- Email: [email protected]
The effects of intra-accumbens neurotensin on sensorimotor gating
Brain Research 760 Ž1997. 80–84
Research report
The effects of intra-accumbens neurotensin on sensorimotor gating David Feifel ) , Kelly L. Minor, Stephanie Dulawa, Neal R. Swerdlow Department of Psychiatry, UniÕersity of California, San Diego, San Diego, California, USA Accepted 18 February 1997
Abstract Neurotensin is a neuropeptide which coexists with mesolimbic dopamine. Previous studies have shown that centrally administered neurotensin can modulate the activity of mesolimbic dopamine with a profile similar to neuroleptics. For example, infusions of neurotensin into the nucleus accumbens inhibit amphetamine-induced hyperlocomotion. Prepulse inhibition ŽPPI. occurs when a weak prestimulus Ž‘prepulse’. inhibits the amplitude of the startle response to an intense stimulus Ž‘pulse’.. PPI is an operational measure of sensorimotor gating which is strongly regulated by mesolimbic dopamine. This study examined the effects of various doses of neurotensin infused into the nucleus accumbens of rats on the prepulse inhibition ŽPPI. of their acoustic startle reflex. Neurotensin Ž0.25–5.0 mg. was infused into the nucleus accumbens of rats. Animals then received subcutaneous injections of amphetamine Ž2 mgrkg. or saline and were placed in startle chambers where measures of startle amplitude and PPI were obtained. Neurotensin increased baseline PPI and blocked amphetamine-induced disruption of PPI in a dose-dependent fashion. The lowest dose of neurotensin tested Ž0.25 mg. significantly increased baseline PPI and both 0.25 and 1.0 mg neurotensin blocked amphetamine-induced decreases in PPI. The 5.0 mg dose of neurotensin had no significant effect on prepulse inhibition. Neurotensin had no effect on the amplitude of the acoustic startle reflex in amphetamine- or saline-treated rats. The results suggest that intra-accumbens neurotensin has a significant, dose-dependent effect on sensorimotor gating in which lower doses Ž0.25–1.0 mg. exhibit a neuroleptic-like action. Keywords: Neurotensin; Sensorimotor gating; Prepulse inhibition; Nucleus accumbens; Dopamine
1. Introduction Neurotensin is tridecapeptide localized in the brain of several species including rat w7x and man w12x and has been shown to act as a neurotransmitter andror neuromodulator w22x. A high concentration of neurotensin is found in mesolimbic neurons, where it is colocalized with dopamine w7,9,10x. Electrophysiological, neurochemical and behavioral studies have demonstrated that central administration of neurotensin produces effects on dopaminergic function similar to that of atypical antipsychotics w2,8x. For example, neurotensin administered into the ventral tegmental area causes release of dopamine in the nucleus accumbens but not the caudate nucleus w2x. In addition, neurotensin administered into the nucleus accumbens or intracerebroventricularly blocks amphetamine-induced locomotion, but does not block amphetamine-induced stereotypy or produce cataplexy w6,16x. These findings have raised inter) Corresponding author. Department of Psychiatry, UCSD Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8620, USA. E-mail:[email protected]
est in the possibility that endogenous neurotensin may play a role in the pathology associated with schizophrenia, and that analogs of neurotensin may have potential as antipsychotic agents w13,14x. In order to further investigate this, we utilized a behavioral paradigm which is regulated by mesolimbic dopamine and which models sensorimotor gating deficiencies exhibited by schizophrenia patients. Prepulse inhibition ŽPPI. is an operational measure of sensorimotor gating in which a startle reflex is inhibited by a weak lead stimulus presented 50–300 ms prior to the startling stimulus. PPI is regulated by a cortical-striatalpallidal-pontine circuit in which mesolimbic dopamine plays a critical regulatory role w17x. PPI is decreased in patients with schizophrenia to a degree that is proportional to their clinical psychopathology w3,4x. PPI is also decreased in rats treated with dopamine agonists such as amphetamine and apomorphine w11,19,20x. Typical and atypical antipsychotics can restore disrupted PPI and their ability to do so is highly correlated with their clinical potency w18,20,21x. The PPI model differs from existing animals models of schizophrenia since it uses identical methods across species to measure a deficit that is integral
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 3 0 6 - 5
D. Feifel et al.r Brain Research 760 (1997) 80–84
to the schizophrenic syndrome and it has been demonstrated to possess face, construct and predictive validity as an animal model of deficient sensorimotor gating in schizophrenia patients w20x. Another advantage of PPI is that it is an easily and objectively quantified graded response that is under tight stimulus control. Thus, PPI is a valuable model for the study of the neuropharmacological substrates of schizophrenia and a useful preclinical screen for the antipsychotic potential of new agents. This study assessed the effects of infusions of neurotensin into the nucleus accumbens of rats on intact and amphetamine-disrupted PPI.
