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The cortical neuropeptide, cortistatin-14, impairs post-training memory processing
Brain Research 775 Ž1997. 250–252
Short communication
The cortical neuropeptide, cortistatin-14, impairs post-training memory processing James F. Flood b
a,b,)
, Kayoko Uezu
a,b
, John E. Morley
a,b
a Geriatric Research Education and Clinical Center (GRECC), VA Medical Center, St. Louis, MO 63106, USA Department of Internal Medicine, DiÕision of Geriatric Medicine, St. Louis UniÕersity School of Medicine, St. Louis, MO 63106, USA
Accepted 26 August 1997
Abstract Cortistatin-14, a neuropeptide, is present primarily in the cortex and hippocampus. In the hippocampus, cortistatin-14 inhibits pyramidal cell firing and co-exists with GABA. To determine if cortistatin-14 would impair retention, saline or cortistatin-14 were injected intracerebroventricularly after footshock avoidance training in CD-1 mice. After 1 week, training was resumed to determine the effect of cortistatin-14 on retention. Cortistatin-14 was found to impair retention relative to the control group at doses of 0.5–5.0 mg. q 1997 Elsevier Science B.V. Keywords: Cortistatin-14; Neuropeptide; Memory; Mice
Cortistatin is a neuropeptide related to somatostatin and is localized within the cortex and hippocampus w6x. It is found in neurons that also contain GABA. Cortistatin-14 inhibited neuronal firing of hippocampal CA1 neurons w6x and blocked acetylcholine induced changes on evoked paired-pulse stimulation of CA1 neurons as well as cortical desynchronization. Increasing acetylcholine turnover or receptor activity is associated with improved retention w1,3– 5,7,8,11x. For these reasons, we determined if cortistatin-14 impaired memory processing. CD-1 male mice Žat least 8 weeks old. obtained from Charles River Breeding Laboratories ŽWilmington, MA. served as subjects. After at least 2 weeks in the laboratory, they were caged individually 48 h prior to training and remained singly housed until retention was tested 1 week later. Animal rooms were on a 12 h light-dark cycle with lights on at 06:00 h. Mice were assigned randomly to groups of 15 and were trained and tested between 08:00 and 14:00 h. Saline or cortistatin-14 ŽPhoenix Pharmaceuticals Inc., Mountain View, CA. was injected intracerebroventricular Ži.c.v.. within 3 min after training. The injection coordinates were 1.0 mm posterior relative to bregma and 1.0 mm to the right of the central suture. The injection depth
)
Corresponding author. VA Medical Center Ž151rJC., 915 N. Grand Blvd., St. Louis, MO 63109, USA. E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 8 4 - 6
was 2.0 mm relative to the skull surface. The scalp was deflected and a hole drilled through the skull on the right side of the central suture. Mice were trained 48 h after surgery. Immediately after training, mice were again placed in the stereotaxic under light enflurane anesthesia. Within 3 min after training, a 2.0 ml solution of saline or cortistatin-14 was injected over 30 s through a 30-ga needle attached to a 10 ml syringe held by a syringe holder attached to the micromanipulator of the stereotaxic instrument. The reliability of the injections was determined histologically by locating the needle tract in frozen brain sections following the retention test. The site of the injection was confirmed using a mouse forebrain stereotaxic atlas w12x. Mice were trained on footshock avoidance in a T-maze w9,10x which consisted of a black plastic alley with a start box at one end and two goal boxes at the other. The start box was separated from the alley by a plastic guillotine door which prevented movement down the alley until training began. An electrifiable stainless steel rod floor ran throughout the maze to deliver scrambled footshock. Mice were not permitted to explore the maze prior to training. A block of training trials began when a mouse was placed into the start box. The guillotine door was raised and a buzzer sounded simultaneously; 5 s later footshock was applied. The goal box entered on the first trial was designated ‘incorrect’ and the footshock was continued until the mouse entered the other goal box,
J.F. Flood et al.r Brain Research 775 (1997) 250–252
which in all subsequent trials was designated as ‘correct’ for the particular mouse. At the end of each trial, the mouse was removed to its home cage until the next trial. Two training conditions were used. The training parameters for one condition, ‘weak’ training, resulted in poor retention test performance of control subject which allowed us to detect improve retention due to peptide administration. For this type of training, mice received four
251
training trials at an intertrial interval of 30 s and the footshock intensity was 0.30 mA. The buzzer intensity was 55 dB. In the second training condition, ‘strong’ training, the parameters reliably resulted in good retention test performance in the control group, thus allowing us to detect impaired retention test performance due to peptide administration. For this type of training, mice received 5 training trials at an intertrial interval of 45 s and the
Fig. 1. Cortistatin failed to improve retention test performance under the weak training condition Žtop. but did impair it relative to the 0 mg group under the stronger training condition Žbottom.. The ) indicates that the mean different from the mean of the control Ž0 mg. group at P - 0.01 by Tukey’s t-test.
