Figure 2. Prefrontal ensemble codes in well-trained rats were selective for relational features.

(A) Example behavior from a single rat. Based on the percentage of trials exhibiting a conditioned response (CR%) and days since asymptotic responding, sessions were divided into five successive stages (vertical lines). Representative pseudocolor plots show normalized firing rate of neurons recorded from the same rat during the learning period (gray) and during the third week after learning (yellow). Top left, ACS-US paired; top right, VCS-US paired; bottom left, ACS alone; bottom right, VCS alone. Neurons were sorted based on the ACS-induced firing rate change during ACS-US pairings from the largest increase (Neuron #1) to the largest decrease (Neuron #357 or 184). During learning, ensemble activity during ACS-US pairings was similar to that during ACS alone presentations and VCS-US pairings; however, during the third post-learning week, it became more similar to that during VCS-US pairings than ACS alone presentations. White lines indicate CS onset and offset, black bars mask US artifacts. (B) During learning (left), binned firing rates of neuron ensembles (PV) during the CS (two vertical lines) were more similar for two conditions with the same modality of CS (ACS-US paired and ACS alone, blue, r ± 95% confidence intervals) than two conditions with the same stimulus relationships (ACS-US paired and VCS-US paired, green). Their similarity became comparable between two condition pairs during subsequent CS-US intervals. During the third post-learning week (right), PV became more similar for two conditions with the shared stimulus relationship than those with the shared CS modality. * indicates p<0.05/10 in random permutation tests. (C) Trial-by-trial display of PVs during CS-US intervals in the third post-learning week. Neurons from four rats were sorted based on the firing rate during ACS-US paired trials from the highest (Neuron #1) to the lowest (Neuron #360)]. A ‘template’ PV was constructed as averaged PVs across 10–80th ACS-US paired trials. The correlation coefficient between the template and PV in each trial (±95% confidence interval) rapidly increased within the first ten ACS-US paired trials (inset). Upon the transition from the epoch with the ACS to the next epoch with the VCS, it abruptly decreased but increased again when the VCS-US pairings began.

DOI: http://dx.doi.org/10.7554/eLife.22177.005

Figure 2—figure supplement 1. Histology and single unit isolation.

(A) Representative image of a tetrode tip (white arrow) located within the prelimbic region of medial prefrontal cortex. (B) Final locations of all usable tetrodes targeted at the prelimbic cortex (black dots). Coordinates adapted from Paxinos and Watson (2007, sixth edition). (C) During a software-assisted spike sorting process (KlustaKwik and MClust), several parameters were extracted from each spike waveform recorded from four wires of a tetrode (Ch. 1–4), and two of all possible parameter combinations (e.g., amplitude recorded in two of four wires) were plotted as a scatter plot. Spikes were separated into several units corresponding to spikes of each neuron based on the similarity of waveform parameters. Three examples that were isolated from one tetrode during a recording session were depicted as a set of averaged spike waveform on four wires (Ch. 1–4). The color of waveforms corresponds to the color used in the scatter plot. (D) Scatter plots show the amplitude of spike waveforms recorded on the channels 1 and 2 of a tetrode across three consecutive days (a.u., arbitrary unit). A few units were isolated from the recording in each day. Pseudocolor plots of binned firing rate (middle) show that some of these units increased (hotter color) or decreased (cooler color) their firing rate upon CS presentations. Based on the shape of spike waveforms recorded on four wires of the tetrode (Ch. 1–4, bottom), the unit 1, 6, and 11 appeared to be recorded from the same neuron which enhanced firing responses to the CS across three days. The unit 3 and 9 appeared to belong to the same neuron, but this neuron was not present in the recording on the third day. Other units, such as the unit 4, 5, and seven were recorded only in one day.

DOI: http://dx.doi.org/10.7554/eLife.22177.006

Figure 2—figure supplement 2. Prefrontal ensemble activity during four conditions with different relational and physical stimulus features.

Pseudocolor plots show normalized firing rates of neurons recorded from each of remaining three rats during learning (left, grey) and the third week after CR% reached asymptote (right, yellow) [top left, ACS-US paired; top right, VCS-US paired; bottom left, ACS alone; bottom right, VCS alone]. In each rat, neurons were sorted based on the ACS-induced firing rate during ACS-US pairings from the largest increase (top) to the largest decrease (bottom). In all three rats, during learning ensemble firing patterns differentiated four conditions while during the third post-learning week, ensemble activity became more similar for two conditions with the same stimulus relationship (i.e. CS-US pairings) than those with the same CS (i.e., auditory or visual CS).

DOI: http://dx.doi.org/10.7554/eLife.22177.007

Figure 2—figure supplement 3. CS-evoked firing patterns during four conditions with different relational and physical stimulus features.

Pseudocolor plots show standard scores of CS-evoked firing rates of neurons recorded from four rats during learning (left) and the third week after CR% reached an asymptote (right; top left, ACS-US paired; top right, VCS-US paired; bottom left, ACS alone; bottom right, VCS alone). Neurons were sorted based on the standard score of firing rates after CS presentations during ACS-US pairings from the largest increase (top) to the largest decrease (bottom). CS-evoked firing patterns were similar between ACS-US paired and ACS alone conditions during learning; however, during the third post-learning week, they became similar between ACS-US paired and VCS-US paired conditions.

DOI: http://dx.doi.org/10.7554/eLife.22177.008

Figure 2—figure supplement 4. Trial-by-trial changes in the similarity of ensemble activity during learning.

Trial-by-trial display of population firing rate vectors (PVs) during CS-US intervals in the learning stage. Neurons from four rats were sorted based on the firing rate during ACS-US paired trials from the highest (Neuron #1) to the lowest (Neuron #813)]. A ‘template’ PV was constructed as averaged PVs across 10–80th ACS-US paired trials. The correlation coefficient between the template and a PV in each trial (±95% confidence interval) gradually increased within the first twenty ACS-US paired trials (inset). It abruptly decreased upon the transition from the epoch with the ACS to the next epoch with the VCS but increased again when the VCS-US pairings began. Note that the difference in the correlation coefficient between the conditions appeared to be smaller than that observed in the third post-learning week shown in Figure 2C.

DOI: http://dx.doi.org/10.7554/eLife.22177.009