Sex Differences in the Activity of Basolateral Amygdalar Neurons That Project to the Bed Nucleus of the Stria Terminalis and Their Role in Anticipatory Anxiety

Abstract

Abnormal fear and anxiety can manifest as psychiatric disorders. The bed nucleus of the stria terminalis (BNST) is implicated in sustained responding to, or anticipation of, an aversive event which can be expressed as anticipatory anxiety. The BLA is also active during anticipatory anxiety and sends projections to the BNST. However, little is known about the role for BLA neurons that project to BNST (BLA-BNST) in anticipatory anxiety in rodents. To address this, we tested whether chemogenetic inactivation of the BLA-BNST pathway attenuates sustained conditioned responses produced by anticipation of an aversive stimulus. For comparison, we also assessed BLA-BNST inactivation during social interaction, which is sensitive to unlearned anxiety. We found that BLA-BNST inactivation reduced conditioned sustained freezing and increased social behaviors, but surprisingly, only in males. To determine whether sex differences in BLA-BNST neuronal activity contribute to the differences in behavior, we used in vivo and ex vivo electrophysiological approaches. In males, BLA-BNST projection neurons were more active and excitable, which coincided with a smaller after-hyperpolarization current (I _AHP) compared with other BLA neurons; whereas in females, BLA-BNST neurons were less excitable and had larger I _AHP compared with other BLA neurons. These findings demonstrate that activity of BLA-BNST neurons mediates conditioned anticipatory anxiety-like behavior in males. The lack of a role of BLA-BNST in females in this behavior, possibly because of low excitability of these neurons, also highlights the need for caution when generalizing the role of specific neurocircuits in fear and anxiety.SIGNIFICANCE STATEMENT Anxiety disorders disproportionately affect women. This hints toward sex differences within anxiety neurocircuitry, yet most of our understanding is derived from male rodents. Furthermore, debilitating anticipation of adverse events is among the most severe anxiety symptoms, but little is known about anticipatory anxiety neurocircuitry. Here we demonstrated that BLA-BNST activity is required for anticipatory anxiety to a prolonged aversive cue, but only in males. Moreover, BLA-BNST neurons are hypoactive and less excitable in females. These results uncover BLA-BNST as a key component of anticipatory anxiety circuitry, and cellular differences may explain the sex-dependent role of this circuit. Uncovering this disparity provides evidence that the assumed basic circuitry of an anxiety behavior might not readily transpose from males to females.

Keywords: BLA; BNST; anxiety; sex differences.

Figure 1.

