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Effects of CNS stimulants on the in vivo release of the colocalized transmitters, dopamine and neurotensin, from rat prefrontal cortex
Neuroscience Lelters. 140 (1992) 129 133 {', 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved {1304-3940/92/$ 05.00
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Effects of CNS stimulants on the in vivo release of the colocalized transmitters, dopamine and neurotensin, from rat prefrontal cortex M a t t h e w J. D u r i n g j, A n d r e w J. Bean-" and R o b e r t H. R o t h D~7~artments ~ff'Pharmacology and Ps)'chiatr.v. Yale Universi O' School q/' Medicine, New Haven, ('T06510 ( USA ) (Received 3(/December 1991; Revised version received 16 March 1992: Accepted 16 March 1992) Key words." Dopamine; Neurotensin: Microdialysis; Amphetamine: Methylphenidate; Nomifensine: Colocalization: Prefrontal Cortex The effect of CNS stimulant drugs on the in vivo release of the colocalized neurotransminers dopamine and neurotensin in rat prefrontal cortex was studied using microdialysis. Amphetamine, methylphenidate and nomifensine all increased exlracellular fluid (ECF) levels of dopamine; however, their effects on neurotensin varied. Amphetamine increased both ECF dopamine (514 _+82% of basal) and neurotensin 1350 ± 49% of basal): however, the neurotensin increase lagged behind the increase in dopamine suggesting a possible trans-synaptic effect. Methylphenidate increased both dopamine and neurotensin (226 +_26% and 151 + 14% of basal respectively) co-synchronously, suggesting exocytosis o1 vesicles containing both dopamine and neurotensin. The nomifensine-induced increase in dopamine {202 _+ 23% of basal) was similar to that of methylphenidate, whereas the increase in neurotensin was significantly delayed and of lower magnitude (134 _+20% of basal). These data suggest that dopamine and neurotensin in part share a common releaseable pool in the prefrontal cortex. Moreover, dopamine may act presynaptically to increase neurotensin release and the different behavioral profiles of these psychostimulants may in parl relate to their different effects on neurotensm release.
Dopamine (DA) and neurotensin (NT) are co-localized in a subset of DAergic neurons which project from the ventral tegmental area (VTA) to the medial prefrontal cortex [11, 19, 20, 23]. The vast majority of, if not all. tyrosine hydroxylase (TH) positively stained fibers of the medial prefrontal cortex also contain neurotensin-like immunoreactivity (NT-LI) and all NT-LI fibers also contain TH immunoreactivity [23]. In addition, we have previously shown that DA and NT are co-localized, in part, in the same storage compartment [2]. Moreover, as there are no intrinsic DAergic or NT-containing neurons within the medial prefrontal cortex [19, 20, 23], this meso-cortico-frontal DA NT projection provides an ideal system to investigate the release and interactions of co-localized transmitters within the CNS. There is evidence from the PNS that co-localized transmitters are released together following electrical stimulation, although the relative increase in release of the co-transmitters may vary according to the firing rate and pattern [1]. ~Present address: Neuroendocrine Program, Yale University School of Medicine, P.O. Box 3333, New Haven, CT 06510, USA. :Present address: Department of Histology and Neurobiology, KaroImska Institute, S-104 01 Stockholm, Sweden. CorresT~ondence: R.H. Roth, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510. LISA.
Recent data from our laboratory suggest that a similar relationship exists within the CNS [3, 6]. The interactions between co-localized transmitters can be further characterized by determining the effect of drugs, whose known actions include neurotransmitter-releasing and uptakeinhibiting properties, on the extracellular levels of colocalized transmitters within the CNS. The CNS stimulants amphetamine, methylphenidate and nomifensine act in part to increase synaptic and extracellular fluid (ECF) levels of DA [8, 9, 13, 22, 25, 26], This enhancement of DAergic neurotransmission is believed to underlie their stimulant properties. These agents, however, both have different mechanisms by which they increase ECF DA and different behavioral profiles. Amphetamine acts to increase DA release from a newly synthesized, reserpine-insensitive pool: whereas methylphenidate and nomifensine increase DA release from a reserpine-sensitive pool: in addition, all these stimulants possess DA-uptake inhibitory activity [7, 14]. These drugs, therefore, provide a means to explore DANT interactions within the medial prefrontal cortex and may differentiate co-release from trans-synaptic effects. We have previously characterized co-release of NT and DA from both rat striatum and medial prefrontal cortex using the technique of brain microdialysis [3 6]. Microdialysis is well suited lk)r the simultaneous moni-
130
