A partial habituation method to test for anterograde and retrograde amnestic treatment effects: Evidence that antagonism of the NMDA receptor can induce anterograde but not retrograde amnestic effects.

BACKGROUND: A progressive decrease in spontaneous locomotion with repeated exposure to a novel environment has been assessed using both within and between-session measures. While both are well-established and reliable measurements, neither are useful alone as methods to concurrently assess treatment effects on acquisition and retention of habituation. NEW METHOD: We report a behavioral method that measures habituation by combining the within and between measurements of locomotion. We used a 30 min session divided into 6 five min blocks. In the first novel environment session activity was maximal in the first 5 min block but was reduced to a low level by the sixth block, indicative of within-session habituation. Using 8 daily sessions, we showed that this terminal block low level of activity progressed incrementally to the first block to achieve complete habituation. RESULTS/COMPARISON WITH EXISTING METHODS: Within-session activity across sessions was used to identify different stages of between session habituation. It was then possible to assess drug treatment effects from partial to complete habituation, so that treatment effects on retention of the previously acquired partial habituation, expressed as a reversion to an earlier within session habituation pattern (retrograde amnesia assessment), as well as the effects on new learning by the failure in subsequent sessions to acquire complete between-session habituation (anterograde amnesia assessment). CONCLUSIONS: The use of spontaneous motor activity to assess learning and memory effects provides the opportunity to assess direct treatment effects on behavior and motor activity in contrast to many learning and memory models.

Morphine and dopamine: Low dose apomorphine can prevent both the induction and expression of morphine locomotor sensitization and conditioning.

The disinhibition of dopamine neurons in the VTA by morphine is considered an important contributor to the reward potency of morphine. In this report, three experiments were conducted in which a low dose of apomorphine (0.05 mg/kg) was used as a pretreatment to reduce dopamine activity. Locomotor hyperactivity was used as the behavioral response to morphine (10.0 mg/kg). In the first experiment, five treatments with morphine induced the development of locomotor and conditioned hyperactivity that were prevented by apomorphine given 10 min prior to morphine. Apomorphine before either vehicle or morphine induced equivalent reductions in locomotion. In the second experiment, the apomorphine pretreatment was initiated after induction of a conditioned hyperactivity and apomorphine prevented the expression of the conditioning. To assess the effects of apomorphine on VTA and the nucleus accumbens, ERK measurements were carried out after the induction of locomotor and conditioned hyperactivity. Increased ERK activation was found and these effects were prevented by the apomorphine in both experiments. A third experiment was conducted to assess the effects of acute morphine on ERK before locomotor stimulation was induced by morphine. Acute morphine did not increase locomotion, but a robust ERK response was produced indicating that the morphine-induced ERK activation was not secondary to locomotor stimulation. ERK activation was again prevented by the apomorphine pretreatment. We suggest that contiguity between the ongoing behavioral activity and the morphine activation of the dopamine reward system incentivizes and potentiates the ongoing behavior generating equivalent behavioral sensitization and conditioned effects.

Context evoked morphine conditioned effects can be equivalent to morphine induced drug effects in terms of behavioral response and ERK activation in reward associated subcortical brain structures.

