Brain Research, 550 (1991) 181-191 © 1991 Elsevier Science Pubhshers B V 0006-8993/91/$03 50 A D ONIS 000689939115697U BRES 16597
181
Research Reports
5-HT3-1ike receptors in the rat medial prefrontal cortex: an electrophysiological study Charles R. Ashby Jr, Yoshlo Mlnabe, Emmehne Edwards and Rex Y Wang Department of Psychtatry and Behaworal Scwnces, State Umverstty of New York at Stony Brook, Stony Brook, NY 11794-8790 (U S A ) (Accepted 11 December 1990) Key words Serotonm3-hke receptor, Medml prefrontal cortex, Mlcro,ontophores]s, 2-Methylserotonm, Serotomn 3 receptor antagomst
In th,s study, we have ]denufied and characterized 5-HTa-hke receptors in the rat medial prefrontal cortex (mPFc), an area with a moderate density of 5-HT3 binding sites, using the techmques of single umt recording and m]cro~ontophoresls The mlcrolontophoresls of the 5-HT3 receptor agomst 2-methyiserotonm (2-Me-5HT), s]mdar to the acUon of 5-HT, produced a current-dependent (10-80 nA) suppression of the firing rate of both spontaneously active and glutamate (GLU)-acuvated (quiescent) mPFc ceils Phenylbiguamde (PBG), another 5-HT3 receptor agomst, suppressed the finng rate of mPFc cells but was less effectwe compared to 2-Me-5HT The continuous lontophoresls (10-20 mm) of 1 M magnesmm chloride markedly attenuated the suppressant effect produced by electrical stlmulaUon of the ascending 5-HT pathway, but &d not alter 2-Me-5HT's action, suggesting that the action of 2-Me-5HT ~s a &rect one The suppressant action of 2-Me-5HT on mPFc cells was blocked by a number of structurally dwerse and selectwe 5-HT3 antagomsts, with a rank order of effectiveness as follows ICS 205930 = (+)-zacopnde > gramsetron = ondansetron = LY 278584 > MDL 72222 Furthermore, the intravenous administration of (+)-zacopnde antagomzed the action of 2-Me-5HT and PBG on mPFc cells In contrast to the effects of the 5-HTs receptors antagomsts, other receptor antagomsts such as metergohne (5-HT]A m l c 2), (+)-pmdolol (5-HT1Am, fl), SCH 23390 (5-HTlc 2, D1), l-sulpmde (D2) or SR 95103 (GABAA) faded to block 2-Me-5HT's action These results combined suggest that 2-Me-5HT's suppressLve action on mPFc cells is medmted &rectly by 5-HT3-hke receptors INTRODUCTION
psychotic properties x8 2z 48 and they also attenuate the
The 5-HT 3 receptor has been pharmacolog]caUy and blochemlcally well defined m the periphery as a result of the d e v e l o p m e n t of selective 5-HT 3 receptor antagonists These receptors me&ate the excitatory response of 5-HT on enteric, afferent and postganghomc sympathetic neurons 43 For example, 5-HT depolar]zes sensory n e u r o n a l
Moreover, it has been shown that the atypical ant]psychotic drug clozapme, but not the typical ant]psychotic
rewarding properties of mcotme
terminals m the heart, releasing acetylchohne from the vagus nerve and producing a reflex bradycardla referred to as the von Bezold-Jansch reflex 9 21 The results from recent studtes have suggested that the 5-HT 3 b m d m g s~te, u n h k e 5-HT~ and 5-HTz sites, ts a hgand gated ,on channel~9 37 55 R a d l o h g a n d binding studies, using 3H-5HT3 antagorests, have shown that 5-HT 3 binding sites are present m the brain 9 33 38 m relatwely high density of 5-HT 3 sites are present m mesocortlcohmblc areas such as the nucleus accumbens, olfactory tubercle, amygdala, entorhmal, cmgulate and frontal cortices 3z 33 Although the exact functional role of central 5-HT 3 binding sites is u n k n o w n , it has been shown that 5-HT 3 receptor antagomsts d~splay ant~emet~c ~5 3s antmnx~ety173~ and ant~-
and m o r p h i n e ]4'27
