Persistent sexually dimorphic effects of adolescent THC exposure on hippocampal synaptic plasticity and episodic memory in rodents

Abstract

There is evidence that cannabis use during adolescence leads to memory and cognitive problems in young adulthood but little is known about effects of early life cannabis exposure on synaptic operations that are critical for encoding and organizing information. We report here that a 14-day course of daily Δ⁹-tetrahydrocannabinol treatments administered to adolescent rats and mice (aTHC) leads to profound but selective deficits in synaptic plasticity in two axonal systems in female, and to lesser extent male, hippocampus as assessed in adulthood. Adolescent-THC exposure did not alter basic synaptic transmission (input/output curves) and had only modest effects on frequency facilitation. Nevertheless, aTHC severely impaired the endocannabinoid-dependent long-term potentiation in the lateral perforant path in females of both species, and in male mice; this was reliably associated with impaired acquisition of a component of episodic memory that depends on lateral perforant path function. Potentiation in the Schaffer-commissural (S-C) projection to field CA1 was disrupted by aTHC treatment in females only and this was associated with both a deficit in estrogen effects on S-C synaptic responses and impairments to CA1-dependent spatial (object location) memory. In all the results demonstrate sexually dimorphic and projection system-specific effects of aTHC exposure that could underlie discrete effects of early life cannabinoid usage on adult cognitive function. Moreover they suggest that some of the enduring, sexually dimorphic effects of cannabis use reflect changes in synaptic estrogen action.

Keywords: CA1; Cannabinoid; Estrogen; Frequency facilitation; Lateral perforant path; Long-term potentiation; Sex differences; Spatial learning; THC; hippocampus.

Fig. 1.

Experimental design and timeline. C57BL6 mice and Long-Evans (L-E) rats of both sexes received daily injections with vehicle or 5 mg/kg THC from postnatal day (P) 30-P43. All groups were allowed at minimum a 3-week washout period before experimental use. After this time, behavior was assessed in 3 tasks (2 odor control, Serial ‘What’ Task, Object Location Memory), one week apart. The specific task order was randomized by cohort (each cohort included equal numbers of vehicle and aTHC-treated animals). Upon completion of behavioral tasks, animals were used for electrophysiological analyses targeting the S—C projection to CA1 apical dendrites and lateral perforant path (LPP) innervation of the dentate gyrus (DG).

Fig. 2.

Input/output (I/O) curves for the S—C and LPP systems were not influenced by adolescent THC treatment. I/O curves were comparable between vehicle (Veh) and aTHC-treated animals for both the S—C CA1 and lateral perforant path (LPP) systems in (A) male mice (CA1: p = 0.38, F_(1,62) = 0.80; LPP: p = 0.05, F_(1,74) = 3.9, N = 5–6), (B) female mice (CA1: p = 0.39, F_(1,73) = 0.75; LPP: p = 0.56, F_(1,56) = 0.35, N = 5–7), (C) male rats (CA1: p = 0.15, F_(1,8) = 2.56; LPP: p = 0.10, F_(1,14) = 3.02, N = 7–8), and (D) female rats (CA1: p = 0.51, F_(1,8) = 0.47; LPP: p = 0.18, F_(1,14) = 1.20, N = 9–13). Linear regression was used to evaluate significance; mean ± SEM values shown. Scale bars for CA1 traces were 2 mV, 5 ms and for LPP traces were 1 mV, 5 ms. N values denote slices per group.

Fig. 3.

Adolescent THC treatment had little effect on frequency facilitation (FF) in rat S—C (CA1) and LPP systems. (A,B) In slices from adult male rats given vehicle (Veh) or THC during adolescence, response profiles were comparable for S—C (A; 10 Hz: p = 0.87, F_(9,135) = 0.51; 20 Hz: p = 0.98, F_(9,135) = 0.29; 40 Hz: p = 0.99, F_(9,135) = 0.12. Veh N = 10, THC N = 7) and LPP (B; 10 Hz: p = 0.86, F_(9,117) = 0.52; 20 Hz: p = 0.99, F_(9,117) = 0.13; 40 Hz: p = 0.69, F_(9,117) = 0.72. Veh N = 7, THC N = 8) systems. (C,D) THC did not influence FF of the female S—C system (C; 10 Hz: p = 0.98, F_(9,234) = 0.29, N = 14/group; 20 Hz: p = 0.92, F_(9,225) = 0.42, Veh N = 11, THC N = 13; 40 Hz: p = 0.43, F_(9,234) = 1.01, N = 14/group) and only modestly depressed LPP FF with 20 and 40 Hz stimulation (D; 10 Hz: p = 0.06, F_(9,270) = 1.88; 20 Hz: **p = 0.009, F_(9,270) = 2.53; 40 Hz: *p = 0.01, F_(9,270) = 2.39, N = 16/group). N values are slices per group; mean ± SEM values shown. Statistics used RM-ANOVA (Interaction). Raw values are reported in Suppl. Figs. 1,2.

Fig. 4.