toring of both peptides and monoamines, and in this study we have applied the method to investigate the pharmacological interactions of cortical DA-NT. Male Sprague-Dawley rats (260-320 g) were housed in a controlled lighting and humidity animal facility and allowed ad libitum access to food (Agway 3000 rat chow) and water. On the morning of the experiment, rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a Kopf stereotactic frame. Rats were maintained at a stable temperature using a homeostatically controlled heating pad and anesthesia was maintained at stable levels by administering small additional doses of chloral hydrate as necessary. The skull was exposed and a hole drilled above the right prefrontal cortex at Paxinos and Watson coordinates AP +3.0, L 2.1 [17]. Microdialysis probes of cannula design [3] and 4 mm exposed membrane were implanted at a 13° medially directed angle at a dorsoventral depth of 5.7 mm (flat skull). The probes were continuously perfused with anartificial ECF [15] at 2.3 pl/min and samples were collected at 20 min intervals. Following a minimum of 120 min after probe implantation, a stable baseline of DA release was established when three consecutive samples varied in their DA concentration by less than 20%. Drugs, dissolved in a 0.9% saline solution, or 0.9% saline alone were administered intraperitoneally. All drugs were given in a volume of 2 ml/kg via a 25 gauge needle. Groups of rats (n=5) received either D-amphetamine sulfate (Sigma, MO) 1 mg/ kg; methylphenidate (Sigma, MO) 20 mg/kg; or nomifensine (Hoechst Roussel Pharm., N J) 10 mg/kg. Dialysis was continued for 120 min following drug or saline administration. At the completion of the experiment, rats were euthanatized and probe localization verified by microscopic examination of coronal sections stained with Neutral red. The 20 rain dialysate samples (total volume 46pl) were split with 20pl analyzed for DA content using HPLC-EC [3] and the remaining 26 pl assayed for NT using radioimmunoassay. In brief, 20 pl of the dialysate was directly injected via a Rheodyne sample loop onto a 10 cm by 2.1 mm narrow bore column, home-packed with 3/lm ods particles. The mobile phase used to achieve separation was a 70 mM phosphate buffer with 2.5 mM sodium octyl phosphate, 0.1 mM EDTA, pH 5.8; methanol 15% v/v. This mobile phase was pumped isocratically using an Altex 110 double piston pump with dual in-line SSI pump suppressors in series at a flow rate of &25 ml/min. The dopamine was selectively retained with all acidic metabolites etuting within the solvent front. An LC-3 (Bioanalytical Systems, West Lafayette, IN) electrochemical detector with a low volume cell and an applied voltage of 0.7 V vs. a Ag/AgC1 reference electrode was used for peak detection. Chromatograms were completed
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Fig. 1. The effects of amphetamine on the in vivo release of DA and NT from the prefrontal cortex. Dialysis probes were implanted into the prefrontal cortex of anesthetized rats. Following the establishment of a stable baseline, either D-amphetamine sulphate (1 mg/kg, i.p.) or saline (0.9%, 2 ml/kg, i.p.) was administered. Twenty-rain samples were analyzed for both DA and NT content (see text). Basal DA release, calculated from 3 consecutive samples preceding drug administration, was 0.58 +_0.08 fmol/min while basal NT was 0.031 _+01005 fmol/min in the amphetamine-treated animals (n=5) and 0.56_+ 0.07 fmot/min and 0.027 + 0.004 fmol/min for DA and NT. respectively, in control animals (n=5). Data are represented as the mean _+S.E.M. relative to basal values. *P<0.05 ANOVA with repeated measures.
within 8 min and peak heights compared with standards for quantification. The sample for NT assay was immediately frozen on dry ice and stored at -70°C for a maximum of 2 weeks prior to assay. The NT was assayed using a highly sensitive, non-equilibrium RIA as previously described [4]. In brief, sodium phosphate buffer (50 mM, pH 7.4) containing EDTA (10 mM), bovine serum albumin (0.1%), and sodium azide (0.02%) was added to unknowns and standards along with NT antiserum that recognizes the COOH terminal of NT [4, 12]. The samples were then incubated at 4°C for 24 h. at which time [125I]NT was added, followed by an additional 24 h incubation period at 4°C. Bound and free [125I]NT were separated using a secondary antibody, and bound and free ratios were corrected for non-specific binding, which was typically 1 2% of total binding. The ICs0 for synthetic NT was 4.1 + 0.7 fmol. while the detection limit of the assay was 0.15 _+0.026 fmol. We have previously shown that this assay recognizes authentic NT from rat brain dialysate by HPLC separation of dialysates [4]. Moreover, the stimulated release of NT was dependent on extracellular levels of calcium [3, 4]. DA levels increased rapidly following the systemic injection of amphetamine with peak levels reached at 40 rain (Fig. 1). The peak elevation of DA at 514 __ 82% of baseline values was less than that generally observed in the striatum [9, 13.24] but consistent with previous reports of amphetamine action in the medial prefrontal
131 cortex [16, 22]. ECF neurotensin also increased following amphetamine; however, the response lagged behind that of DA by 20 min with the peak increase of 350 _+ 49% above baseline levels reached at 60 min post drug administration. Both DA and NT had returned to basal values at 100 min. In the group of rats which received methylphenidate (Fig. 2), DA concentrations increased in the first 20 rain interval after drug administration and reached peak levels of 226 +_ 26% of baseline at 40 rain, returning to baseline values at 80 rain. N T rose in parallel with DA in the first 20 min collect (to a peak of 151 + 14% of basal); however, levels returned to baseline values in all subsequent samples. In the nomifensine-treated rats, DA levels rose with a peak increase to 202 +_ 23% of basal levels reached at 60 rain following drug administration. N T levels were unchanged for the first 80 rain but increased at the 100 and 120 min time points (by 128 + 12% and 134 + 20% of basal, respectively). These data reveal that systemic administration of CNS stimulants affects both DA and N T ECF concentrations in rat medial prefrontal cortex. Co-localized transmitters, including monoamines and neuropeptides, are typically found within different intracellular storage compartments. Agents which act preferentially on specific storage compartments would therefore be expected to have differential effects on the release and extracellular concentrations of these co-localized transmitters. Our data confirm this hypothesis as