Prolonged cued-conditioned freezing is lower in females compared with males. A, Schematic representation of the prolonged cued fear paradigm where rats are conditioned to the prolonged tone (CS; 5 kHz, 80 dB, 8 min; gray boxes) and receive 5 randomized foot shocks (0.3 mA, 0.5 ms) in context A (Cxt A). Four days later in a separate context (Cxt B), percent time freezing during the CS in the absence of footshocks was tested. B, Percent time spent freezing (1 min bin averages) in naive male (black, N = 19) and female (gray, N = 24) rats during prolonged cued fear conditioning was analyzed, but there was only a significant main effect of time (F_{(3.705,151.9)} = 74.27, p < 0.0001, two-way RM ANOVA) but no main effect of sex (F_(1,41) = 0.8288, p = 0.3679, two-way RM ANOVA) nor an interaction between sex and time (F_(10,410) = 0.5015, p = 0.8889, two-way RM ANOVA). There were no differences in the total time spent freezing for the first 4 min of the tone (1; t₍₄₁₎ = 1.190, p = 0.21, unpaired t test) and the last 4 min of the tone (2; t₍₄₁₎ = 0.334, p =0.740, unpaired t test) between males and females during prolonged cued fear training. C, During testing, there was a main effect of time (F_(3.657,150) = 24.31, p < 0.0001), and a trend for a main effect of sex (F_(1,41) = 3.497, p = 0.0687, two-way RM ANOVA), but no significant interaction between time and sex (F_(10,410) = 1.613, p = 0.1004, two-way RM ANOVA). Females had significantly lower total time spent freezing during both the first half of the tone (1; t₍₄₁₎ = 3.903, **p = 0.0003, unpaired t test) and second half of the tone (2; t₍₄₁₎ = 5.424, **p < 0.0001, unpaired t test) compared with males during prolonged cued fear testing. Percent time freezing was also calculated and compared by estrous cycle stage (black, N = 11 proestrous; and gray, N = 13 metestrous) and time (D). There were main effects of both time (F_{(4.376,96.27)} = 38.09, p < 0.0001, two-way RM ANOVA) and estrous cycle stage (F_(1,22) = 7.127, p = 0.014, two-way RM ANOVA), with all animals spending more time freezing as the experiment progressed, and proestrous females freezing less overall compared with metestrous females. There was also a significant interaction between time and estrous cycle stage (F_(10,220) = 1.947, *p = 0.0404, two-way RM ANOVA). E, A significant interaction between time and estrous cycle phase was found during testing (F_(10,220) = 2.573, p = 0.0058, two-way RM ANOVA) as proestrous rats froze significantly less than metestrous females during testing. There was also a main effect of time (F_(10,220) = 11.44, p < 0.0001, two-way RM ANOVA), but not cycle stage (F_(1,22) = 0.4649, p = 0.5024, two-way RM ANOVA) as both groups reduced freezing over time. F, A separate group of male (black, N = 6) and female (gray, N = 12) rats were conditioned to the tone in the absence of footshock. There was no main effect of time (F_{(3.311,33.11)} = 1.224, p = 0.3177, two-way RM ANOVA), sex (F_(1,10) = 1.317, p = 0.2779, two-way RM ANOVA), nor interaction between the two (F_(10,100) = 1.224, p = 0.2853, two-way RM ANOVA) during training. G, These same rats were then tested in Cxt B to ensure that there were no sex differences in freezing in response to tone alone. While there was no main effect of sex (F_(1,16) = 2.274, p = 0.151, two-way RM ANOVA), we did have a main effect of time (F_{(4.294,68.71)} = 15.72, p < 0.0001, two-way RM ANOVA) as rats began to freeze at the end of the experiment. Furthermore, there was also a significant interaction (F_(10,160) = 2.548, p = 0.007, two-way RM ANOVA) between time and sex as male rats froze near the end of the experiment more than females.

Figure 2.

Intersectional chemogenetic targeting of BLA-BNST neurons with Cre-dependent DREADDs. A, CAV2-Cre was injected bilaterally into the BNST (top left) in combination with Cre-dependent inhibitory DREADDs in the BLA (top right). Photomicrograph illustrating Cre-recombinase (bottom left; green) and mCherry (bottom right; red) immunoreactivity in the BLA of virus-injected rats. B, Photomicrograph examples of Cre-recombinase (green) and mCherry (red) coexpression in the BLA at low (top) and high magnification (bottom). White arrows indicate double-labeled cells. C, DREADD⁺ neurons were identified for ex vivo recordings based on mCherry fluorescence. CNO (bath-applied, 10 μm) significantly decreased excitability of DREADD⁺ neurons from females (N = 5 neurons, p < 0.001, F_(1,20) = 70.4, two-way RM ANOVA) and males (N = 4 neurons, p < 0.01, F_(1,15) = 40.6, two-way RM ANOVA). D, In contrast, CNO did not significantly impact the excitability of non-DREADD⁺ neurons (N = 5 neurons, p > 0.05, F_(1,20) = 0.31, two-way RM ANOVA).

Figure 3.

