Nicotinic Acetylcholine Receptors Control Encoding and Retrieval of Associative Recognition Memory through Plasticity in the Medial Prefrontal Cortex

Summary Nicotinic acetylcholine receptors (nAChRs) expressed in the medial prefrontal cortex have critical roles in cognitive function. However, whether nAChRs are required for associative recognition memory and the mechanisms by which nAChRs may contribute to mnemonic processing are not known. We demonstrate that nAChRs in the prefrontal cortex exhibit subtype-specific roles in associative memory encoding and retrieval. We present evidence that these separate roles of nAChRs may rely on bidirectional modulation of plasticity at synaptic inputs to the prefrontal cortex that are essential for associative recognition memory.

. (Related to Figure 1) Total object exploration in OiP with nAChR inactivation Table S2. (Related to Figure 1) Total object exploration in OL with nAChR inactivation Table S3. (Related to Figure 3) Total object exploration in object-in-place task with AMPA receptor trafficking disruption Table S1: Total object exploration in OiP with nAChR inactivation. Related to Figure 1 Total exploration of the four objects did not differ between control or drug condition at any time point as analysed by paired t-test. Data is presented as mean ± SEM.

Infusion Condition Sample Phase Exploration (s) P Value Test Phase Exploration (s) P Value
Pre-Sample  Total exploration of the two objects did not differ between control or drug condition at any time point as analysed by paired t-test. Data is presented as mean ± SEM.  Total exploration of the four presented objects did not differ between active and inactive peptide conditions at any time point as analysed by paired t-test. Data is presented as mean ± SEM.

Supplemental Experimental Procedures Animals
Male Lister Hooded rats (Harlan Laboratories, UK) were used in all experiments. Surgical procedures and behavioural tests were conducted on 34 adult rats (>300g). Subjects were removed from further testing if the implanted cannula became damaged or dislodged. Rats aged post-natal day 30 were used for slice electrophysiology. All animals were group housed under a 12hour light/dark cycle (light phase 18:00-06:00), with experimental procedures conducted during the dark phase of the cycle. Water and food was available ad libitum. All procedures were conducted in accordance with the UK Animals Scientific Procedures Act (1986), in addition to local rules and regulations of the University of Bristol.

Stereotaxic Surgery
Chronically indwelling guide cannula were implanted under inhaled isoflurane anaesthetic (Induction 4%, maintenance 2-3%; Merial, Harlow, UK). Following exposure of the skull, small burr holes were drilled and stainless steel guide cannula (26 gauge; Plastics One, Semat, UK) were targeted bilaterally into the mPFC (From bregma, AP +3.20mm; ML ±0.75mm; DV -3.5mm). Implanted cannula were secured to the skull by three stainless steel screws (Plastics One, Semat, UK) and dental cement. Dummy cannulae were inserted into guide cannula and dust caps fitted to minimise risk of postsurgical infection. Saline (5ml subcutaneous) and analgesic solution (0.05ml intramuscular; Vetergesic 0.3mg/ml) were administered for hydration and pain management on completion of the surgery. Rats were single housed for 7 days following surgery and allowed 2 weeks recovery before behavioural testing.

Behavioural Procedures
Associative recognition memory was assessed using an object-in-place (OiP) task and spatial discrimination by object-location (OL) tasks (Fig 1A,D). Tests were conducted in an open field arena (length; 100cm, width; 90cm, height; 50cm) with global spatial cues available. A video recorder was positioned above the arena to enable recording of exploration data. All rats were habituated to handling and the arena across 5 consecutive daily sessions.
The OiP and OL tasks consist of a sample and test phase, separated by 24h retention delay. In the OiP sample phase, rats were individually positioned in the centre of the arena and permitted free exploration of four different objects for 5mins. The objects (> 9 × 8 × 7 cm) were constructed from duplo® and were presented in the corners of the arena, 15cm from the arena walls. In the test phase (3min) two of the objects were switched in position to introduce novelty in the configuration. In the OL sample phase the animals were presented with two identical duplo® objects for 3min. Following the retention delay, the animal was replaced in the arena for the test phase, in which one object was moved into a novel position within the arena.
Exploration of each object was measured in both the sample and test phases. Exploration was counted when a rat was positioned with its nose directed towards and <2cm from an object. A minimum criterion of 15secs of total exploration during the sample phase and 10secs during the test phase was required for inclusion in statistical analysis. Exploration was scored by an experimenter blind to the drug-condition and the configuration of objects presented and moved (left or right pairs) counterbalanced within each experiment.
Novelty preference is reflected in a positive discrimination ratio (DR), calculated as the difference in the time spent exploring the moved vs unmoved objects divided by the total amount of exploration. DR=0 signifies equal exploration of moved and unmoved objects, and thus chance.

