Experience-Dependent Effects of Muscimol-Induced Hippocampal Excitation on Mnemonic Discrimination

Abstract

Memory requires similar episodes with overlapping features to be represented distinctly, a process that is disrupted in many clinical conditions as well as normal aging. Data from humans have linked this ability to activity in hippocampal CA3 and dentate gyrus (DG). While animal models have shown the perirhinal cortex is critical for disambiguating similar stimuli, hippocampal activity has not been causally linked to discrimination abilities. The goal of the current study was to determine how disrupting CA3/DG activity would impact performance on a rodent mnemonic discrimination task. Rats were surgically implanted with bilateral guide cannulae targeting dorsal CA3/DG. In Experiment 1, the effect of intra-hippocampal muscimol on target-lure discrimination was assessed within subjects in randomized blocks. Muscimol initially impaired discrimination across all levels of target-lure similarity, but performance improved on subsequent test blocks irrespective of stimulus similarity and infusion condition. To clarify these results, Experiment 2 examined whether prior experience with objects influenced the effect of muscimol on target-lure discrimination. Rats that received vehicle infusions in a first test block, followed by muscimol in a second block, did not show discrimination impairments for target-lure pairs of any similarity. In contrast, rats that received muscimol infusions in the first test block were impaired across all levels of target-lure similarity. Following discrimination tests, rats from Experiment 2 were trained on a spatial alternation task. Muscimol infusions increased the number of spatial errors made, relative to vehicle infusions, confirming that muscimol remained effective in disrupting behavioral performance. At the conclusion of behavioral experiments, fluorescence in situ hybridization for the immediate-early genes Arc and Homer1a was used to determine the proportion of neurons active following muscimol infusion. Contrary to expectations, muscimol increased neural activity in DG. An additional experiment was carried out to quantify neural activity in naïve rats that received an intra-hippocampal infusion of vehicle or muscimol. Results confirmed that muscimol led to DG excitation, likely through its actions on interneuron populations in hilar and molecular layers of DG and consequent disinhibition of principal cells. Taken together, our results suggest disruption of coordinated neural activity across the hippocampus impairs mnemonic discrimination when lure stimuli are novel.

Keywords: CA3; aging; dentate gyrus; epilepsy; object recognition; perirhinal cortex.

FIGURE 1

(A) Timeline of experimental manipulations and histological assessment of dorsal CA3/DG cannulae placements from Experiments 1 and 2. (B) Representative images show DAPI-stained sections (converted to grayscale) with tracts of 22G stainless steel guide cannulae positioned above the dorsal CA3/DG at target coordinates, relative to Bregma: AP –4 mm, ML ±3mm, DV –2.6 mm from skull surface. While no visible damage was observed in most rats, 28G microinjectors protruded an additional 1 mm below cannulae tips, centering the infusion between DG upper and lower blades. (C) Representative image from Experiment 1 brain section processed with fluorescence in situ hybridization (FISH) to label Arc mRNA (Cy3; red channel), counter-stained with DAPI. Strong induction of the immediate-early gene Arc is evident in both upper and lower blades of DG. (D) Schematics show position of foci of infusions based on cannulae tracts in each rat from Experiment 1 (n = 10), Experiment 2 (n = 17), and separate study carried out to verify effects of vehicle vs. muscimol infusion on Arc mRNA expression as a read-out of neural activity (n = 6). Numerical values on brain sections indicate AP coordinate, relative to Bregma.

FIGURE 2

Apparatus and object stimuli used in procedural training and the rodent version of the mnemonic similarity task, as previously described by Johnson et al. (2017). (A) Object discrimination training and testing were carried out in an L-shaped maze. Food rewards were hidden in recessed wells in the choice platform, covered by object stimuli. Rats alternated between the start area and choice platform on each discrimination trial. (B) Object pairs used in procedural training. All rats were first trained to criterion of ≥81.3% (≥26/32 correct trials) in distinguishing a pair of standard unrelated objects that shared no feature overlap, then were trained to the same criterion on a distinct pair of LEGO objects that shared 38% visible front-facing features, and a similar pair of LEGO objects that shared 63% visible front-facing features. The object of each pair serving as the target (S+) and the order of training on distinct vs. similar LEGO pairs was counter-balanced across rats in each experiment. (C) Objects used in the rodent version of the mnemonic similarity task. Left side of panel shows target (S+) object and LEGO pre-training lure object (S–) that shared 38% front-facing visible features. Rats were trained to discriminate this target from the pre-training lure to a criterion of ≥81.3% (≥26/32 correct trials) before moving on to mnemonic discrimination tests. Right side of panel shows each of 4 LEGO test lure objects in order of increasing similarity, or percent visible feature overlap: a distinct, standard lure object that shared 0% overlap with the target, and LEGO lure objects that shared 50, 70, and 90% overlap with the target.