2. Materials and methods 2.1. Subjects Male Sprague–Dawley rats Ž225–250 g on arrival. were housed in groups of two or three and maintained on a reversed 12-h:12-h lightrdark schedule Žlights off at 0700 h., with food and water provided ad libitum. Behavioral testing occurred between 0900 and 1500 h. Animals were handled individually within 3 days of arrival and daily thereafter.
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treatments Žsaline vs. amphetamine. were alternated in a counterbalanced fashion resulting in a within Žamphetamine condition. = between Žneurotensin dose. design. 2.4. Startle testing Ten minutes after receiving their subcutaneous injections, animals undergoing startle testing Ž n s 34. were placed in separate startle chambers ŽSR-LAB, San Diego Instruments, San Diego.. Startle chambers consisted of a Plexiglas cylinder 8.2 cm in diameter resting on a 12.5 = 25.5 cm Plexiglas frame within a ventilated enclosure Startle chambers were housed in a sound-attenuated room exposed to a 65 db wAx background noise. After a 5 min acclimation period, acoustic noise bursts were presented via a speaker mounted 24 cm above the animal. A piezoelectric accelerometer mounted below the Plexiglas frame detected and transduced the motion within the cylinder. Rats were exposed to five different stimuli conditions: a 120 dB startle pulse alone ŽP-alone., P-Alone preceded 100 ms by a 3, 5 or 10 dB acoustic prepulse or no stimulus. Stimuli were presented in five blocks, arranged in a pseudorandomized order. There was an average of 15 s between stimuli. 2.5. Locomotor testing
2.2. Surgery Surgery began 8–14 days after introduction of animals to their home cages. Each animal was anesthetized with pentobarbital sodium and stereotaxically implanted with 23 gauge stainless steel guide-cannula aimed 3 mm dorsal to the nucleus accumbens and fixed with dental cement and skull screws. Stereotaxic coordinates used ŽA: q3.8 mm, M-L: "1.2 mm, V: y4.0 mm relative to bregma, flat skull. were adapted from previous published studies of the behavioral effects of neurotensin infused into the nucleus accumbens w6,16x. Testing began after a 7–10 day postoperative recovery period for rats. 2.3. Drug administration Prior to each test session rats were given intra-accumbens infusions of either 0 Žsaline., 0.25, 1.0, or 5.0 mg of neurotensin ŽSigma Chemicals.. A 30 gauge microinjector needle which extended 3 mm beyond the guide cannula was used to deliver neurotensin into the nucleus accumbens. Injection volume of 1 ml per side was infused over 60 s via polyurethane tubing attached to a microsyringe by an electrical syringe pump. Microinjector needles were left in place for an additional 60 s after infusions and then replaced with metal wire stylets. Rats were then given subcutaneous injections of either amphetamine Ž2 mgrkg. or saline. Each animal was tested on two separate occasions one week apart. Central neurotensin doses were kept constant during both test sessions, whereas peripheral
A separate group of rats was habituated for 90 min prior to testing in locomotor cages that were equipped with two pairs of photobeam sensors. Ten minutes following administration of drugs, they were returned to these cages for 20 min of activity recording measured as the total number of single and crossover beam breaks. The duration of locomotor testing closely approximated the duration of startle test sessions. Comparable findings emerged from measures of single beam breaks and crossovers, therefore, only data from single beam breaks are reported. 2.6. Histology Following testing, animals had 1.0 ml methylene blue infused via their guide cannulae. Each animal was subsequently sacrificed by sedative overdose and perfused with a 10% formalin solution. Brain histology was performed to verify cannulae placement. Two animals were found to have misplaced cannulae and data from these animals were not included in the statistical analysis. 2.7. Statistical analyses Prepulse inhibition was calculated as the percent reduction in startle amplitude compared to the startle amplitude under pulse only conditions using the formula, w1-Žprepulse amplituderP-alone amplitude.x = 100. A preliminary analysis revealed no differential drug effects across the various prepulse intensities tested. PPI was therefore averaged over
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all prepulse intensities and this averaged data was analyzed using a two-way ANOVA Žneurotensin= amphetamine condition.. Significant effects were followed up with comparisons of individual group means by post-hoc t-tests. Similar analyses were conducted on startle amplitudes on P-alone trials and on activity counts as measured by the number of photocell beam breaks.