BLA-BNST inactivation has opposing effects on freezing to a prolonged conditioned cue in male and female rats. A, Rats were conditioned as described previously (Fig. 1A), then injected with either saline (N = 21, black circles) or CNO (N = 22, gray circles) before testing. B, There was only a main effect of time (F_{(4.557,186.8)} = 81.06, p < 0.0001, two-way RM ANOVA) during prolonged cued fear training. There was no significant main effect of CNO treatment (F_(1,41) = 0.3874, p = 0.5371, two-way RM ANOVA), nor was there a main interaction between time and CNO (F_(10,410) = 0.4609, p = 0.9146, two-way RM ANOVA). C, During extinction, when all rats were grouped together, there was only a main effect of time (F_{(2.9868,121.7)} = 12.31, p < 0.0001, two-way RM ANOVA) on freezing behavior, but no main effect of drug alone (F_(1,41) = 0.1296, p = 0.7207, two-way RM ANOVA) nor interaction between drug and time (F_(10,410) = 0.3397, p = 0.9699, two-way RM ANOVA). D, For male rats that received either saline (N = 9, black circles) or CNO (N = 10, gray circles) injections before testing, there was only a main effect of time (F_(3.33,56.61) = 46.98, p < 0.0001, two-way RM ANOVA) on freezing during training, but no main drug effect (F_(1,17) = 0.06733, p = 0.7984, two-way RM ANOVA) nor significant interaction (F_(10,170) = 0.7074, p = 0.7167, two-way RM ANOVA). E, During testing, there was only a main effect of time (F_{(2.802,47.63)} = 3.528, p = 0.024, two-way RM ANOVA), but not of drug (F_(1,17) = 2.176, p = 0.1584, two-way RM ANOVA) nor an interaction (F_(10,170) = 1.143, p = 0.3331, two-way RM ANOVA). F, Female rats that were injected with either saline (N = 12, black circles) or CNO (N = 12, gray circles) had a main effect of time on freezing during training (F_{(4.794 105.5)} = 38.18, p < 0.0001, two-way RM ANOVA), but not for drug (F_(1,22) = 0.8739, p = 0.36, two-way RM ANOVA) nor an interaction between drug and time (F_(10,220) = 0.7041, p = 0.7201, two-way RM ANOVA). G, Similarly, during testing sessions, there was only a main effect of time (F_{(2.868,63.10)} = 10.75, p < 0.0001, two-way RM ANOVA), but not drug alone (F_(1,22) = 0.8374, p = 0.3701, two-way RM ANOVA) nor an interaction between time and drug (F_(10,220) = 1.239, p = 0.2673, two-way RM ANOVA). H, Total time spent freezing during 2 min baseline, the first 4 min of the prolonged tone (I), the second 4 min of the prolonged tone (J), and the postshock recovery period (K) of the testing session were separately analyzed. There was a significant interaction between drug and sex during the first half of the tone (H; F_(1,39) = 4.636, *p = 0.0375, two-way ANOVA) as CNO treatment reduced freezing in males, but increased freezing in females during the first half of the tone. There was no main effect of drug (F_(1,39) = 0.1044, p = 0.7483, two-way ANOVA) or sex (F_(1,39) = 0.6896, p = 0.4114, two-way ANOVA) alone.

Figure 4.

CNO administration during prolonged fear extinction does not alter freezing behavior in naive rats. A, Naive rats were conditioned as described previously (Fig. 1A). Rats were injected with either saline (N = 10; black circles) or CNO (N = 12; gray circles) before extinction. There was only a main effect of time on freezing behavior during training (B; F_(2.489,22.4) = 34.74, p < 0.0001, two-way RM ANOVA) and testing (C; F_{(3.291,29.62)} = 7.985, p = 0.0003, two-way RM ANOVA) in males. There was no significant effect of drug (F_(1,9) = 0.08397, p = 0.7786, two-way RM ANOVA) nor interaction between time and drug (F_(10,90) = 0.6511, p = 0.7661, two-way RM ANOVA) during training. The lack of effect of drug (F_(1,9) = 0.3623, p = 0.5621, two-way RM ANOVA) or interaction (F_(10,90) = 0.6585, p = 0.7596, two-way RM ANOVA) was also observed during testing. Similar results were observed in females with only a main effect of time during both training (D; F_(3.74,33.66) = 31.44, p < 0.0001, two-way RM ANOVA) and testing (E; F_{(3.631,32.68)} = 7.177, p = 0.0004, two-way RM ANOVA). Females also did not have a main effect of drug (F_(1,9) = 0.0623, p = 0.8085, two-way RM ANOVA) nor interaction (F_(10,90) = 0.8144, p = 0.6155, two-way ANOVA) during training and testing (main effect of drug; F_(1,9) = 0.02609, p = 0.8752, two-way RM ANOVA; main interaction; F_(10,90) = 0.2640, p = 0.9873, two-way RM ANOVA).

Figure 5.

