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1.
FIGURE 6

FIGURE 6. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

PCE increases the surface expression of GABABR1a receptor subunits in the dorsal striatum. (A) Representative immunoblots of lysates from the dorsal striatum of 60-day-old SPF and cocaine-exposed mice compare levels of total and surface membrane-bound fractions of GABABR1, GABABR2, and actin. Antibody against GABABR1 labeled 2 bands, GABABR1a and GABABR1b. (B) Densitometry measurements of receptor protein bands presented as the ratio of densities for SPF and cocaine-exposed mice measured in the same gel (*p<0.05, t-test).

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.
2.
FIGURE 2

FIGURE 2. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

PCE reduces growth. (A) Cocaine (18.38±0.6 g/d) and SPF (18.44±0.6 g/d) dams consumed less food than saline dams (21.33±0.5 g/d). For panels A, B and D, **p<0.01, ***p<0.001 for saline compared to SPF or cocaine, t-test. (B) The weight of cocaine-treated and SPF dams were similar throughout pregnancy and both weighed less than saline-exposed dams. (C) Cocaine pups weighed less than saline pups through week 6 and weighed less than SPF pups through week 7. SPF pups weighed less than saline pups on weeks 1 and 2 (p<0.05, t-test), but weighed more than saline pups after week 3 (p<0.01, t-test). (D) Saline pups had a larger cranial (bi-parietal) diameter than either cocaine or SPF pups after week 1 and there was no difference in the cranial diameter of cocaine and SPF mice (p>0.1 t-test).

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.
3.
FIGURE 5

FIGURE 5. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

(A) Representative traces show that CGP52432 had no effect on the frequency of mEPSC in cells from SPF mice (3.2±0.8 Hz in vehicle vs. 3.3±0.9 Hz following CGP52432; p=0.1, paired t-test) and the cumulative distribution of mEPSC amplitudes was unchanged. (B) CGP52432 increased the frequency of mEPSCs in cells from cocaine mice (3.1±0.8 Hz in vehicle vs. 3.9±0.8 Hz following CGP52432) and the cumulative mEPSC amplitude distribution was unchanged. #p<0.05, ##p<0.01, paired t-test. (C) Representative traces show the average responses to paired-pulses delivered at 50 ms every 30 sec before (above, left) and 5 to 7.5 min following bath application of baclofen (above, right). Graph shows the normalized amplitude of the first eEPSC (of the pair) and the normalized PPR. Baclofen reduced the amplitude of the first eEPSC (−92±23 pA for vehicle vs. −21±5 pA following baclofen) and increased the PPR (1.1±0.1 in vehicle to 1.6±0.2 following baclofen) in MSNs from SPF mice. (D) In cells from cocaine-exposed mice, baclofen reduced the amplitude of the first eEPSC (−59±6 pA for vehicle vs. −13±4 pA in baclofen) and increased the PPR (1.2±0.1 in vehicle vs. 1.8±0.4 in baclofen). (E) Concentration-curves demonstrate that baclofen (100 nM – 50 μM) reduced the amplitude of the first eEPSC and (F) increased the PPR to a greater degree in MSNs from cocaine-exposed mice than in SPF controls (IC50= 4.2 μM;48 n=6, 5, 12, 5, 6 cells for SPF and n=4, 5, 5, 4 for cocaine. *p<0.05, **p<0.01, ANOVA). Bars: A and B, 10 pA, 250 ms; C and D, 100 pA, 12.5 ms.

