Weiqin Zhao

Complex Roles of Glutamate in the Gibbs—Ng Model of One-trial Aversive Learning in the New-born Chick

Glutamate is the most widespread excitatory transmitter in the CNS and is probably involved in LTP, a neural phenomenon which may be associated with learning and memory formation. Intracerebral injection of large amounts of glutamate between 5 min and 2.5 min after passive avoidance learning in young chicks inhibits short-term memory, which occurs between 0 and 10 min post-learning in a three-stage model of memory formation first established by Gibbs and Ng(25) [Physiol. Behav. 23:369-375; 1979]. This effect may be attributed to non-specific excitation. Blockade of glutamate uptake by L-aspartic and beta-hydroxamate also abolishes this stage of memory, provided the drug is administered within 2.5 min of learning. Interference with either production of percursors for transmitter glutamate in astrocytes or with glutamate receptors is also detrimental to memory formation, but the effects appear much later. After its release from glutamatergic neurons, glutamate is, to a large extent, accumulated into astrocytes where it is converted to glutamine, which can be returned to glutamatergic neurons and reutilized for synthesis of transmitter glutamate, and partly oxidized as a metabolic substrate. The latter process leads to a net loss of transmitter glutamate which can be compensated for by de novo synthesis of a glutamate precursor alpha-ketoglutarate (alpha KG) in astrocytes, a process which is inhibited by the astrocyte-specific toxin fluoroacetate (R. A. Swanson, personal communication). Intracerebral injection of this toxin abolishes memory during an intermediate stage of memory processing occurring between 20 and 30 min post-training (50) [Cog. Brain Res, 2:93-102; 1994]. Injection of methionine sulfoximine (MSO), a specific inhibitor of glutamine synthetase, which interferes with the re-supply of transmitter glutamate to neurons by inhibition of glutamine synthesis in astrocytes, has a similar effect. This effect of MSO is prevented by intracerebral injection of glutamate, glutamine, or a combination and alpha KG and alanine. MSO must be administered before learning, but does not interfere with acquisition since short-term memory remains intact. Administration of either the NMDA antagonist AP5, the AMPA antagonist DNQX, or the metabotropic receptor antagonist MCPF, also induces amnesia. Memory loss in each case does not occur until after 70 min post-training, during a protein synthesis-dependent long-term memory stage which begins at 60 min following learning. However, to be effective, AP5 must be administered within 60 s following learning, MCPG before 15 min post-learning, and DNQX between 15 and 25 min after learning. Together, these findings suggest that learning results in an immediate release of glutamate, followed by a secondary release of this transmitter at later stages of processing of the memory trace, and that one or both of these increases in extracellular glutamate concentration are essential for the consolidation of long-term memory. Since both fluoroacetate and MSO act exclusively on glial cells, the findings also show that neuronal-glial interactions are necessary during the establishment of memory.

Effect of PKC inhibitors and activators on memory

Changes in the activity of the enzyme protein kinase C (PKC) have been implicated in learning and memory consolidation, and in the induction of long-term potentiation. The precise role of PKC in memory processing is still unknown. Using 1-day-old chicks trained on a single-trial passive avoidance task, we demonstrate that inhibition of PKC activity by melittin induced retention loss, in a dose-dependent manner, in the second stage of a three-stage sequence of memory processing. The effect was lateralized to the left hemisphere of the chick forebrain. This effect of melittin was prevented by high concentrations (16-320 microM) of the PKC activator, phorbol 12-myristate 13-acetate (PMA). Furthermore, concentrations of PMA in the range 1.6 to 40 microM were shown to induce long-term memory consolidation following a weakly reinforced version of the learning task, which normally does not lead to formation of long-term memory. That these actions of PMA are attributable to PKC activation is supported by the further finding that the inactive phorbol ester 4 alpha-PDD had no effect either on melittin-induced amnesia or on memory consolidation following weakly reinforced learning. Paradoxically, concentrations of 16 microM or higher of PMA inhibited memory consolidation for the normal strongly reinforced learning trial, an effect again not observed with 40 alpha-PDD. The results are consistent with the view that PKC activity may be implicated in a pre-long-term stage of memory processing.