FIGURE 3

Verification of the effect of muscimol infusions on neural activity across hippocampal subregions with fluorescence in situ hybridization (FISH) for the immediate-early genes (IEGs) Arc and Homer 1a. (A) Regions of interest from which z-stacks were captured in CA1 and CA3 for quantification of IEGs by manual counting in ImageJ. (B) Representative images from a muscimol-infused rat (left) and vehicle-infused rat (right) show schematic of cursors aligned across upper and lower blades of DG for densitometric quantification of IEGs using ImageJ software. (C–E) Representative z-stacks show Arc (fluorescein; green channel) and Homer1a (Cy3; red channel) labeling from a vehicle (veh)-infused and muscimol (mus)-infused rat; (C) CA1, (D) CA3, and (E) DG. (F) Plots show mean ± SEM percent IEG-positive DAPI-stained neuronal nuclei reflecting cells active at baseline (base), 60 min prior to sac (Homer1a cytosol), versus cells activated by the infusion (INF), 30 min prior to sac (Homer1a nuclear foci + Arc cytosol). Both infusion conditions increased percent neurons active in CA1 and CA3 relative to baseline [main effect time point: F_(1,4) = 24.3, p < 0.008; no main effect infusion condition: F_(1,4) = 0.39, p = 0.565]. (G) Mean ± SEM integrated density of Arc mRNA signal quantified in DG upper and lower blades. Muscimol infusion led to greater Arc expression in both DG subregions [main effect infusion condition: F_(1,3) = 16.7, p < 0.027].

FIGURE 4

Procedural object discrimination training required to reach a criterion performance level of ≥81.3% (≥26/32 correct responses) on a single daily session in Experiments 1 and 2. (A) Number of incorrect responses required to reach criterion on object pairs used for procedural discrimination training in rats from Experiment 1, and rats assigned to groups that received vehicle infusions first (veh) or muscimol infusions first (mus) across two test blocks in Experiment 2. Irrespective of experiment or group, all rats learned to accurately identify the target of the standard object pair after fewer incorrect trials, relative to all other pairs (simple contrasts, p’s < 0.001). Conversely, all rats required a greater number of incorrect trials to reach criterion on the similar LEGO pair (simple contrasts, p’s < 0.001). (B) Number of incorrect responses made prior to reaching criterion in identifying the target (S+) object relative to the distinct lure object in pre-training for mnemonic discrimination tests. Amount of training required did not differ by experiment or group. Additionally, amount of training required to reach criterion on this pair, after cannulation surgery, did not differ from that required to reach criterion on the distinct LEGO pair prior to surgery (p = 0.60).

FIGURE 5

(A) Timeline of infusions and mnemonic discrimination tests administered in Experiment 1. All rats (n = 10) received a vehicle (veh) and muscimol (mus) infusion in each of three tests blocks. Order of veh and mus infusions in each test block was randomized across rats with a Latin square design, therefore this timeline shows one example permutation of infusion order. Tests took place every 3 days, with 2 wash-out days on which rats remained in their home cages and did not complete any behavioral training or testing. Infusions were administered 30 min prior to the beginning of each test. (B,C) Performance (% correct trials) on mnemonic discrimination tests in Experiment 1 plotted by trial type, with each of the 4 lure objects sharing 0, 50, 70, or 90% visible front-facing features (target-lure overlap). Test performance on days with vehicle infusions (veh) designated by open circles, and on days with muscimol infusions (mus) by filled circles. (B) Mean ± SEM performance on mnemonic discrimination tests collapsed across 3 test blocks, to a total of 30 trials with each lure object under each infusion condition. Rats made fewer correct responses on trials with LEGO lures (50–90% target-lure overlap), relative to the standard object lure [0% overlap; main effect lure: F_(3,27) = 44.1, p < 0.001; simple contrasts: p’s < 0.01]. (C) Mean ± SEM performance plotted for each of the 3 test blocks (10 trials/lure object/infusion condition). Muscimol impaired discrimination when lure objects were novel, in block 1, across all lures [main effect infusion: F_(1,7) = 16.7, p < 0.005]. By block 2, muscimol impaired performance only on the 70% target-lure overlap problem [infusion × lure: F_(3,27) = 2.95, p < 0.05]. No difference in performance between infusion conditions was observed in block 3.