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.
4.
FIGURE 7

FIGURE 7. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

D2Rs provoke GABAAR-dependent paradoxical responses following PCE. (A) In MSNs from SPF mice, representative traces (above) and graph demonstrate that the D2R agonist quinpirole reduces the amplitude of the first eEPSC (in each pair; −160±27 pA for vehicle vs. −140±29 pA following quinpirole) and increases the PPR (1.2±0.1 in vehicle to 1.6±0.2 in quinpirole). (B) In MSNs from cocaine mice, quinpirole increased the amplitude of the first eEPSC (−81±18 pA for vehicle vs. −106±20 pA for quinpirole) and the PPR decreased (1.4±0.2 in vehicle vs. 1.0±0.1 in quinpirole). (C) In MSNs from SPF mice, quinpirole diminished the frequency (inset, left; 5.1±0.9 Hz in Veh vs. 4.1±0.8 Hz in quinpirole), but not amplitude (inset, right) of low-release probability (5–10 pA) inward currents. For panels C and D, #p<0.05, ##p<0.01, paired t-test. (D) In MSNs from cocaine-exposed mice, quinpirole increased the frequency (3.4±0.7 Hz in Veh vs. 4.7±1 Hz in quinpirole) of low-release probability mEPSCs, while having no effect on the cumulative amplitude distribution. (E) Amphetamine (left) and quinpirole (right) decreased FM1-43 destaining in slices from saline and SPF mice, but increased release in slices from cocaine mice. *p<0.05, **p<0.01, Mann-Whitney. (F) In cells from SPF mice, the GABABR antagonist CGP52432 did not change the eEPSC amplitude (−132±20 pA for Veh vs. −122±16 pA for CGP52432) or the PPR (1.3±0.1 in Veh vs. 1.4±0.1 in CGP52432). When quinpirole was added to CGP52432, the eEPSC amplitude decreased (−115±15 pA) and the PPR increased (1.6±0.1). (G) In MSNs from cocaine-exposed mice, the GABABR antagonist increased the eEPSC amplitude (−94±23 pA for Veh vs. −121±26 pA for CGP52432) and decreased the PPR (1.5±0.1 in Veh vs. 1.0±0.1 in CGP52432). When quinpirole was added to CGP52432, there was a slight reduction in eEPSC amplitude (−112±12 pA) and an increase in the PPR (1.3±0.1). (H) In MSNs from SPF mice, bicuculline did not change the amplitude of the first evoked current (−143±31 pA for Veh vs. −148±33 pA for bicuculline; p=0.1) or the PPR (1.1±0.1 in Veh vs. 1.2±0.2 in bicuculline; p=0.06, paired t-test). When quinpirole was added to bicuculline, the eEPSC amplitude decreased (−132±22 pA) and the PPR increased (1.5±0.1). (I) In MSNs from cocaine mice, bicuculline did not change the amplitude of the eEPSC (−103±24 pA for Veh vs. −101±21 pA for bicuculline) or the PPR (0.9±0.1 in Veh vs. 0.9±0.1 in bicuculline). When quinpirole was added to bicuculline, the eEPSC amplitude decreased (−82±21 pA) and the PPR increased (1.3±0.1). Bars: A, B, F, and G–I, 100 pA, 12.5 ms; C and D, 10 pA, 250 ms.