DA and NT clearly have different release kinetics following the administration of these CNS stimulants. However, our data also suggest that these transmitters may in part share a common releasable compartment, as the initial time course of the methylphenidate-induced increase in extracellular levels of both transmitters was identical. These data support our previous results which demonstrated a common reserpine-sensitive pool of DA and N T in the rat medial prel¥ontal cortex [2]. The magnitude of the increase in extracellular DA following methylphenidate is, however, much greater than that of the N T increase. This is likely to be due to the much greater concentration of DA within these reserpine- and methylphenidate-sensitive vesicles, as well as the effect of methylphenidate to inhibit DA uptake, thereby further increasing ECF levels. The amphetamine data clearly demonstrate a lag in the response of NT as compared to the DA release. This suggests that the amphetamine-releaseable DA pool, predominantly newly synthesized reserpine-insensitive cytosolic DA, is released by these neurons independently from the co-localized transmitter NT. Although the effect of amphetamine on NT release may be direct, it is likely that trans-synaptic and specifically presynaptic of-
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Fig. 2. The effects of methylphenidate on the in vivo release of DA and N T t'rom the prefrontal cortex. Dialysis probes were implanted into the prefrontal cortex of anesthetized rats. Following the establishment of a stable baseline, methylphenidate (20 mg/kg, i.p.) was administered. Twenty-min samples were analyzed for both DA and NT content (see text). Basal DA release, calculated from 3 consecutive samples preceding drug administration, was 0.60 +_ 0.07 fmol/min while basal N F was 0.03 + 0.004 fmol/min. Data are represented as the mean + S.E.M. relative to basal ~alues 02-5). *P<0.05 ANOVA with repeated measures.
fects may be largely responsible for the effect. The nomifensine data also supports this potential mechanism. The peak extracellular DA concentrations reached following nomifensine (10 mg/kg) are approximately 30% of the amphetamine-stimulated peak levels. Likewise, the increase in N T following nomifensine is also relatively small, only 34% above basal values and shows even a greater delay compared to the time course of the DA release. This suggests that a DA trans-synaptic mechanism may also account for the nomifensine effect on NT. The reduced potency of nomifensine in elevating ECF DA may also be in part responsible for the greater lag time and the small effect on NT release. The late rise in extracellular N T is also consistent with a normal drift in N T concentrations during the experiment (a 20% fluctuation in N T levels was observed following saline injection in Fig. 1) and may therefore be unrelated to the nomifensine administration. In addition, amphetamine may also have some weak action on the vesicular compartment, particularly at higher concentrations [7, 25]. The more rapid increase in NT with amphetamine may therefore result in part from its release from this grantdar pool. We have recently reported the in vivo presence of functional release-modulating DA autoreceptors in the rat medial pre-frontal cortex [5]. The activation of these receptors by the specific autoreceptor agonist EMD-23448 or low dose apomorphine produced a decrease in extracellular DA as measured by microdialysis and all hTcrease in NT release. Thus, nerve terminal autoreceptors in the prefrontal cortex appear to exert reciprocal effects
132
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Fig. 3. The effects of nomifensine on the in vivo release of DA and NT from the prefrontal cortex. Dialysis probes were implanted into the prefrontal cortex of anesthetized rats. Following the establishment of a stable baseline, nomifensine (10 mg/kg, i.p.) was administered. Twentymin samples were analyzed for both DA and NT content (see text). Basal DA release, calculated from 3 consecutive samples preceding drug administration, was 0.65 + 0.08 fmol/min while basal NT was 0.035 _+0.005 fmol/min. Data are represented as the mean _+S.E.M. relative to basal values (n=5). *P<0.05 ANOVA with repeated measures.
on DA and NT release. The delayed increase in NT following the administration of amphetamine and nomifensine is consistent with this DA autoreceptor-mediated effect elicited by the elevated synaptic DA. The co-release of neurotensin with DA following psychostimulant administration is likely to modulate the postsynaptic effects of DA. DA predominantly generates inhibitory postsynaptic potentials when applied iontophoretically in the prefrontal cortex [21]. In contrast, NT probably has excitatory postsynaptic activity [18]. The functional effect of such co-release is further complicated by the presynaptic effect of NT on DA cells to facilitate DA release [10]. Thus, the co-release of DA and NT may under certain situations facilitate DAergic neurotransmission via a NT presynaptic action, whereas an excitatory postsynaptic effect of NT may disinhibit DA neurotransmission under other conditions. The stimulatory effect of DA on NT release via presynapatic receptors is likely to further modulate net transmission. It is probable that the action of these psychomotor stimulants is not simply due to their effects on monoaminergic transmission and that NT and perhaps other co-localized peptide transmitters, such as cholecystokinin, may also play a significant role in their behavioral effects. These studies were supported in part by USPHS Grants MH-14092, GM-07324 and NS-28227. t Bartfai, T., Iverfelt, K., Fisone, G. and Serfozo, P., Regulation of the release of coexisting neurotransminers, Annu. Rev. Pharmacol. Toxicol., 28 (1988) 285 310.