FIGURE 6

(A) Timeline of infusions and mnemonic discrimination tests administered in Experiment 2. Rats were randomly assigned after surgery to either receive vehicle infusions in the first test block (veh-first group; n = 9) or muscimol infusions in the first test block (mus-first group; n = 8). Each test block comprised 3 tests with the same infusion condition. In block 1, veh-first rats received vehicle and mus-first rats received muscimol prior to tests 1–3. In block 2, infusion conditions reversed; veh-first rats received muscimol and mus-first rats received vehicle prior to tests 4–6. (B–E) Performance (% correct trials) on mnemonic discrimination tests in Experiment 2 plotted by trial type, with objects sharing 0, 50, 70, or 90% target-lure overlap. Test performance on days with vehicle infusions (veh) designated by open circles, and on days with muscimol infusions (mus) by filled circles. Data for block 1 are shown in (B,C). Data for block 2 are shown in (D,E). (B,D) Mean ± SEM performance on mnemonic discrimination tests collapsed across blocks 1 and 2, with a total of 30 trials per lure object for each infusion group. As in Experiment 1, performance decreased as target-lure overlap increased [main effect lure: F_(3,45) = 61.8, p < 0.001]. Muscimol impaired discrimination across all lures in block 1 (B), but did not impair discrimination in block 2 (D) [main effect block: F_(1,15) = 41.2, p < 0.001; block × group: F_(1,15) = 116.3, p < 0.001]. (C) Mean ± SEM performance plotted for each test of block 1 (tests 1–3; 10 trials/lure object/infusion group). Muscimol infusions impaired discrimination across all tests of block 1, when lures were relatively novel [main effect group: F_(1,16) = 27.2, p < 0.001]; however, rats in the mus-first group showed selective improvement across tests on trials with the more distinct lure objects [main effect test: F_(2,32) = 11.2, p < 0.001; test × lure: F_(6,96) = 6.13, p < 0.001; 3-way interaction: F_(6,96) = 2.86, p < 0.013]. (E) Mean ± SEM performance plotted for each test of block 2 (tests 4–6; 10 trials/lure object/infusion group). Muscimol infusions did not impair discrimination across tests of block 2, when rats of the veh-first group had gained prior experience with lure objects [main effect lure: F_(3,45) = 58.5, p < 0.001; all other effects: p’s > 0.11].

FIGURE 7

Measures of response selection behavior in Experiment 2. Data from days with vehicle infusions (veh) designated by open circles, and on days with muscimol infusions (mus) by filled circles. Data are plotted by test day (tests 1–6; t1–t6) across each of the two test blocks. (A) Mean ± SEM side bias index values across tests. A side bias index of 1 reflects a complete bias to select the object presented on either the left or right side of the choice platform, while a side bias of 0 reflects no side bias and an equal number of responses made to the left and right sides. In block 1, side bias index values were greater after muscimol relative to vehicle [main effect infusion: F_(1,15) = 33.4, p < 0.001], though decreased across tests as rats gained experience with lure objects and were increasingly able to suppress their inherent bias to correctly select the target object [main effect test: F_(2,30) = 6.38, p < 0.005]. Conversely, in block 2, mean side bias index values did not differ based on infusion condition or test (p’s > 0.25). (B) Mean ± SEM response latency values for correct discrimination responses made to rats’ inherently biased side. Latencies did not differ by infusion condition [main effect infusion: F_(1,9) = 0.61, p = 0.455] or across test days [main effect test: F_(5,45) = 0.81, p = 0.547; infusion × test: F_(5,45) = 0.21, p = 0.956].

FIGURE 8

Effect of dorsal CA3/DG muscimol infusions on spatial alternation performance, as a behavioral control condition in rats from Experiment 2 (n = 8). Plots show the total number of spatial memory errors (i.e., return to same arm of the figure-8 maze on 2 consecutive laps, rather than alternating between the two arms) on 10 pre-training trials and 10 post-infusion trials on test day 1 (all rats infused with muscimol; mus) and test day 2 (all rats infused with vehicle; veh). Mean ± SEM number of errors for pre-training and post-vehicle trials designated by open circles, and for post-muscimol trials with filled circle. Muscimol impaired alternation performance, even after completing all infusions and mnemonic discrimination tests of Experiment 2 [t₍₇₎ = –3.22, p < 0.01]; vehicle did not alter rats’ alternation performance [t₍₇₎ = –0.15, p = 0.86].