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.
5.
FIGURE 1

FIGURE 1. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

The striatal ‘microcircuit’ and the proposed mechanism for over-inhibition of corticostriatal activity following PCE. (A) This simplified striatal circuit is composed of medium spiny neurons (MSNs) that are excited by glutamate and inhibited by dopamine and GABA. Glutamate (green) released from cortical afferents stimulates MSNs through post-synaptic AMPA receptors (R).30 Dopamine (purple) modulates corticostriatal activity through D1- or D2-class dopamine receptors on MSNs and ‘filters’ corticostriatal activity though D2Rs acting on corticostriatal terminals.16 Synaptic ‘filtering’ occurs when dopamine inhibits a subset of cortical terminals with a low probability of release.12,16 GABA (blue) is capable of providing strong inhibition of corticostriatal activity through ionotropic GABAARs on MSNs, and also through metabotropic GABABRs located on corticostriatal terminals. GABABRs are relatively inactive in the resting state and provide little tonic inhibition.41 GABAergic fast-spiking and PLTS-type interneurons are excited by glutamate and are inhibited by dopamine D2Rs,32 as well as by GABAA autoreceptors33 that are tonically-activated by GABAergic inputs from the pallidum and other sources.41 (A1) In saline and SPF mice, D2R stimulation with an agonist inhibits glutamate release from a subset of cortical terminals (red box). D2R stimulation slightly depolarizes (activates) PLTS interneurons (red oval), but provides no downstream modulation of corticostriatal activity via GABABRs on cortical terminals. (B) PCE reduces GABA interneuron migration38 and hyperpolarizes (inhibits) both FS and PLTS-type interneurons (double blue arrow). The putative reduction in GABA availability promotes overexpression of GABABR1a-receptor subunits which sensitizes GABABRs on corticostriatal terminals (red arrow) to produce tonic over-inhibition of glutamate release (blue arrow). Phasic dopamine release is also suppressed. (B1) Stimulation of D2Rs (red oval) suppresses GABA interneurons, by reducing glutamate release from cortical afferents (blue arrow) and by hyperpolarizing PLTS-type GABA interneurons. The reduction in GABA availability (double blue arrow) relieves tonic inhibition at GABABRs on corticostriatal terminals to produce a paradoxical increase in glutamate release (red arrow). D2Rs located on corticostriatal terminals (red box) remain inhibitory, but since GABABRs are more potent modulators of presynaptic release,30 corticostriatal activity is dominated by the change in GABA. (B2) Blockade of inhibitory GABAA autoreceptors (blue oval) has little effect on interneuron function following PCE, but competes with the D2R-dependent reduction in GABA inhibition (red oval) and prevents dopamine-dependent corticostriatal excitation following PCE. Therefore, when D2R are stimulated in the presence of a GABAAR antagonist, the synapse may remain suppressed by tonic inhibition at GABABRs, but dopamine filtering (red box) is restored.

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.
6.
FIGURE 3

FIGURE 3. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

PCE causes abnormal behaviors. (A) The latency for removing the tail from warm water was higher in cocaine-exposed mice over three trials, while the flick-latencies of saline and SPF mice were similar (p=0.48, Mann-Whitney). For panels A and C, **p<0.01, ***p<0.001 for cocaine compared with saline or SPF, Mann-Whitney test. (B) Saline, SPF and cocaine-exposed mice spent a similar amount of time on the rotarod over the first 15 trials (p=0.1, t-test), but the falling latency decreased in cocaine mice after trial 15, while latencies of saline and SPF mice increased. For panels B, D, and E, *p<0.05, **p<0.01, ***p<0.001 for cocaine compared with either saline or SPF mice, t-test. (C) Instead of falling, some mice would grip onto, and rotate with the rod. Cocaine mice gripped the rotarod more than saline or SPF mice, while there was no difference between the SPF and saline control groups (p=0.1, Mann-Whitney). (D) Open-field ambulations of saline, SPF and cocaine-exposed mice were similar on test day 1. A saline injection, administered immediately following the first test increased ambulations in cocaine-exposed mice on test day 2. (E) Saline, SPF, and cocaine mice were treated with saline for 2 days and then received amphetamine (2 mg/kg, i.p.) for 5 consecutive days. Mice were later challenged with amphetamine on days 10 and 28. Locomotor activity measured for 90 min following each amphetamine treatment revealed no stereotypic behaviors (data not shown). Locomotor activity increased in response to repeated amphetamine in all treatment groups. Ambulations in saline and SPF mice remained similar throughout testing (p=0.07, repeated-measures ANOVA). Cocaine-exposed mice demonstrated reduced ambulations on experiment days 4–7, but not following a drug challenge in withdrawal (days 10 and 28), as locomotor activity was similar in all groups (p=0.1, ANOVA). (F) When suspended by the tail, saline, SPF, and cocaine-exposed mice showed an equivalent increase in immobility over time, as there was no significant Treatment*Time effect (p=0.16, two-way ANOVA). These groups of mice also spent a similar amount of time immobile at most time points during the test (min 1, 2, 4, and 6). However, compared to saline, both cocaine and SPF mice spent a longer time immobile during min 3 and min 5. *p=0.03 and **p=0.01 for saline compared to either SPF or cocaine mice, t-test.

