Peptides, Vol. 11, pp. 933-938. PergamonPress plc, 1990. Printed in the U.S.A.

0196-9781/90 $3.00 + .00

Vasoactive Intestinal Peptide (VIP): An Amnestic Neuropeptide J A M E S F. F L O O D , t J O N S. G A R L A N D

A N D J O H N E. M O R L E Y

St. Louis University, Division of Geriatric Medicine, St. Louis, MO 63104 and Geriatric Research Education and Clinical Center Veterans Administration Medical Center, St. Louis, MO 63106 R e c e i v e d 18 A p r i l 1990

FLOOD, J. F., J. S. GARLAND AND J. E. MORLEY. Vasoactiveintestinalpeptide (VIP): An amnestic neuropeptide. PEPTIDES 11(5) 933-938, 1990--Vasoactive intestinal peptide (VIP) is a neuropeptide present in high concentrations in the hippocampus. The studies reported here demonstrate that VIP administered into the third ventricle of the brain caused amnesia in mice trained on a left-right footshock avoidance task in a T-maze. VIP resulted in amnesia when administered directly into the rostral portion of the hippocampus at a 10-fold lower dose than was needed to produce amnesia when VIP was administered intracerebroventricularly. When VIP was administered 24 hr after training, it failed to impair retention measured a week later. VIP receptor antagonist ([4-C1-D-Phe6,LeulV]VIP) enhanced retention when administered into the rostral portion of the hippocampus, suggesting that VIP plays a physiological role in memory modulation. VIP receptor antagonist administered 24 hr after training did not facilitate retention. To gain some insight as to how VIP may be affecting memory processing, we determined if some memory-improving compounds showed a selective ability to block amnesia induced by VIP. The amnestic effect of VIP was blocked by peripheral administration of the memory-enhancing agents, arecoline, naloxone and ST 587 (a noradrenergic receptor agonist) but not by cholecystokinin octapeptide. Central administration of arecoline, but not neuropepide Y, blocked the amnestic effect of VIP. It is concluded that VIP is a potent amnestic peptide. Mice

Memory

Neuropeptides

Retention

VIP

VASOACTIVE intestinal peptide (VIP) was first isolated from pig intestine (30). The neuropeptide is widely distributed throughout the peripheral and central nervous system in many species (21). VIP exerts effects on a variety of physiological functions including: regional blood flow, cardiovascular response, smooth muscle tone, secretion of water, immune and neuroendocrine function, brain metabolism by promoting the hydrolyis of glycogen (29). Evidence has been obtained that VIP coexists in neurons also containing acetylcholine, gamma amino butyric acid (GABA), norepinephrine, neuropeptide Y (NPY), cholecystokinin and dynorphin (7). VIP meets the commonly agreed criterion for established neurotransmitter function in the central and pheripheral nervous system (1,29). It has been shown to effect neuronal excitability (8,26), exert a powerful excitatory effect on CA1 neurons of the hippocampus (6), increase choline acetyltransferase activity in hippocampal tissue by increasing the enzyme's affinity for choline rather than acetyl-CoA (19), enhance muscarinic binding (20), activate adenylate cyclase and cyclic-AMP (4) and act synergistically with norepinephrine (8) and acetylcholine (27). Evidence indicates that most VIP neurons act within a specific local neuronal population to regulate activity of the area but projections through the limbic cortex, hippocampal collateral projections, hypothalamus and central gray of the midbrain have

been identified. VIP exists in high concentrations in the CA 1, CA3 and the dentate gyrus of the hippocampus, the amygdala and the cerebral cortex (5,21). We determined the effect of VIP on retention since it was found to be coexistent with neurotransmitters, which are known to modulate memory processing, and because of its localization within the limbic structures. The effect of posttraining administration of VIP on memory processing in the central nervous system as a whole and in the hippocampus was studied. METHOD

Subjects and Drugs After 1 week in the laboratory, CD-1 male mice, obtained at 6 weeks of age from Charles River Breeding Laboratories, Wilmington, MA, were individually caged 24-48 hr prior to training and remained singly housed until retention was tested one week later. The median body weight was 35 g, with a range of 33 to 38 g. Animal rooms are maintained on a 12-hr light-dark cycle with light on at 0600. The mice were trained between 0700 and 1500. Mice were assigned randomly to coded groups to prevent experimenter bias. Vasoactive intestinal peptide (VIP), VIP receptor antagonist

1Requests for reprints should be addressed to Dr. James F. Flood (151/JC), VA Medical Center, 915 N. Grand Blvd., St. Louis, MO 63106.

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([4-C1-D-Phe6,LeuIT]VIP), neuropeptide Y (NPY) and cholecystokinin octapeptide (CCK-8) were purchased from Peninsula Laboratories, Inc., Belmont, CA. Arecoline hydrobromide (ARE) was purchased from Sigma Chemical Company, St. Louis, MO. Naloxone (NLX) was provided by DuPont Chemical Company, Wilmington, DE and ST 587 by Boehringer Engelheim Pharmaceuticals, Inc. All drug solutions were prepared in saline. The doses of ARE, NLX and ST 587 are expressed on the basis of the salt. The dosages of drugs and peptides are given in the appropriate experiments below.

