Research reportIdentification of the opioid receptors involved in passive-avoidance learning in the day-old chick during the second wave of neuronal activity
Introduction
One-trial passive-avoidance training in the day-old chick is an attractive model to study long-term memory formation. This paradigm exploits the precocity of newly hatched chicks, who explore their environment by pecking and rapidly learn to distinguish between edible and distasteful objects. If a chick is presented with a bead coated in the bitter-tasting substance methylanthranilate (MeA), it will peck once, show a characteristic disgust response, and subsequently avoid a similar but dry bead presented later [6], [25]. This paradigm has the advantage that since it takes only a single brief training trial, the exact time of memory inception is known and hence the sequence of events that occur during memory consolidation. Using this paradigm, Freeman et al. [21] have shown the existence of two distinct waves of protein synthesis. The first occurs up to 90 min post-training and the other between 4 and 5 h after training. Two phases of neuronal activity following training have also been demonstrated in the chick. Electrophysiological studies have shown a dramatic increase in spontaneous high frequency neuronal bursting [49]. Initially, this bursting activity is distributed between left and right intermediate medial hyperstriatum ventrale (IMHV), but within 4–7-h shifts to the right IMHV and to the lobus parolfactorius (LPO) [26], [27]. A series of lesion studies [28], [56], [57] has shown that the IMHVs are involved in the acquisition of memory but not its retention, whereas the LPOs are involved in retention and recall but not the acquisition of memory for the passive-avoidance training. Studies using c-Fos and c-Jun as markers of neuronal activity have also demonstrated a biphasic pattern of activity, where first the IMHV is activated followed by the LPO [19]. These findings fit in with the concept of two phases of neuronal activity with information being processed in one area of the brain (e.g. IMHV) before being redistributed to other brain regions (e.g. LPO).
There are three well studied groups of opioid receptors μ, δ, and κ [37]. Endogenous opioid peptides and exogenous non-peptides (alkaloids or opiates) are known to interact with their receptors to mediate a number of biological events which include the modulation of pain, analgesia, behavioural and locomotor activity and effects on the neuroendocrine systems [44]. All three receptor classes are G-protein coupled receptors which have been shown to inhibit adenylyl cyclase, to decrease the conductance of volted gated Ca2+ channels or activate K+ channel current thereby reducing membrane excitability and hence transmitter release [7]. A novel member of the opioid receptor family, the ORL1 (opioid receptor-like) receptor is expressed in relatively high abundance in rat hippocampus [1]. This receptor is closely homologous to the μ, δ and κ-receptors, but does not bind with appreciable affinity the classical opioid ligands [3], [5], [24], [39], [45], [52], [76]. An endogenous ligand for the ORL1-receptor, orphanin FQ (also known as nociceptin), has been isolated and found to have hyperalgesic rather than analgesic properties [50], [59], suggesting an anti-opioid role of this peptide in nociceptive processing. Orphanin FQ has been found to impair spatial learning and inhibit synaptic transmission and LTP in rat [64], [77]. Other, putative members of the opioid family of receptors include the ϵ- and ζ-opioid receptors [55]. However, little is known about the pharmacology of these receptors.
Injection of various agonists and antagonists of opioid receptor function into the IMHV around the time of training have identified an involvement of the μ-, δ- and κ-opioid receptors in passive-avoidance memory formation in the chick [9], [10], [11], [58]. However, electrophysiological studies have also identified a second distinct wave of neuronal activity that occurs between 4 and 7 h post-training in the LPO [26], [27] which must reflect a change in neurotransmission. We therefore chose to look at the role of opioid receptors during this phase of memory consolidation. In addition, we presumed that opioid receptor activity involves protein synthesis and since protein synthesis is required between 4 and 5 h post-training [21] we chose to study the process 5 h after training in order to allow some time for translation to occur. In this study we examine the role of opioid receptors during the second wave of neuronal activity by injecting agonists and/or antagonists of the μ-, δ-, κ- and ORL1-receptors directly into the LPO 5 h post-training. In addition, since both cytosolic and mitochondrial protein synthesis has been shown to be important in memory consolidation during this second wave of neuronal activity [22] we have injected agonists of the μ- and δ-receptors to see if they can reverse the amnesia due to protein synthesis inhibition.
Section snippets
Preparation and injection of drugs
All opiates/peptides were initially dissolved in water and their salinity adjusted to 0.9% with 2× saline. A solution of chloramphenicol (CAP) was freshly prepared by first dissolving the solid in a minimal quantity of ethanol and the solution was adjusted to a final concentration of 7.4 mM (2.4 mg/ml) with saline. A 30-mM (8 mg/ml) solution of anisomycin (ANI) was prepared by dissolving the solid in a minimal quantity of 3 M HCl, after which the pH was adjusted to 7 by the addition of 3 M
Results
None of the drugs tested produced any effect on the ability of the chicks to discriminate between an aversive (chrome bead) and non-aversive (white bead) stimulus, indicating a lack of effect on normal neuronal functioning (results not shown).
The role of the μ-opioid receptor in passive-avoidance learning
Fig. 1B shows that bilateral intracranial injections of 100 μM β-FAN, a specific antagonist of the μ-opioid receptor [36], [72], [74], administered 5 h post-training, caused amnesia for the learning task. This finding indicates an involvement of the μ-opioid receptor in long-term memory formation for a passive-avoidance learning task during the second wave of neuronal activity. Endo-2, an endogenous and highly specific agonist of the μ-opioid receptor [79] failed to disrupt memory formation
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