Intra-amygdala injections of CREB antisense impair inhibitory avoidance memory: role of norepinephrine and acetylcholine

Abstract

Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed brain sites, through mechanisms that include the release of norepinephrine and acetylcholine within the amygdala. Thus, CREB antisense injections may affect memory by interfering with mechanisms of modulation, rather than storage, of memory. In the present experiment, rats received bilateral intra-amygdala infusions of CREB antisense (2 nmol/1 microL) 6 h prior to inhibitory avoidance training. In vivo microdialysis samples were collected from the right amygdala before, during, and following training. CREB antisense produced amnesia tested at 48 h after training. In addition, CREB antisense infusions dampened the training-related release of norepinephrine, and to a lesser extent of acetylcholine, in the amygdala. Furthermore, intra-amygdala infusions of the beta-adrenergic receptor agonist clenbuterol administered immediately after training attenuated memory impairments induced by intra-amygdala injections of CREB antisense. These findings suggest that intra-amygdala treatment with CREB antisense may affect processes involved in modulation of memory in part through interference with norepinephrine and acetylcholine neurotransmission in the amygdala.

Figure 1.

Experiment timelines. (Top) Design for CREB antisense and microdialysis experiment. (Bottom) Design for CREB antisense and clenbuterol effect on memory.

Figure 2.

Baseline levels of release of norepinephrine (NE) (left) and acetylcholine (ACh) (right) in the amygdala. Note that there were no differences in baseline levels of release of either neurotransmitter following CREB antisense treatment in the amygdala.

Figure 3.

Increases in release of norepinephrine (NE) in the amygdala after inhibitory avoidance training. Note that training resulted in increased release of NE in both controls and CREB antisense groups. However, the magnitude and duration of release was reduced in rats treated with CREB antisense.

Figure 4.

Increases in release of acetylcholine (ACh) in the amygdala after inhibitory avoidance training. Note that training resulted in increased release of ACh in both controls and CREB antisense groups. Also, the duration but not initial magnitude of release was attenuated in rats treated with CREB antisense.

Figure 5.

Effects of intra-amygdala injections of clenbuterol on memory. Immediate post-training injections of clenbuterol (10 ng) significantly enhanced memory assessed 48 h after training. (*) P < 0.05.

Figure 6.

Effects of intra-amygdala injections of CREB antisense and clenbuterol on memory. CREB antisense injections 6 h prior to training resulted in significantly lower latencies on memory tests 48 h after training (rOGN vs. a-CREB). Immediate post-training injections of clenbuterol, at a 10-ng but not a 1-ng dose, into the amygdala attenuated the effects of CREB antisense (a-CREB vs. a-CREB + CLEN 10). (rOGN) Randomized oligodeoxynucleotide; (a-CREB) CREB antisense; (CLEN 1) clenbuterol, 1 ng; (CLEN 10) clenbuterol, 10 ng; (*) P < 0.05.

Figure 7.

Expression of CREB and pCREB in the amygdala after CREB antisense or PBS control injections into opposite sides of the amygdala 6 h prior to training. Brains were collected 45 min after training. Sections shown are ∼100 μm posterior to the infusion sites. Note that expression of both CREB and pCREB was depressed in the antisense versus control side. The Atlas figures are adapted from Paxinos and Watson (2005) (reprinted with permission from Elsevier ©2005). Bregma −3.24.

Figure 8.

Expression of c-Fos after CREB antisense or PBS control injections into opposite sides of the amygdala 6 h prior to training. Brains were collected 90 min after training. Sections shown are ∼100 μm rostral to the infusion sites. Note the decreased c-Fos expression in the lateral amygdala (outlined) on the antisense-injected versus control side of the amygdala.

Figure 9.

c-Fos protein expression 6 h after intra-amygdala infusions of control or CREB antisense, and 1.5 h after inhibitory avoidance training. Note the decreased expression of c-Fos protein in the lateral amygdala following CREB antisense treatment; (*) P < 0.05.

Figure 10.

Representative photomicrograph showing guide cannula placement dorsal to the amygdala and microinjection cannula placement in the amygdala. Microinjections were administered bilaterally, and microdialysis samples were collected from the right amygdala.