Distinct roles for the deacetylase domain of HDAC3 in the hippocampus and medial prefrontal cortex in the formation and extinction of memory

Abstract

Histone deacetylases (HDACs) are chromatin modifying enzymes that have been implicated as powerful negative regulators of memory processes. HDAC3has been shown to play a pivotal role in long-term memory for object location as well as the extinction of cocaine-associated memory, but it is unclear whether this function depends on the deacetylase domain of HDAC3. Here, we tested whether the deacetylase domain of HDAC3has a role in object location memory formation as well as the formation and extinction of cocaine-associated memories. Using a deacetylase-dead point mutant of HDAC3, we found that selectively blocking HDAC3 deacetylase activity in the dorsal hippocampus enhanced long-term memory for object location, but had no effect on the formation of cocaine-associated memory. When this same point mutant virus of HDAC3 was infused into the prelimbic cortex, it failed to affect cocaine-associated memory formation. With regards to extinction, impairing the HDAC3 deacetylase domain in the infralimbic cortex had no effect on extinction, but a facilitated extinction effect was observed when the point mutant virus was delivered to the dorsal hippocampus. These results suggest that the deacetylase domain of HDAC3 plays a selective role in specific brain regions underlying long-term memory formation of object location as well as cocaine-associated memory formation and extinction.

Keywords: Chromatin; Conditioned place preference; Dorsal hippocampus; Epigenetics; Long-term memory; Object location.

Figure 1

Dominant negative point mutant virus (HDAC3(Y298H)-v5 expresses effectively in the dorsal hippocampus. (A) V5 epitope was added to the point mutant virus. (B) Representative immunofluorescence image showing expression of the V5 epitope tag (green) in the dorsal hippocampus (DH) after infusion of AAV-HDAC3(Y298H)-v5. No V5 staining was observed with the AAV-EV control virus. Cells were counterstained with DAPI (blue). (C) V5 mRNA was significantly increased in the DH of mice infused with AAV-HDAC3(Y298H)-v5, AAV-EV n = 5, HDAC3(Y298H)-v5 n = 4. (D) HDAC3 was also significantly increased in the DH of mice infused with AAV-HDAC(Y298H)-v5 compared to AAV-EV controls, AV-EV n = 6, HDAC3(Y298H)-v5 n = 6. *p < 0.05.

Figure 2

Blocking HDAC3 activity in the DH enhanced memory for object location (OLM), but not for object recognition (ORM). (A) Mice received subthreshold training (3 min) in an environment with two identical objects and received a retention test 24 hours later in which one object is moved to a new location. Schematic describes methods for B and C. (B) Mice given AAV-HDAC3(Y298H)-v5 showed a significant preference for the novel object location 24h after training compared with EV controls, AAV-EV n = 8, HDAC3(Y298H)-v5 n = 6. (C) Groups did not differ in total exploration time of the two objects. (D) Mice received subthreshold training (3 min) in an environment with two identical objects and received a retention test 24 hours later in which one object was replaced with a novel one (ORM), AAV-EV n = 9, HDAC3(Y298H)-v5 n = 7. Schematic describes methods for E and F. (E) Neither AAV-EV control or AAV-HDAC3(Y298H)-v5 mice exhibited significant preference for the novel object. (F) Groups did not differ in total exploration time of the two objects. *p < 0.05.

Figure 3

Blocking HDAC3 activity in the dorsal hippocampus has no effect on the formation of cocaine-induced CPP memory. (A) Schematic of the cocaine-CPP procedure. (B) Cocaine-CPP expression indicated by mean CPP score (time spent in cocaine-paired (CS+) minus saline-paired (CS−) ± s.e.m). At 5 mg/kg cocaine conditioning dose, AAV-HDAC3(Y298H)-v5 mice exhibited similar CPP score to EV controls during the post-test, AAV-EV n = 15, HDAC3(Y298H)-v5 n = 15.