The impairment of long-term memory formation by the phosphatase inhibitor okadaic acid

While there is considerable evidence that protein kinase activity is involved in memory formation, there has been, as yet, no direct investigation of a role for protein phosphatases. However, phosphatases have been implicated in the effects of the activation of glutamate receptors of the NMDA type, in long-term depression, and in the regulation of transmitter release and membrane ion channel activities, phenomena which have been shown to be possibly involved in cellular memorial processes. In the present paper, inhibition of protein phosphatase by 0.5 nM okadaic acid, a selective inhibitor of phosphatases 1 and 2A, is demonstrated to prevent memory consolidation in day-old chicks trained on a single trial passive avoidance task. Retention losses first occurred after 30 min post-learning, at an intermediate stage of memory formation preceding a protein synthesis-dependent long-term stage. It is suggested that protein phosphatase activity is involved in precursor processes to long-term memory consolidation.

Concentration-dependent effects of protein phosphatase (PP) inhibitors implicate PP1 and PP2A in different stages of memory formation.

Numerous studies have demonstrated roles for protein phosphorylation and for specific kinases in memory formation; however, a role for specific protein phosphatases has not been established. Previous studies using pharmacobehavioral methods to implicate protein phosphatase activity in memory formation have been unable to discriminate between protein phosphatases 1 (PP1) and 2A (PP2A), as available cell-permeable agents generally inhibit both enzyme classes. To address this difficulty the present study exploited differences in the potency of the selective phosphatase inhibitor, okadaic acid, toward PP1 and PP2A. Within the context of a temporally precise animal model of memory, developed using the day-old chick (Gallus domesticus), acute administration of various concentrations of okadaic acid was found to disrupt two temporally distinct stages of memory formation. When administered bilaterally into an area of the chick brain implicated in memory formation, concentrations of okadaic acid known to selectively inhibit PP2A in vitro disrupted memory from 50 min posttraining. Higher concentrations, reported to inhibit both PP2A and PP1 in vitro, produced significant retention deficits from 20 min posttraining. Identical temporally specific effects were also obtained by varying the concentration and time of administration of calyculin A, a phosphatase inhibitor with equal potency toward both enzyme classes. Hence, different phosphatase enzymes may contribute to different stages of the enzymatic cascade believed to underlie memory formation.

Peptidyl‐prolyl‐cis/trans‐isomerase activity may be necessary for memory formation

At present, evidence for a plethora of physiological roles for the different classes of peptidyl‐prolyl‐cis/trans‐isomerases (PPIases, EC 5.2.1.8) is emerging. Cyclosporin A (CyA) has been previously reported to disrupt memory formation in a temporally specific manner, when administered intracranially to day‐old chicks trained on a single‐trial, passive‐avoidance task [Bennett, P.C., Zhao, W., Lawen, A. and Ng, K.T. (1996) Brain Res. 730, 107–117]. CyA is known to inhibit both the PPIase activity of cyclophilin and, indirectly, the protein phosphatase activity of calcineurin. Therefore to begin to distinguish between these two functions we studied the effects on memory formation of three non‐immunosuppressive CyA analogues, in order to study the involvement of cyclophilins. These drugs retain the capacity to bind to and inhibit the PPIase activity of cyclophilin, but do not bind in the complex with cyclophilin to calcineurin and, therefore, do not inhibit its phosphatase activity. All three drugs exert effects on memory formation comparable to those induced by CyA, significantly inhibiting memory formation when injected intracranially (50 fmol per hemisphere) immediately following training. Brain extracts from chicks treated with [MeVal4]CyA show a strong inhibition of cyclophilin activity. These data show a requirement for the PPIase activity of a cyclophilin for successful memory formation and constitute the first set of data establishing a physiological role for a cyclophilin.

Changes in phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in processing of short-term and long-term memories after passive avoidance learning