drugs h a l o p e n d o l and chlorpromazme, possesses 5-HT 3 receptor antagomzmg properties 5 45 We have begun to identify and characterize 5-HT3-hke receptors m the rat brain O u r p r e h m m a r y results suggest that 5-HT3-hke receptors m the med]al prefrontal cortex (mPFc) are different from those m the periphery Thus, mlcro]ontophoresls of 2-Me-5HT produces a currentd e p e n d e n t suppression of m P F c cells' firing and this action ~s blocked by selectwe 5-HT 3 receptor antagomsts ICS 205930 and gramsetron but not by non-selective 5-HT antagomsts such as ( + ) - p l n d o l o l or metergohne z In a d d m o n , we have d e m o n s t r a t e d that the 5-HT3-hke receptors m fronto-cmgulate and e n t o r h m a l cortices might be coupled to p h o s p h o m o s m d e 4 20 In this paper, we attempted to further characterize 5-HT3-hke receptors in the rat mPFc, using the 5-HT 3 agomsts 2-Me-5HT and P B G and a n u m b e r of structurally dwerse 5-HT 3 antagomsts
Correspondence R Y Wang, Department of Psychiatry and Behavioral Sciences, Putnam Hall-South Campus, State Umverslty of New York at Stony Brook, Stony Brook, NY 11794-8790, U S A Fax (1) (516) 632-8953
182 MATERIALS AND METHODS
Ammals and surgery Male albino Sprague-Dawley rats (200-300 g, Tacomc, Germantown, NY) were anesthetized w~th chloral hydrate (400 mg/kg, ~ p ) and placed in a stereotax~c instrument The body temperature oI the rats was maintained at 37-38 °C using an electric heating pad A lateral tall veto was cannulated w~th a 25 g needle for the i v injection of addltmnal anesthetic as reqmred or mject~on of drug soluhon A burr hole was drilled over the mPFc 11-12 0 mm anterior to lambda and 0 3-1 0 mm lateral to the mldhne suture according to the atlas of Paxlnos and Watson'u~
Smgle umt recording and mwrmontophorests Standard extracellular single cell recording and mlcrolontophoret~c techmques were used as previously described4~ so Five or 6 barrel glass m~croplpettes were pulled with a vertical electrode puller (Nanshlge PE2) and broken back under a hght microscope to a dmmeter of 5-8pro The central (recording) barrel was filled with a 2 M NaCI solutmn saturated with 1% Fast Green dye (Fisher) The m vitro ~mpedance of the central barrel was 2-6 M.Q when measured at 135 Hz (Winston BL-1000B) One side barrel was filled with a 2 M NaCI solutton (impedance 5-15 M~2) The remaining side barrels (20-30 MI2) contained a combination of the following compounds (10 mM, pH 4-4 5 in 1 mM NaCI) 2-Me-5HT (2-methylserotonln, Research Biochemical I n c , Natlck, MA, Sandoz. Basel. SwRtzerland and Glaxo. Ware. U K ), PBG (phenylblguamde. Sigma. St Lores. MO). metergohne (Farm Itaha), (_+)-plndolol (Sandoz), l-sulplnde (Ravlzza), SR 95103 ([2-(3carboxypropyl)-3-amlno-4-methyl-6-phenylpyndazmum chloride], SANOFI, Montpelher. France), gramsetron (Beecham Pharmaceuucals, Essex. U K ). ondansetron (Glaxo), (R) and (S)-zacopnde, (+)-zacopnde (Wyeth-Ayerst Co ), LY 278584 (1-(methyl-n-(8methyl-8-azab~cyclo [3.2,1]-oct-3-yl)-IH-mdazole-3-carboxam~de, Eh Lilly C o r p . Indlanapohs, IN), ICS 205930 ([3-2-tropanyl1H-mdazole-3-carboxyhc acid ester]. Sandoz) and MDL 72222 ([1-H-3a-5a-tropan-3-yl]-3,5-d~chlorobenzoate, MerreU Dow Research Institute, Strasbourg, France) The concentratmn of MgC1z and l-glutamate (S~gma) were one molar (pH 4-4 5) and 10 mM (pH 8). respectively MDL 72222 was dissolved m a few drops of 2% lacuc acid plus demmzed, degassed water The remaining compounds were &ssolved m demmzed, degassed water The pH of the drug soluuons were adjusted w~th 0 1 N HCI and 0 1 N NaOH All drug solutions were prepared fresh or stored at -70 °C Retaining currents (posmve for glutamate and negauve for the remaining compounds) of 8-10 nA were apphed to each