Inactivation of BLA-BNST neurons during conditioning does not affect sustained freezing to a prolonged cue. A, Rats were conditioned as described previously (Fig. 1A). B, Rats were injected with either saline (N = 24; black circles) or CNO (N = 24; gray circles) before conditioning. There was a main effect of time (B; F_(4.46,205.2) = 104, p < 0.0001, two-way RM ANOVA) on freezing behavior during training and testing (C; F_{(3.489,160.5)} = 19.95, p < 0.0001, two-way RM ANOVA). There was no main effect of drug (F_(1,46) = 1.144, p = 0.2904, two-way RM ANOVA) nor interaction (F_(10,460) = 1.356, p = 0.1983, two-way RM ANOVA) during training or testing (main effect of drug, F_(1,46) = 0.7682, p = 0.3853, two-way RM ANOVA; main interaction, F_(10,460) = 1.245, p = 0.2596, two-way RM ANOVA). When animals were separated out by sex, BLA-BNST inhibition during prolonged cued fear training had only a main effect of time during both training (D; F_{(4.243,93.35)} = 57.8, p < 0.0001, two-way RM ANOVA) and testing (E; F_{(3.761,82.74)} = 13.48, p < 0.0001, two-way RM ANOVA) in males. No main effect of drug nor interaction between drug and time was observed during training (main effect of drug, F_(1,22) = 0.1057, p = 0.7482, two-way RM ANOVA; main interaction, F_(10,220) = 1.028, p = 0.4205, two-way RM ANOVA) nor testing (main effect of drug, F_(1,22) = 0.002366, p = 0.9616, two-way RM ANOVA; main interaction, F_(10,220) = 0.5052, p = 0.8854, two-way RM ANOVA) in males. Similar results were observed in females with only a main effect of time during both training (F; F_(3.82,99.32) = 53.38, p < 0.0001, two-way RM ANOVA) and testing (G; F_{(3.255,84.63)} = 9.072, p < 0.0001, two-way RM ANOVA) but no main effect of drug nor interaction during training (main effect of drug, F_(1,26) = 3.528, p = 0.0716, two-way RM ANOVA; main interaction, F_(10,260) = 0.7071, p = 0.7176, two-way RM ANOVA) or testing (main effect of drug, F_(1,26) = 2.010, p = 0.1682, two-way RM ANOVA; main interaction, F_(10,260) = 1.105, p = 0.3589, two-way RM ANOVA).

Figure 6.

OF exploration is not affected by inactivation of BLA-BNST. OF exploration was examined in rats following either CNO (N = 22, gray) or saline (N = 27, black) injections. There were no significant differences in total distance traveled (A1; t₍₄₇₎ = 0.4362, p = 0.6647, unpaired t test), average speed (B1; t₍₄₇₎ = 0.2639, p = 0.7930, unpaired t test), number of center entries (C1; t₍₄₇₎ = 0.2722, p = 0.7866, unpaired t test), time spent in the center (D1; t₍₄₇₎ = 0.03786, p = 0.97, unpaired t test), and distance traveled in the center (E1; t₍₄₇₎ = 0.5061, p = 0.6152, unpaired t test) during the novel OF task. The same parameters were also analyzed by sex and drug treatment (N = 15 saline males, N = 8 CNO males, N = 12 saline females, and N = 14 CNO females). There were main effects of sex when total distance traveled (A2; F_(1,45) = 39.56, p < 0.0001, two-way ANOVA), average speed (B2; F_(1,45) = 39.37, **p < 0.0001, two-way ANOVA), number of center entries (C2; F_(1,45) = 7.703, **p = 0.008, two-way ANOVA), and distance traveled in the center (E2; F_(1,45) = 7.997, **p = 0.007, two-way ANOVA) were analyzed. All other effects and interactions were not significant when total distance traveled (A2; main interaction, F_(1,45) = 0.3066, p = 0.5825; main effect of drug, F_(1,45) = 3.324, p = 0.0749, two-way ANOVA), average speed (B2; main interaction, F_(1,45) = 0.3327, p = 0.5669; main effect of drug, F_(1,45) = 3.327, p = 0.0748, two-way ANOVA), number of center entries (C2; main interaction, F_(1,45) = 0.366, p = 0.5482; main effect of drug, F_(1,45) = 0.03995, p = 0.8425, two-way ANOVA), time spent in the center (D2; main interaction, F_(1,45) = 0.0009681, p = 0.9753; main effect sex, F_(1,45) = 0.8749, p = 0.3546; main effect of drug, F_(1,45) = 0.04559, p = 0.8319, two-way ANOVA), and distance traveled in the center (E2; main interaction, F_(1,45) = 1.1, p = 0.2998; main effect of drug, F_(1,45) = 0.9853, p = 0.3262, two-way ANOVA) were calculated. The times spent in the center (D2) were not significantly different between sex or by drug treatment. The same parameters were also analyzed by estrous cycle stage and drug treatment (proestrous, N = 11; and metestrous, N = 15). Total distance traveled (A3; main effect cycle stage, F_(1,22) = 0.111, p = 0.7421; main effect of drug, F_(1,22) = 0.028, p = 0.8686; main interaction, F_(1,22) = 0.9335, p = 0.3445, two-way ANOVA), average speed (B3; main interaction, F_(1,22) = 0.03899, p = 0.8453; main effect cycle, F_(1,22) = 0.1992, p = 0.6597; main effect of drug, F_(1,22) = 0.8147, p = 0.3765, two-way ANOVA), number of center entries (C3; main interaction, F_(1,22) = 0.0005892, p = 0.9809; main effect of cycle, F_(1,22) = 0.04773, p = 0.8291; main effect of drug, F_(1,22) = 0.2939, p = 0.5932, two-way ANOVA), time in the center (D3; main interaction, F_(1,22) = 0.113, p = 0.7399; main effect of cycle, F_(1,22) = 1.088, p = 0.3083; main effect of drug, F_(1,22) = 0.1594, p = 0.6936, two-way ANOVA), and distance traveled in the center (E3; main interaction, F_(1,22) = 0.4916, p = 0.4905; main effect of cycle, F_(1,22) = 1.298, p = 0.2668; main effect of drug, F_(1,22) = 2.875, p = 0.1041, two-way ANOVA) were calculated and determined to be comparable between all groups.