3. Results 3.1. PPI Fig. 1 illustrates PPI results. A two way ANOVA of PPI results revealed a significant amphetamine effect Ž F Ž1,30. s 8.69, P - 0.01. and a significant neurotensin effect Ž F Ž3,30. s 6.51, P - 0.01.. There was no significant amphetamine = neurotensin interaction effect Ž F Ž3,30. s 1.51, n.s... Post-hoc individual comparisons indicated that 0.25 mg Ž t Ž16. s y4.12, P - 0.001. and 1.0 mg Ž t Ž16. s y3.09, P - 0.01., but not 5.0 mg of neurotensin, significantly increased PPI in amphetaminetreated rats and blocked amphetamine-induced disruption of PPI. Baseline PPI was increased by 0.25 mg Ž t Ž16. s y3.26, P - 0.01. neurotensin but no other tested dose. 3.2. Pulse-alone startle amplitude Fig. 2 illustrates pulse-alone startle amplitude. A similar analysis applied to the pulse-alone amplitude indicated that amphetamine significantly decreased startle amplitude Ž F Ž1,30. s 12.54, P - 0.001., but there was no significant main effect of neurotensin Ž F Ž3,30. s 0.21, n.s.. and there
Fig. 1. Prepulse inhibition of acoustic startle reflex exhibited by rats treated with nucleus accumbens infusions of various doses of neurotensin and subcutaneous injections of saline or amphetamine Ž2 mgrkg.. ) , Significantly different from treatment with same neurotensin dose and no amphetamine. q, Significantly different from treatment with same amphetamine dose and 0 mg neurotensin group. ns8–10 for all treatment conditions.
Fig. 2. P-alone startle amplitude in rats treated with nucleus accumbens infusions of various doses of saline or neurotensin and subcutaneous injections of amphetamine Ž2 mgrkg.. ) , Significantly Ž P - 0.05. different from treatments with no amphetamine and same dose of neurotensin. ns8–10 for all treatment conditions.
was no significant amphetamine= neurotensin interaction effect Ž F Ž3,30. s 0.56, n.s... 3.3. Locomotor actiÕity Fig. 3 illustrates locomotor activity results. A two-way ANOVA of locomotor activity indicates that amphetamine significantly increased locomotor activity as measured by the number of beam-breaks Ž F Ž1, 21. s 21.02, P - 0.001.. Neurotensin also had a significant effect on locomotion Ž F Ž3,21. s 9.11, P - 0.001. and there was a significant neurotensin= amphetamine effect Ž F Ž3,21. s 6.34, P 0.01.. Each dose of neurotensin tested significantly de-
Fig. 3. Locomotor activity, as measured by total number of beam breaks in 20 min, exhibited in rats treated with nucleus accumbens infusions of various doses of neurotensin and subcutaneous injections of saline or amphetamine Ž2 mgrkg.. ) P - 0.05, ) ) P - 0.01, ) ) ) P - 0.001 compared with amphetamine plus 0 mg neurotensin group. ns6–7 for all treatment conditions.
D. Feifel et al.r Brain Research 760 (1997) 80–84
creased locomotion in amphetamine-treated animals based on individual post-hoc comparisons. The highest dose, 5.0 mg, completely blocked amphetamine-induced locomotor activation resulting in locomotor activity that was comparable to baseline levels. Neurotensin had no significant effect on baseline Žsystemic saline. levels of locomotor activity.
4. Discussion Neurotensin’s ability to block amphetamine-induced hyperlocomotion is consistent with previous reports w6,16x. The results from startle testing suggest that neurotensin has a somewhat more complex effect on sensorimotor gating. The lower doses of neurotensin, 0.25 and 1.0 mg, significantly blocked the amphetamine-induced disruption of PPI, which is consistent with a neuroleptic-like action w18,20,21x. The low dose of neurotensin facilitated baseline PPI. Facilitation of baseline PPI is produced by a number of clinically effective antipsyhotics w18x. This effect on baseline behavior stands in contrast to the lack of effect on baseline locomotor activity observed in this study. This may be due to the low baseline levels of spontaneous locomotor activity in habituated rats. Neurotensin in the dose range of 5.0 mg, the highest dose tested in this study, has been shown to effectively block amphetamine-induced hyperlocomotion in previous reports w6,16x. In the current study too, this dose had a potent anti-amphetamine effect on locomotion. However, 5.0 mg neurotensin did not block the amphetamine-induced disruption of PPI and had no effect on baseline PPI. In fact, a non-significant trend toward potentiation of amphetamine’s effect was exhibited by this dose of neurotensin. The overall dose-response curve exhibited by neurotensin on PPI suggests the descending limb of an inverted U-shaped relationship in which maximal neuroleptic-like actions on PPI are produced at doses lower than for locomotor activity and other behaviors. The reason for the differential effect of this dose of neurotensin on different amphetamine-induced behaviors is not clear. A dissociation of neurotensin’s effect on various behaviors has also been previously reported by others. Robledo et al. w15x found intra-accumbens neurotensin blocked cocaine-induced hyperlocomotion but not cocaine self-administration. It is possible that the neural substrates responsible for neurotensin’s modulation of psychostimulant-induced effects on sensorimotor processing, reward and locomotion are somewhat distinct resulting in differential sensitivity to modulation by neurotensin. The exact nature these putative substrate differences underlying neurotensin’s differential actions on dopamine-agonist behavior is not known. However, possibilities include distinct neuroanatomical mediation within the nucleus accumbens Že.g. core vs. shell. andror distinct dopamine receptor subtype mediation w5x.