2 Bean, A.J., Adrian, T.E., Modtin, 1.M. and Roth, R.H., Dopamine and neurotensin storage in colocalized neuronal populations, J. Pharmacol. Exp. Ther., 249 (1989) 681-687. 3 Bean, A.J., During, M.J. and Roth, R.H., Stimulation-induced release of coexistent neurotransmitters in the prefrontal cortex: an in vivo microdialysis study of dopamine and neurotensin release, J. Neurochem., 53 (1989)655-657. 4 Bean, A.J., During, M.J., Deutch, A.Y. and Roth, R,H., The effects of dopamine depletion on striatal neurotensin: biochemical and immunohistochemical studies, J. Neurosci., 9 (1989) 4430 4438. 5 Bean, A.J., During, M J . and Roth, R.H., Effects of dopamine autoreceptor stimulation on the release of colocalized transmitters: in vivo release of dopamine and neurotensin from rat prefrontal cortex, Neurosci. Lett., 108 (1990) 143-148. 6 Bean, A.J., During, M.J. and Roth, R.H., Extracellular dopamine and neurotensin in rat prefrontal cortex in vivo: effects of median tbrebrain bundle stimulation frequency, stimulation pattern, and dopamine autoreceptors, J. Neurosci., 11 (1991) 2694-2702. 7 Braestrup, C , Biochemical differentiation of" amphetamine vs. methylphenidate and nomifensine in rats. J. Pharm. Pharmacol., 29 (1977) 463-470. 8 Butcher, S.R, Liptrot, J. and Arbuthnott, G.W., Characterisation of methylphenidate and nomifeusine induced dopamine release in rat striatum using in vivo microdialysis, Neurosci. Lett, 122 (1991/ 245 248. 9 Butcher. S.R, Fairbrother, I.S., Kelly, J.S. and Arbuthnott, G.W., Amphetamine-induced dopamine release in the rat striatum: An in vivo microdialysis study, J. Neurochem., 50 (1988) 346- 355. 10 Heiter, E.. Boireau, A., Dubedat, R and Blanchard, J.C., Neurotensin effects on evoked release of dopamine in slices from striatum, nucleus accumbens, and prefrontal cortex in rat, Naunyn-Schmiedeberg's Arch. Pharmacol., 337 (1988) 13 17. t I Hokfelt, T., Everitt, B.J., Theodorsson-Norheim, E: and Goldstein, M., Occurrence of neurotensin-like immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons, J. Comp. Neurol., 222 (1984) 543- 559. 12 Jennes, L., Stumpf, W.E. and Kalivas, P.W., Neurotensin: topographical distribution in rat brain by immunohistochemistry, J. Comp. Neurol.. 210 (1982) 211-224. 13 KuczenskL R. and Segal, R.. Concomitant characterization of behavioral and striatal neurotransmitter response to amphetamine using in vivo microdialysis, J. Neurosci., 9 (1989) 2051-2065. 14 McMillen, B.A., CNS stimulants: two distinct mechanisms of action for amphetamine-like drugs, Trends Pharmacol. Sci., (1983) 429 432. 15 Moghaddam, B. and Bunney, B.S., Ionic composition of microdialysis perfusing solution alters the pharmacological responsiveness and basal outflow of striatal dopamine, J. Neurochem., 53 (1989) 652 654. 16 Moghaddam, B., Roth, R.H. and Bunney, B.S., Characterization of dopamine release in the rat medial prefrontal cortex by in vivo microdialysis: comparison to the striatum, Neuroscience, 36 (1990) 669 676. 17 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986. 18 Pinnock, R.D., Neurotensin depolarizes substantia nigra dopamine neurons, Brain Res., 338 (1985) 151-154. 19 Seroogy, K.B,, Ceccatelli, S., Shalling, M., Hokfelt, T., Frey, R, Dockray, G., Buchan, A. and Goldstein, M., A subpopulation of dopaminergic neurons in the rat ventral mesencephalon contain both neurotensin and cholecystokinin, Brain Res., 455 (1988) 8 8 98.
133 20 Seroogy, K.B., Mehta, A. and Fallon, J.H., Neurotensin and cholecystokinin coexistence within neurons of the ventral mesencephalon: projections to the forebrain, Exp. Brain Res.. 68 (1987) 277 289. 21 Sesack, S.R. and Bunney, B.S., Pharmacological characterization of the receptor mediating electrophysiological responses to dopamine in the rat medial prefrontal cortex: a microiontophoretic study, J. Pharmacol. Exp. Ther., 248 (1989) 1323 1333. 22 Sharp, T., Zetterstrom, T., Ljunberg. T. and Ungerstedt, U., A direct comparison of amphetamine-induced behaviors and regional brain dopamine release in the rat using inlracerebral dialysis, Brain Res.. 410 (1987) 322 330.
23 Studler, J.M., Kitabgi, E, Tramu, G., Herve, D., Glowinski, J. and Tassin, J.P., Extensive colocalization of neurotensin with dopamine in rat meso-cortico-frontal dopaminergic neurons, Neuropeptides, 11 (1988) 95 100. 24 Zetterstrom, T., Sharp, T., Marsden, C.A. and Ungerstedt, L!., In vivo measurement of dopamine and its metabolites by intracerebral microdialysis: changes after D-amphetamine, J. Neurochem., 41 (1983) 1769 1773. 25 Zetterstrom, T., Sharp, T. and Ungerstedt, U., Further evaluation of the mechanism by which amphetamine reduces striatal dopamine metabolism: an in vivo brain dialysis study, Eur. J. Pharmacol., 132 (1986) 1 9.