Figure 7.

Inhibition of BLA-BNST neurons increases social interaction in males, but not females. A, The total time spent interacting was compared within individual animals (N = 63) following both CNO (black) and saline (gray) injections administered before SI tests and found to be similar (t₍₆₂₎ = 0.5433, p = 0.5889, paired t test). B, Rats were separated out by sex and drug treatment, and analysis determined a main effect of sex (F_(1,61) = 7.949, **p = 0.0065, two-way RM ANOVA) as males spent more time interacting than females, but no main effect of drug (F_(1,61) = 0.4725, p = 0.4944, two-way RM ANOVA). There was also a significant interaction between sex and drug (F_(1,61) = 6.484, *p = 0.0134, two-way RM ANOVA) as males interacted more following BLA-BNST inhibition, but females did not. C, The total time spent interacting was compared within individual metestrous (N = 21) and proestrous (N = 12) rats following both CNO (gray) and saline (black) injections trended toward metestrous females interacting more than proestrous females (main interaction, F_(1,31) = 0.1113, p = 0.741; main effect of cycle, F_(1,31) = 4.012, p = 0.054; main effect of drug, F_(1,31) = 1.833, p = 0.1856, two-way RM ANOVA). D, Latency to interact was comparable between saline- and CNO-treated rats (t₍₆₂₎ = 1.326, p = 0.1897, paired t test). E, However, when animals were separated out by sex, the latency to interact did have a significant interaction between sex and drug (F_(1,61) = 9.342, **p = 0.0033, two-way RM ANOVA) with males that received CNO having a shorter latency to interact compared with males that received saline (Sidak test for multiple comparisons, ^##p = 0.0045, 95% CI (1.665, 10.210)). There was no main effect of sex (F_(1,61) = 1.997, p = 0.1627, two-way RM ANOVA) or drug alone (F_(1,61) = 2.422, p = 0.1248, two-way RM ANOVA). F, The latency to interact in female rats following either a saline or CNO injection was also not different between cycle stage (main interaction, F_(1,31) = 0.001501, p = 0.9693; main effect of cycle, F_(1,31) = 2.702, p = 0.1103; main effect of drug, F_(1,31) = 1.537, p = 0.2244, two-way RM ANOVA). G, Baseline SI time was analyzed in a subset of male (N = 14) and female (N = 14) rats and compared 4 d later to SI time following saline treatment. The total time spent interacting during the baseline SI task (black bars) compared with total interaction following saline injection (gray bars) was found to be similar (main interaction, F_(1,26) = 0.2521, p = 0.6198; main effect of drug, F_(1,26) = 0.1795, p = 0.6753, two-way RM AVOVA), but there was a main effect of sex (F_(1,26) = 9.680, **p = 0.0045, two-way RM ANOVA) as males interacted more than females. A subset of rats (N = 4 males and N = 4 females) had the same viral injection procedure but did not receive CAV2-Cre injections and therefore had nonfunctional DREADDs. When these animals were injected with CNO, there were no significant changes in latency to interact (H; main interaction, F_(1,7) = 0.2847, p = 0.6101; main effect of sex, F_(1,7) = 0.4408, p = 0.528; main effect of drug, F_(1,7) = 0.1279, p = 0.7312, two-way RM ANOVA) nor SI time (I; main interaction, F_(1,7) = 0.1315, p = 0.7276; main effect of sex, F_(1,7) = 1.555, p = 0.2525; main effect of drug, F_(1,7) = 0.001323, p = 0.972, two-way RM ANOVA).