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In this study, amphetamine significantly decreased pulse-alone startle amplitude. Although several previous studies have also demonstrated a reduction in pulse-alone startle amplitude by psychostimulants w1,11,20,21x, other studies have not shown this effect w18x. It is interesting that the only the groups treated with 0 and 5.0 mg neurotensin show a statistically significant amphetamine-induced decrease in pulse-alone startle amplitude by post-hoc analysis. While this is a similar statistical pattern to that seen with PPI, neurotensin does not seem to produce any changes in startle amplitude among amphetamine-treated animals. The selective post-hoc results are due to small differences in variability and small differences in baseline startle amplitude among different groups. Furthermore, since PPI was calculated using a percentage score, such a decrease in pulse-alone amplitude would not be expected to artificially increase the amount of PPI. Antipsychotic agents do not have a consistent effect on pulse-alone amplitude but some studies have found that typical w20x and atypical w18x antipsychotics can reduce it. Since neurotensin had no significant effect on the pulse-alone startle amplitude, it is unlikely that any of the observed effects on PPI are due to effects on the primary startle reflex itself. Rather, neurotensin seems to specifically alter the ‘higherorder’ modulation of the startle reflex represented in this study by PPI which is critically regulated by mesolimbic dopamine. In summary, these results support previous studies suggesting an antipsychotic-like profile for neurotensin. However, they suggest that optimal neuroleptic effects on prepulse inhibition may occur at a different neurotensin dose than for other behaviors regulated by mesolimbic dopamine. These findings also support the use of PPI as a useful pre-clinical test paradigm for agents with putative antipsychotic potential and may provide important information relevant to clinical effects not obtained by other behavioral paradigms.
Acknowledgements This research was supported by a Scottish Rite Research Award and a National Alliance for Research in Schizophenia and Depression ŽNARSAD. Young Investigator Award to D.F. N.R.S. was supported by MH42228. The authors thank Pam Auerbach and Navid Taiid for their technical assistance and Tammi Reza for her help in preparing this paper.
References w1x VP Bakshi, MA Geyer, N Taiid, NR Swerdlow, A comparison of the effects of amphetamine, strychnine and caffeine on prepulse inhibition and latent inhibition, Behav. Pharmacol. 6 Ž1995. 801–809. w2x CD Blaha, AG Phillips, HC Fibinger, RF Lane, Effects of neu-
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D. Feifel et al.r Brain Research 760 (1997) 80–84 rotensin on dopamine release in the nucleus accumbens: Comparisons with atypical antipsychotic drug action, Ann. N.Y. Acad. Sci. 668 Ž1992. 478–480. DL Braff, MA Geyer, Sensorimotor gating and schizophrenia: Human and animal model studies, Arch. Gen. Psychiatry 47 Ž1990. 181–188. R.W. Butler, M.A. Jenkins, M.A. Geyer and D.L. Braff, Wisconsin card sorting deficits and diminished sensorimotor gating in a discrete subgroup of schizophrenic patients, in: C.A. Tamminga and S.C. Schulz ŽEds.., Advances in Neuropsychiatry and Psychopharm, vol. 1: Schizophrenia Research, Raven Press, New York, 1991, pp. 163–168. J Diaz, D Levesque, N Griffon, CH Lammers, MP Martres, P Sokoloff, JC Schwartz, Opposing roles for D2 and D3 receptors on neurotensin MRNA expression in nucleus accumbens, Eur. J. Neurosci. 6 Ž1994. 1384–1387. GN Ervin, LS Birkemo, CV Nemeroff, AJ Prange Jr., Neurotensin blocks certain amphetamine-induced behaviours, Nature 291 Ž1981. 73–76. T Hokfelt, BJ Everitt, E Theodorsson-Norheim, M Goldstein, Occurence of neurotensin-like immunoreactivity in subpopulations of hypothalamic, mesencephalic, and medullary catecholamine neurons, J. Comp. Neurol. 222 Ž1984. 543–559. FB Jolicoeur, MA Gagne, R Rivest, A Drumheller, S St-Pierre, Atypical neuoroleptic-like behavioral effects of neurotensin, Brain Res. Bull. 32 Ž1993. 481–487. PW Kalivas, JS Miller, Neurotensin neurons in the ventral tegmental area project to the medial nucleus accumbens, Brain Res. 300 Ž1984. 157–160. PW Kalivas, CB Nemeroff, AJ Prange Jr., Neuroanatomical site specific modulation of spontaneous motor activity by neurotensin, Eur. J. Pharmacol. 78 Ž1982. 471–474. RS Mansbach, MA Geyer, DL Braff, Dopaminergic stimulation disrupts sensorimotor gating in the rat, Psychopharmacology 94 Ž1988. 507–514. PJ Manberg, WW Youngblood, CB Nemeroff, MN Rossor, LL
