1
SUPPLEMENTAL RESULTS
2
Firing patterns between control and nicotine exposed rats
3
The average firing pattern over increasing- and decreasing-type neurons, broken down by
4
trial-type and direction, is illustrated in figures S1A,B and S2A,B for control (A) and PNE rats
5
(B). Neural activity is aligned to port exit (left) and fluid well entry (right). Since approximately
6
equal numbers of neurons fired more strongly for contra- and ipsilateral movements, we
7
segregated each cell’s activity into preferred and nonpreferred response directions. Preferred
8
direction is defined as the direction that elicited the strongest firing rate during the response
9
epoch (averaged over correct STOP and GO trials). As defined by our analysis, average activity
10
in the preferred direction (thick) is always stronger than activity in the nonpreferred direction
11
(thin) during the response epoch. Direction on STOP trials always refers to the direction the
12
animal responded to.
13
When comparing average firing between control and nicotine rats the most striking
14
difference between them is the overall reduction in mPFC firing. This is more readily apparent in
15
the population histograms averaged across trial-types (Fig. 2F,G).
16
17
Impact of prenatal nicotine exposure on stop-signal encoding by increasing-type neurons
18
Although firing in the increasing-type neurons of nicotine exposed animals was
19
attenuated (Fig. 2F) and the number of single direction correlated cells was reduced (Fig. 3D),
20
examination of average population histograms (Fig. S1A,B) suggests the strength of selectivity
21
for the two trial-types appear largely unaffected. For both the control and PNE groups, activity
22
appeared higher for STOP relative to GO trials for responses made in the preferred direction
23
(Fig. S1A, B; thick red vs. thick blue). Although overall activity was reduced in nicotine rats,
24
the difference between STOP and GO trials in the preferred direction did not appear to be weaker
25
in nicotine rats. To quantify differences between STOP and GO trials, we computed a stop index
26
defined as the difference between STOP and GO trial activity (STOP-GO/STOP+GO) in both
27
directions for each neuron. The distributions of these indices for preferred and nonpreferred
28
directions are plotted in figure S1C-F.
29
In these stop index plots, a shift in the positive direction indicates that neuronal firing was
30
stronger for STOP relative to GO trials than the opposite effect (stronger firing for GO relative to
31
STOP trials). In the preferred direction, the stop index was not significant for either group and
1
1
the two distributions did not significantly differ from each other (Fig. S1C,E; Wilcoxon; p =
2
0.23). In the nonpreferred direction, stop indices for control and nicotine rats were not
3
significantly shifted from zero (Fig. S1D; Wilcoxon; p = 0.84; Fig. S1F; Wilcoxon; p = 0.47) and
4
were not significantly different from one another (Fig. S1D,F; Wilcoxon; p = 0.42).
5
6
Impact of prenatal nicotine on directional selectivity of increasing-type neurons
7
As described previously, firing of neurons in mPFC was highly directional. To further
8
assess the directional encoding for each trial-type we computed a directional index (preferred-
9
nonpreferred / preferred+nonpreferred) during the response epoch independently for STOP and
10
GO trials. By defining preference based on the average over STOP and GO trials, this analysis
11
allows us to ask if the distribution of directional indices is different between the two trial types.
12
In control and nicotine cells, directional index distributions were shifted significantly above zero
13
during both GO and STOP trials (Fig. S1G,H,J,K) but there was no significant difference
14
between directionality of GO and STOP trials in either group (control; Fig.S1G,H; Wilcoxon; p
15
= 0.50; PNE; Fig. S1J,K; Wilcoxon; p = 0.36).
16
Directional responding implies that mPFC is involved in executive functions pertaining
17
to the spatial location of the response. If directional signals in mPFC are important for directing
18
behavior, they should be attenuated on errors. Consistent with this hypothesis, the distribution of
19
direction indices was significantly reduced on STOP errors compared to correct STOP trials,
20
suggesting that without substantial directional selectivity, errors were made. Interestingly, this
21
reduction in directional selectivity during error trials was only present in the PNE group (Fig.
