P. Gerhardt

Sequence-specific effects of neurokinin substance P on memory, reinforcement, and brain dopamine activity.

There is ample evidence that the neurokinin substance P (SP) can have neurotrophic as well as memory-promoting effects. This paper outlines a recent series of experiments dealing with the effects of SP and its N- and C-terminal fragments on memory, reinforcement, and brain monoamine metabolism. It was shown that SP, when applied peripherally (IP), promotes memory (inhibitory avoidance learning) and is reinforcing (place preference task) at the same dose of 37 nmol/kg. Most important, however, is the finding that these effects seemed to be encoded by different SP sequences, since the N-terminal SP1-7 (185 nmol/kg) enhanced memory, whereas C-terminal hepta- and hexapeptide sequences of SP proved to be reinforcing in a dose equimolar to SP. These differential behavioral effects were paralleled by selective and site-specific changes in dopamine (DA) activity, as both SP and its C-, but not N-terminus, increased extracellular DA in the nucleus accumbens (NAc), but not in the neostriatum. The neurochemical changes lasted at least 2 h after injection. These results show that the reinforcing action of peripheral administered SP may be mediated by its C-terminal sequence, and that this effect could be related to DA activity in the NAc. Direct application of SP (0.74 pmol) into the region of the nucleus basalis magnocellularis (NBM) was also memory-promoting and reinforcing, and again, these effects were differentially produced by the N-terminus and C-terminus, supporting the proposed structure-activity relationship for SPs effects on memory and reinforcement. These results may provide a hypothetical link between the memory-modulating and reinforcing effects of SP and the impairment in associative functioning accompanying certain neurodegenerative processes.

Positively reinforcing effects of the neurokinin substance P in the basal forebrain: Mediation by its C-terminal sequence

The conditioned corral preference paradigm was used to assess reinforcing effects of substance P (SP) and its N- and C-terminal fragments injected unilaterally into the region of the nucleus basalis magnocellularis (NBM) in rats. Behavioral testing was carried out in a circular open field, consisting of 4 quadrants equally preferred by the animals prior to conditioning. A single conditioning trial was performed. Rats received one microinjection (0.5 microliter) of SP (0.74 pmol), of the N-terminal fragment SP (1-7) and the C-terminal fragment analog DiMe-C7 (each at doses of 0.074, 0.74, and 74 pmol), or vehicle (phosphate-buffered saline; PBS). After injection the rats were placed into the open field with the four quadrants being separated by Plexiglas barriers (closed corral). During the test for conditioned corral preference, when provided a choice between the four quadrants, only those rats injected with SP and the equimolar dose of DiMe-C7 (0.74 pmol) spent more time in the treatment corral, indicative of a positively reinforcing action. None of the other doses of DiMe-C7 and of SP(1-7) influenced the preference behavior. For rats injected with 0.74 pmol SP, SP (1-7), and DiMe-C7, a behavioral analysis was performed for the 15 min conditioning trial. SP and DiMe-C7 reduced rearing and grooming behavior, whereas DiMe-C7 and SP(1-7) increased locomotor activity. However, the acute behavioral effects of SP and its fragments were not correlated with the subsequent place preference behavior during the test trial. The results are discussed in the framework of a structure/activity relationship for the positively reinforcing properties of SP in the region of the NBM. Furthermore, neuropathological implications of the present data are considered, since the homologous nucleus basalis of Meynert in man is known to degenerate in Alzheimers disease, which is characterized behaviorally by a progressive deterioration in associative functioning.

Evidence for mnemotropic action of cholecystokinin fragments Boc-CCK-4 and CCK-8S

Memory-modulating and reinforcing effects of the cholecystokinin (CCK) fragments, CCK-8S and Boc-CCK-4, after systemic application in rats were investigated. Habituation to the novelty of environmental stimuli was used to test for mnemonic effects using two different tasks (rearing behavior in an open field; head-dips in a hole-board). Immediate posttrial administration of CCK-8S and Boc-CCK-4 resulted in a reduction of rearing and head-dip behavior during testing, indicative of enhanced habituation and, thus, facilitation of memory. In contrast, administration of CCK-8S and Boc-CCK-4 with a delay of 2.5 or 5 h after training or pretrial injection of CCK-8S did not enhance habituation. No evidence for reinforcing or aversive properties of CCK-8S and Boc-CCK-4 was observed in a conditioned place preference task. In summary, the results indicate memory-enhancing effects of peripherally, posttrial-administered CCK-8S and Boc-CCK-4.