T-Maze Apparatus and Training The T-maze apparatus has been previously described (11). It consisted of a black plastic start alley (46 cm long) with a start box at one end and two goal boxes (17.5 cm long) at the other and a depth of 12.5 cm and width of 9.8 cm throughout. The floor consisted of stainless steel rods. The start box was separated from the start alley by a plastic guillotine door which prevented the mouse from moving down the alley until the training started. A training trial started when a mouse was placed into the start box. The guillotine door was raised and the buzzer sounded simultaneously, then 5 sec later the footshock was applied. The goal box that the mouse first entered on this trial was designated as "incorrect" and the footshock was continued until the mouse entered the other goal box, which on all subsequent trials was designated "correct" for that particular mouse. At the end of each trial, the mouse was removed from the goal box and returned to its home cage. A new trial began by placing the mouse in the start box, at which time the buzzer was sounded and the guillotine door was raised. Footshock was given 5 sec later if the mouse did not move into its correct goal box. Entry into the correct goal box terminated the buzzer and footshock. Two training conditions were used in these studies. Weak training was used to induce poor retention (mean trials to criterion of greater than 9) in the control group so that enhanced retention could be detected. Under this training condition mice received 4 training trials with an intertrial interval of 30 sec. A doorbell-type buzzer (55 dB) served as the conditioned stimulus. The unconditioned stimulus was 0.30 mA of footshock (Coulbourn Instruments scrambled grid floor shocker model E3-08). In the second training condition, the training strength was increased so that the control group showed good retention (mean trials to criterion less than 7), enabling detection of impaired retention test performance. This was accomplished by giving 5 training trials with a 45-sec intertrial interval, an unconditioned stimulus of 65 dB and 0.35 mA footshock. Under weak or strong training conditions, retention was tested one week later by continuing to train the mice until they made 5 avoidances in 6 consecutive trials. The overall significance of the drug treatment effect was determined by a one-way or two-way analysis of variance (15). Dunnett's t-test was used to make multiple comparisons between each drug group and the control group (15).

Drug Administration The surgical procedures used to prepared mice for bilateral injections into the hippocampus or ICV injection into the third ventricle have been described in detail previously (1 I). In brief, mice were anesthetized with methoxyflurane, placed in a stereotaxic instrument and holes were drilled through the skull over the injection sites after deflecting the scalp. The coordinates for bilateral injections into the rostral portion of the hippocampus were - 1.6 mm with respect to bregma, 1.6 mm right and left of

FLOOD

the central suture and 1.6 mm deep. The coordinates for the ICV injection were - 0 . 5 mm with respect to bregma, 0.5 mm to the right of the central suture and 2.8 mm deep. The coordinates were confirmed using a stereotaxic atlas (31). Mice were trained 24-48 hr after surgery. Immediately after training mice were again placed in the stereotaxic under enflurane anesthesia. Within three minutes after training, a 0.5-txl solution of drug or saline was injected into the target structure over 60 sec through a 31-gauge needle fixed to a 10-txl syringe with PE-10 tubing and driven by a Sage Syringe Pump (Model 341A). This method of injection resulted in reliable administration into the hippocampus (Fig. 1). The reliability of the injections was determined by injecting dye into the sites and determining the location of dye in frozen brain sections. In addition, 31-gauge tubing was implanted and the mice were sacrificed and frozen brain sections examined to determine placement. After technicians practiced on between 50 to 200 mice, depending on the brain structure, the accuracy of the injection was determined by histological verification of the location of dye or the cannula tip. Accuracy was 100% over 20 to 30 tests. Mice received ICV injections in a similar manner except that 2 ILl of drug solution or saline was administered. All drugs and peptides were administered after training so that they could not infuence acquisition by altering sensorimotor ability of motivation to avoid the footshock. The long one-week retention interval was used to measure effects on long-term retention so that the drugs and peptides would not have direct effect on retention test performance per se. Thus in a paradigm where compounds cannot directly affect performance either at the time of training or testing, we conclude that altered retention is due to altered memory processing. RESULTS

Experiment 1. Effect of Intracerebroventricularly Administered VIP on Retention Since a preliminary study failed to find improvement of retention, the purpose of this experiment was to determine if VIP impaired retention in a dose-dependent manner. Mice were trained so that the control group would show good retention as described above. Immediately after training, separate groups of mice (N = 15) received 0.5, 1.0, 2.5, or 5.0 ixg of VIP or saline ICV. Retention was tested one week later by continuing the training until a mouse made 5 avoidance responses in 6 consecutive training trials. As the dose of VIP increased so did the mean trials to criterion. A one-way ANOVA run on trials to criterion indicated a significant treatment effect, F(4,70)= 5.42, p

Vasoactive intestinal peptide (VIP): an amnestic neuropeptide.

Vasoactive intestinal peptide (VIP) is a neuropeptide present in high concentrations in the hippocampus. The studies reported here demonstrate that VI...
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