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Effects of CNS stimulants on the in vivo release of the colocalized transmitters, dopamine and neurotensin, from rat prefrontal cortex M a t t h e w J. D u r i n g j, A n d r e w J. Bean-" and R o b e r t H. R o t h D~7~artments ~ff'Pharmacology and Ps)'chiatr.v. Yale Universi O' School q/' Medicine, New Haven, ('T06510 ( USA ) (Received 3(/December 1991; Revised version received 16 March 1992: Accepted 16 March 1992) Key words." Dopamine; Neurotensin: Microdialysis; Amphetamine: Methylphenidate; Nomifensine: Colocalization: Prefrontal Cortex The effect of CNS stimulant drugs on the in vivo release of the colocalized neurotransminers dopamine and neurotensin in rat prefrontal cortex was studied using microdialysis. Amphetamine, methylphenidate and nomifensine all increased exlracellular fluid (ECF) levels of dopamine; however, their effects on neurotensin varied. Amphetamine increased both ECF dopamine (514 _+82% of basal) and neurotensin 1350 ± 49% of basal): however, the neurotensin increase lagged behind the increase in dopamine suggesting a possible trans-synaptic effect. Methylphenidate increased both dopamine and neurotensin (226 +_26% and 151 + 14% of basal respectively) co-synchronously, suggesting exocytosis o1 vesicles containing both dopamine and neurotensin. The nomifensine-induced increase in dopamine {202 _+ 23% of basal) was similar to that of methylphenidate, whereas the increase in neurotensin was significantly delayed and of lower magnitude (134 _+20% of basal). These data suggest that dopamine and neurotensin in part share a common releaseable pool in the prefrontal cortex. Moreover, dopamine may act presynaptically to increase neurotensin release and the different behavioral profiles of these psychostimulants may in parl relate to their different effects on neurotensm release.
Dopamine (DA) and neurotensin (NT) are co-localized in a subset of DAergic neurons which project from the ventral tegmental area (VTA) to the medial prefrontal cortex [11, 19, 20, 23]. The vast majority of, if not all. tyrosine hydroxylase (TH) positively stained fibers of the medial prefrontal cortex also contain neurotensin-like immunoreactivity (NT-LI) and all NT-LI fibers also contain TH immunoreactivity [23]. In addition, we have previously shown that DA and NT are co-localized, in part, in the same storage compartment [2]. Moreover, as there are no intrinsic DAergic or NT-containing neurons within the medial prefrontal cortex [19, 20, 23], this meso-cortico-frontal DA NT projection provides an ideal system to investigate the release and interactions of co-localized transmitters within the CNS. There is evidence from the PNS that co-localized transmitters are released together following electrical stimulation, although the relative increase in release of the co-transmitters may vary according to the firing rate and pattern [1]. ~Present address: Neuroendocrine Program, Yale University School of Medicine, P.O. Box 3333, New Haven, CT 06510, USA. :Present address: Department of Histology and Neurobiology, KaroImska Institute, S-104 01 Stockholm, Sweden. CorresT~ondence: R.H. Roth, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510. LISA.
Recent data from our laboratory suggest that a similar relationship exists within the CNS [3, 6]. The interactions between co-localized transmitters can be further characterized by determining the effect of drugs, whose known actions include neurotransmitter-releasing and uptakeinhibiting properties, on the extracellular levels of colocalized transmitters within the CNS. The CNS stimulants amphetamine, methylphenidate and nomifensine act in part to increase synaptic and extracellular fluid (ECF) levels of DA [8, 9, 13, 22, 25, 26], This enhancement of DAergic neurotransmission is believed to underlie their stimulant properties. These agents, however, both have different mechanisms by which they increase ECF DA and different behavioral profiles. Amphetamine acts to increase DA release from a newly synthesized, reserpine-insensitive pool: whereas methylphenidate and nomifensine increase DA release from a reserpine-sensitive pool: in addition, all these stimulants possess DA-uptake inhibitory activity [7, 14]. These drugs, therefore, provide a means to explore DANT interactions within the medial prefrontal cortex and may differentiate co-release from trans-synaptic effects. We have previously characterized co-release of NT and DA from both rat striatum and medial prefrontal cortex using the technique of brain microdialysis [3 6]. Microdialysis is well suited lk)r the simultaneous moni-
130
toring of both peptides and monoamines, and in this study we have applied the method to investigate the pharmacological interactions of cortical DA-NT. Male Sprague-Dawley rats (260-320 g) were housed in a controlled lighting and humidity animal facility and allowed ad libitum access to food (Agway 3000 rat chow) and water. On the morning of the experiment, rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a Kopf stereotactic frame. Rats were maintained at a stable temperature using a homeostatically controlled heating pad and anesthesia was maintained at stable levels by administering small additional doses of chloral hydrate as necessary. The skull was exposed and a hole drilled above the right prefrontal cortex at Paxinos and Watson coordinates AP +3.0, L 2.1 [17]. Microdialysis probes of cannula design [3] and 4 mm exposed membrane were implanted at a 13° medially directed angle at a dorsoventral depth of 5.7 mm (flat skull). The probes were continuously perfused with anartificial ECF [15] at 2.3 pl/min and samples were collected at 20 min intervals. Following