MODULATION OF MEMORY, REINFORCEMENT AND ANXIETY PARAMETERS BY INTRA-AMYGDALA INJECTION OF CHOLECYSTOKININ-FRAGMENTS BOC-CCK-4 AND CCK-8S

This series of experiments examined the effects of the cholecystokinin (CCK) fragments Boc-CCK-4 and CCK-8s on memory, reinforcement and anxiety following unilateral injection into the central nucleus of the amygdala (CeA). In experiment 1, rats with chronically implanted cannulae were injected with CCK-8s or Boc-CCK-4 and were tested on a one-trial uphill avoidance task. Post-trial injection of 20 ng Boc-CCK-4 or 1 ng CCK-8s was found to improve the retention performance, whereas lower and higher doses had no effect. The hypermnestic effects of Boc-CCK-4 and CCK-8s were no longer evident when injection was performed 5 h, rather than immediately, after the learning trial. In experiment 2, the elevated plus-maze was used to gauge anxiogenous properties of intra-amygdala injections of Boc-CCK-4 and CCK-8s in memory-enhancing doses. The treatment with 20 ng Boc-CCK-4 and 1 ng CCK-8s did not influence the number of entries into and time spent on the open and enclosed arms of the maze as well as other anxiety-related behaviors. In experiment 3, possible reinforcing effects of the CCK-fragments were examined. After intra-amygdala injection of Boc-CCK-4 or CCK-8s in memory-enhancing doses the rats were placed into one of four restricted quadrants of a circular open field (closed corral) for a single conditioning trial. Subsequent tests for conditioned corral preference revealed no evidence for reinforcing or aversive effects of the CCK-fragments. In sum, these findings indicate that Boc-CCK-4 and CCK-8s facilitate memory processing upon injection into the CeA without exerting reinforcing or anxiogenous effects.

Substance P enhancement of inhibitory avoidance learning: Mediation by the N-terminal sequence

Experiments were performed to investigate the effects of intraperitoneally administered undecapeptide substance P (SP), its N-terminal fragment SP(1-7) (SPN) and the C-terminal analog [pGlu6]-SP(6-11) (SPC) on inhibitory avoidance learning, using a one-trial up-hill avoidance task. In Experiment 1 rats were injected with either SP (50 micrograms/kg), SPN (3.3, 33, 167, 333 micrograms/kg) or SPC (2.7, 27, 134, 268 micrograms/kg) immediately after the training trial. Controls received the diluent vehicles. When tested 24 hr later, rats injected with 50 micrograms/kg SP (37 nmol/kg) and 167 micrograms/kg SPN (185 nmol/kg) exhibited longer step-up latencies than vehicle-treated controls. None of the other doses of SPN nor of the C-terminal fragment influenced performance. In Experiment 2, 167 micrograms/kg SPN or vehicle was injected posttrial either immediately or 5 hr after the training trial. Retention latencies 24 hr later were longer for rats treated with 167 micrograms/kg SPN immediately after the training trial. Performance of the SPN 5-hr delay group did not differ from that of the vehicle-injected controls, ruling out proactive effects of SPN on recall.

Naloxone blocks conditioned place preference induced by substance P and [pGlu6]-SP(6–11)

The effect of prior treatment with the opioid receptor (opioceptor) antagonist naloxone on conditioned place preference produced by the neurotachykinin substance P (SP) and its C-terminal hexapeptide analog [pGlu6]-SP(6-11) (SPC) was investigated in rats. Place conditioning was assessed using a circular open field partitioned into four quadrants that were equally preferred by the rats prior to drug treatment. On three successive days, rats received an intraperitoneal (i.p.) injection of naloxone-HCl (1 mg/kg) or vehicle 15 min before an i.p. injection of either 37 nmol/kg SP, equimolar dosed SPC or corresponding diluent vehicle. After injection the rats were placed into their assigned treatment corral for 15 min. During the test for conditioned corral preference (CCP), when provided a choice between the four quadrants, rats injected with SP or SPC spent more time in the treatment corral compared to vehicle controls, indicative of a positive reinforcing action of these peptides. The pre-treatment with naloxone blocked the positive reinforcing effects of both SP and SPC; when injected alone, naloxone did not influence the preference behavior. Gross locomotor activity was affected by neither treatment. Thus, the positive reinforcing effects of SP and SPC may be mediated via interactions with the endogenous opioid system(s).