of the drug barrels to prevent passwe dfffusmn between ejection periods For recording mPFc cells, the m~crop~pette was lowered to touch the cortex and then moved 0 5-3 5 mm below the cortical surface using a hydrauhc m~crodnve Electrical s~gnals were passed through a h~gh ~mpedance amphfier, displayed on an oscilloscope and momtored by an audmamphfier The s~gnals were then fed into a window &scnm~nator (Fmtromcs WDR 420), which was set such that the standard output was triggered by mdw~dual action potentials The integrated rate histograms generated by the analog output of the window dtscnmmator were plotted on a polygraph recorder (Gould 220)
Electrtcal snmulauon of the caudal hnear raphe nucleus A concentric electrode (NE-100, David Kopf Instruments) was implanted m the caudal hnear raphe nucleus (CLI, anterior 2 2 mm to Lambda, lateral 0 0 mm to the mldhne and ventral 6 8 mm to the dura), a structure through which the majority of the ascending 5-HT fibers pass6 36 Pulse trams were dehvered at a rate of 15 Hz, with a 1 ms pulse width and with an intensity of 0 1-1 0 mA using a &gltal pulse generator and an lsopulser (WPI, pulsemaster A300) It has been demonstrated that 10 Hz is an opumal frequency for stimulation of the m~dbram raphe to produce a maximum turnover of 5-HT in the forebraln ~ At the end of each experiment, the final site of the recording mlcrop~pette was marked by the mntophoretlc ejectmn (-25/xA for
20-30 mms) ot 1% Fast Green dye, which results m the deposmon of a Fast Green spot The location of this spot is verified m h~stologlcally prepared tissue The site of the stimulating electrodc ~as marked by passing a 0 05 mA posltwe direct current lor 20 through the concentric electrode This procedure subsequently lesions the area and th~s is verified using hlstologv
Assessment of drug-mduced effects on mPFc cell~ GLU was used to activate quiescent neurons (firing rate
181
Research Reports
5-HT3-1ike receptors in the rat medial prefrontal cortex: an electrophysiological study Charles R. Ashby Jr, Yoshlo Mlnabe, Emmehne Edwards and Rex Y Wang Department of Psychtatry and Behaworal Scwnces, State Umverstty of New York at Stony Brook, Stony Brook, NY 11794-8790 (U S A ) (Accepted 11 December 1990) Key words Serotonm3-hke receptor, Medml prefrontal cortex, Mlcro,ontophores]s, 2-Methylserotonm, Serotomn 3 receptor antagomst
In th,s study, we have ]denufied and characterized 5-HTa-hke receptors in the rat medial prefrontal cortex (mPFc), an area with a moderate density of 5-HT3 binding sites, using the techmques of single umt recording and m]cro~ontophoresls The mlcrolontophoresls of the 5-HT3 receptor agomst 2-methyiserotonm (2-Me-5HT), s]mdar to the acUon of 5-HT, produced a current-dependent (10-80 nA) suppression of the firing rate of both spontaneously active and glutamate (GLU)-acuvated (quiescent) mPFc ceils Phenylbiguamde (PBG), another 5-HT3 receptor agomst, suppressed the finng rate of mPFc cells but was less effectwe compared to 2-Me-5HT The continuous lontophoresls (10-20 mm) of 1 M magnesmm chloride markedly attenuated the suppressant effect produced by electrical stlmulaUon of the ascending 5-HT pathway, but &d not alter 2-Me-5HT's action, suggesting that the action of 2-Me-5HT ~s a &rect one The suppressant action of 2-Me-5HT on mPFc cells was blocked by a number of structurally dwerse and selectwe 5-HT3 antagomsts, with a rank order of effectiveness as follows ICS 205930 = (+)-zacopnde > gramsetron = ondansetron = LY 278584 > MDL 72222 Furthermore, the intravenous administration of (+)-zacopnde antagomzed the action of 2-Me-5HT and PBG on mPFc cells In contrast to the effects of the 5-HTs receptors antagomsts, other receptor antagomsts such as metergohne (5-HT]A m l c 2), (+)-pmdolol (5-HT1Am, fl), SCH 23390 (5-HTlc 2, D1), l-sulpmde (D2) or SR 95103 (GABAA) faded to block 2-Me-5HT's action These results combined suggest that 2-Me-5HT's suppressLve action on mPFc cells is medmted &rectly by 5-HT3-hke receptors INTRODUCTION