a minimum of 120 min after probe implantation, a stable baseline of DA release was established when three consecutive samples varied in their DA concentration by less than 20%. Drugs, dissolved in a 0.9% saline solution, or 0.9% saline alone were administered intraperitoneally. All drugs were given in a volume of 2 ml/kg via a 25 gauge needle. Groups of rats (n=5) received either D-amphetamine sulfate (Sigma, MO) 1 mg/ kg; methylphenidate (Sigma, MO) 20 mg/kg; or nomifensine (Hoechst Roussel Pharm., N J) 10 mg/kg. Dialysis was continued for 120 min following drug or saline administration. At the completion of the experiment, rats were euthanatized and probe localization verified by microscopic examination of coronal sections stained with Neutral red. The 20 rain dialysate samples (total volume 46pl) were split with 20pl analyzed for DA content using HPLC-EC [3] and the remaining 26 pl assayed for NT using radioimmunoassay. In brief, 20 pl of the dialysate was directly injected via a Rheodyne sample loop onto a 10 cm by 2.1 mm narrow bore column, home-packed with 3/lm ods particles. The mobile phase used to achieve separation was a 70 mM phosphate buffer with 2.5 mM sodium octyl phosphate, 0.1 mM EDTA, pH 5.8; methanol 15% v/v. This mobile phase was pumped isocratically using an Altex 110 double piston pump with dual in-line SSI pump suppressors in series at a flow rate of &25 ml/min. The dopamine was selectively retained with all acidic metabolites etuting within the solvent front. An LC-3 (Bioanalytical Systems, West Lafayette, IN) electrochemical detector with a low volume cell and an applied voltage of 0.7 V vs. a Ag/AgC1 reference electrode was used for peak detection. Chromatograms were completed
600
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Time (minutes)
Fig. 1. The effects of amphetamine on the in vivo release of DA and NT from the prefrontal cortex. Dialysis probes were implanted into the prefrontal cortex of anesthetized rats. Following the establishment of a stable baseline, either D-amphetamine sulphate (1 mg/kg, i.p.) or saline (0.9%, 2 ml/kg, i.p.) was administered. Twenty-rain samples were analyzed for both DA and NT content (see text). Basal DA release, calculated from 3 consecutive samples preceding drug administration, was 0.58 +_0.08 fmol/min while basal NT was 0.031 _+01005 fmol/min in the amphetamine-treated animals (n=5) and 0.56_+ 0.07 fmot/min and 0.027 + 0.004 fmol/min for DA and NT. respectively, in control animals (n=5). Data are represented as the mean _+S.E.M. relative to basal values. *P<0.05 ANOVA with repeated measures.
within 8 min and peak heights compared with standards for quantification. The sample for NT assay was immediately frozen on dry ice and stored at -70°C for a maximum of 2 weeks prior to assay. The NT was assayed using a highly sensitive, non-equilibrium RIA as previously described [4]. In brief, sodium phosphate buffer (50 mM, pH 7.4) containing EDTA (10 mM), bovine serum albumin (0.1%), and sodium azide (0.02%) was added to unknowns and standards along with NT antiserum that recognizes the COOH terminal of NT [4, 12]. The samples were then incubated at 4°C for 24 h. at which time [125I]NT was added, followed by an additional 24 h incubation period at 4°C. Bound and free [125I]NT were separated using a secondary antibody, and bound and free ratios were corrected for non-specific binding, which was typically 1 2% of total binding. The ICs0 for synthetic NT was 4.1 + 0.7 fmol. while the detection limit of the assay was 0.15 _+0.026 fmol. We have previously shown that this assay recognizes authentic NT from rat brain dialysate by HPLC separation of dialysates [4]. Moreover, the stimulated release of NT was dependent on extracellular levels of calcium [3, 4]. DA levels increased rapidly following the systemic injection of amphetamine with peak levels reached at 40 rain (Fig. 1). The peak elevation of DA at 514 __ 82% of baseline values was less than that generally observed in the striatum [9, 13.24] but consistent with previous reports of amphetamine action in the medial prefrontal
131 cortex [16, 22]. ECF neurotensin also increased following amphetamine; however, the response lagged behind that of DA by 20 min with the peak increase of 350 _+ 49% above baseline levels reached at 60 min post drug administration. Both DA and NT had returned to basal values at 100 min. In the group of rats which received methylphenidate (Fig. 2), DA concentrations increased in the first 20 rain interval after drug administration and reached peak levels of 226 +_ 26% of baseline at 40 rain, returning to baseline values at 80 rain. N T rose in parallel with DA in the first 20 min collect (to a peak of 151 + 14% of basal); however, levels returned to baseline values in all subsequent samples. In the nomifensine-treated rats, DA levels rose with a peak increase to 202 +_ 23% of basal levels reached at 60 rain following drug administration. N T levels were unchanged for the first 80 rain but increased at the 100 and 120 min time points (by 128 + 12% and 134 + 20% of basal, respectively). These data reveal that systemic administration of CNS stimulants affects both DA and N T ECF concentrations in rat medial prefrontal cortex. Co-localized transmitters, including monoamines and neuropeptides, are typically found within different intracellular storage compartments. Agents which act preferentially on specific storage compartments would therefore be expected to have differential effects on the release and extracellular concentrations of these co-localized transmitters. Our data confirm this hypothesis as DA and NT clearly have different release kinetics following the administration of these CNS