252
J.F. Flood et al.r Brain Research 775 (1997) 250–252
footshock intensity was 0.35 mA. The buzzer intensity was 65 dB. After either type of training, saline or cortistatin-14 solution was administered i.c.v. within 3 min after training. One week later, T-maze training was resumed until each mouse made five avoidance responses in six consecutive training trials. Trials to this criterion was taken as a measure of retention. In this experiment, mice were randomly assigned to groups which received 0, 0.05, 0.5, or 5 mg of cortistatin-14. The 0 mg dose received saline. Half of the mice in each of the dose groups were trained under the weak training conditions and the remainder under the strong training conditions. There were 15 mice in each of 8 groups. A 2-way analysis of variance ŽANOVA; dose of cortistatin = training condition., run on the trials to criterion from the retention test, indicated that the effect of dose Ž F3, 112 s 16.24., training condition Ž F1, 112 s 28.26. and their interaction Ž F3, 112 s 16.26. were all significant at P - 0.001. Fig. 1 indicates that under the ‘weak’ training condition cortistatin-14 did not effect retention test performance. This was confirmed by a 1-way ANOVA Ž F - 1.. However, under the ‘stronger’ training condition cortistatin-14 yielded a dose-dependent increase in the mean trials to criterion. Under this training condition, the means of the 0.5 and 5.0 mg dose groups were significantly greater than the mean of the control ŽTukey’s t-test: t s 8.03, t s 12.73, respectively.. When a drug is administered after training, it cannot directly effect learning. Since, we used a 1 week retention period, and cortistatin is promptly metabolized, the drugs cannot directly affect performance on the retention test. If the drugs, by virtue of the time of the injection, cannot directly influence learning or retention test performance, any improvement or impairment in retention test performance relative to the control, we interpreted as being the result of altered neurotransmitter receptor activity occurring shortly after training Ži.e., memory processing.. Most drugs that improve retention under training conditions where control performance is poor Žmean between 9 and 10 trials to criterion. will also impair retention under training conditions were control performance is good Žmean between 6 and 7 mean trials to criterion.. Therefore, it can be difficult to determine if a compound is truly an amnestic if the only data available is a test for impaired retention test performance. Cortistatin-14 impaired memory processing under the strong training condition where controls had a relatively low mean trials to criterion. Since cortistatin-14 failed to improve retention under the weak training condition where control retention test performance was poor, we conclude that cortistatin-14 is a pure amnestic neuropeptide. Increases in GABAergic activity are associated with
impaired retention w2–4,8x. In the hippocampus, GABA may have this effect post synaptically by inhibiting pyramidal cell firing or by presynaptic inhibition of cholinergic terminals which innervate the pryamidal cell. Cortistatin-14 is co-localized with GABA in the hippocampus and inhibits CA1 neuronal fining w6x. This leads us to postulate that cortistatin-14 impaired memory processing by reducing neurotransmitter activity by facilitating GABA inhibition of aceytlcholine activity in the hippocampus.
Acknowledgements This research was supported by the Medical Research Service of the Department of Veterans Affairs.