22
S1K,L; Wilcoxon; p < 0.01) although there was a trend toward significance in the control group
23
(control; Fig. S1H,I; Wilcoxon; p = 0.09). Furthermore, the distribution of directional indices on
24
error trials for control rats was not significantly different than the PNE group (Wilcoxon; p =
25
0.42). Despite being weaker, the STOP error direction index distributions were still significantly
26
positive suggesting that activity in mPFC better reflected the nature of the movement, not the
27
sensory stimulus that triggered it. There was no difference between control and PNE direction
28
distributions during GO trials (Fig. S1G,J; Wilcoxon; p = 0.18) or STOP trials (Fig. S1H,K;
29
Wilcoxon; p = 0.61).
30
31
Lastly, the timing of the direction signaling in mPFC neurons was subtly different
between groups. Significant direction signaling, as a function of time, was determined using a
2
1
sliding window analysis. For STOP and GO trials independently, activity between the preferred
2
and nonpreferred directions was compared in 100ms epochs that slid 10ms after each iteration.
3
The results of these analyses are presented as tick marks in figures S1A left and B left where
4
epochs of significant direction signaling (Wilcoxon; p < 0.01) are marked in red for STOP trials
5
and blue for GO trials. Increasing-type cells showed significant modulation of direction during
6
GO trials swiftly (around port exit) in both groups. During STOP trials, significant directional
7
tuning was not achieved until after the stop change reaction time (SCRT) in both groups
8
suggesting that mPFC is not initiating the correct action during STOP trials but is modifying or
9
monitoring behavior as it is occurring. This is consistent with the ‘conflict monitoring’ function
10
that we propose in the main text. The differences between the two groups shed some light on
11
STOP trial deficits observed in PNE rats. Relative to control neurons, PNE cells encoded the
12
correct direction on GO trials much sooner (a difference of 140ms) but provided later directional
13
tuning during STOP trials (a difference of 40ms). With neurons in the PNE group driving the
14
directional response on GO trials earlier, it theoretically complemented a reduction in STOP trial
15
directional firing and, by extension, performance since STOP trials require the inhibition of the
16
initial GO behavior. Earlier direction differentiation during GO trials in PNE animals fits with
17
the greater proportion of premature responses (Fig. 2C) and reduced accuracy on gS trials (Fig.
18
5E) observed in PNE animals. Thus at the population level, directional signals on STOP trials
19
were weaker and delayed. This finding complements the reduction in single neurons that
20
showed reduced directional sensitivity in the main text (Fig. 3D).
21
22
23
Impact of prenatal nicotine exposure on decreasing-type neurons
The average firing over all decreasing-type neurons, separated by trial-type, is illustrated
24
in figure S2A and B for control and PNE groups. As discussed in the main text, average mPFC
25
firing was attenuated in the PNE group.
26
As in increasing-type neurons, neural activity appeared higher for STOP compared to GO
27
trials for responses made in the preferred direction for both control and PNE groups (Fig.
28
S2A,B). As above, stop indices (Fig. S2C-F) and directional indices (Fig. S2G-L) were
29
computed for each decreasing-type neuron during the response epoch for both the control and
30
PNE group.
3
1
The shifts in stop index distributions in the preferred direction (Fig. S2C,E; Wilcoxon; ps
2
< 0.01) were significant and positive for both the control and PNE groups. Interestingly, stop
3
index distributions were significantly negatively shifted from zero in the nonpreferred direction
4
in both groups (Fig. S2D,F; Wilcoxon; ps < 0.05). Stop index distributions did not significantly
5
differ between groups in either the preferred (Fig. S2C,E; Wilcoxon; p = 0.23) or nonpreferred
6
(Fig. S2D,F; Wilcoxon; p = 0.42) direction. Thus in the majority of decreasing-type neurons,
7
firing on STOP trials was higher in the preferred direction and lower in the nonpreferred
8
direction. This reflects a heightened directional signal on STOP relative to GO trials. Although
9
prenatal exposure to nicotine attenuates firing of decreasing-type neurons, it does not appear to
10
11
alter these neural correlates (i.e. STOP vs. GO).