Figure 8.

Inhibition of BLA-BNST neurons before testing does not alter freezing in female rats. A, Rats were conditioned as described previously (see Fig. 1A), and CNO was administered before testing. Proestrous females spent significantly less time freezing compared with metestrous females during the training session (data not shown; main effect of time, F_{(4.748,94.97)} = 38.83, p < 0.0001; main effect of estrous cycle stage, F_(1,20) = 12.47, p = 0.0021; main interaction, F_(10,200) = 2.315, p = 0.0135, two-way RM ANOVA). When females were separated by estrous cycle stage, there was only a main effect of time for proestrous (B; main interaction, F_(10,100) = 0.3377, p = 0.9687; main effect of time, F_{(3.774,37.74)} = 14.74, p < 0.0001; main effect of drug, F_(1,10) = 1.12, p = 0.3148, two-way RM ANOVA), and metestrous (D; main interaction, F_(10,80) = 0.8596, p = 0.5738; main effect of time, F_(2.638,21.1) = 20.69, p < 0.0001; main effect of drug, F_(1,8) = 0.9287, p = 0.3634, two-way RM ANOVA) during the training session. During testing, there was also only a main effect of time in proestrous (C; main interaction, F_(10,100) = 0.7198, p = 0.7041; main effect of time, F_{(2.419,24.19)} = 4.056, p = 0.0242; main effect of drug, F_(1,10) = 1.346, p = 0.273, two-way RM ANOVA) and metestrous (E; main interaction, F_(10,80) = 0.5362, p = 0.8595; main effect of time, F_{(22.542,20.33)} = 6.842, p = 0.0033; main effect of drug, F_(1,8) = 2.237, p = 0.1731, two-way RM ANOVA) rats. The total time spent freezing during baseline (F; main interaction, F_(1,18) = 1.332, p = 0.2635; main effect of cycle stage, F_(1,18) = 0.4348, p = 0.518; main effect of drug, F_(1,18) = 0.07242, p = 0.7909, two-way ANOVA), the first half of the tone (G; main interaction, F_(1,18) = 2.48, p = 0.1327; main effect of cycle, F_(1,18) = 6.143, *p = 0.0233; main effect of drug, F_(1,18) = 1.009, p = 0.3285, two-way ANOVA), second half of the tone (H; main interaction, F_(1,18) = 2.122, p = 0.1624; main effect of cycle, F_(1,18) = 0.0789, p = 0.782; main effect of drug, F_(1,18) = 0.1433, p = 0.7095, two-way ANOVA), and post-test (I; main interaction, F_(1,18) = 3.83, p = 0.066; main effect of cycle, F_(1,18) = 1.558, p = 0.228; main effect of drug, F_(1,18) = 0.08564, p = 0.7731, two-way ANOVA) were also analyzed separately. During the first half of the tone, metestrous females froze significantly more that proestrous females regardless of BLA-BNST inhibition.

Figure 9.

Inhibition of BLA-BNST neurons during prolonged cued fear training reduces freezing in metestrous females. A, Proestrous (N = 9) and metestrous (N = 19) female rats were injected with either saline (black) or CNO (gray) before training as described previously (Fig. 1A). BLA-BNST inactivation had no effect on freezing behavior in proestrous females (B; main interaction, F_(10,70) = 0.5992, p = 0.8093; main effect of time, F_{(3.120,21.84)} = 11.29, p < 0.0001; main effect of drug, F_(1,7) = 0.1793, p = 0.6847, two-way RM ANOVA) and testing sessions (C; main interaction, F_(10,70) = 0.7499, p = 0.6754; main effect of time, F_{(2.409,16.86)} = 3.528, p = 0.0455; main effect of drug, F_(1,7) = 0.8542, p = 0.3861, two-way RM ANOVA). D, However, in metestrous females, BLA-BNST inhibition significantly reduced freezing during training (main interaction, F_(10,170) = 1.817, p = 0.0609; main effect of time, F_{(4.085,69.44)} = 4.41 = 18.31, p < 0.0001; main effect of drug, F_(1,17) = 6.172, *p = 0.0237, two-way RM ANOVA). E, Interestingly, this significant impact on training did not affect metestrous female freezing behavior during testing (main interaction, F_(10,170) = 0.6373, p = 0.7804; main effect of time, F_{(23.547,60.3)} = 8.395, p < 0.0001; main effect of drug, F_(1,17) = 1.170, p = 0.2944, two-way RM ANOVA).