psychotic properties x8 2z 48 and they also attenuate the
The 5-HT 3 receptor has been pharmacolog]caUy and blochemlcally well defined m the periphery as a result of the d e v e l o p m e n t of selective 5-HT 3 receptor antagonists These receptors me&ate the excitatory response of 5-HT on enteric, afferent and postganghomc sympathetic neurons 43 For example, 5-HT depolar]zes sensory n e u r o n a l
Moreover, it has been shown that the atypical ant]psychotic drug clozapme, but not the typical ant]psychotic
rewarding properties of mcotme
terminals m the heart, releasing acetylchohne from the vagus nerve and producing a reflex bradycardla referred to as the von Bezold-Jansch reflex 9 21 The results from recent studtes have suggested that the 5-HT 3 b m d m g s~te, u n h k e 5-HT~ and 5-HTz sites, ts a hgand gated ,on channel~9 37 55 R a d l o h g a n d binding studies, using 3H-5HT3 antagorests, have shown that 5-HT 3 binding sites are present m the brain 9 33 38 m relatwely high density of 5-HT 3 sites are present m mesocortlcohmblc areas such as the nucleus accumbens, olfactory tubercle, amygdala, entorhmal, cmgulate and frontal cortices 3z 33 Although the exact functional role of central 5-HT 3 binding sites is u n k n o w n , it has been shown that 5-HT 3 receptor antagomsts d~splay ant~emet~c ~5 3s antmnx~ety173~ and ant~-
and m o r p h i n e ]4'27
drugs h a l o p e n d o l and chlorpromazme, possesses 5-HT 3 receptor antagomzmg properties 5 45 We have begun to identify and characterize 5-HT3-hke receptors m the rat brain O u r p r e h m m a r y results suggest that 5-HT3-hke receptors m the med]al prefrontal cortex (mPFc) are different from those m the periphery Thus, mlcro]ontophoresls of 2-Me-5HT produces a currentd e p e n d e n t suppression of m P F c cells' firing and this action ~s blocked by selectwe 5-HT 3 receptor antagomsts ICS 205930 and gramsetron but not by non-selective 5-HT antagomsts such as ( + ) - p l n d o l o l or metergohne z In a d d m o n , we have d e m o n s t r a t e d that the 5-HT3-hke receptors m fronto-cmgulate and e n t o r h m a l cortices might be coupled to p h o s p h o m o s m d e 4 20 In this paper, we attempted to further characterize 5-HT3-hke receptors in the rat mPFc, using the 5-HT 3 agomsts 2-Me-5HT and P B G and a n u m b e r of structurally dwerse 5-HT 3 antagomsts
Correspondence R Y Wang, Department of Psychiatry and Behavioral Sciences, Putnam Hall-South Campus, State Umverslty of New York at Stony Brook, Stony Brook, NY 11794-8790, U S A Fax (1) (516) 632-8953
182 MATERIALS AND METHODS
Ammals and surgery Male albino Sprague-Dawley rats (200-300 g, Tacomc, Germantown, NY) were anesthetized w~th chloral hydrate (400 mg/kg, ~ p ) and placed in a stereotax~c instrument The body temperature oI the rats was maintained at 37-38 °C using an electric heating pad A lateral tall veto was cannulated w~th a 25 g needle for the i v injection of addltmnal anesthetic as reqmred or mject~on of drug soluhon A burr hole was drilled over the mPFc 11-12 0 mm anterior to lambda and 0 3-1 0 mm lateral to the mldhne suture according to the atlas of Paxlnos and Watson'u~
