following injections of 0, 0.I25, 0.25 or 0.5 gg (0, 0.63, 1.26, 2.5t nmole) of quisqualate, and subjects in the kainate group received injections of 0, 5, or
10 ng (0, 23.4 or 46.9 pmole) of kainate. All injections were administered in a randomized order for individual animals. Three groups of seven to eight subjects were used to study the effects of EAA antagonists on ingestive behavior in nondeprived animals. All subjects received an initial "habituation" run in which they received injections of normal saline and were then allowed to feed for I h. After this, subjects in the GAMS group received intra-MR injections of 0, 5, 10 or 20 gg (0, 20.8, 41.6 or 83.2 nmole) of GAMS, subjects in the pBB-PZDA group received injections of 0, 1.25, or 2.5 gg (0, 3.6 or 7.1 nmole) of pBBPZDA, and subjects in the third group received injections of saline, 7.1 nmole pBBpZDA, or 7.1 or 47.3 nmole AP6. All injections were separated from each other by at least 3 days and were administered in a randomized order for individual animals. A final group of eight subjects was used as a control for the tonicity of the infusates. These subjects received injections of either normal saline or of normal saline containing an additional 2.5 gg/0.5 gl sodium chloride. This tatter solution is approximately isotonic with the 20 gg/0.5 gl solution of GAMS, which was the most concentrated solution used in the present study. Histology. Following the completion of behavioral studies, rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with saline followed by 10% formalin. Brains were then removed and stored in formalin for at least 2 weeks at which time 64 g frozen sections were taken through the extent of the cannula tracks and stained with cresyl violet. Statistics. Data were analyzed either by repeated measures analyses of variance (ANOVAs) followed, where appropriate, by NewmanKeuls tests, or by planned comparison ANOVAs (Kirk 1968).
Results All cannulae terminated within the region o f the M R at sites c o m p a r a b l e to those we have illustrated in previous publications (Wirtshafter et aI. 1987, 1988; Wirtshafter a n d Trifunovic 1989). Feeding and drinking by deprived animals following i n t r a - M R injections o f quisqualate are shown in Fig. 1. Analysis o f these d a t a by means o f repeated measures A N O V A s d e m o n s t r a t e d a significant effect o f quisqualate injections on b o t h f o o d [ F ( 2 , 2 1 ) = 17.18, P < 0 . 0 1 ] and water [ F ( 2 , 2 1 ) = 9 . 8 4 , P < 0 . 0 1 ] intake. As can be seen in Fig. 2, i n t r a - M R injections o f kainate also produced a significant attenuation o f b o t h f o o d [ F ( 2 , 1 0 ) = 5.57, P < 0 . 0 5 ] and water [ F ( 2 , 1 0 ) = 7 . 3 9 , P < 0 . 0 5 ] intake. In contrast to the effects o f the agonists, i n t r a - M R injections o f the kainate/quisqualate antagonist G A M S p r o d u c e d significant feeding [ F ( 3 , 2 1 ) = 4 . 2 7 , P < 0 . 0 5 ] and drinking [F(3,21) =3.19, P < 0 . 0 5 ] in nondeprived rats (Fig. 3). Similar increases in f o o d intake were produced by p B B - P Z D A [ F ( 2 , 1 4 ) = 8.88, P < 0 . 0 1 ] (Fig. 4) a n d this d r u g also tended to p r o d u c e an increase in water intake, b u t this effect fell short o f the usual criteria o f statistical significance [F(2,14) = 3.48, P < 0.08]. Figure 5 displays the c o m p a r a t i v e effects o f pBBP Z D A and A P 6 on feeding and drinking. Analysis o f these d a t a by means o f planned c o m p a r i s o n A N O V A s d e m o n s t r a t e d that 2.5 gg (7.1 nmole) o f p B B - P Z D A p r o d u c e d a significant increase in b o t h f o o d [ F ( 1 , 9 ) = 18.75, P < 0 . 0 1 ] and water [ F ( 1 , 9 ) = 8 . 3 1 , P < 0 . 0 2 ] in-
1t
'o 1 g4 8 v t.,1J
v
z Ci 0 0
oi
L=
7 6 5 4
3 2 I
@
o
0 0
.125
.25
QUISOUALKrE
DOSE
.5
5
o
lO
20
CAMS DOSE (ug)
(ug) 14
1211-
12 11 10
10v
E
t,t
z bJ
87gS-
O
ml
N
nbJ
4-
21
0
.125
.25
0
.5
QUISQUALATE DOSE (ug)
Fig. 1. Effects of intra-median raphe injections of quisqualic acid on food and water intake following 24 h food deprivation. Vertical bars indicate SEMs and stars indicate P < 0 . 0 5 versus vehicle injections
10
5
2O
GAMS DOSE ( u g )
Fig. 3. Effects of intra-median raphe injections of G A M S on food and water intake in nondeprived rats. Vertical bars indicate SEMs and stars indicate P < 0.05 versus vehicle injections
a-
10. g m
v
7.
ILl
e.
