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Prenatal cocaine exposure (PCE) can cause persistent neuropsychological and motor abnormalities in affected children, but the physiological consequences of PCE remain unclear. Conclusions drawn from clinical studies can sometimes be confounded by poly-substance abuse and nutritional deprivation. However, existing observations suggest that cocaine exposure in utero, as in adults, increases synaptic dopamine and promotes enduring dopamine-dependent plasticity at striatal synapses, altering behaviors and basal ganglia function.
We used a combination of behavioral measures, electrophysiology, optical imaging, and biochemical and electrochemical recordings to examine corticostriatal activity in adolescent mice exposed to cocaine in utero.
We show that PCE caused abnormal dopamine-dependent behaviors, including heightened excitation following stress and blunted locomotor augmentation to repeated treatment with amphetamine. These abnormal behaviors were consistent with abnormal GABA interneuron function, which promoted a reversible depression in corticostriatal activity. PCE hyperpolarized and reduced tonic GABA currents in both fast-spiking and PLTS-type GABA interneurons to increase tonic inhibition at GABAB receptors on presynaptic corticostriatal terminals. While D2 receptors paradoxically increased glutamate release following PCE, normal corticostriatal modulation by dopamine was reestablished with a GABAAR antagonist.
The dynamic alterations at corticostriatal synapses that occur in response to PCE parallel the reported effects of repeated psychostimulants in mature animals, but differ in being specifically generated through GABA. Our results indicate that approaches which normalize GABA and D2 receptor-dependent synaptic plasticity may be useful for treating the behavioral effects of PCE and other developmental disorders that are generated through abnormal GABAergic signaling.
PCE can cause debilitating neuropsychological and motor abnormalities in humans and is an important public health concern.1 In spite of prevention-related measures, over 4% of pregnant women in the United States use illicit drugs,2 with up to 1% using cocaine.1 Cocaine readily crosses the placenta3 and can cause withdrawal symptoms in neonates and cognitive and behavioral abnormalities in adolescence.4,5 Unfortunately, the prospect for treating affected children remains poor, as the drug’s effect on the developing nervous system remains unclear.6
Observations in the clinic1,4,5 and laboratory7–10 suggest that abnormal symptoms and signs following PCE reflect alterations in corticostriatal function. The striatum is the primary nucleus for cortical information entering the basal ganglia and regulates motor control, cognition, and habit learning.11,12 Attention to salient behavioral cues, as well as the selection and execution of movements and decisions, requires the striatum to collect cortical signals, modulate information, and activate appropriate corticofugal pathways.13 Signal integration is thought to occur at a ‘striatal microcircuit’, where glutamate from cortical inputs, dopamine from nigrostriatal terminals, and gamma-aminobutyric acid (GABA) from striatal interneurons interact at the dendritic spines of striatal medium spiny projection neurons (MSNs) to select behaviorally-relevant synapses (Fig 1A).12,14–16 Here we show that PCE causes abnormal dopamine-dependent motor behaviors and striatal synaptic plasticity in adolescent mice through abnormal GABA interneuron function, providing insights into novel therapeutic approaches that may improve outcome in children with PCE.
Procedures were approved by the University of Washington Institutional Animal Care and Use Committee and are detailed in the Supplementary Methods. Timed-pregnant Swiss Webster-strain (n=78), C57Bl6-strain (n=9), and Lhx6-GFP BAC transgenic (n=10; MMRRC-GENSAT project, www.gensat.org) dams received either cocaine (20mg/kg, s.q.) or saline twice daily from embryonic day 8 through 18.7 As cocaine causes anorexia,7 controls included a saline-treated, pair-fed group (SPF) whose nutritional intake was matched to that of cocaine-treated dams, and a saline-treated group that were allowed to feed ad libitum (saline). Litters were fostered to an untreated surrogate dam at birth (P0), weaned by P22, and utilized as indicated below.
Tissue was extracted from the dorsal striatum of 14 mice, aged 63–70 days and biotinylated for both total and surface receptor (R) expression of GABABR subunit proteins, as described23 in the Supplementary Methods.
Values given in the text and in the figures are Mean ± SEM. Differences, considered significant if p<0.05, were assessed with appropriate t-tests, ANOVAs or the non-parametric Mann-Whitney test (see Supplementary Methods).