Infusions
Infusions of drug or vehicle were given to a volume of 1μL per PFC hemisphere through implanted guide cannula. Infusions delivered 15mins prior to sample phase to study effects on memory acquisition, immediately after sample exploration to study effects on memory consolidation, or 15mins before the test phase to study effects on memory retrieval. In line with previous work, infusions of TAT conjugated peptides were delivered 1hour before sample or test phases (Cazakoff & Howland., 2011). A within-subjects experimental design was used with the order of treatment (i.e. drug or vehicle) counter-balanced within experiment.
Excitatory postsynaptic currents (EPSCs) were evoked through extracellular stimulation of the hippocampal fibre bundle at 0.1Hz by a concentric bipolar electrode. Plasticity at the hippocampal-mPFC input was investigated using a paradigm of spike timing-dependent plasticity induction. A stable 5min baseline was obtained within 10mins of attaining whole-cell configuration to reduce dialysis. Cells were then held in current-clamp configuration and 80 trains of pre-post pairings were delivered at theta (5Hz) frequency. Each train consisted of 5 EPSPs at 100Hz, paired with a 7.5ms delay from the 3 rd EPSP to 3 postsynaptic action potentials (APs) evoked by suprathreshold current injections (Parent et al., 2010).

Drugs
Compounds infused in vivo were dissolved in sterile 0.9% saline solution. Drugs used in vitro were dissolved in aCSF and bath applied or added to the intracellular solution for postsynaptic loading. Nicotine ditartrate, PNU-282987, RJR-2403 Oxalate, and Methyllycaconitine (MLA) were purchased from Abcam (Cambridge Science Park, UK). Dihydro-β-erythroidine hydrobromide (DHβE), Zeta inhibitory peptide (ZIP; SIYRRGARRWRKL) and scrambled ZIP (RLYRKRIWRSAGR) were purchased from Tocris (Bristol, UK). The peptides TAT-GluR3γ (YGRKKRRQRRRYKEGYNVTG), scrambled TAT-GluR23γ (YGRKKRRQRRRVYKYGGYNE), GluR23γ (YKEGYNVYG) and GluR23A (AKEGANVAG) were purchased from Eurogentec (LIEGE Science Park, Belgium. In vivo GluR23γ and its scrambled peptide control were delivered attached to the transduction domain of the TAT protein to enable cell permeability (Brebner et al., 2005). GluR23A was used for postsynaptic infusion as scrambled GluR23γ was not commercially available without a conjugated TAT domain.

Statistical Analysis
Discrimination ratios (DR) were used to analyse performance in all behavioural experiments. The mean DR for each condition were compared using one-sample two-tailed t-tests against zero (chance) discrimination. Paired two-tailed t-tests were used to compare DRs between vehicle and drug conditions in individual animals. Paired t-tests were also used to compare the total time spent exploring objects in both sample and test phases of the tasks between vehicle and drug conditions.
For in vitro electrophysiology, the mean peak amplitude of responses recorded in the last 5mins of the baseline were compared against that of the final 5mins of plasticity by paired two-tail t-tests or repeated-measures ANOVA with Bonferroni corrected post-hoc comparisons. Unpaired two-tailed ttests were used to compare the normalised response amplitudes following post-synaptic loading of active and inactive peptide. Except as stated, statistical analysis was conducted using the raw data collected, graphs are presented as mean values (±SEM) normalised to the baseline. All statistical analysis was performed using IBM SPSS 21 software package, with statistical significance was taken as P<0.05.