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Iversen, AJ Prange Jr., JS Kizer, Regional distribution of neurotensin in human brain, J. Neurochem. 38 Ž1982. 1777–1780. CB Nemeroff, G Bissette, Neuropeptides, dopamine and schizophrenia, Ann. N.Y. Acad. Sci. 668 Ž1992. 273–291. CB Nemeroff, B Levant, B Myers, G Bissette, Neurotensin, antipsychotic drugs, and schizophrenia, Ann. N.Y. Acad. Sci. 668 Ž1992. 147–156. R Robledo, R Maldonado, GF Koob, Neurotensin injected into the nucleus accumbens blocks the psychostimulant effects of cocaine but does not atttenuate cocaine self-administration in the rat, Brain Res. 622 Ž1993. 105–112. KM Skoog, ST Cain, CB Nemeroff, Centrally administered neurotensin suppresses locomotor hyperactivity induced by Damphetamine but not by scopolamine or caffeine, Neuropharmacology 25 Ž1986. 777–782. NR Swerdlow, SB Caine, DL Braff, MA Geyer, The neural substrates of sensorimotor gating of the startle reflex: A review of recent findings and their implications, J. Psychopharmacol. 6 Ž1992. 176–190. NR Swerdlow, MA Geyer, Clozapine and haloperidol in an animal model of sensorimotor gating deficits in schizophrenia, Pharmacol. Biochem. Behav. 44 Ž1993. 741–744. NR Swerdlow, RS Mansbach, MA Geyer, L Pulvirenti, GF Koob, DL Braff, L Pulvirenit, Amphetamine disruption of prepulse inhibition of acoutstic startle in rats is reversed by depletion of mesolimbic dopamine, Psyhcopharmacology 100 Ž1990. 413–416. NR Swerdlow, DL Braff, N Taaid, MA Geyer, Assessing the validity of an animal model of deficient sensorimotor gating in schizophrenic patients, Arch. Gen. Psychiatry 51 Ž1994. 139–154. NR Swerdlow, D Zisook, N Taaid, Seroquel ŽICI 204,636. restores prepulse inhibition of acoustic startle in apomorphine-treated rats: Similarities to clozapine, Psychopharmachology 114 Ž1994. 675– 678. GR Uhl, RR Goodman, SH Snyder, Neurotensin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat: Immunohistochemical mapping, Brain Res. 167 Ž1979. 77–91.
Research report
The effects of intra-accumbens neurotensin on sensorimotor gating David Feifel ) , Kelly L. Minor, Stephanie Dulawa, Neal R. Swerdlow Department of Psychiatry, UniÕersity of California, San Diego, San Diego, California, USA Accepted 18 February 1997
Abstract Neurotensin is a neuropeptide which coexists with mesolimbic dopamine. Previous studies have shown that centrally administered neurotensin can modulate the activity of mesolimbic dopamine with a profile similar to neuroleptics. For example, infusions of neurotensin into the nucleus accumbens inhibit amphetamine-induced hyperlocomotion. Prepulse inhibition ŽPPI. occurs when a weak prestimulus Ž‘prepulse’. inhibits the amplitude of the startle response to an intense stimulus Ž‘pulse’.. PPI is an operational measure of sensorimotor gating which is strongly regulated by mesolimbic dopamine. This study examined the effects of various doses of neurotensin infused into the nucleus accumbens of rats on the prepulse inhibition ŽPPI. of their acoustic startle reflex. Neurotensin Ž0.25–5.0 mg. was infused into the nucleus accumbens of rats. Animals then received subcutaneous injections of amphetamine Ž2 mgrkg. or saline and were placed in startle chambers where measures of startle amplitude and PPI were obtained. Neurotensin increased baseline PPI and blocked amphetamine-induced disruption of PPI in a dose-dependent fashion. The lowest dose of neurotensin tested Ž0.25 mg. significantly increased baseline PPI and both 0.25 and 1.0 mg neurotensin blocked amphetamine-induced decreases in PPI. The 5.0 mg dose of neurotensin had no significant effect on prepulse inhibition. Neurotensin had no effect on the amplitude of the acoustic startle reflex in amphetamine- or saline-treated rats. The results suggest that intra-accumbens neurotensin has a significant, dose-dependent effect on sensorimotor gating in which lower doses Ž0.25–1.0 mg. exhibit a neuroleptic-like action. Keywords: Neurotensin; Sensorimotor gating; Prepulse inhibition; Nucleus accumbens; Dopamine
1. Introduction Neurotensin is tridecapeptide localized in the brain of several species including rat w7x and man w12x and has been shown to act as a neurotransmitter andror neuromodulator w22x. A high concentration of neurotensin is found in mesolimbic neurons, where it is colocalized with dopamine w7,9,10x. Electrophysiological, neurochemical and behavioral studies have demonstrated that central administration of neurotensin produces effects on dopaminergic function similar to that of atypical antipsychotics w2,8x. For example, neurotensin administered into the ventral tegmental area causes release of dopamine in the nucleus accumbens but not the caudate nucleus w2x. In addition, neurotensin administered into the nucleus accumbens or intracerebroventricularly blocks amphetamine-induced locomotion, but does not block amphetamine-induced stereotypy or produce cataplexy w6,16x. These findings have raised inter) Corresponding author. Department of Psychiatry, UCSD Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8620, USA. E-mail:[email protected]