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.
7.
FIGURE 4

FIGURE 4. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

PCE causes presynaptic depression through GABABRs. (A) Current clamp recordings in MSNs from SPF (n=15) and cocaine (n=11) mice displayed similar current-voltage curves with inward rectification, typical for MSNs (responses were measured at arrows in panel B). (B) Representative traces (above) demonstrate that fewer action potentials were generated in cells from cocaine mice in response to depolarizing current pulses (below). (C) The corticostriatal slice stained with FM1-43 and diaminobenzidine shows the areas of stimulation and recording. Corticostriatal activity was provoked using a bipolar stimulating electrode placed over cortical layers V–VI. Electrophysiological, optical, and biochemical recordings were obtained from the corresponding motor striatum (recording region), located 1.5–2.0 mm from the site of stimulation. (D) Representative traces (above) of voltage-clamp recordings show that similar cortical stimulation intensities evoked lower amplitude currents in MSNs from cocaine-exposed mice, compared to SPF. The AMPAR antagonist NBQX prevented evoked currents. Graph (below) shows the mean peak current evoked by the series of increasing cortical stimulation intensities in MSNs from SPF (n=10) and cocaine (n=12) mice. The GABABR antagonist CGP52432 had no effect on SPF cells, but blocked the reduction in eEPSC amplitudes in cocaine MSNs. *p<0.05, **p<0.01, t-test. Cells were voltage clamped at −70 mV to minimize post-synaptic GABAAR-mediated conductances (calculated ECl = −74.2 mV).47 (E) Representative traces of mEPSCs (above; recorded in the presence of tetrodotoxin (1μM) show a reduction in low-release probability (5–20 pA) inward currents in cells from cocaine mice. The average frequency of mEPSCs (inset, left) was lower in cocaine MSNs (3.4±0.4 Hz), compared to SPF (4.2±0.6 Hz), while the cumulative mEPSC amplitude distributions (inset, right) were similar. *p<0.05, t-test. (F) Stimulation of axons or cell bodies of projection neurons in layers V–VI of the cortex overlying the motor striatum resulted in endocytosis of FM1-43 dye by recycling synaptic vesicles, characteristic of corticostriatal afferents.16 Following dye loading, cortical stimulation at 20 Hz (beginning at t=0) resulted in exocytosis of FM1-43 dye from the terminals, which decreased in a manner approximated by a single exponent, characteristic of synaptic vesicle fusion.22 Feedback from MSNs was prevented using glutamatergic receptor antagonists (see Supplemental Methods). FM1-43 destaining was activity and calcium-dependent since no stimulation (n=30) or bath-applied cadmium (200μM; n=25) prevented stimulated release of the dye from presynaptic terminals. As FM1-43 destaining generally followed first-order kinetics, corticostriatal release was characterized by the halftime (t1/2) of release, defined as the time required for terminal fluorescence to decay to half its initial value.22 (G) Mean ± SEM halftimes of FM1-43 release for destaining curves shown in panel F. FM1-43 destining was similar in slices from saline (t1/2=205 sec) and SPF (t1/2=200 sec) mice (p=0.6, Mann-Whitney), but was reduced in slices from cocaine mice (t1/2=233 sec; *p<0.05, Mann-Whitney). (H) An advantage of this optical technique is that we are able to examine vesicular release kinetics from individual cortical terminals. When the halftimes of individual terminals are presented relative to their standard deviation from the median value, a straight line indicates a normally-distributed (or single) population.15 Normal probability plots of individual terminal halftimes of release for experiments in panel F show that PCE decreased exocytosis from the slowest-releasing terminals (those with the highest t1/2). This depression in corticostriatal release following PCE was not due to an inadequate innervation since the number of active corticostriatal terminals was higher in cocaine mice (61.4±10 puncta vs. 41.9±5 for saline; p=0.02, ANOVA). Bars: B, 40 mV, 25 ms; C, 1 mm; D, 50 pA, 5 ms; E, 10 pA, 250 ms. Curves were fit with a Hill equation.

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.
8.
FIGURE 8

FIGURE 8. From: Over-inhibition of Corticostriatal Activity following Prenatal Cocaine Exposure.

