Persistent inhibitory circuit defects and disrupted social behaviour following in utero exogenous cannabinoid exposure

G A Vargish, K A Pelkey, X Yuan, R Chittajallu, D Collins, C Fang, C J McBain, G A Vargish, K A Pelkey, X Yuan, R Chittajallu, D Collins, C Fang, C J McBain

Abstract

Placental transfer of Δ9-tetrahydrocannabinol (THC) during pregnancy has the potential to interfere with endogenous cannabinoid (CB) regulation of fetal nervous system development in utero. Here we examined the effect of maternal CB intake on mouse hippocampal interneurons largely focusing on cholecystokinin-expressing interneurons (CCK-INTs), a prominent CB subtype-1 receptor (CB1R) expressing neuronal population throughout development. Maternal treatment with THC or the synthetic CB1R agonist WIN55,212-2 (WIN) produced a significant loss of CCK-INTs in the offspring. Further, residual CCK-INTs in animals prenatally treated with WIN displayed decreased dendritic complexity. Consistent with these anatomical deficits, pups born to CB-treated dams exhibited compromised CCK-INT-mediated feedforward and feedback inhibition. Moreover, pups exposed to WIN in utero lacked constitutive CB1R-mediated suppression of inhibition from residual CCK-INTs and displayed altered social behavior. Our findings add to a growing list of potential cell/circuit underpinnings that may underlie cognitive impairments in offspring of mothers that abuse marijuana during pregnancy.

Conflict of interest statement

The authors have no conflicts of interest.