The strength of directional encoding for decreasing-type neurons was unaffected by
12
prenatal nicotine exposure. For both groups, the directional index was significantly shifted for
13
both GO and STOP trial-types (Fig. S2G,H,J,K) and direction indices were significantly greater
14
under STOP trials than GO trials (control; Wilcoxon; p < 0.01; PNE; Wilcoxon; p < 0.01).
15
There was no difference between control and PNE direction distributions (GO trials; Fig. S2G,J;
16
Wilcoxon; p = 0.18; STOP trials; Fig. S2H,K; Wilcoxon; p = 0.76).
17
The strength of directional tuning was significantly reduced on STOP errors compared to
18
correct STOP trials in both groups (Fig. S2H,I,K,L; control; Wilcoxon; p < 0.01; PNE;
19
Wilcoxon; p < 0.01) and there was no significant difference between control and PNE directional
20
index distributions for incorrect trials (Wilcoxon; p = 0.52). We conclude that overall firing of
21
decreasing-type cells in mPFC was attenuated after prenatal exposure to nicotine, but directional
22
encoding remained intact at the population level.
23
Directional differentiation as a function of time was largely similar between groups
24
during STOP trials; significant directional encoding was achieved slightly after unpoke and
25
before the SCRT in both groups (tick marks). Significant directional differentiation did appear
26
earlier on GO trials in PNE compared to control cells suggesting a prepotency toward the correct
27
GO direction. This is in complement to the larger proportion of premature responses in PNE
28
animals. We conclude that the overall reduction in firing rate in PNE decreasing-type cells did
29
not largely change either trial-type or directional encoding at the neuronal or population level.
30
31
Multiple regression analysis of single-unit activity
4
1
To complement the analyses above and the larger effects originally shown in figure 3D,
2
we have displayed the results of the multiple regression analysis on each increasing- and
3
decreasing-type cell in figure S3. The model used was designed to determine if neuronal firing
4
correlates significantly and uniquely with movement speed, direction, and/or type of trial at the
5
single-cell level. The yellow circle in figure S3A indicates the number and percentage of
6
increasing-type mPFC cells in control animals that were significantly modulated by the direction
7
of the response when variance for the other two parameters was factored out (partial r2). Forty
8
neurons (33%) were significantly modulated by direction and of these 40 neurons, 18 β-values of
9
the direction parameter were positive (greater firing for the contralateral direction) whereas 22
10
were negative, values not different from 50/50 split (binomial sign test; p = 0.64). Forty neurons
11
(33%) were significantly and uniquely correlated with movement time and of these 40, the
12
number of negative β-values (greater firing for faster movement speeds) did not differ from the
13
number of positive β-values (Fig. S3A; movement time; 25 vs. 15; binomial sign test; p = 0.15).
14
A substantial portion of the aforementioned neurons exhibited significant partial r2 values for
15
both the direction and movement time parameters (Fig. S3A; orange + brown; n = 13)
16
highlighting the role of mPFC in functions regarding spatial response and movement latency. A
17
small proportion of control increasing-type neurons featured significant partial r2 values for the
18
trial-type (STOP vs. GO) parameter (Fig. S3A; trial-type; n = 14; 12%). Of these 14 neurons,
19
equal numbers showed higher firing for STOP trials over GO trials and vice versa (binomial sign
20
test; p = 1).
21
In direct comparison to increasing-type control neurons, we examined increasing-type
22
neurons in the PNE group. Of these cells, 23% (n = 29) were significantly modulated by
23
direction. Nineteen of these 29 had associated positive β-values (greater firing in the
24
contralateral direction) which was not statistically greater than the number of neurons with
25
associated negative β-values (Fig. S3C; direction; 19 vs. 10; binomial sign test; p = 0.14). Of
26
the neurons that were significantly and uniquely correlated with movement speed (Fig. S3C;
27
movement time; n = 39; 13%), a significant proportion featured positive β-values (Fig. S3C;
28
movement time; 38 vs. 1; binominal sign test; p < 0.01). Lastly, increasing-type neurons of PNE
29
rats had 8 neurons that were modulated by the trial-type parameter; of which, statistically more
30
featured positive β-values than negative β-values (Fig. S3C; trial-type; 8 vs. 0; binomial sign
31
test; p < 0.01).