Timed-pregnant cocaine-exposed (n=19) and SPF dams (n=17) consumed similar quantities of food throughout gravidity (p=0.95), but 14% less than saline dams (n=20; p<0.001, t-test; Fig 2A). Maternal weights reflected these differences in food intake, as saline dams gained more weight than either cocaine or SPF dams (p<0.001, repeated-measures ANOVA; Fig 2B). Following birth, cocaine-exposed pups (n=59) weighed less than either saline (n=96) or SPF pups (n=51) until week 6 (p<0.01, t-test; Fig 2C), while the cranial diameter of saline pups (n=100) was greater than that of SPF (n=24) or cocaine pups (n=35) after week 1 (p<0.01, t-test; Fig 2D). The reduction in postnatal weight was dependent on PCE, while the smaller head size was a consequence of nutrition.
Dopamine-dependent reflexes and behaviors were assessed in adolescent mice. Cocaine-exposed mice (n=19) showed increased nociceptive latencies on tail-flick testing compared to either saline (n=27) or SPF mice (n=20; p<0.001, Mann-Whitney; Fig 3A). Rotarod testing for motor coordination revealed increasing falling latencies for saline (n=26), SPF (n=20), and cocaine-exposed mice (n=8; p<0.001, repeated-measures ANOVA; Fig 3B), consistent with motor learning.19 However, falling latencies in cocaine-exposed mice decreased in later trials (p<0.05 compared to either saline or SPF mice, ANOVA) and cocaine mice gripped the rod more frequently than either saline or SPF mice (p<0.001, Mann-Whitney; Fig 3C).
Locomotion in the open-field was similar in saline (n=35), SPF (n=38), and cocaine-exposed mice (n=24; Fig 3D). Following a saline injection, the ambulations of saline and SPF mice remained similar while cocaine-exposed mice became more active (p<0.001, t-test). In the absence of injection, similar responses were found in all groups (not shown).
To test for dopamine-dependent plasticity,20 locomotor responses were measured in response to repeated amphetamine (2mg/kg/d i.p.). Saline (n=14), SPF (n=20), and cocaine-exposed mice (n=12) all demonstrated locomotor sensitization with an increase in locomotor activity following repeated amphetamine (p<0.01, repeated-measures ANOVA). Similar to prior studies,25 cocaine-exposed mice had blunted locomotor augmentation to repeated amphetamine (p<0.001, repeated-measures ANOVA; Fig 3E), but comparable sensitized responses to amphetamine challenges in withdrawal.
As a control experiment to test for hypodopaminergia21 and depression,26 mice were suspended by the tail and time spent immobile was measured. While there was no overall difference between saline (n=49), SPF (n=29), and cocaine mice (n=29; Fig 3F), both SPF and cocaine mice displayed small increases in immobility compared to saline mice, suggesting an effect of nutritional deficiency as opposed to PCE.
These behaviors suggested that PCE might alter dopamine-dependent excitatory neurotransmission within the dorsal (motor) striatum.27 Whole-cell electrophysiological recordings in MSNs from SPF and cocaine-exposed mice revealed similar passive and active membrane properties (Fig 4A and Supplementary Table S1). However, compared to SPF mice, neurons from cocaine-exposed mice were 36% less responsive to current injection (p<0.001, ANOVA; Fig 4B) and cortical stimulation >0.6 mA evoked lower amplitude excitatory postsynaptic currents (eEPSC; p<0.05, two-way ANOVA; Fig 4C and D). This inhibition of corticostriatal activity following PCE was likely of presynaptic origin,28 since the eEPSC paired-pulse ratio (PPR) was higher in cocaine (1.2±0.05; n=49 cells) than in SPF (1.07±0.03; n=60; p=0.01) and the frequency of miniature (m) EPSCs was 18% less in cocaine-exposed cells (n=19) compared to SPF (n=14; p=0.03, t-test), while the cumulative mEPSC amplitude distributions were unchanged (Fig 4E).
Exocytosis from corticostriatal terminals was directly examined by multiphoton microscopy using the endocytic tracer FM1-43.15 The halftime of FM1-43 release was 14% lower in cocaine-treated mice (n=125 puncta; p=0.02, Mann-Whitney) and compared to saline (n=102) or SPF (n=100), PCE selectively inhibited the subset (~50%) of cortical terminals with a low probability of release, while the faster-releasing terminals remained unperturbed (Fig 4F–H). This synaptic depression was long-lasting, since exocytosis in younger 30-day-old mice was 18% lower in cocaine (t1/2=245 sec; n=114) compared to saline (t1/2=201 sec; n=68; p=0.01, Mann-Whitney not shown).
We tested whether this presynaptic depression might occur through GABAergic neurotransmission, which regulates presynaptic corticostriatal activity14 via metabotropic GABABRs located on corticostriatal terminals.29 Tonic inhibition by GABABRs was absent in SPF mice, since the GABABR antagonist CGP52432 (10μM) did not change the eEPSC amplitude (Fig 4D) or the frequency of mEPSCs (10±14%; n=7 cells; Fig 5A). However, CGP52432 prevented the tonic inhibition of MSNs from cocaine-exposed mice (Fig 4D) and increased the frequency of mEPSCs (19±3%; n=6; p=0.02, paired t-test; Fig 5B).