References w1x R.T. Bartus, R.L. Dean, J.A. Goas, A.S. Lippa, Age-related changes in passive avoidance retention: modulation with dietary choline, Science 209 Ž1980. 301–303. w2x J.D. Brioni, M.W. Decker, L.P. Gamboa, I. Izquierdo, J.L. McGaugh, Muscimol injections in the medial septum impair spatial learning, Brain Res. 522 Ž1990. 227–234. w3x C. Castellano, J.L. McGaugh, Oxotremorine attenuates retrograde amnesia induced by post-training administration of the GABAergic agonists muscimol and baclofen, Behav. Neural Biol. 56 Ž1991. 25–31. w4x J.J. Chrobak, R.W. Stackman, T.J. Walsh, Intraseptal administration of muscimol produces dose-dependent memory impairments in the rat, Behav. Neural Biol. 52 Ž1989. 357–369. w5x C. Dalmaz, I.B. Introini-Collison, J.L. McGaugh, Noradrenergic and cholinergic interactions in the amygdala and the modulation of memory storage, Behav. Brain Res. 58 Ž1993. 167–174. w6x L. de Lecea, J.R. Criado, O. Prospero-Garcia, K.M. Gautvik, P. Schweitzer, P.E. Danielson, C.L.M. Dunlop, G.R. Siggins, S.J. Henriksen, J.G. Sutcliffe, A cortical neuropeptide with neuronal depressant and sleep-modulating properties, Nature 381 Ž1996. 242– 245. w7x J.F. Flood, D.W. Landry, M.E. Jarvik, Cholinergic receptor interactions and their effects on long-term memory processing, Brain Res. 215 Ž1981. 177–185. w8x J.F. Flood, F.J. Harris, J.E. Morley, Age-related changes in hippocampal drug facilitation of memory processing in SAMP8 mice, Neurobiol. Aging 17 Ž1996. 15–24. w9x J.F. Flood, J.E. Morley, E. Roberts, Amnestic effects in mice of four synthetic peptides homologous to amyloid b protein from patients with Alzheimer disease, Proc. Natl. Acad. Sci. 88 Ž1991. 3363–3366. w10x J.F. Flood, J.E. Morley, T.H. Lanthorn, Modulation of memory processing by D-cycloserine acting on the NMDA receptor complex, Eur. J. Pharmacol. 221 Ž1992. 249–254. w11x I. Izquierdo, C. Da Cunha, R. Rosat, D. Jerusalinsky, M.B.C. Ferreira, J.H. Medina, Neurotransmitter receptor involved in posttraining memory processing by the amygdala, medial septum and hippocampus of the rat, Behav. Neural Biol. 58 Ž1992. 16–26. w12x B.M. Slotnick, C.M., Leonard. A stereotaxic atlas of the albino mouse forebrain, US Govt. Printing Office, Washington, DC, 1975.
Short communication
The cortical neuropeptide, cortistatin-14, impairs post-training memory processing James F. Flood b
a,b,)
, Kayoko Uezu
a,b
, John E. Morley
a,b
a Geriatric Research Education and Clinical Center (GRECC), VA Medical Center, St. Louis, MO 63106, USA Department of Internal Medicine, DiÕision of Geriatric Medicine, St. Louis UniÕersity School of Medicine, St. Louis, MO 63106, USA
Accepted 26 August 1997
Abstract Cortistatin-14, a neuropeptide, is present primarily in the cortex and hippocampus. In the hippocampus, cortistatin-14 inhibits pyramidal cell firing and co-exists with GABA. To determine if cortistatin-14 would impair retention, saline or cortistatin-14 were injected intracerebroventricularly after footshock avoidance training in CD-1 mice. After 1 week, training was resumed to determine the effect of cortistatin-14 on retention. Cortistatin-14 was found to impair retention relative to the control group at doses of 0.5–5.0 mg. q 1997 Elsevier Science B.V. Keywords: Cortistatin-14; Neuropeptide; Memory; Mice
Cortistatin is a neuropeptide related to somatostatin and is localized within the cortex and hippocampus w6x. It is found in neurons that also contain GABA. Cortistatin-14 inhibited neuronal firing of hippocampal CA1 neurons w6x and blocked acetylcholine induced changes on evoked paired-pulse stimulation of CA1 neurons as well as cortical desynchronization. Increasing acetylcholine turnover or receptor activity is associated with improved retention w1,3– 5,7,8,11x. For these reasons, we determined if cortistatin-14 impaired memory processing. CD-1 male mice Žat least 8 weeks old. obtained from Charles River Breeding Laboratories ŽWilmington, MA. served as subjects. After at least 2 weeks in the laboratory, they were caged individually 48 h prior to training and remained singly housed until retention was tested 1 week later. Animal rooms were on a 12 h light-dark cycle with lights on at 06:00 h. Mice were assigned randomly to groups of 15 and were trained and tested between 08:00 and 14:00 h. Saline or cortistatin-14 ŽPhoenix Pharmaceuticals Inc., Mountain View, CA. was injected intracerebroventricular Ži.c.v.. within 3 min after training. The injection coordinates were 1.0 mm posterior relative to bregma and 1.0 mm to the right of the central suture. The injection depth