Figure 10.

BNST-projecting BLA neurons are less active in females compared with males. Single-unit in vivo electrophysiological recordings were performed in male (N = 13) and female (N = 11) rats. A, Representative trace recording of an antidromically stimulated BLA-BNST neuron in which BNST stimulation (0.9 mA) successively elicits a corresponding BLA AP. Antidromic responses were identified as having 100% firing response probability and a <1 ms variability in latency during repeated stimulation (0.1 Hz). B, Spontaneously firing BLA-BNST neurons fired at a higher frequency in males compared with females (t_(33.97) = 1.859, p = 0.0717, unpaired t test with Welch's correction). C, The average number of BLA-BNST neurons per track was significantly higher in males compared with females (t₍₁₆₎ = 4.206, **p < 0.001, unpaired t test with Welch's correction). E, There was a significant interaction between sex and neuron type when comparing BNST-projecting neurons with other BLA neurons as this specific subpopulation of neurons fired at a higher frequency compared with other BLA neurons in males, but at a lower frequency in females (main interaction, F_(1,132) = 4.357, *p = 0.041; main effect of sex, F_(1,132) = 0.3048, p = 0.5818; main effect of neuron type, F_(1,132) = 0.2541, p = 0.6152, two-way ANOVA). D, This is also represented by the individual trace recordings from spontaneously active BLA neurons in male (top left) and female (top right) rats as well as an example BLA-BNST neuron from a male (bottom left) and female (bottom right) rat. F, Location of BNST stimulation probe during recordings for males (N = 26, blue) and females (N = 11, red) and example of histologic verification of stimulation probe within the BNST (right). G, Location for BNST-projecting BLA neurons for males (N = 26, blue) and females (N = 11, red) and example of histologic verification of Pontamine Sky Blue dye ejected in the BLA recording site at 10× (right). Numbers indicate the bregma coordinates from the atlas of Paxinos and Watson (2007).

Figure 11.

BLA neurons are more excitable and have smaller AHPs in females. A, Representative traces of BLA neuron firing responses in response to a range of depolarizing current steps in BLA neurons from male (top) and female rats (bottom). B, Representative traces of AHPs recorded from BLA neurons of male (black) and female rats (gray) following a train of 5 APs. C, BLA neurons recorded from females were more excitable than males over a range of current steps (left; main effect of sex, p = 0.0002, F_(1,217) = 14.18, N = 17 neurons males, N = 16 neurons females, two-way ANOVA), and this coincided with BLA neurons from females having a smaller medium and slow afterhyperpolarization amplitude (mAHP and sAHP, respectively; main effect of sex, p = 0.0034, F_(1,42) = 9.65, N = 12 neurons males, N = 11 neurons females, two-way ANOVA). D, Representative traces of the AHP current (I_AHP) measured in BLA neurons from male (black) and female rats (gray) evoked by a brief voltage step. E, The amplitude of the medium and slow IAHP currents (I_AHP(m) and I_AHP(s)) is significantly lower in BLA neurons recorded from female rats compared with males (main effect of sex, p = 0.0022, F_(1,29) = 11.32, N = 17 neurons males, N = 14 neurons females, two-way ANOVA).

Figure 12.

BLA-BNST neurons are more excitable and have smaller AHPs in males. A, Representative traces of BLA-BNST neuron firing in response to a range of depolarizing current steps recorded from male (top) and female rats (bottom). B, BLA-BNST neurons recorded from males (N = 11 neurons, white) are more excitable than adjacent unlabeled BLA neurons (N = 11 neurons, black; left; p = 0.018, F_(1,140) = 5.749, two-way ANOVA) and have smaller I_AHP amplitude (right; p = 0.001, F_(1,28) = 15.44, N = 8 neurons/group, two-way ANOVA). C, BLA-BNST neurons recorded from females (N = 8, white) are less excitable (left) compared with adjacent BLA neurons (N = 8, black; p = 0.028, F_(1,98) = 5.0, two-way ANOVA) and show larger I_AHP amplitude (right, p = 0.015, F_(1,22) = 7.0, N = 6 or 7 neurons/group, two-way ANOVA).