To determine if PCE might change the sensitivity of presynaptic GABABRs, eEPSCs from MSNs were measured in response to cortical stimulation with 50 ms paired-pulses, applied every 30 sec.30 In MSNs from SPF mice, the GABABR agonist baclofen (5μM) decreased the amplitude of the first current of the pair (−67±3%; p=0.002) and the PPR increased (44±8%; n=14; p<0.001, paired t-test; Fig 5C), consistent with strong presynaptic inhibition by GABABRs.30 In cocaine-exposed mice, baclofen also decreased the amplitude of the first eEPSC (−79±4%; n=8; p=0.03) and the PPR increased (61±6%; p=0.01, paired t-test; Fig 5D). Over a range of concentrations, baclofen depressed corticostriatal activity to a greater degree in cocaine-exposed MSNs (p<0.05, 2-way ANOVA; Fig 5E and F), suggesting that PCE increases GABABR sensitivity.
Western blots determined striatal GABABR subunit expression in the dorsal striatum and the membrane surface protein pool was detected with biotinylation. Results showed a 25±5% increase in the GABABR1a-receptor subunit surface protein membrane pool from cocaine-exposed striatum (1.41±0.3) compared to SPF (1±0.1; n=7 mice each, p=0.02, t-test; Fig 6). The total protein level of GABABR1a and levels of both GABABR1b and GABABR2 were unchanged, indicating that PCE selectively increases the expression of GABABR1a-receptor subunits, which are preferentially expressed on corticostriatal terminals.31
Dopamine modulates corticostriatal activity through D2Rs16 and in MSNs from SPF mice, the D2R agonist quinpirole decreased the amplitude of the first eEPSC (−15±7%; n=9 cells; p=0.004) and increased the PPR (30±8%; p=0.01, paired t-test; Fig 7A). In cocaine mice, quinpirole paradoxically increased the eEPSC amplitude (43±19%; n=8; p=0.02) and decreased the PPR (−25±6%; p=0.05, paired t-test; Fig 7B). This aberrant excitation of corticostriatal activity by D2Rs following PCE was likely presynaptic since quinpirole reduced the frequency of mEPSCs in SPF cells (−18±6%; n=7; p=0.003; Fig 7C), but enhanced their frequency in cocaine-exposed neurons (38±14%; n=8; p=0.04, paired t-test; Fig 7D). Similar plasticity was found in MSNs from mice exposed to half the dose of cocaine and also in younger 30-day-old C57Bl6-strain mice (Supplementary Data and Fig S1).
These changes in corticostriatal activity were accompanied by long-term adaptations in dopamine transmission, as measurements of electrically-evoked dopamine release and reuptake using cyclic voltammetry showed that PCE produced region-specific alterations in phasic dopamine release without affecting clearance (Supplementary Data and Fig S2). However, optical experiments in slices from saline and SPF mice, confirmed that both the dopamine releaser amphetamine15 (10μM) and quinpirole reduced exocytosis by specifically modulating glutamatergic inputs with a low probability of release (Fig 7E, Supplementary Data and Fig S3), but boosted release from those same synapses in slices from cocaine-exposed mice. Consistent with the lack of D1Rs on cortical terminals within the dorsal striatum,15 D1R ligands did not change exocytosis or the modulatory response to amphetamine (Supplementary Data and Fig S4).