stimulants. However, our data also suggest that these transmitters may in part share a common releasable compartment, as the initial time course of the methylphenidate-induced increase in extracellular levels of both transmitters was identical. These data support our previous results which demonstrated a common reserpine-sensitive pool of DA and N T in the rat medial prel¥ontal cortex [2]. The magnitude of the increase in extracellular DA following methylphenidate is, however, much greater than that of the N T increase. This is likely to be due to the much greater concentration of DA within these reserpine- and methylphenidate-sensitive vesicles, as well as the effect of methylphenidate to inhibit DA uptake, thereby further increasing ECF levels. The amphetamine data clearly demonstrate a lag in the response of NT as compared to the DA release. This suggests that the amphetamine-releaseable DA pool, predominantly newly synthesized reserpine-insensitive cytosolic DA, is released by these neurons independently from the co-localized transmitter NT. Although the effect of amphetamine on NT release may be direct, it is likely that trans-synaptic and specifically presynaptic of-
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Fig. 2. The effects of methylphenidate on the in vivo release of DA and N T t'rom the prefrontal cortex. Dialysis probes were implanted into the prefrontal cortex of anesthetized rats. Following the establishment of a stable baseline, methylphenidate (20 mg/kg, i.p.) was administered. Twenty-min samples were analyzed for both DA and NT content (see text). Basal DA release, calculated from 3 consecutive samples preceding drug administration, was 0.60 +_ 0.07 fmol/min while basal N F was 0.03 + 0.004 fmol/min. Data are represented as the mean + S.E.M. relative to basal ~alues 02-5). *P<0.05 ANOVA with repeated measures.
fects may be largely responsible for the effect. The nomifensine data also supports this potential mechanism. The peak extracellular DA concentrations reached following nomifensine (10 mg/kg) are approximately 30% of the amphetamine-stimulated peak levels. Likewise, the increase in N T following nomifensine is also relatively small, only 34% above basal values and shows even a greater delay compared to the time course of the DA release. This suggests that a DA trans-synaptic mechanism may also account for the nomifensine effect on NT. The reduced potency of nomifensine in elevating ECF DA may also be in part responsible for the greater lag time and the small effect on NT release. The late rise in extracellular N T is also consistent with a normal drift in N T concentrations during the experiment (a 20% fluctuation in N T levels was observed following saline injection in Fig. 1) and may therefore be unrelated to the nomifensine administration. In addition, amphetamine may also have some weak action on the vesicular compartment, particularly at higher concentrations [7, 25]. The more rapid increase in NT with amphetamine may therefore result in part from its release from this grantdar pool. We have recently reported the in vivo presence of functional release-modulating DA autoreceptors in the rat medial pre-frontal cortex [5]. The activation of these receptors by the specific autoreceptor agonist EMD-23448 or low dose apomorphine produced a decrease in extracellular DA as measured by microdialysis and all hTcrease in NT release. Thus, nerve terminal autoreceptors in the prefrontal cortex appear to exert reciprocal effects
132
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Fig. 3. The effects of nomifensine on the in vivo release of DA and NT from the prefrontal cortex. Dialysis probes were implanted into the prefrontal cortex of anesthetized rats. Following the establishment of a stable baseline, nomifensine (10 mg/kg, i.p.) was administered. Twentymin samples were analyzed for both DA and NT content (see text). Basal DA release, calculated from 3 consecutive samples preceding drug administration, was 0.65 + 0.08 fmol/min while basal NT was 0.035 _+0.005 fmol/min. Data are represented as the mean _+S.E.M. relative to basal values (n=5). *P<0.05 ANOVA with repeated measures.
on DA and NT release. The delayed increase in NT following the administration of amphetamine and nomifensine is consistent with this DA autoreceptor-mediated effect elicited by the elevated synaptic DA. The co-release of neurotensin with DA following psychostimulant administration is likely to modulate the postsynaptic effects of DA. DA predominantly generates inhibitory postsynaptic potentials when applied iontophoretically in the prefrontal cortex [21]. In contrast, NT probably has excitatory postsynaptic activity [18]. The functional effect of such co-release is further complicated by the presynaptic effect of NT on DA cells to facilitate DA release [10]. Thus, the co-release of DA and NT may under certain situations facilitate DAergic neurotransmission via a NT presynaptic action, whereas an excitatory postsynaptic effect of NT may disinhibit DA neurotransmission under other conditions. The stimulatory effect of DA on NT release via presynapatic receptors is likely to further modulate net transmission. It is probable that the action of these psychomotor stimulants is not simply due to their effects on monoaminergic transmission and that NT and perhaps other co-localized peptide transmitters, such as cholecystokinin, may also play a significant role in their behavioral effects. These studies were supported in part by USPHS Grants MH-14092, GM-07324 and NS-28227. t Bartfai, T., Iverfelt, K., Fisone, G. and Serfozo, P., Regulation of the release of coexisting neurotransminers, Annu. Rev. Pharmacol. Toxicol., 28 (1988) 285 310.