est in the possibility that endogenous neurotensin may play a role in the pathology associated with schizophrenia, and that analogs of neurotensin may have potential as antipsychotic agents w13,14x. In order to further investigate this, we utilized a behavioral paradigm which is regulated by mesolimbic dopamine and which models sensorimotor gating deficiencies exhibited by schizophrenia patients. Prepulse inhibition ŽPPI. is an operational measure of sensorimotor gating in which a startle reflex is inhibited by a weak lead stimulus presented 50–300 ms prior to the startling stimulus. PPI is regulated by a cortical-striatalpallidal-pontine circuit in which mesolimbic dopamine plays a critical regulatory role w17x. PPI is decreased in patients with schizophrenia to a degree that is proportional to their clinical psychopathology w3,4x. PPI is also decreased in rats treated with dopamine agonists such as amphetamine and apomorphine w11,19,20x. Typical and atypical antipsychotics can restore disrupted PPI and their ability to do so is highly correlated with their clinical potency w18,20,21x. The PPI model differs from existing animals models of schizophrenia since it uses identical methods across species to measure a deficit that is integral
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 3 0 6 - 5
D. Feifel et al.r Brain Research 760 (1997) 80–84
to the schizophrenic syndrome and it has been demonstrated to possess face, construct and predictive validity as an animal model of deficient sensorimotor gating in schizophrenia patients w20x. Another advantage of PPI is that it is an easily and objectively quantified graded response that is under tight stimulus control. Thus, PPI is a valuable model for the study of the neuropharmacological substrates of schizophrenia and a useful preclinical screen for the antipsychotic potential of new agents. This study assessed the effects of infusions of neurotensin into the nucleus accumbens of rats on intact and amphetamine-disrupted PPI.
2. Materials and methods 2.1. Subjects Male Sprague–Dawley rats Ž225–250 g on arrival. were housed in groups of two or three and maintained on a reversed 12-h:12-h lightrdark schedule Žlights off at 0700 h., with food and water provided ad libitum. Behavioral testing occurred between 0900 and 1500 h. Animals were handled individually within 3 days of arrival and daily thereafter.
81
treatments Žsaline vs. amphetamine. were alternated in a counterbalanced fashion resulting in a within Žamphetamine condition. = between Žneurotensin dose. design. 2.4. Startle testing Ten minutes after receiving their subcutaneous injections, animals undergoing startle testing Ž n s 34. were placed in separate startle chambers ŽSR-LAB, San Diego Instruments, San Diego.. Startle chambers consisted of a Plexiglas cylinder 8.2 cm in diameter resting on a 12.5 = 25.5 cm Plexiglas frame within a ventilated enclosure Startle chambers were housed in a sound-attenuated room exposed to a 65 db wAx background noise. After a 5 min acclimation period, acoustic noise bursts were presented via a speaker mounted 24 cm above the animal. A piezoelectric accelerometer mounted below the Plexiglas frame detected and transduced the motion within the cylinder. Rats were exposed to five different stimuli conditions: a 120 dB startle pulse alone ŽP-alone., P-Alone preceded 100 ms by a 3, 5 or 10 dB acoustic prepulse or no stimulus. Stimuli were presented in five blocks, arranged in a pseudorandomized order. There was an average of 15 s between stimuli. 2.5. Locomotor testing
2.2. Surgery Surgery began 8–14 days after introduction of animals to their home cages. Each animal was anesthetized with pentobarbital sodium and stereotaxically implanted with 23 gauge stainless steel guide-cannula aimed 3 mm dorsal to the nucleus accumbens and fixed with dental cement and skull screws. Stereotaxic coordinates used ŽA: q3.8 mm, M-L: "1.2 mm, V: y4.0 mm relative to bregma, flat skull. were adapted from previous published studies of the behavioral effects of neurotensin infused into the nucleus accumbens w6,16x. Testing began after a 7–10 day postoperative recovery period for rats. 2.3. Drug administration Prior to each test session rats were given intra-accumbens infusions of either 0 Žsaline., 0.25, 1.0, or 5.0 mg of neurotensin ŽSigma Chemicals.. A 30 gauge microinjector needle which extended 3 mm beyond the guide cannula was used to deliver neurotensin into the nucleus accumbens. Injection volume of 1 ml per side was infused over 60 s via polyurethane tubing attached to a microsyringe by an electrical syringe pump. Microinjector needles were left in place for an additional 60 s after infusions and then replaced with metal wire stylets. Rats were then given subcutaneous injections of either amphetamine Ž2 mgrkg. or saline. Each animal was tested on two separate occasions one week apart. Central neurotensin doses were kept constant during both test sessions, whereas peripheral