)
Corresponding author. VA Medical Center Ž151rJC., 915 N. Grand Blvd., St. Louis, MO 63109, USA. E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 8 4 - 6
was 2.0 mm relative to the skull surface. The scalp was deflected and a hole drilled through the skull on the right side of the central suture. Mice were trained 48 h after surgery. Immediately after training, mice were again placed in the stereotaxic under light enflurane anesthesia. Within 3 min after training, a 2.0 ml solution of saline or cortistatin-14 was injected over 30 s through a 30-ga needle attached to a 10 ml syringe held by a syringe holder attached to the micromanipulator of the stereotaxic instrument. The reliability of the injections was determined histologically by locating the needle tract in frozen brain sections following the retention test. The site of the injection was confirmed using a mouse forebrain stereotaxic atlas w12x. Mice were trained on footshock avoidance in a T-maze w9,10x which consisted of a black plastic alley with a start box at one end and two goal boxes at the other. The start box was separated from the alley by a plastic guillotine door which prevented movement down the alley until training began. An electrifiable stainless steel rod floor ran throughout the maze to deliver scrambled footshock. Mice were not permitted to explore the maze prior to training. A block of training trials began when a mouse was placed into the start box. The guillotine door was raised and a buzzer sounded simultaneously; 5 s later footshock was applied. The goal box entered on the first trial was designated ‘incorrect’ and the footshock was continued until the mouse entered the other goal box,
J.F. Flood et al.r Brain Research 775 (1997) 250–252
which in all subsequent trials was designated as ‘correct’ for the particular mouse. At the end of each trial, the mouse was removed to its home cage until the next trial. Two training conditions were used. The training parameters for one condition, ‘weak’ training, resulted in poor retention test performance of control subject which allowed us to detect improve retention due to peptide administration. For this type of training, mice received four
251
training trials at an intertrial interval of 30 s and the footshock intensity was 0.30 mA. The buzzer intensity was 55 dB. In the second training condition, ‘strong’ training, the parameters reliably resulted in good retention test performance in the control group, thus allowing us to detect impaired retention test performance due to peptide administration. For this type of training, mice received 5 training trials at an intertrial interval of 45 s and the
Fig. 1. Cortistatin failed to improve retention test performance under the weak training condition Žtop. but did impair it relative to the 0 mg group under the stronger training condition Žbottom.. The ) indicates that the mean different from the mean of the control Ž0 mg. group at P - 0.01 by Tukey’s t-test.
252
J.F. Flood et al.r Brain Research 775 (1997) 250–252
footshock intensity was 0.35 mA. The buzzer intensity was 65 dB. After either type of training, saline or cortistatin-14 solution was administered i.c.v. within 3 min after training. One week later, T-maze training was resumed until each mouse made five avoidance responses in six consecutive training trials. Trials to this criterion was taken as a measure of retention. In this experiment, mice were randomly assigned to groups which received 0, 0.05, 0.5, or 5 mg of cortistatin-14. The 0 mg dose received saline. Half of the mice in each of the dose groups were trained under the weak training conditions and the remainder under the strong training conditions. There were 15 mice in each of 8 groups. A 2-way analysis of variance ŽANOVA; dose of cortistatin = training condition., run on the trials to criterion from the retention test, indicated that the effect of dose Ž F3, 112 s 16.24., training condition Ž F1, 112 s 28.26. and their interaction Ž F3, 112 s 16.26. were all significant at P - 0.001. Fig. 1 indicates that under the ‘weak’ training condition cortistatin-14 did not effect retention test performance. This was confirmed by a 1-way ANOVA Ž F - 1.. However, under the ‘stronger’ training condition cortistatin-14 yielded a dose-dependent increase in the mean trials to