Amnesia produced by altered release of neurotransmitters after intraamygdala injections of a protein synthesis inhibitor

Abstract

Amnesia produced by protein synthesis inhibitors such as anisomycin provides major support for the prevalent view that the formation of long-lasting memories requires de novo protein synthesis. However, inhibition of protein synthesis might disrupt other neural functions to interfere with memory formation. Intraamygdala injections of anisomycin before inhibitory avoidance training impaired memory in rats tested 48 h later. Release of norepinephrine (NE), dopamine (DA), and serotonin, measured at the site of anisomycin infusions, increased quickly by approximately 1,000-17,000%, far above the levels seen under normal conditions. NE and DA release later decreased far below baseline for several hours before recovering at 48 h. Intraamygdala injections of a beta-adrenergic receptor antagonist or agonist, each timed to blunt effects of increases and decreases in NE release after anisomycin, attenuated anisomycin-induced amnesia. In addition, similar to the effects on memory seen with anisomycin, intraamygdala injections of a high dose of NE before training impaired memory tested at 48 h after training. These findings suggest that altered release of neurotransmitters may mediate amnesia produced by anisomycin and, further, raise important questions about the empirical bases for many molecular theories of memory formation.

Fig. 1.

Effects of intraamygdala injections of anisomycin (ANI) on memory and on c-Fos immunoreactivity. (Left) Memory assessed 48 h after training in rats pretreated with intraamygdala injections of anisomycin or vehicle. Anisomycin significantly impaired retention latencies (P < 0.05 vs. controls). (Center and Right) c-Fos immunoreactivity after intraamygdala infusions of either vehicle (Center) or anisomycin (Right). The sections shown were taken one to two sections posterior to the cannula tract, i.e., within 100 μm of the injection. Amygdala c-Fos immunoreactivity after anisomycin treatment was markedly reduced to nearly undetectable levels. n = 8, vehicle; n = 7, anisomycin.

Fig. 2.

Effects of pretraining intraamygdala injections of anisomycin (ANI) (n = 5) or vehicle (n = 5) on release of NE, DA, and 5-HT near the site of injection. Microdialysis samples were collected for analyses every 15 min beginning 45 min before and ending 2 h after injections of anisomycin. (Left) Note that NE, DA, and 5-HT each exhibited very large increases in release in the samples collected soon after injection. (Right) The y axis was expanded to show, for the same data, the decreases in release of the neurotransmitters toward the end of the dialysis session. Note that the magnitude of release decreased with time after the initial large increase. Release levels were significantly below baseline for NE and DA at the time microdialysis was terminated. B, baseline; Inj., injection; T, training; P, posttraining.

Fig. 3.

Effects of intraamygdala anisomycin (ANI) (n = 4) and vehicle (n = 4) injections on release of NE, DA, and 5-HT. Microdialysis samples were collected every 45 min beginning 135 min before and ending 8 h after injections. As in Fig. 2, the y axis was expanded in Right to show, for the same data, the decreases in release of the neurotransmitters toward the end of the dialysis session. Note that 5-HT but not NE and DA levels had returned to baseline even 8+ h after anisomycin injections. B, baseline; Inj., injection; P, postinjection.

Fig. 4.

Levels of NE, DA, and 5-HT assessed 48 h after anisomycin (ANI) injections. All mean values were comparable with those of controls, showing recovery of neurotransmitter levels to baseline values at this time.

Fig. 5.

Attenuation and induction of a amnesia with noradrenergic drugs. (Upper) Design for attenuation of anisomycin-induced amnesia with propranolol and clenbuterol. (Lower) Intraamygdala injections of anisomycin 2 h before training impaired memory tested 48 h after training. Propranolol (PROP) was administered 10 min before anisomycin (ANI) injections to reduce the effects on memory of increased release of NE soon after anisomycin treatment. Propranolol significantly attenuated amnesia on test trials 48 h after training administered 2 h after anisomycin treatment (P < 0.05 vs. anisomycin). In addition, clenbuterol (CLEN) was administered 10 min before training (110 min after anisomycin) to reduce the effects of decreased release of NE at this interval after anisomycin treatment. Clenbuterol also attenuated anisomycin-induced amnesia (P < 0.05 vs. anisomycin). To mimic the pulse of NE release after training, a high dose of NE was injected into the amygdala before training. The high dose of NE itself was sufficient to impair memory tested at 48 h (P < 0.05 vs. vehicle controls). The NE treatment was as effective as anisomycin in producing amnesia (NE vs. anisomycin, P > 0.8). n = 23, vehicle with or without propranolol or clenbuterol; n = 25, anisomycin amnesia controls; n = 8, propranolol plus anisomycin; n = 9, anisomycin plus clenbuterol; n = 7, NE.

Fig. 6.

Inhibition of c-Fos immunoreactivity in the basolateral amygdala after anisomycin (ANI) and coadministration of propranolol (PROP) or clenbuterol (CLEN). The sections shown were taken one to two sections posterior to the cannula tract, i.e., within 100 μm of the injection. The additional infusion of propranolol or clenbuterol into the amygdala did not substantially affect c-Fos inhibition caused by intraamygdala anisomycin treatment.