Characteristic autophosphorylation of calcium/calmodulin‐dependent protein kinase II (CaMKII) and its consequences have made this kinase an interesting target in studying the molecular pathway for important neuronal functions including learning and memory formation. In this article, we use immunoprecipitation and immunoblotting methods to detect changes in phosphorylation of CaMKII during memory formation in 1‐day‐old chicks trained in a single trial passive avoidance task. A 60‐kDa protein has been immunoprecipitated from the chick brain with an anti‐rabbit CaMKII antibody. This protein shows strong and specific immunoactivities with antibodies against the calmodulin binding site of CaMKII, and the N and C terminals of β‐CaMKII. Commercially available anti‐phosphoserine and anti‐phosphothreonine antibodies are shown to sensitively detect phosphorylation of purified CaMKII. The basal phosphorylation of CaMKII from the intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) regions of the chick brain is shown to be largely right hemisphere‐lateralized. When chicks are subjected to a passive avoidance training experience, a specific increase in CaMKII phosphorylation is induced in the IMHV and LPO of the left hemisphere from those chicks whose memory for the training experience is successfully retrieved. While this specific increase in CaMKII phosphorylation is seen in both the left IMHV and left LPO in short‐term memory, it is detectable only in the left LPO associated with long‐term memory retrieval. The present results provide evidence that in vivo changes in phosphorylation of CaMKII are associated specifically with processing of distinct memory stages, which take place in specific brain regions. J. Neurosci. Res. 55:557–568, 1999.

2-deoxygalactose interferes with an intermediate processing stage of memory.

The effect of 2-deoxygalactose (2-D-gal), an inhibitor of glycoprotein synthesis, on memory formation was investigated with the day-old chick trained on a single-trial passive discrimination task. 2-D-gal (10 mumol/chick) was shown to inhibit memory formation at a time before the emergence of an antibiotic-sensitive long-term memory stage. The amnestic effect of 2-D-gal was successfully prevented by galactose, and more significantly by noradrenaline. In contrast, anisomycin-induced amnesia was resistant to challenge by either galactose or noradrenaline. The results are consistent with the view that some glycoprotein involvement in memory formation occurs prior to the formation of protein synthesis-dependent long-term memory, and this role of glycoproteins may be associated with the triggering of long-term memory formation by noradrenaline.

Passive avoidance learning induced change in GAP43 phosphorylation in day-old chicks

Day-old chicks trained on a single trial passive discriminated avoidance task demonstrated a significant increase in in vitro phosphorylation of a 50 kDa protein in P2M fractions of total forebrain. The increase occurred 30 min posttraining, at a time when previous reports suggest that mechanisms for triggering protein synthesis-dependent long-term memory consolidation are activated. These changes in phosphorylation rates were accompanied by a substantial enhancement of total kinase activity. Immunoblotting studies with monoclonal anti-GAP43 antibody indicate that this protein is GAP43. These results contradict previous reports of a decrease in in vitro GAP43 phosphorylation following the same learning paradigm. A number of procedural differences may account for this discrepancy. The results suggest that changes in the phosphorylation state may be associated with mechanisms triggering long-term memory consolidation.

Phosphorylation changes following weakly reinforced learning and ACTH-induced memory consolidation for a weak learning experience

The formation of a protein synthesis-dependent long-term memory stage in day-old chicks trained on a passive discriminated avoidance task has been shown to occur only with an adequate level of reinforcement, and is preceded by a significant change in the phosphorylation state of the forebrain synaptosomal membrane protein GAP43 protein. In the present study, it is shown that weakly reinforced training did not lead to formation of a long-term memory stage or to any change in phosphate incorporation into forebrain P2M protein bands. However, administration of ACTH immediately posttraining led to both the formation of the long-term memory stage and a preceding significant increase in the phosphorylation of GAP43. These findings are consistent with the view that a reinforcement-dependent neurohormone-mediated change to the phosphorylation of this synaptosomal membrane protein may be implicated in the triggering of long-term memory consolidation.

The Involvement of Ca2+/Calmodulin-Dependent Protein Kinase in Memory Formation in Day-Old Chicks

Day-old chicks trained on a single trial passive avoidance learning task showed a significant increase, relative to untrained controls, in activity of the Ca2+/calmodulin-dependent protein kinase (CaMK) in the particulate fraction from tissues from the intermediate medial hyperstriatum ventrale region of the forebrain. The increased kinase activity was observed within 10 min following training and persisted for at least 70 min posttraining. Amnesia for the task was induced by micromolar concentrations of the specific CAMK II antagonist, KN-62, administered into the neostriatal/hyperstriatal region of the forebrain. The effect of KN-62 was lateralized. In the right hemisphere, KN-62 induced amnesia only when injected within 2. 5 min following training, with memory loss evident by 5 min posttraining. In contrast, in the left hemisphere amnesia was induced by KN-62 administered as late as 5 min posttraining, with onset of amnesia occurring after 10 min posttraining. The findings were interpreted within the context of a three-stage model of memory formation.