2 Bean, A.J., Adrian, T.E., Modtin, 1.M. and Roth, R.H., Dopamine and neurotensin storage in colocalized neuronal populations, J. Pharmacol. Exp. Ther., 249 (1989) 681-687. 3 Bean, A.J., During, M.J. and Roth, R.H., Stimulation-induced release of coexistent neurotransmitters in the prefrontal cortex: an in vivo microdialysis study of dopamine and neurotensin release, J. Neurochem., 53 (1989)655-657. 4 Bean, A.J., During, M.J., Deutch, A.Y. and Roth, R,H., The effects of dopamine depletion on striatal neurotensin: biochemical and immunohistochemical studies, J. Neurosci., 9 (1989) 4430 4438. 5 Bean, A.J., During, M J . and Roth, R.H., Effects of dopamine autoreceptor stimulation on the release of colocalized transmitters: in vivo release of dopamine and neurotensin from rat prefrontal cortex, Neurosci. Lett., 108 (1990) 143-148. 6 Bean, A.J., During, M.J. and Roth, R.H., Extracellular dopamine and neurotensin in rat prefrontal cortex in vivo: effects of median tbrebrain bundle stimulation frequency, stimulation pattern, and dopamine autoreceptors, J. Neurosci., 11 (1991) 2694-2702. 7 Braestrup, C , Biochemical differentiation of" amphetamine vs. methylphenidate and nomifensine in rats. J. Pharm. Pharmacol., 29 (1977) 463-470. 8 Butcher, S.R, Liptrot, J. and Arbuthnott, G.W., Characterisation of methylphenidate and nomifeusine induced dopamine release in rat striatum using in vivo microdialysis, Neurosci. Lett, 122 (1991/ 245 248. 9 Butcher. S.R, Fairbrother, I.S., Kelly, J.S. and Arbuthnott, G.W., Amphetamine-induced dopamine release in the rat striatum: An in vivo microdialysis study, J. Neurochem., 50 (1988) 346- 355. 10 Heiter, E.. Boireau, A., Dubedat, R and Blanchard, J.C., Neurotensin effects on evoked release of dopamine in slices from striatum, nucleus accumbens, and prefrontal cortex in rat, Naunyn-Schmiedeberg's Arch. Pharmacol., 337 (1988) 13 17. t I Hokfelt, T., Everitt, B.J., Theodorsson-Norheim, E: and Goldstein, M., Occurrence of neurotensin-like immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons, J. Comp. Neurol., 222 (1984) 543- 559. 12 Jennes, L., Stumpf, W.E. and Kalivas, P.W., Neurotensin: topographical distribution in rat brain by immunohistochemistry, J. Comp. Neurol.. 210 (1982) 211-224. 13 KuczenskL R. and Segal, R.. Concomitant characterization of behavioral and striatal neurotransmitter response to amphetamine using in vivo microdialysis, J. Neurosci., 9 (1989) 2051-2065. 14 McMillen, B.A., CNS stimulants: two distinct mechanisms of action for amphetamine-like drugs, Trends Pharmacol. Sci., (1983) 429 432. 15 Moghaddam, B. and Bunney, B.S., Ionic composition of microdialysis perfusing solution alters the pharmacological responsiveness and basal outflow of striatal dopamine, J. Neurochem., 53 (1989) 652 654. 16 Moghaddam, B., Roth, R.H. and Bunney, B.S., Characterization of dopamine release in the rat medial prefrontal cortex by in vivo microdialysis: comparison to the striatum, Neuroscience, 36 (1990) 669 676. 17 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, New York, 1986. 18 Pinnock, R.D., Neurotensin depolarizes substantia nigra dopamine neurons, Brain Res., 338 (1985) 151-154. 19 Seroogy, K.B,, Ceccatelli, S., Shalling, M., Hokfelt, T., Frey, R, Dockray, G., Buchan, A. and Goldstein, M., A subpopulation of dopaminergic neurons in the rat ventral mesencephalon contain both neurotensin and cholecystokinin, Brain Res., 455 (1988) 8 8 98.
133 20 Seroogy, K.B., Mehta, A. and Fallon, J.H., Neurotensin and cholecystokinin coexistence within neurons of the ventral mesencephalon: projections to the forebrain, Exp. Brain Res.. 68 (1987) 277 289. 21 Sesack, S.R. and Bunney, B.S., Pharmacological characterization of the receptor mediating electrophysiological responses to dopamine in the rat medial prefrontal cortex: a microiontophoretic study, J. Pharmacol. Exp. Ther., 248 (1989) 1323 1333. 22 Sharp, T., Zetterstrom, T., Ljunberg. T. and Ungerstedt, U., A direct comparison of amphetamine-induced behaviors and regional brain dopamine release in the rat using inlracerebral dialysis, Brain Res.. 410 (1987) 322 330.
23 Studler, J.M., Kitabgi, E, Tramu, G., Herve, D., Glowinski, J. and Tassin, J.P., Extensive colocalization of neurotensin with dopamine in rat meso-cortico-frontal dopaminergic neurons, Neuropeptides, 11 (1988) 95 100. 24 Zetterstrom, T., Sharp, T., Marsden, C.A. and Ungerstedt, L!., In vivo measurement of dopamine and its metabolites by intracerebral microdialysis: changes after D-amphetamine, J. Neurochem., 41 (1983) 1769 1773. 25 Zetterstrom, T., Sharp, T. and Ungerstedt, U., Further evaluation of the mechanism by which amphetamine reduces striatal dopamine metabolism: an in vivo brain dialysis study, Eur. J. Pharmacol., 132 (1986) 1 9.