A separate group of rats was habituated for 90 min prior to testing in locomotor cages that were equipped with two pairs of photobeam sensors. Ten minutes following administration of drugs, they were returned to these cages for 20 min of activity recording measured as the total number of single and crossover beam breaks. The duration of locomotor testing closely approximated the duration of startle test sessions. Comparable findings emerged from measures of single beam breaks and crossovers, therefore, only data from single beam breaks are reported. 2.6. Histology Following testing, animals had 1.0 ml methylene blue infused via their guide cannulae. Each animal was subsequently sacrificed by sedative overdose and perfused with a 10% formalin solution. Brain histology was performed to verify cannulae placement. Two animals were found to have misplaced cannulae and data from these animals were not included in the statistical analysis. 2.7. Statistical analyses Prepulse inhibition was calculated as the percent reduction in startle amplitude compared to the startle amplitude under pulse only conditions using the formula, w1-Žprepulse amplituderP-alone amplitude.x = 100. A preliminary analysis revealed no differential drug effects across the various prepulse intensities tested. PPI was therefore averaged over
82
D. Feifel et al.r Brain Research 760 (1997) 80–84
all prepulse intensities and this averaged data was analyzed using a two-way ANOVA Žneurotensin= amphetamine condition.. Significant effects were followed up with comparisons of individual group means by post-hoc t-tests. Similar analyses were conducted on startle amplitudes on P-alone trials and on activity counts as measured by the number of photocell beam breaks.
3. Results 3.1. PPI Fig. 1 illustrates PPI results. A two way ANOVA of PPI results revealed a significant amphetamine effect Ž F Ž1,30. s 8.69, P - 0.01. and a significant neurotensin effect Ž F Ž3,30. s 6.51, P - 0.01.. There was no significant amphetamine = neurotensin interaction effect Ž F Ž3,30. s 1.51, n.s... Post-hoc individual comparisons indicated that 0.25 mg Ž t Ž16. s y4.12, P - 0.001. and 1.0 mg Ž t Ž16. s y3.09, P - 0.01., but not 5.0 mg of neurotensin, significantly increased PPI in amphetaminetreated rats and blocked amphetamine-induced disruption of PPI. Baseline PPI was increased by 0.25 mg Ž t Ž16. s y3.26, P - 0.01. neurotensin but no other tested dose. 3.2. Pulse-alone startle amplitude Fig. 2 illustrates pulse-alone startle amplitude. A similar analysis applied to the pulse-alone amplitude indicated that amphetamine significantly decreased startle amplitude Ž F Ž1,30. s 12.54, P - 0.001., but there was no significant main effect of neurotensin Ž F Ž3,30. s 0.21, n.s.. and there
Fig. 1. Prepulse inhibition of acoustic startle reflex exhibited by rats treated with nucleus accumbens infusions of various doses of neurotensin and subcutaneous injections of saline or amphetamine Ž2 mgrkg.. ) , Significantly different from treatment with same neurotensin dose and no amphetamine. q, Significantly different from treatment with same amphetamine dose and 0 mg neurotensin group. ns8–10 for all treatment conditions.
Fig. 2. P-alone startle amplitude in rats treated with nucleus accumbens infusions of various doses of saline or neurotensin and subcutaneous injections of amphetamine Ž2 mgrkg.. ) , Significantly Ž P - 0.05. different from treatments with no amphetamine and same dose of neurotensin. ns8–10 for all treatment conditions.
was no significant amphetamine= neurotensin interaction effect Ž F Ž3,30. s 0.56, n.s... 3.3. Locomotor actiÕity Fig. 3 illustrates locomotor activity results. A two-way ANOVA of locomotor activity indicates that amphetamine significantly increased locomotor activity as measured by the number of beam-breaks Ž F Ž1, 21. s 21.02, P - 0.001.. Neurotensin also had a significant effect on locomotion Ž F Ž3,21. s 9.11, P - 0.001. and there was a significant neurotensin= amphetamine effect Ž F Ž3,21. s 6.34, P 0.01.. Each dose of neurotensin tested significantly de-
Fig. 3. Locomotor activity, as measured by total number of beam breaks in 20 min, exhibited in rats treated with nucleus accumbens infusions of various doses of neurotensin and subcutaneous injections of saline or amphetamine Ž2 mgrkg.. ) P - 0.05, ) ) P - 0.01, ) ) ) P - 0.001 compared with amphetamine plus 0 mg neurotensin group. ns6–7 for all treatment conditions.