While D2Rs inhibit corticostriatal release in untreated mice,15 they also suppress GABA interneuron function,32 and might increase corticostriatal activity by reducing tonic inhibition at sensitized GABABRs. In MSN from SPF mice, inhibition by D2Rs was not dependent on GABABRs, as the GABABR antagonist CGP52432 did not change the eEPSC amplitude (−5±3%; n=6) or the PPR (13±5%), whereas CGP52432 with quinpirole decreased the eEPSC amplitude (−23±4%; p<0.03) and increased the PPR (33±11%; p<0.02, compared with vehicle or CGP52432, paired t-test; Fig 7F). In MSNs from cocaine-exposed mice however, CGP52432 was excitatory, as the eEPSC amplitude increased (38±15%; n=8; p=0.03) and the PPR decreased (−26±6%; p=0.02, paired t-test; Fig 7G). CGP52432 with quinpirole remained excitatory, but the eEPSC amplitude decreased (−18±8%; p<0.04) and the PPR increased (19±12%; p<0.05, compared with vehicle or CGP52432, paired t-test), possibly due to concurrent corticostriatal inhibition by D2Rs.30
GABAARs promote tonic inhibition of GABA interneurons,33 but are absent from presynaptic corticostriatal terminals.29 In MSNs from SPF mice, the GABAAR antagonist bicuculline (10μM) did not change the eEPSC amplitude (5±3%; n=9) or the PPR (12±7%; Fig 7H) and D2Rs remained inhibitory, as quinpirole in the presence of bicuculline decreased the eEPSC amplitude (−15±3%; p=0.02) and increased the PPR (50±9%; p<0.01, compared with vehicle or bicuculline, paired t-test). In cocaine cells, bicuculline did not change the eEPSC amplitude (2±7%; n=9; p=0.3) or the PPR (3±4%; p=0.6), but bicuculline blocked excitation by D2Rs since quinpirole with bicuculline reduced the eEPSC amplitude (−25±8%; p<0.02) and increased the PPR (38±7%; p<0.04, compared with vehicle or bicuculline, paired t-test; Fig 7I).
As results indicated that cocaine-induced corticostriatal plasticity is generated through GABA interneurons, we used Lhx6-GFP transgenic mice to target striatal fast-spiking (FS) and persistent-low-threshold-spiking (PLTS) interneurons. Whole-cell voltage- and current-clamp recordings identified FS and PLTS interneurons by their distinctive physiological properties14,34 and showed that PCE lowered their resting membrane potentials (FS, −11.6%, p=0.04; PLTS, −18%, p=0.02) and action potential thresholds (FS, −18%, p=0.02; PLTS, −27%, p=0.001, compared to saline, t-test; Supplementary Table S2–S4, Fig 8A and B).
In saline-exposed mice, ambient GABA inhibited interneurons as the GABAAR antagonist bicuculline depolarized both FS (32±2%, n=4, p<0.001) and PLTS cells (26±8%, n=5, p=0.04, paired t-test; Fig 8C and D). However, cocaine-exposed FS and PLTS cells demonstrated little depolarization following bicuculline (FS, 2±3%, p=0.4; PLTS, 4±2%, p=0.1), consistent with the reported reduction in GABAAR subunit expression following PCE.23,27
D2Rs can modify tonic GABA inhibition through G-protein-coupled phosphorylation of GABAAR subunits by PKA.35 The D2R agonist quinpirole did not change the membrane potential in FS interneurons from saline- (1±1%, n=7) and cocaine-exposed mice (1±1%, n=7), but it depolarized PLTS cells from saline-exposed mice (5±1%, n=8, p=0.003) and hyperpolarized PLTS cells from cocaine-exposed mice (4±1%, n=6; p=0.02, paired t-test; Fig 8E and F). While quinpirole reduced the extent of depolarization by bicuculline in saline-exposed FS (29%, n=7, p=0.003, t-test) and had little effect on quinpirole in PLTS interneurons (4%, n=8), quinpirole promoted substantial depolarization by bicuculline in both cocaine-exposed FS (29±8%, p<0.001) and PLTS interneurons (24±8%; p=0.02, paired t-test). Thus, the paradoxical reduction in GABA interneuron function following D2R activation was alleviated by the GABAAR antagonist.
PCE also provoked abnormal excitatory, AMPA receptor-mediated responses in GABA interneurons. In saline-exposed mice, quinpirole had no effect on the eEPSC amplitude (7±10%; n=11; p=0.4) or the PPR (0.5±9%; p=0.9). The addition of bicuculline to quinpirole did not change the PPR (−0.2±10%), but enhanced cell excitability as the eEPSC amplitude increased (55±11%; p<0.02, compared with vehicle or quinpirole, paired t-test; Fig 8G). Following PCE, quinpirole reduced the eEPSC amplitude (−18±5%; n=8; p=0.02) and increased the PPR (27±7%; p=0.01). The addition of bicuculline to quinpirole increased the eEPSC amplitude (29±2%; p=0.01), while the PPR approached baseline (−2±5%; Fig 8H; p=0.9, compared with vehicle; p=0.02, compared to quinpirole, paired t-test).
PCE can produce long-lasting behavioral and motor disturbances in affected children,4,5 and it reduced dopamine-dependent reflex and motor task performance in mice. These abnormal behaviors were paralleled by a long-lasting and reversible depression of corticostriatal activity that was generated though abnormally-functioning GABA interneurons (Fig 1B). PCE promoted inappropriate dopamine filtering of corticostriatal activity; rather than boosting stronger cortical connections by inhibiting the weak,15 D2Rs strengthened weaker glutamatergic synapses while stronger signals remained unperturbed (Fig 1).