Conditioned drug cues can evoke brief drug-like responses. In this report we show that using brief test sessions, contextual cues can induce conditioned hyperlocomotion and ERK responses equivalent to morphine induced responses. To assess acute unconditioned effects, rats that received morphine (MOR-1) or vehicle (VEH-1) were immediately placed onto an arena for a 5-min locomotion recording session after which ERK was measured in the ventral tegmental area (VTA) and nucleus accumbens (NAc). There were no differences in locomotion between the groups. However, the MOR-1 group had strong ERK activation in VTA and NAc. To assess MOR-conditioned effects, a chronic phase was carried out according to a Pavlovian conditioning protocol. There were two MOR paired groups (MORP), one MOR unpaired (MOR-UP) group and two VEH groups. The treatments were administered over 5 daily five minute test sessions. The final conditioning test was on day 6, in which one of the MOR-P groups and one of the VEH groups received VEH (MOR-P/VEH-6 and VEH/VEH-6, respectively). The other MOR-P group and VEH group received MOR (MOR-P/MOR; VEH/MOR-6, respectively). The MOR-UP group received VEH (MOR-UP/VEH-6). Rats received the treatments immediately prior to a 5-minute arena test, and after the session ERK was measured. No morphine induced locomotor stimulation was observed on day 1 but on days 2 to 5, hyperlocomotion in both MOR-P groups occurred. On test day 6, the MOR-P/VEH-6 and the MOR-P/MOR-6 groups had comparable locomotor stimulant responses and similar ERK activity in the VTA and NAc. The MOR-UP group did not differ from the VEH group. We suggest that ERK activation evoked by acute morphine served as a Pavlovian unconditioned stimulus to enable the contextual cues to acquire morphine conditioned stimulus properties and increase the incentive value of the contextual cues.

A new method to study learning and memory using spontaneous locomotor activity in an open-field arena.

BACKGROUND: Reduced locomotion with repeated exposure to a novel environment is often used as a measure of the basic adaptive learning process of habituation. While this is a well-established and reliable measure of habituation, it is not useful for the investigation of neurobiological changes before and after habituation because of the uncontrolled differential activity levels in a novel versus habituated environment. In this study we report a behavioral method that uses spontaneous locomotion to measure habituation, in which the total spontaneous locomotion in an initially novel environment does not change with repeated testing but, the ratio of central to peripheral activity does change and is indicative of habituation. The test sessions are brief (5 min) and the locomotion is measured in 2 separate zones. The peripheral zone comprises 8/9 of the test arena and the central zone 1/9 of the arena. RESULTS/COMPARISON WITH EXISTING METHODS: In contrast to methods that use between-session reductions in locomotion to assess habituation, this method employs brief test sessions in which overall activity between sessions does not change, but the distribution of locomotion in the periphery versus the central zone of the arena does change. The brevity of the test session also enables us to utilize post-trial drug treatment protocols to impact memory consolidation. CONCLUSIONS: The progressive change in the central/peripheral activity ratio with repeated testing can be determined independently of total activity and provides a habituation acquisition function that permits the measurement of neurobiological changes without the complication of effects related to changes in locomotor activity per se. The present report also presents evidence that this method can be used with post-trial drug treatment protocols to study the learning and memory effects of the post-trial treatments without the use of explicit rewards and punishments.

Morphine reward effects and morphine behavioral sensitization: The adventitious association of morphine activation of brain reward effects with ongoing spontaneous activity.

The development of sensitization is one of the hallmarks of addictive drugs such as morphine. We administered morphine (10 mg/kg; MOR) to induce locomotor sensitization and ERK activation in the VTA and NAc. In the first experiment, four groups of rats received five daily 30 min sessions in an open-field, and locomotion was measured. For the first four sessions, one group received MOR pre-test (MOR-P); a second group received vehicle pre-test (MOR-UP) and MOR 30 min post-test; the remaining 2 groups received vehicle (VEH) pre-test. On the fifth session, the MOR-P, MOR-UP, and one VEH group received MOR pre-test and the remaining VEH group received VEH. Sensitization emerged in the first 5 min and progressed over to the second and third 5 min blocks only in the MOR-P group. For the second experiment, 4 groups received MOR and 4 groups VEH, and were then returned to their home cage and after 5, 15, 30 or 60 min post-injection, were euthanized for ERK measurements in VTA and NAc. ERK activation increased and peaked at 5 min post injection in the MOR group and then declined to VEH levels by 30 min. Another two groups received either MOR or VEH immediately before a 5 min arena test and ERK was measured immediately post-test. MOR had no effect on locomotion but increased ERK in the VTA and NAc. The peak ERK activation in VTA reflected activation of reward systems by morphine that reinforced locomotor behavior and with repeated treatments, induced a sensitization effect.