5
1
Figure S3B and D detail the relatively similar results of the regression procedure for
2
control (B) and PNE (D) decreasing-type mPFC cells, which have been compared generally in
3
figure 3D. More precisely, 24% (n = 63) and 20% (n = 45) of cells in control and PNE
4
decreasing-type cells respectively exhibit significant partial r2 values for the direction parameter,
5
neither of which feature β-values different from 50/50 split (Fig. S3B; direction; 35 vs. 28;
6
binominal sign test; p = 0.45; Fig. S3D; direction; 29 vs. 16; binominal sign test; p = 0.07). Like
7
the direction parameter, decreasing-type neurons in control and PNE animals did not differ in the
8
proportions of cells modulated by movement speed (control; n = 85; 33%; PNE; n = 74; 32%).
9
Additionally, movement time modulated cells showed significantly more cells with positive β-
10
values (Fig. S3B; movement time; 79 vs. 6; binomial sign test; p < 0.01; Fig. S3D; movement
11
time; 70 vs. 4; binomial sign test; p < 0.01). Lastly, as in the two previous parameters, equal
12
proportions of decreasing-type cells in control and nicotine rats exhibited significant partial r2
13
values for type of trial (control; n = 24; 9%; PNE; n = 17; 7%) and neither groups of significant
14
trial-type cells featured β-values different from 50/50 split (Fig. S3B; trial-type; 16 vs. 8;
15
binomial sign test; p = 0.15; Fig. S3D; trial-type; 10 vs. 7; binomial sign test; p = 0.63).
16
17
18
Firing in mPFC is not dependent on the degree of conflict on trials preceding GO trials
In the main text we claim that mPFC, while not particularly sensitive to response
19
inhibition, was able to detect the degree of conflict on STOP trials based on the previous trial.
20
That is, STOP trials have elevated directional conflict when preceded by a GO trial (gS trials)
21
due to the lack of preparation for conflict resolution as compared to STOP trials preceded by a
22
STOP trial (sS trials). However, an alternative explanation for this effect cannot be discounted;
23
increased activity on gS trials might reflect elevated conflict or it may simply reflect that the
24
previous trial was a GO trial. If the latter is true, activity should have also been high when the
25
previous trial was a GO and the current trial was a GO trial (gG trials). If this increase in
26
activity is due to the conflict induced by the previous trial, gG trial firing should not differ from
27
sG trial firing as there is no conflict on current GO trials.
28
In figure S4, we plotted gG trial and sG trial firing of increasing-type neurons of the
29
control (Fig. S4A) and PNE groups (Fig. S4B) aligned on port exit. We compared gG and sG
30
trial firing by calculating a firing index ((gG-sG)/gG+sG)) for every neuron during the 1s epoch
31
beginning at port exit (Fig. S4C,D). Neither distribution was significantly shifted from zero
6
1
suggesting no differential encoding of these two trial-types. This suggests that mPFC activity
2
plays an important role in monitoring conflict from previous trials and not simply the trial-type
3
identity in the recent past.
4
5
6
Effects of PNE on encoding properties of mPFC was not solely due to hypoactivation
Figures 2E and F in the main text show decreases of mPFC activity when PNE rats are
7
either executing a trial or in a relative state of quiescence (baseline). We also observed
8
reductions in performance and movement speed correlations with prefrontal firing as well as the
9
inability to distinguish STOP trials based on prior conflict in the PNE group (conflict
10
monitoring). It may be argued that these decrements in task-based encoding are simply due to a
11
hypoactive mPFC. That is, would reductions in mPFC activity of healthy subjects recreate
12
encoding deficits seen in the PNE group? To answer this question, we matched firing rates
13
between the two groups and reanalyzed the aforementioned task-encoding properties.