GABA interneurons are created in the medial ganglionic eminence36 and become potent regulators of corticostriatal signalling,37 but their tangential migration is reduced following PCE.38 PCE reduced FS and PLTS interneuron excitability and the downstream increase in GABABR1a-receptor subunit sensitivity would inhibit corticostriatal activity when extracellular GABA surges during synaptic activity.39
While normally promoting inhibition at corticostriatal synapses,15,30 D2Rs boosted corticostriatal activity in cocaine-exposed mice. D2Rs likely reduced tonic inhibition of corticostriatal GABABRs by inhibiting excitatory inputs on GABA interneurons and by hyperpolarizing PLTS interneurons. Because only small currents are required to activate PLTS interneurons, minor decreases in the membrane potential become physiologically significant, requiring recruitment of additional excitatory inputs39, which are also aberrantly controlled by D2Rs.
We also found that a GABAAR antagonist can prevent this paradoxical excitation of corticostriatal activity by D2Rs. While GABAAR blockade had no direct effect on corticostriatal activity,29 it prevented the reduction in tonic GABA currents that occurred in response to D2R activation. Inhibition of GABA interneuron activity by GABAA autoreceptors33 is dependent on ambient GABA supplied by striatal neurons and inputs from the external pallidum.33,40,41 In controls, GABAAR blockade suppressed inhibitory tonic GABA currents, the interneuron depolarized, but any increase in synaptic GABA remained undetected at corticostriatal GABABRs. PCE hyperpolarized GABA interneurons and also suppressed bicuculline-sensitive tonic GABA currents, which were reactivated by the D2R agonist. Thus, the combination of GABAAR antagonist and D2R agonist would strengthen corticostriatal inhibition at GABABRs and oppose any reduction in tonic GABA caused by the D2R agonist alone.
D2Rs located on GABA interneurons33 modulate tonic GABA currents through PKA35 which is essential to the physiological states of both dopamine30 and GABAA receptors.14,34 In striatal MSNs, tonic GABAAR currents are dependent on β3-subunit phosphorylation which is inhibited by D2R inactivation of PKA through intracellular Gi/o protein coupling.35 PCE down-regulates cortical β3-GABAAR subunits,23 suggesting that these aberrant responses to D2R and GABAAR activation may be due to alterations in receptor subunit expression, G-protein coupling, phosphorylation, or GABA transport.35,40
Synaptic depression and dopamine-dependent excitation at corticostriatal synapses are also elicited by repeated psychostimulant exposure in adult mice.20 In contrast to PCE, these phenomena in mature mice are produced through D1 and cholinergic receptors. PCE failed to elicit excitatory D1R responses, perhaps due to D1R uncoupling42 or because the dopamine-releasing effects of cocaine in rodents may not be possible until the first week of life.43 Differences in plasticity were also reflected in the observed behaviors, as PCE reduced the magnitude of augmented locomotor responses to repeated amphetamine, but achieved comparable locomotion with controls in withdrawal. Thus, PCE did not spare the adaptive behavior that is linked to incentive saliency and drug dependence.44
Other observations following PCE might also arise from paradoxical D2R responses generated by over-inhibited synapses. The reduction in body weight is consistent with dysregulated D2R-mediated hypophysiotrophic function45 (as well as through reduced uteroplacental blood flow6) and decreased thermal reactivity suggests an abnormal D2R-dependent reflex arc.17,18 Cocaine-exposed mice also performed poorly on late rotarod trials and demonstrated a heightened response to aversive stimulation, suggesting that stress or unanticipated environmental changes cause inconsistent behaviors, similar to those reported in adolescent boys with a history of PCE.1,9
These findings support and extend reports of abnormal GABA function in the cortex and hippocampus of mouse models for PCE.23 Since GABA receptor ligands can reverse synaptic depression and allow appropriate D2R filtering at corticostriatal synapses, these findings provide insight into novel therapeutic approaches that may correct behaviors in cocaine-exposed mice and improve outcome in children with PCE, as well as a broad range of disorders that involve abnormal GABAergic signaling.46
Supported by the University of Washington Alcohol & Drug Abuse Institute (GPS and NSB), NS052536, NS060803, NS060803-2S1, HD02274 (NSB), Mary Gates Endowment for Students research scholarship (IN and JSL), F31MH08626 (JCL), 5T32DA007278 (GPS), DA016782 (PEMP), the University of Washington Vision Research Center and Seattle Children’s Hospital, Seattle, WA. We thank Drs. Mu-ming Poo, Laura Jansen and Claire Walker for advice on the Western blot and Drs. Richard Palmiter and Kyle Steinman for their critical review.