Application of ‘nose-poke habituation’ validation with post-trial diazepam- and cholecystokinin-induced hypo- and hypermnesia

The present study describes the use of nose-poke habituation as a memory task and demonstrates that it is sensitive to hypo- and hypermnestic pharmacological treatments administered post-trial. Habituation of nose-poke behavior of rats was defined as a reduction in number of nose-pokes compared to baseline. It was measured using a board with 16 holes, to which animals were exposed on 2 consecutive days (baseline and test) for 10 min, respectively. After the first exposure, rats were injected intraperitoneally (i.p.) immediately or with a delay of 2.5 h with doses of diazepam (0.9-4.5 mg/kg) known to be hypomnestic, or cholecystokinin (CCK-8S; 0.2-25 micrograms/kg), which was reported to have memory facilitating effects. An enhancement of habituation in comparison with vehicle controls was interpreted in terms of a hypermnestic effect of the treatment. Conversely, hypomnestic action of the drug treatment was inferred from a reduced habituation. The results show that when diazepam was injected immediately post-trial, the normal reduction in number of nose-pokes during test was prevented, indicative of a failure to habituate presumably due to an amnesia for the baseline/training trial. In contrast, enhanced habituation (facilitation of memory) was induced when CCK-8S was injected immediately post-trial, as reflected by a decrease in number of nose-pokes during test compared to control animals. The effects were not due to enduring proactive effects of the compounds on performance during test, since post-trial injections of diazepam or CCK-8S with a delay of 2.5 h did not have the effects that immediate post-trial injection had.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced learning produced by injection of neurokinin substance P into the region of the nucleus basalis magnocellularis : mediation by the N-terminal sequence

The effect of unilateral injection of the neurokinin substance P (SP) and of certain N- or C-terminal SP-fragments into the region of the nucleus basalis magnocellularis (NBM) on inhibitory avoidance learning was investigated. Rats with chronically implanted cannulae were tested on a one-trial uphill avoidance task. Immediately after the training trial, rats were injected with 0.74 pmol SP or equimolar dosed SP(1-7), DIME-C7, or SP(7-11). Control groups included vehicle-injected rats and a group given an injection of SP(1-7) 5-h after the trial. When tested 24 h later, rats treated with SP or SP(1-7), but not with DIME-C7 or SP(7-11), exhibited longer step-up latencies than vehicle-treated controls. The retention latencies for rats in the SP(1-7) 5-h delay group did not differ from those of vehicle-injected animals, ruling out proactive effects of SP(1-7) on performance. The results show that SP facilitates retention of an inhibitory avoidance response when injected into the NBM. Furthermore, the amino acid sequence that encodes this effect may be located in the N-terminal part of the SP-molecule.

Mnemogenic effects of injecting RA-octil, a CE-inhibitor derivate, systemically or into the basal forebrain

The aim of this study was to investigate the effects of systemically or intracerebrally administered RA-octil, a derivate of the angiotensin converting enzyme (CE)-inhibitor ramipril, on memory and reinforcement and to compare its effectiveness with that of the neurokinin substance P (SP). In the first experiment systemic post-trial application of RA-octil and SP in the rat enhanced habituation, a learning task which does not require motivational treatments. Unlike SP, injection of RA-octil did not have reinforcing effects as measured with a conditioned place preference task. In the second experiment, a facilitation of inhibitory avoidance learning was obtained by injection of RA-octil or SP unilaterally into the basal forebrain immediately after the learning trial. In contrast, a 5 h delayed injection of RA-octil had no effects on learning. The results demonstrate memory-enhancing effects of RA-octil after systemic application as well as after injection into the basal forebrain. Furthermore, the mnemogenic effects of SP after central and peripheral administration were confirmed. Since RA-octil, although being structurally closely related to CE-inhibitors, does not affect plasma CE, yet exhibits mnemogenic effects, it is possible that “cognition-enhancing” actions of CE-inhibitors are dissociable from their action within the renin-angiotensin system.

Comparison of neurokinin substance P with morphine in effects on food-reinforced operant behavior and feeding

In the present study, substance P (SP) was injected intraperitoneally (IP), and its effects on operant behavior were assessed in rats, which had been trained to bar press for food reward on a fixed-ratio (FR) 20 schedule. These effects were compared with IP injection of morphine sulfate, which had previously been shown to strongly suppress operant responding on FR schedules. The IP injection of SP resulted in a dose-related decrement in response rates. SP in a dose range of 250-500 micrograms/kg decreased operant responding, whereas SP in a dose range of 5-50 micrograms/kg did not influence response rates. The IP injection of morphine (10 mg/kg) markedly suppressed operant responding. However, in contrast to the rate-decreasing effects of SP, this suppression was not selective for the reinforced lever as responding on the nonreinforced lever, used as a control, was also decreased. Furthermore, both injection of 10 mg/kg morphine and SP in a dose range of 250-500 micrograms/kg was found to reduce food intake when the animals had free access to food subsequent to the operant conditioning session. The present results provide the first evidence that systemically administered neurokinin SP can affect operant responding for food reward. The suppressive effects on operant behavior and feeding obtained with systemic SP or morphine are discussed with respect to recent findings showing that both drugs can modulate mesolimbic dopamine activity after systemic drug injection.