CREB regulates memory allocation in the insular cortex

Abstract

The molecular and cellular mechanisms of memory storage have attracted a great deal of attention. By comparison, little is known about memory allocation, the process that determines which specific neurons in a neural network will store a given memory. Previous studies demonstrated that memory allocation is not random in the amygdala; these studies showed that amygdala neurons with higher levels of the cyclic-AMP-response-element-binding protein (CREB) are more likely to be recruited into encoding and storing fear memory. To determine whether specific mechanisms also regulate memory allocation in other brain regions and whether CREB also has a role in this process, we studied insular cortical memory representations for conditioned taste aversion (CTA). In this task, an animal learns to associate a taste (conditioned stimulus [CS]) with the experience of malaise (such as that induced by LiCl; unconditioned stimulus [US]). The insular cortex is required for CTA memory formation and retrieval. CTA learning activates a subpopulation of neurons in this structure, and the insular cortex and the basolateral amygdala (BLA) interact during CTA formation. Here, we used a combination of approaches, including viral vector transfections of insular cortex, arc fluorescence in situ hybridization (FISH), and designer receptors exclusively activated by designer drugs (DREADD) system, to show that CREB levels determine which insular cortical neurons go on to encode a given conditioned taste memory.

Fig. 1. Selective modulation of a set of genetically tagged insular cortex neurons

(A) Schematic map of the lentivirus construct designed to express CREB and hM4Di. The hM4Di and GFP/CREB (or GFP/dTomato) genes located on both side of the T2A sequence are under the control of a Camk2a promoter. (B) Representative picture showing localized GFP expression in the insular cortex by CREB virus infection. Scale bar indicates 500 µm. (C) Representative pictures showing co-expression of CREB and hM4Di in the same insular cortical neurons. CREB and hM4Di were detected by immunohistochemical staining using specific antibodies to GFP (green) and HA-tag (red). Merged images show co-localized expression of either GFP/CREB or GFP/dTomato with hM4Di. GFP/CREB and hM4Di proteins show different subcellular localization, as expected. Scale bar indicates 50 µm. (D) Representative traces showing that CNO (10 µM) inactivated transduced (GFP+) neurons in the insular cortex, whereas it had no effect on GFP− neurons. The dashed line represents the resting membrane potential (RMP) before drug administration. The currents injected are shown underneath associated traces. ACSF, artificial cerebrospinal fluid. (E and F) Summary of the selective effect of CNO on (E) changes in resting membrane potential (RMP) and (F) changes in input resistance (IR) of the insular cortex neurons. * p < 0.05 and ** p < 0.01. Data represent mean ± s.e.m. (GFP− cells, black columns; GFP+ cells, green columns).

Fig. 2. Silencing of CREB+ neurons in the insular cortex impairs CTA memory retrieval

CREB or control lentiviruses were infused into the insular cortex 3 weeks before CTA training. Saline or CNO was systemically injected 45 min before CTA memory retrieval. (A) CTA memory was significantly impaired by selective silencing of CREB positive neurons by CNO (Saline group, n = 8; CNO group, n = 9). (B) CNO injection did not impair CTA memory retrieval in control mice (Saline group, n = 9; CNO group, n = 9). LI was normalized to 100 for a saline group in each experiment. Data represent mean ± s.e.m. *p < 0.05. (Saline-injected mice, white columns; CNO-injected mice, black columns).

Fig. 3. Insular cortex neurons with increased CREB level are preferentially recruited into encoding conditioned taste memory

(A) Representative images showing expression of gfp (green) and arc (red). Yellow arrows indicate double-labeled neurons (gfp+ and arc+). Nuclei were stained using DAPI. Scale bar indicates 50 µm. (B) Probability of cells expressing arc following taste memory retrieval over cells transduced with the CREB or control viruses in the insular cortex (n = 4 mice per group). (C) Overall arc+ cells in the analyzed area (n = 4 mice per group). (D) Probability of cells expressing arc in home cage mice in cells transduced with the CREB or control viruses in the insular cortex (n = 4 mice per group). (E) Probability of cells expressing arc following taste memory retrieval with saline or CNO in vCREB-positive cells (n = 6 saline group and n = 7 CNO group.) Data represent mean ± s.e.m., * p < 0.05 and ** p < 0.01. (B,C and D, control virus group, white columns; CREB virus group, black columns; E, saline group, white columns; CNO group, black columns).