14
To match firing patterns between the two groups, we sub-selected neurons from the
15
control group to match session-wide baseline and response epoch firing rate and variance in the
16
PNE group (increasing-type; baseline; 4.3 ± 3.71 spks/s vs. 4.15 ± 4.03 spks/s; Wilcoxon; ps >
17
0.50; response epoch; 6.26 ± 4.84 spks/s vs. 6.27 ± 4.94 spks/s; Wilcoxon; ps > 0.50; decreasing-
18
type; baseline; 4.75 ± 4.34 spks/s vs. 4.58 ± 4.22 spks/s; Wilcoxon; ps > 0.50; response epoch;
19
3.27 ± 3.22 spks/s vs. 3.19 ± 3.40 spks/s; Wilcoxon; ps > 0.50). We intentionally excluded a low
20
number neurons to maintain our large sample size and not disrupt the relative proportions of
21
increasing- or decreasing-type neurons (or neurons per rat) as these effects were not different
22
between groups in figures 2D and E (χ2; Wilcoxon; ps > 0.50).
23
The original results in figure 4 depicted decreases in correlations between mPFC firing
24
and both accuracy (percent correct) and movement time in the PNE group. We subjected our
25
sub-selected groups of neurons to the same analyses and found that the results remained largely
26
unchanged. For increasing-type cells, firing during the response epoch was positively correlated
27
with percent correct. The regression was significant for the control (r = 0.39; p < 0.01) and the
28
PNE group (r = 0.19; p < 0.05) but these effects do not significantly differ (t-test; p = 0.05). The
29
correlation between movement time and firing rate was significant for increasing-type cells only
30
in the PNE group (r = 0.25; p < 0.01) but this correlation did not differ from the control group (t-
31
test; p = 0.30). However, for decreasing-type neurons, there was a positive correlation between
7
1
movement time and firing rate, which was only significant in the control group (r = 0.31; p <
2
0.01) and was different from the PNE group (t-test; p < 0.05). Finally, the correlation between
3
firing rate and percent correct in decreasing-type cells was not significant in the control group (r
4
= 0.12, p = 0.08), but was significantly negatively correlated in the PNE group (r = -0.15; p <
5
0.05). These correlations differed significantly (t-test; p < 0.01).
6
The main text shows that while the mPFC in the control group is capable of dissociating
7
STOP trials based on previous conflict once the response was commenced, mPFC neurons in the
8
PNE group were largely incapable of this conflict detection leading to severe deficits on gS trials
9
(Fig. 5). In the sub-selected group of control neurons, this differential encoding of gS and sS
10
trials remained (gS-sS divided by the sum; Wilcoxon; p < 0.01) whereas PNE neurons did not
11
show this encoding (gS-sS divided by the sum; Wilcoxon; p = 0.89). Importantly, these two
12
distributions in the sub-selected group of neurons sustained their significant difference
13
(Wilcoxon; p < 0.05).
14
Upon comparison of these firing rate matched neurons relative to their original
15
populations, it appears as though hypoactivation of mPFC in the PNE group complemented, but
16
did not cause, disrupted encoding our task.
17
18
19
FIGURE LEGENDS
20
Figure S1. (a-b) Average firing rate (spikes/s) over time aligned on port exit (left) and well entry
21
(right) for all control (a) and nicotine exposed (b) neurons that fired more strongly during the
22
‘response epoch’ (port exit to well entry) relative to baseline (1s epoch beginning 2s prior to trial
23
initiation). The time necessary to inhibit a response (stop change reaction time; SCRT) is
24
defined as the difference between STOP trial movement time and GO trial movement time.