Adolescent THC (aTHC) treatment enhanced frequency facilitation (FF) of S—C (CA1) projections in male mice. Ten pulse trains of 10, 20, or 40 Hz stimulation were applied to S—C or LPP systems and initial fEPSP slopes were normalized to the first response. (A) For males, S—C FF was greater at all frequencies for aTHC vs Vehicle (Veh) groups (10 Hz: **p = 0.002, F_(9,108) = 3.20; 20 Hz: ****p < 0.0001, F_(9,108) = 5.76; 40 Hz: ****p < 0.0001, F_(9,108) = 9.59, N = 7/group). (B) For male LPP, FF was comparable between Veh- and aTHC-treatment groups (10 Hz: p = 0.13, F_(9,117) = 1.56; 20 Hz: p = 0.08, F_(9,108) = 1.76; 40 Hz: p = 0.94, F(9,117) = 0.39, Veh N = 8, THC N = 6–7). (C,D) Among females, aTHC did not alter S—C FF (C; 10 Hz: p = 0.99, F_(9,108) = 0.22, N = 14/group; 20 Hz: p = 0.31, F_(9,108) = 1.20, Veh N = 11, THC N = 13; 40 Hz: p = 0.98, F_(9,108) = 0.26, N = 7/group), but enhanced LPP FF with 20 and 40 Hz stimulation (D; 10 Hz: p = 0.74, F_(9,90) = 0.67; 20 Hz: *p = 0.02, F_(9,90) = 2.3; 40 Hz: *p = 0.049, F_(9,90) = 1.99, N = 6/group). N denotes slices per group; mean ± SEM values shown. Statistics used RM-ANOVA (Interaction). Raw values for these datasets are presented in Suppl. Figs. 1,2.

Fig. 5.

Adolescent THC treatment had modest effects on CA1 responses to burst stimulation and TBS trains. (A) Representative traces for a single burst response (100 Hz, 4 pulses). Scale bar: 1 mV, 10 ms. (B) The single burst response profile (fEPSP slopes, normalized to first pulse) did not differ between THC- and Veh-treated rats (Male: p = 0.44, F_(3,36) = 0.93, N = 7/group; Female: p = 0.96, F_(3,69) = 0.10, Veh N = 11, THC N = 14). (C) In mice, aTHC had no effect on the single burst response in males (p = 0.35, F(_3,30) = 1.15, n = 6/group) but significantly enhanced responses to later pulses in the burst in females (*p = 0.02 vs Veh, F_(3,36) = 3.68, N = 7/group). (D) Representative trace from rat showing responses to the first 5 theta bursts (200 ms inter-burst). Scale bar: 1 mV, 100 ms. (E) In rat, THC did not influence within theta train facilitation in males (burst response area, normalized to first burst; p = 0.99 vs Veh, F_(9,108) = 0.23, N = 7/group) but caused a slight decline later in the theta train in females (*p = 0.03. F_(9,207) = 2.13, Veh N = 11, THC N = 14). (F) In mice, aTHC enhanced within theta train facilitation in males (*p = 0.02 vs Veh, F_(3,30) = 3.63, N = 6/group) without significant effect in females (p = 0.20, F_(3,36)) = 1.62, N = 7/group). N denotes slices per group; mean ± SEM values shown. R-M ANOVA (Interaction) for all analyses. Raw measures are reported in Suppl. Fig. 3.

Fig. 6.

Adolescent THC impaired both S—C and LPP LTP with greatest effects in females. Potentiation was induced using a single train of near threshold TBS (5 bursts for mice; 10 bursts for rat) for S—C projections or 1 s of 100 Hz stimulation for the LPP. (A,B) In males, aTHC did not influence CA1-LTP in rats (A, p = 0.66, Veh N = 6 vs THC N = 7sliees/group) or mice (B, p = 0.70; N = 6/group). (C,D) In females, aTHC impaired S—C LTP in both rats (C, ****p < 0.0001, N = 13/group) and mice (D, *p = 0.01, N = 7/group). (E) In slices from Veh-treated female mice, infusion of estradiol (E2, 1 nM) increased the slopes of S—C fEPSPs and this effect was blocked in the presence of ERα antagonist MPP (3 μM) (One-way ANOVA: p = 0.004, F_(2,21) = 7.03; Bonferroni post-hoc: *p < 0.05 Veh + E2 vs E2 + MPP, N = 7/group). E2 failed to increase response size in slices from aTHC-treated female mice (post-hoc: **p < 0.01 Veh E2 vs THC + E2, N = 10). (F,G) In males, LPP LTP was unaffected by aTHC in rats (F, p = 0.77, N = 7/group) but was absent after aTHC treatment in mice (G, **p = 0.002; Veh N = 8 vs THC N = 7). (H,I) In female rats and mice, aTHC severely impaired LPP LTP (H, Rat: **p = 0.006, Veh N = 12 vs THC N = 14; I, Mouse: ***p = 0.0004, Veh N = 6 vs THC N = 7). For all panels, superimposed representative traces at right show mean baseline (min 18–20) (solid) and post-induction (min 78–80) (dashed) responses. N denotes slices per group. Mean ± SEM values shown. Scale bars: 1 mV, 5 ms. LTP statistics were analyzed with 2-tail unpaired t-test. Raw values are reported in Suppl. Fig. 3.