Figure 4

Focal homozygous gene deletion of Hdac3 in the dorsal hippocampus has no effect on the formation of cocaine-induced CPP memory. (A) Schematic of the cocaine-CPP procedure. (B) Cocaine-CPP expression indicated by mean CPP score (time spent in cocaine-paired (CS+) minus saline-paired (CS−) ± s.e.m). At 5 mg/kg cocaine conditioning dose, Hdac3^flox/flox mice exhibited similar CPP score to Hdac3^+/+ controls during the post-test. (C) Representative immunofluorescence image showing expression of HDAC3 (green) in the dorsal hippocampus after infusion of AAV2.1 Cre infused in Hdac3^+/+ and Hdac3^flox/flox mice. Cells were counterstained with DAPI (blue). (D) Quantification of immunostaining confirmed that HDAC3 was significantly reduced in Hdac3^flox/flox mice compared to Hdac3^+/+ controls, Hdac3^+/+ n = 12, Hdac3^flox/flox n = 11. *p < 0.05.

Figure 5

Blocking HDAC3 activity in the prelimbic cortex (PrL) has no effect on the formation of cocaine-induced CPP memory. (A) Schematic of the cocaine-CPP procedure. (B) Cocaine-CPP expression indicated by mean CPP score (time spent in cocaine-paired (CS+) minus saline-paired (CS−) ± s.e.m). At 5 mg/kg cocaine conditioning dose, AAV-HDAC3(Y298H)-v5 mice exhibited similar CPP score to EV controls during the post-test, AAV-EV n = 12, HDAC3(Y298H)-v5 n = 12. (C) Representative immunofluorescence image showing expression of the V5 epitope tag (green) after infusion of AAV-HDAC3(Y298H)-v5 targeting the PrL. No V5 staining was observed with the AAV-EV control virus. Cells were counterstained with DAPI (blue) and neurons were counterstained with NeuroTrace (red). V5 expression was largely confined to the prelimbic region of the medial prefrontal cortex. (D) Targeted viral infusion in the PrL. The shaded regions of the mouse atlas images illustrate the representative extend of viral infusion.

Figure 6

Blocking HDAC3 activity in the infralimbic cortex (IL) has no effect on the extinction of cocaine-induced CPP memory. (A) Schematic of the cocaine-CPP procedure. (B) Cocaine-CPP expression indicated by mean CPP score (time spent in cocaine-paired (CS+) minus saline-paired (CS−) ± s.e.m). Animals were initially trained on 20 mg/kg cocaine. Subsequently, animals received intra-IL AAV-EV or AAV-HDAC3(Y298H)-v5 viral infusions and extinction memory was examined. AAV-HDAC3(Y298H)-v5 mice exhibited similar CPP score to EV controls during post-tests 2–5, AAV-EV n = 17, HDAC3(Y298H)-v5 n = 16. (C) Representative immunofluorescence image showing expression of the V5 epitope tag (green) after infusion of AAV-HDAC3(Y298H)-v5 targeting the IL. No V5 staining was observed with the AAV-EV control virus. Cells were counterstained with DAPI (blue) and neurons were counterstained with NeuroTrace (red). V5 expression was largely confined to the infralimbic region of the medial prefrontal cortex. (D) Targeted viral infusion in the IL. The shaded regions of the mouse atlas images illustrate the representative extend of viral infusion.

Figure 7

Blocking HDAC3 activity in the dorsal hippocampus (DH) enhances extinction of cocaine-induced CPP memory. (A) Schematic of the cocaine-CPP procedure. (B) Cocaine-CPP expression indicated by mean CPP score (time spent in cocaine-paired (CS+) minus saline-paired (CS−) ± s.e.m). Animals were initially trained on 20 mg/kg cocaine. Subsequently, animals received intra-DH AAV-EV or AAV-HDAC3(Y298H)-v5 viral infusions and extinction memory was examined. Animals infused with AAV-HDAC3(Y298H)-v5 in the DH showed significantly reduced cocaine-CPP on post-test 2 compared to animals given intra-DH EV control. ⊗ indicates a significant treatment by test interaction and * indicates a significant decrease in PS of AAV-HDAC3(Y298H)-v5 compared to AAV-EV control animals during post-test 2, Bonferroni post-hoc, p<0.05, AAV-EV n = 10, HDAC3(Y298H)-v5 n = 12.