25
SCRT is marked as the vertical dotted line at 120ms in control and 98ms in PNE animals. Blue
26
lines refer to GO trials, red lines refer to STOP trials, and dashed lines refer to errant trials
27
(incorrect direction). Due to the heterogeneous direction specificity of individual cells, each cell
28
was characterized as having a preferred direction and a nonpreferred direction. This preference
29
was determined by asking the direction (contra- or ipsilateral to the recorded hemisphere) that
30
elicited the highest firing rate during the response epoch for each cell. Therefore, as defined by
8
1
our analysis, preferred direction (thick lines) is always higher than the nonpreferred direction
2
(thin lines) during the response epoch. Tick marks represent significant p-values in temporal
3
space after preferred direction was compared to nonpreferred direction in the population for GO
4
trials (blue ticks) and STOP trials (red ticks) in 100ms epochs that slid by 10ms after each
5
iteration (Wilcoxon; p < 0.01). Arrowheads denote the average movement time (well entry)
6
during GO trials (blue arrowheads; control = 584ms; PNE = 456ms) and STOP trials (red
7
arrowheads; control = 704ms; PNE = 554ms) for the analyzed sessions. (c-f) Stop indices for
8
preferred (c,e) and nonpreferred (d,f) directions from control (c,d) and PNE (e,f) cells. Stop
9
indices are calculated by taking the activity during the response epoch from STOP trials,
10
subtracting activity during the response epoch on GO trials, and dividing it by the sum of the two
11
((STOP-GO/(STOP+GO)) in each direction for every cell. Significant shifts from zero (as
12
calculated by Wilcoxon) denote that neuronal activity is significantly different between STOP
13
and GO trials in a given direction. (g-l) Direction index distributions defined as activity during
14
the response epoch in the preferred direction minus activity during the response epoch in the
15
nonpreferred direction divided by the sum ((preferred-nonpreferred)/(preferred+nonpreferred)) in
16
every cell. These calculations are specific to GO trials (g,j), STOP trials (h,k), and STOP errors
17
(i,l) for control (g-i) and PNE (j-l) cells. Significant shifts from zero (as calculated by Wilcoxon)
18
signify that activity is greater in one direction than the other at the neuronal level. Distributions
19
are directly compared via Wilcoxon.
20
21
Figure S2. (a-l) All conventions as in figure S1 for decreasing-type neurons. SCRT values are
22
149ms for control cells and 128ms for PNE cells. Average movement times (as marked by
23
arrowheads) for GO trials are 546ms for control and 505ms for PNE cells whereas movement
24
times for STOP trials are 713ms for control and 633ms for PNE cells.
25
26
Figure S3. (a-d) Results of the multiple regression procedure detailed in the Methods section for
27
increasing- (a,c) and decreasing-type (b,d) cells in control (a,b) and PNE groups (c,d). Yellow
28
circle encompasses the number (and percentage) of neurons that show significant partial r2
29
values for the direction parameter. Conventions as above for the movement time (red circle) and
30
trial-type (blue circle) parameters. Overlapping portions denote the number (and percentage) of
9
1
single cells that exhibited significant partial r2 values for two (orange, green, purple) or all three
2
(brown) parameters.
3
Figure S4. (a-b) Population histograms of all mPFC neurons in the control (a) and PNE (b)
4
groups that fired significantly above baseline between port exit to well entry (termed ‘response
5
epoch’). Activity is aligned to port exit. All lines represent accurate GO trials that either
6
followed a GO trial (‘gG’; dark blue) or followed a STOP trial (‘sG’; light blue). Thick lines
7
refer to the preferred direction defined as the direction that elicited the greatest firing rate during
8
the response epoch. Thin lines refer to the nonpreferred direction. (c-d) Distributions calculate
9
the difference between firing during the 1s epoch following port exit on gG versus sG trials
10
divided by the sum for control (c) and PNE (d) groups. Significance is determined via Wilcoxon
11
(p < 0.05). (e-f) Percentage of correct responses (e) and movement latencies (f) defined as the
12
duration between port exit and well entry on gS and sG trials independently for control (black)
13
and PNE (gray) groups. Asterisks indicate planned comparisons (Wilcoxon; p < 0.05).
14
Table S1. Comparisons of physical characteristics between the nicotine group (0.06mg/ml of
15
nicotine in the drinking water) and the control group (unadulterated drinking water). All values
16
are compared using a t-test (p < 0.05) except “Number of Pregnant Females” which was
17
compared using a χ2 test.
10