Morphine administered post-trial induces potent morphine conditioned effects if the context is novel but not if the context is familiar.

Morphine administered shortly after exposure to a novel environment induces potent locomotor stimulant conditioning. Environmental novelty is important as pre-exposure (PE) to a stimulus can attenuate the capacity to acquire conditioned stimulus (CS). Here, the importance of environmental novelty for the efficacy of an open-field to become a CS for elicitation of a morphine conditioned response was assessed by comparing the effects of morphine administered post-trial following a 5 min exposure to a novel environment versus a PE environment. Four groups of rats (2 vehicle and 2 morphine groups) were used. Two groups received ten daily 5 min non-drug PEs to an open-field arena and the other two groups were not pre-exposed to the environment. Subsequently, all groups received post-trial injections of either vehicle or morphine immediately after each of five daily 5 min sessions in the open-field. Importantly, on the first day of testing prior to the first post-test morphine administration, the locomotor activity of the novel and PE groups was not different. Over the 5 post-trial morphine treatments, the activity of the PE morphine group, the PE vehicle and the novel environment vehicle groups did not change and were equivalent. In contrast, in the novel environment morphine group, a conditioned hyper-activity response increased with repeated post-trial morphine treatments. For the morphine group it is suggested that the novel environment initiated a post-trial stimulus trace that occurred in temporal contiguity with the post-trial drug response and enabled the trace to become a CS for the morphine unconditioned response. In contrast, PE induced a latent inhibition effect in the PE morphine group, thus the post-trial CS trace was insufficient to become associated to the morphine response and no conditioning occurred. In addition to conventional drug induced Pavlovian delay conditioning, the findings are suggestive of drug induced Pavlovian trace conditioning.

Reversal of morphine conditioned behavior by an anti-dopaminergic post-trial drug treatment during re-consolidation.

Morphine has potent pro-dopamine effects that can be manifested as hyper-locomotion and these behavioral effects can undergo conditioning and sensitization. The aim of the present study was to assess whether an inhibitory dopaminergic post-trial treatment (0.05 mg/kg apomorphine) given during re-consolidation could reduce morphine conditioning. To induce conditioned morphine hyperactivity and control for morphine exposure, a paired/unpaired Pavlovian conditioning protocol was used. The morphine paired groups received morphine in the open-field test arena and the unpaired groups received the same morphine (10 mg/kg) treatments but in a different environment. The morphine treatments were administered once per day for 5 days. With repeated treatments, the paired morphine groups developed a sensitized hyper-locomotion response whereas the unpaired morphine groups did not differ from vehicle groups. Subsequently, the paired, unpaired and vehicle groups were given four daily non-drug 5 min conditioning tests. In these conditioning tests, the paired but not the unpaired and vehicle groups exhibited a conditioned locomotor stimulant response. These groups were subdivided into matched groups and received either vehicle or 0.05 mg/kg apomorphine either during re-consolidation immediately post-test or after re-consolidation 15 min post-test. In the immediate post-trial treatment groups, the morphine conditioned response in the paired group was eliminated after only one post-trial apomorphine treatment. The same immediate 0.05 mg/kg apomorphine post-trial treatments had no effect on the unpaired morphine or vehicle groups. In the paired group that received vehicle immediately post-trial, the conditioned response remained robust and unchanged over the four conditioning tests. In the post-trial 15 min delay treatment groups, the post-trial apomorphine treatments had no effect on the morphine conditioned response. These results showed that the inhibition of dopamine activity by apomorphine during the re-consolidation of a cue activated morphine conditioned response eliminated morphine conditioned effects. In that morphine conditioned effects are important for the initiation of addiction and in triggering drug craving and relapse, this finding has potential relevance to opioid addiction treatment.