D. Feifel et al.r Brain Research 760 (1997) 80–84
creased locomotion in amphetamine-treated animals based on individual post-hoc comparisons. The highest dose, 5.0 mg, completely blocked amphetamine-induced locomotor activation resulting in locomotor activity that was comparable to baseline levels. Neurotensin had no significant effect on baseline Žsystemic saline. levels of locomotor activity.
4. Discussion Neurotensin’s ability to block amphetamine-induced hyperlocomotion is consistent with previous reports w6,16x. The results from startle testing suggest that neurotensin has a somewhat more complex effect on sensorimotor gating. The lower doses of neurotensin, 0.25 and 1.0 mg, significantly blocked the amphetamine-induced disruption of PPI, which is consistent with a neuroleptic-like action w18,20,21x. The low dose of neurotensin facilitated baseline PPI. Facilitation of baseline PPI is produced by a number of clinically effective antipsyhotics w18x. This effect on baseline behavior stands in contrast to the lack of effect on baseline locomotor activity observed in this study. This may be due to the low baseline levels of spontaneous locomotor activity in habituated rats. Neurotensin in the dose range of 5.0 mg, the highest dose tested in this study, has been shown to effectively block amphetamine-induced hyperlocomotion in previous reports w6,16x. In the current study too, this dose had a potent anti-amphetamine effect on locomotion. However, 5.0 mg neurotensin did not block the amphetamine-induced disruption of PPI and had no effect on baseline PPI. In fact, a non-significant trend toward potentiation of amphetamine’s effect was exhibited by this dose of neurotensin. The overall dose-response curve exhibited by neurotensin on PPI suggests the descending limb of an inverted U-shaped relationship in which maximal neuroleptic-like actions on PPI are produced at doses lower than for locomotor activity and other behaviors. The reason for the differential effect of this dose of neurotensin on different amphetamine-induced behaviors is not clear. A dissociation of neurotensin’s effect on various behaviors has also been previously reported by others. Robledo et al. w15x found intra-accumbens neurotensin blocked cocaine-induced hyperlocomotion but not cocaine self-administration. It is possible that the neural substrates responsible for neurotensin’s modulation of psychostimulant-induced effects on sensorimotor processing, reward and locomotion are somewhat distinct resulting in differential sensitivity to modulation by neurotensin. The exact nature these putative substrate differences underlying neurotensin’s differential actions on dopamine-agonist behavior is not known. However, possibilities include distinct neuroanatomical mediation within the nucleus accumbens Že.g. core vs. shell. andror distinct dopamine receptor subtype mediation w5x.
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In this study, amphetamine significantly decreased pulse-alone startle amplitude. Although several previous studies have also demonstrated a reduction in pulse-alone startle amplitude by psychostimulants w1,11,20,21x, other studies have not shown this effect w18x. It is interesting that the only the groups treated with 0 and 5.0 mg neurotensin show a statistically significant amphetamine-induced decrease in pulse-alone startle amplitude by post-hoc analysis. While this is a similar statistical pattern to that seen with PPI, neurotensin does not seem to produce any changes in startle amplitude among amphetamine-treated animals. The selective post-hoc results are due to small differences in variability and small differences in baseline startle amplitude among different groups. Furthermore, since PPI was calculated using a percentage score, such a decrease in pulse-alone amplitude would not be expected to artificially increase the amount of PPI. Antipsychotic agents do not have a consistent effect on pulse-alone amplitude but some studies have found that typical w20x and atypical w18x antipsychotics can reduce it. Since neurotensin had no significant effect on the pulse-alone startle amplitude, it is unlikely that any of the observed effects on PPI are due to effects on the primary startle reflex itself. Rather, neurotensin seems to specifically alter the ‘higherorder’ modulation of the startle reflex represented in this study by PPI which is critically regulated by mesolimbic dopamine. In summary, these results support previous studies suggesting an antipsychotic-like profile for neurotensin. However, they suggest that optimal neuroleptic effects on prepulse inhibition may occur at a different neurotensin dose than for other behaviors regulated by mesolimbic dopamine. These findings also support the use of PPI as a useful pre-clinical test paradigm for agents with putative antipsychotic potential and may provide important information relevant to clinical effects not obtained by other behavioral paradigms.
Acknowledgements This research was supported by a Scottish Rite Research Award and a National Alliance for Research in Schizophenia and Depression ŽNARSAD. Young Investigator Award to D.F. N.R.S. was supported by MH42228. The authors thank Pam Auerbach and Navid Taiid for their technical assistance and Tammi Reza for her help in preparing this paper.
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