Figures

Figure 1. Loss of CCK-INTs following in…
Figure 1. Loss of CCK-INTs following in utero THC or WIN exposure
A) Representative images of CCK, PV, SOM, CR and VIP labeling in hippocampal sections from mice born to VH or WIN treated mothers. B) Summary plot of the density of labeled cells for each marker in paired VH/WIN treated litters. 11 mice from 11 litters/treatment were examined for CCK and PV, 10 mice from 10 litters/treatment for SOM, and 12 mice from 12 litters/treatment for CR and VIP (*p=5×10−6). C) Summary plot of the density of labeled cells for CCK, PV and SOM in paired VH/THC treated litters. Three mice from three litters/treatment were examined for each marker (*p=0.001). D) Representative images (left) and summary plot (right) of GFP+ cell labeling in 5HT3AR-GFP and Nkx2.1-Cre:RCE-GFP mice born to VH or WIN treated mothers. Dashed lines indicate the region of interest for cell counts. For 5HT3AR-GFP 7 mice from seven WIN or VH treated litters were examined (*p=3.9×10−9) and for Nkx2.1-Cre:RCE-GFP mice 3 mice from 3 litters/treatment were analyzed. E) Representative images (left) and group data counts (right) for CCK-INT bouton stains with CB1R after prenatal treatment with VH or WIN. 4 mice from 4 litters/treatment were counted (*p=0.02). F) Drawings of reconstructed CCK-BCs (left) and SCAs (right) recorded VH (top) and WIN (bottom) treated animals. Dendrites are black for both treatment groups while axons are colored red and blue for VH and WIN treated animals, respectively. Dashed lines indicate the borders of SP. G) Quantification of terminal density along the axon of CCK-BCs and SCAs in VH and WIN treated animals (CCK-BCs: VH: n=5, WIN=4; SCAs: n=4/treatment). H) Quantification of total dendrite length for CCK-BS and SCAs in VH and WIN treated animals (*p=0.03). I) Sholl analyses for dendrites of CCK-BCs (left) and SCAs (right) in VH or WIN treated mice (*p=0.03, for both). Values are plotted as mean±SEM.
Figure 2. WIN exposure does not affect…
Figure 2. WIN exposure does not affect PC morphology or physiology
A) Images of L1NCAM (red) and Hoechst (blue) staining in both VH (top) and WIN (bottom) treated animals. Images show normal patterning of projections in both the corpus collosum (left) and fimbria (right). B) Left panel shows sample images of Hoechst stain in CA1 of VH (top) and WIN (bottom) treated animals. Right panel shows summarized data of PC density in CA1 of VH and WIN treated animals (VH: n=4 mice/4 litters, WIN: n=4 mice/4 litters). C) Left panel shows drawings of reconstructed PCs in VH (left) and WIN (right) treated mice. Right panel shows summarized plot of PC dendritic length in VH and WIN treated animals (VH: n=6 cells/3 mice/2 litters, WIN: n=6 cells/3 mice/2 litters). D) Continuous traces (left) and representative waveform (middle) of sEPSCs recorded in CA1 PCs of VH (top) and WIN (bottom) treated mice. Right panel shows summarized data for sEPSC amplitude and frequency in VH and WIN mice (VH: n=9 cells/2 mice/2 litters, WIN: n=9 mice/2 mice/2 litters). E) Left panel shows representative traces of field EPSPs recorded in CA1 stratum radiatum of VH (top) and WIN (bottom) animals following stimulation of the Schaffer collateral pathway at increasing intensity. Left panel shows summarized plot of input/out dynamics in VH and WIN mice depicting field EPSP amplitude at various stimulation intensities (VH: n=9 slices/3 mice/2 litters, WIN: n=9 slices/3 mice/2 litters).
Figure 3. Deficient CCK-INT mediated inhibitory drive…
Figure 3. Deficient CCK-INT mediated inhibitory drive in prenatally WIN treated mice
A) At left are continuous traces of representative CA1 PC recordings showing sIPSCs before and after DSI induction (+70mV, truncated) in VH or WIN treated mice. Middle panel shows averaged sIPSC waveforms for events obtained for 5s before and after DSI induction. Group data plotted at right highlights changes in amplitude, frequency and charge of sIPSCs following DSI induction expressed as percentage of pre-DSI levels (VH: n=8 cells/3 animals/2 litters, WIN: n=11 cells/3 animals/2 litters; *Amplitude p=0.03, *Charge: p=0.03. B) Schematic of feedback inhibition evoked by alvear stimulation (left) and averaged CA1 IPSCS (right) from representative recordings illustrating sensitivity to both GABA (Bicuculline, 10μM) and glutamate (DNQX, 10μM/dl-AP5, 50μM) antagonism confirming their disynaptic nature. C) At left are averaged disynaptically evoked IPSC waveforms from representative recordings before and after acute WIN (1μM) application in VH and WIN treated mice. Group data plots to the right show acute WIN effects on IPSC amplitudes for each recording. D) Summarized data shown as % of control amplitudes obtained prior to acute WIN application (right; VH: n=20 cells/11 animals/5 litters, WIN: n=16 cells/10 animals/6 litters; * p=0.01). E) Representative example traces (left) and group data summary (right) illustrating the effect of acute WIN application on 5 pulse trains of disynaptically evoked IPSCs in VH and WIN treated mice (VH: n=20 cells/11 animals/5 litters, WIN: n=16 cells/10 animals/6 litters; p=0.01, 0.02, 0.10, 0.03, and 0.05 for pulses 1–5 respectively). F) Schematic of feedforward inhibition evoked by Schaffer collateral stimulation (left). At middle are averaged feedforward evoked waveforms showing the inward monosynaptic excitatory current and outward disynaptic inhibitory current. Right panel shows summarized plot of the % of disynaptic inhibition that is sensitive to DSI in both VH and WIN treated mice (VH: n=5 cells/3 animals/2 litters, WIN: n=6 cells/3 animals/3 litters; p=0.03).
Figure 4. Loss of tonic eCB mediated…
Figure 4. Loss of tonic eCB mediated inhibition of CCK-BC release in WIN treated mice
A) Schematic diagram (left), averaged IPSC traces from representative recordings (middle), and group data summary plots (right) illustrating the effects of the CB1R antagonist AM251 (10 μM) on basal CCK-BC to CA1 PC transmission in VH and WIN treated mice (VH: n=10 cells/4 animals/1 litter, WIN: n=9 cells/3 animals/2 litters; * p=0.01). B) Effect of AM251 on trains of monosynaptically evoked CCK-BC to PC IPSCs in VH and WIN treated mice. Shown are averaged IPSC trains from representative recordings (left) and group data summaries for changes in total charge during the train (middle, *p=0.03) or amplitudes of each event during the trains (right, p=0.01, 0.11, 0.06, 0.13, and 0.03 for pulses 1–5 respectively) expressed as %s of control responses obtained before AM251 (VH: n=10 cells/4 animals/1 litters, WIN: n=9 cells/3 animals/2 litters,). C) Schematic diagram of CCK-INT to PC paired recording (left), representative traces of unitary events before and after DSI induction (middle) and group data summary of DSI amplitude changes expressed as % inhibition of pre-DSI levels (right) for connected pairs in VH and WIN treated mice (VH: n=6 pairs/4 animals/2 litters), WIN: n=6 pairs/4 animals/2 litters). D) Single trial traces from representative CCK-INT to PC pairs (left) and group data summary (right) illustrating the degree of asynchronous release from CCK-INTs during train activity in VH and WIN treated mice. Asynchronicity was measured as the charge transfer during a 300ms window centered on the peak of the last AP in the train (boxed region; VH: n=9 pairs/6 animals/2 litters, WIN: n=8 pairs/6 animals/2 litters).
Figure 5. Reduced social interaction in WIN…
Figure 5. Reduced social interaction in WIN treated mice
A) Schematic diagram of three-chamber social interaction test. B) Group data summarizing the number entries mice from each treatment group made into chambers containing the novel mouse or object. C) Group data showing the total time mice from each treatment group spent interacting with the novel mouse and novel object (*p=0.01). D–E) Elevated plus maze group data summarizing the number of entries (D) and total time spent (E) in the open and closed arms of the maze for VH and WIN treated mice. F) Summary of the number of head dips mice from each treatment made during elevated plus maze testing. 11 mice from 3 litters were evaluated per treatment group for each behavioral test.

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