Smgle umt recording and mwrmontophorests Standard extracellular single cell recording and mlcrolontophoret~c techmques were used as previously described4~ so Five or 6 barrel glass m~croplpettes were pulled with a vertical electrode puller (Nanshlge PE2) and broken back under a hght microscope to a dmmeter of 5-8pro The central (recording) barrel was filled with a 2 M NaCI solutmn saturated with 1% Fast Green dye (Fisher) The m vitro ~mpedance of the central barrel was 2-6 M.Q when measured at 135 Hz (Winston BL-1000B) One side barrel was filled with a 2 M NaCI solutton (impedance 5-15 M~2) The remaining side barrels (20-30 MI2) contained a combination of the following compounds (10 mM, pH 4-4 5 in 1 mM NaCI) 2-Me-5HT (2-methylserotonln, Research Biochemical I n c , Natlck, MA, Sandoz. Basel. SwRtzerland and Glaxo. Ware. U K ), PBG (phenylblguamde. Sigma. St Lores. MO). metergohne (Farm Itaha), (_+)-plndolol (Sandoz), l-sulplnde (Ravlzza), SR 95103 ([2-(3carboxypropyl)-3-amlno-4-methyl-6-phenylpyndazmum chloride], SANOFI, Montpelher. France), gramsetron (Beecham Pharmaceuucals, Essex. U K ). ondansetron (Glaxo), (R) and (S)-zacopnde, (+)-zacopnde (Wyeth-Ayerst Co ), LY 278584 (1-(methyl-n-(8methyl-8-azab~cyclo [3.2,1]-oct-3-yl)-IH-mdazole-3-carboxam~de, Eh Lilly C o r p . Indlanapohs, IN), ICS 205930 ([3-2-tropanyl1H-mdazole-3-carboxyhc acid ester]. Sandoz) and MDL 72222 ([1-H-3a-5a-tropan-3-yl]-3,5-d~chlorobenzoate, MerreU Dow Research Institute, Strasbourg, France) The concentratmn of MgC1z and l-glutamate (S~gma) were one molar (pH 4-4 5) and 10 mM (pH 8). respectively MDL 72222 was dissolved m a few drops of 2% lacuc acid plus demmzed, degassed water The remaining compounds were &ssolved m demmzed, degassed water The pH of the drug soluuons were adjusted w~th 0 1 N HCI and 0 1 N NaOH All drug solutions were prepared fresh or stored at -70 °C Retaining currents (posmve for glutamate and negauve for the remaining compounds) of 8-10 nA were apphed to each of the drug barrels to prevent passwe dfffusmn between ejection periods For recording mPFc cells, the m~crop~pette was lowered to touch the cortex and then moved 0 5-3 5 mm below the cortical surface using a hydrauhc m~crodnve Electrical s~gnals were passed through a h~gh ~mpedance amphfier, displayed on an oscilloscope and momtored by an audmamphfier The s~gnals were then fed into a window &scnm~nator (Fmtromcs WDR 420), which was set such that the standard output was triggered by mdw~dual action potentials The integrated rate histograms generated by the analog output of the window dtscnmmator were plotted on a polygraph recorder (Gould 220)
Electrtcal snmulauon of the caudal hnear raphe nucleus A concentric electrode (NE-100, David Kopf Instruments) was implanted m the caudal hnear raphe nucleus (CLI, anterior 2 2 mm to Lambda, lateral 0 0 mm to the mldhne and ventral 6 8 mm to the dura), a structure through which the majority of the ascending 5-HT fibers pass6 36 Pulse trams were dehvered at a rate of 15 Hz, with a 1 ms pulse width and with an intensity of 0 1-1 0 mA using a &gltal pulse generator and an lsopulser (WPI, pulsemaster A300) It has been demonstrated that 10 Hz is an opumal frequency for stimulation of the m~dbram raphe to produce a maximum turnover of 5-HT in the forebraln ~ At the end of each experiment, the final site of the recording mlcrop~pette was marked by the mntophoretlc ejectmn (-25/xA for
20-30 mms) ot 1% Fast Green dye, which results m the deposmon of a Fast Green spot The location of this spot is verified m h~stologlcally prepared tissue The site of the stimulating electrodc ~as marked by passing a 0 05 mA posltwe direct current lor 20 through the concentric electrode This procedure subsequently lesions the area and th~s is verified using hlstologv
Assessment of drug-mduced effects on mPFc cell~ GLU was used to activate quiescent neurons (firing rate