criterion. Under this training condition, the means of the 0.5 and 5.0 mg dose groups were significantly greater than the mean of the control ŽTukey’s t-test: t s 8.03, t s 12.73, respectively.. When a drug is administered after training, it cannot directly effect learning. Since, we used a 1 week retention period, and cortistatin is promptly metabolized, the drugs cannot directly affect performance on the retention test. If the drugs, by virtue of the time of the injection, cannot directly influence learning or retention test performance, any improvement or impairment in retention test performance relative to the control, we interpreted as being the result of altered neurotransmitter receptor activity occurring shortly after training Ži.e., memory processing.. Most drugs that improve retention under training conditions where control performance is poor Žmean between 9 and 10 trials to criterion. will also impair retention under training conditions were control performance is good Žmean between 6 and 7 mean trials to criterion.. Therefore, it can be difficult to determine if a compound is truly an amnestic if the only data available is a test for impaired retention test performance. Cortistatin-14 impaired memory processing under the strong training condition where controls had a relatively low mean trials to criterion. Since cortistatin-14 failed to improve retention under the weak training condition where control retention test performance was poor, we conclude that cortistatin-14 is a pure amnestic neuropeptide. Increases in GABAergic activity are associated with
impaired retention w2–4,8x. In the hippocampus, GABA may have this effect post synaptically by inhibiting pyramidal cell firing or by presynaptic inhibition of cholinergic terminals which innervate the pryamidal cell. Cortistatin-14 is co-localized with GABA in the hippocampus and inhibits CA1 neuronal fining w6x. This leads us to postulate that cortistatin-14 impaired memory processing by reducing neurotransmitter activity by facilitating GABA inhibition of aceytlcholine activity in the hippocampus.
Acknowledgements This research was supported by the Medical Research Service of the Department of Veterans Affairs.
References w1x R.T. Bartus, R.L. Dean, J.A. Goas, A.S. Lippa, Age-related changes in passive avoidance retention: modulation with dietary choline, Science 209 Ž1980. 301–303. w2x J.D. Brioni, M.W. Decker, L.P. Gamboa, I. Izquierdo, J.L. McGaugh, Muscimol injections in the medial septum impair spatial learning, Brain Res. 522 Ž1990. 227–234. w3x C. Castellano, J.L. McGaugh, Oxotremorine attenuates retrograde amnesia induced by post-training administration of the GABAergic agonists muscimol and baclofen, Behav. Neural Biol. 56 Ž1991. 25–31. w4x J.J. Chrobak, R.W. Stackman, T.J. Walsh, Intraseptal administration of muscimol produces dose-dependent memory impairments in the rat, Behav. Neural Biol. 52 Ž1989. 357–369. w5x C. Dalmaz, I.B. Introini-Collison, J.L. McGaugh, Noradrenergic and cholinergic interactions in the amygdala and the modulation of memory storage, Behav. Brain Res. 58 Ž1993. 167–174. w6x L. de Lecea, J.R. Criado, O. Prospero-Garcia, K.M. Gautvik, P. Schweitzer, P.E. Danielson, C.L.M. Dunlop, G.R. Siggins, S.J. Henriksen, J.G. Sutcliffe, A cortical neuropeptide with neuronal depressant and sleep-modulating properties, Nature 381 Ž1996. 242– 245. w7x J.F. Flood, D.W. Landry, M.E. Jarvik, Cholinergic receptor interactions and their effects on long-term memory processing, Brain Res. 215 Ž1981. 177–185. w8x J.F. Flood, F.J. Harris, J.E. Morley, Age-related changes in hippocampal drug facilitation of memory processing in SAMP8 mice, Neurobiol. Aging 17 Ž1996. 15–24. w9x J.F. Flood, J.E. Morley, E. Roberts, Amnestic effects in mice of four synthetic peptides homologous to amyloid b protein from patients with Alzheimer disease, Proc. Natl. Acad. Sci. 88 Ž1991. 3363–3366. w10x J.F. Flood, J.E. Morley, T.H. Lanthorn, Modulation of memory processing by D-cycloserine acting on the NMDA receptor complex, Eur. J. Pharmacol. 221 Ž1992. 249–254. w11x I. Izquierdo, C. Da Cunha, R. Rosat, D. Jerusalinsky, M.B.C. Ferreira, J.H. Medina, Neurotransmitter receptor involved in posttraining memory processing by the amygdala, medial septum and hippocampus of the rat, Behav. Neural Biol. 58 Ž1992. 16–26. w12x B.M. Slotnick, C.M., Leonard. A stereotaxic atlas of the albino mouse forebrain, US Govt. Printing Office, Washington, DC, 1975.