Fig. 7.

Adolescent THC treatment caused enduring impairments in episodic and spatial memoiy. (A) Schematic of the serial odor ‘what’ task for rat. (B) Vehicle (Veh) and aTHC-treated males similarly distinguished novel odor E from previously sampled odor A (Veh n = 9, t₈ = 3.42, **p = 0.009 on bar for within group comparison time with novel vs familiar odor; THC n = 10, t₉ = 3.13, **p = 0.006; t₁₇ = 0.38, p = 0.71 between groups). Veh-female rats discriminated the novel cue but those receiving aTHC did not (Veh n = 16, t₁₅ = 3.42, *p = 0.019; THC n = 15, t₁₄ = 2.26, p = 0.47; *p = 0.036 between groups). (C) Rat 2-odor discrimination task. (D) In the 2-odor task, all rat groups preferentially explored the novel odor (Male; Veh n = 8, t₇ = 9.46, ****p < 0.0001 on bar; THC n = 8, t₇ = 4.45, **p = 0.003. Female; Veh n = 8, t₇ = 5.5, ***p < 0.001; THC n = 7, t₅ = 8.5, ***p < 0.001). (E) Left. Schematic of‘what’ task for mice. Right. Veh-treated male and female mice preferentially explored novel odor D vs familiar odor A whereas those receiving aTHC did not (Male: Veh n = 15, t₁₄ = 4.68, ***p = 0.0004; THC n = 12, t₁₁ = 0.98, p = 0.35; t₂₅ = 3.57; **p = 0.0016 between groups; Female: Veh n = 11, t₁₀ = 4.03, **p = 0.002; THC n = 11, t₁₀ = 1.21, p = 0.30; t₂₉ = 1.86, **p = 0.002 between groups). (F) Left. 2-odor discrimination task for mice. Right. All groups preferentially explored the novel odor at testing (Male: Veh n = 12, t₁₁ = 3.77, **p = 0.003; THC n = 12, t₁₁ = 5.73, ***p = 0.0001; p = 0.29 between groups; Female: Veh n = 13, t₁₂ = 3.84, **p = 0.0024; THC n = 12, t₁₁ = 3.25, **p = 0.008; p = 0.68 between groups). (G) Object Location Memory (OLM) task. (H) For male rats, OLM discrimination indexes (DIs) did not differ between Veh and aTHC groups (Veh n = 14, THC n = 15; t₂₀ = 2.58, p = 0.33); both groups preferentially explored the moved object (Suppl. Table 1). For female rats, those that received aTHC had significantly lower DIs than Veh-controls (Veh n = 12, THC n = 10; t₂₀ = 2.58, *p = 0.018) and failed to discriminate the moved object (Suppl. Table 1). (I) For male mice, Veh and aTHC groups similarly discriminated the moved object (n = 12/group; t₂₂ = 1.51, p = 0.15; Suppl. Table 1) whereas for females, Veh-treated mice discriminated the moved object whereas aTHC-treated mice did not (n = 9/group; t₁₆ = 3.06, **p = 0.007; Suppl. Table 1). For panels B, D, E, F asterisks on the bars denote within group comparisons for time sampling the novel vs familiar odor using the 2-tailed paired t-test, and asterisks above bars denote between group comparisons that used the 2-tailed unpaired t-test. Mean ± SEM values shown. See Suppl. Figs. 4 and 5 for individual animal sampling times and estrous cycle staging.

Fig. 8.

Rats and mice show no preference for individual odors used at testing in the Serial What task. (A) To assure that rats exhibited no preference for either of the two odors used at testing in the Serial What task, exploration times of odors A and E were measured for rats with only prior exposure to habituation (empty) odor cups. (B) Rats exhibited no preference for odor A or E with both males and non-proestrus females spending comparable amounts of time sampling the two odors (Male: Veh: n = 6, t₅ = 0.20, p = 0.85; THC: n = 6, t₅ = 0.89, p = 0.41; Female: Veh: n = 6, t₅ = 0.004, p = 0.99; THC n = 6, t₅ = 0.18, p = 0.87; paired t-test within groups). (C) Similarly mice with prior habituation to empty cups were tested for time exploring odors A and D, the same testing pair used in the mouse Serial What task. (D) Untrained male and female mice exhibited no preference for odor A or D (Male: Veh: n = 5, t₄ = 0.02, p = 0.99; THC: n = 5, t₄ = 0.22, p = 0.98; Female: Veh: n = 5, t₄ = 0.74, p = 0.49; THC n = 12, t₄ = 1.32, p = 0.26 paired t-test within groups). Individual animal measures shown; the lines link measures for the same rodent.