Abnormal GABA interneuron function. (A) Current clamp recordings show characteristic responses of FS and (B) PLTS interneurons from Lhx6-GFP transgenic mice to hyperpolarizing and depolarizing current injections (below). Both FS and PLTS interneurons from cocaine cells had lower resting membrane potentials (RMP) than saline cells. Current-voltage plots (right) show similar responses in saline and cocaine-exposed cells after subtraction of their resting membrane potentials (responses were measured at arrows). FS cells were silent at rest, and displayed a high firing rate with little adaptation following depolarizing current injection. Spikes were short and followed by a large after-hyperpolarization. PLTS interneurons exhibited a marked time-dependent sag in response to hyperpolarizing current injections and a rebound persistent low-threshold spike and/ or a plateau potential persisted after termination of hyperpolarizing current. During current injections, both FS and PLTS interneurons displayed a variable pattern of spike bursts (1–48 action potentials) interspersed by membrane oscillations. Compared to FS interneurons, PLTS interneurons exhibited a much higher input resistance, a lower resting membrane potential (RMP), and a much lower input current was required to produce action potentials (Supplementary Tables S3–S5), with values similar to those reported previously.14,49 (C) Representative current-clamp recordings in FS and (D) PLTS interneurons from saline-(above) and cocaine-exposed mice (below) demonstrate typical responses to the GABAAR antagonist bicuculline before and after bath application of the sodium channel blocker tetrodotoxin. In FS and PLTS interneurons, bicuculline depolarized saline-exposed cells (the RMP became more positive) to a much greater degree than cocaine-exposed cells. GABA likely produced tonic inhibition at GABAA autoreceptors14 since the change in membrane potential by bicuculline in saline-exposed mice (FS, 37±2%, p<0.001; PLTS, 23±7%, p=0.03) and cocaine-exposed mice (FS, 3±1%, p=0.09; PLTS, 4±1%, p=0.003, paired t-test) persisted when synaptic transmission was blocked by tetrodotoxin. Note that the cellular input resistance was monitored by (250 ms, 100 pA) current pulses applied every 10 sec. Changes in input resistance during depolarization were measured after transiently repolarizing the cell to resting membrane potential levels. Interestingly, bicuculline reduced the input resistance (Supplementary Fig S5), suggesting recruitment of additional ion channels with depolarization that are critical for sustained high-frequency firing.39 (E) Representative current-clamp recordings in FS and (F) PLTS interneurons from saline-(above) and cocaine-exposed mice (below) demonstrate typical responses to the D2 receptor agonist quinpirole before and after bicuculline. Quinpirole had no effect in FS interneurons, but slightly depolarized saline-exposed PLTS cells, while hyperpolarizing cocaine-exposed PLTS cells. For all interneurons, the membrane potential became more positive and the cell depolarized when bicuculline was added to quinpirole. A summary of membrane potentials and input resistance for FS and PLTS interneurons under all conditions tested can be found in Supplementary Fig S5. (G) Excitatory inputs onto GFP+ fluorescent interneurons from Lhx6-GFP transgenic mice were activated with paired-pulses using cortical bipolar stimulating electrodes. The PPR was similar in cells from saline (1.35±0.11; n=11) and cocaine-exposed mice (1.13±0.09; n=8; p=0.2, t-test). In saline-exposed mice, representative traces (above) and graph show that quinpirole did not change the amplitude of the first eEPSC (−32±3 pA in vehicle vs. −35±6 pA following quinpirole) or the PPR (1.3±0.1 in vehicle vs. 1.3±0.1 in quinpirole). When quinpirole was combined with bicuculline, the eEPSC amplitude increased (−45±6 pA), but the PPR remained unchanged (1.3±0.3). The AMPA-receptor antagonist NBQX (10 μM) abolished the eEPSC. (H) In GFP+ interneurons from cocaine-exposed mice, quinpirole reduced the amplitude of the first eEPSC (−62±5 pA in vehicle vs. −49±9 pA following quinpirole) and the PPR increased (1.1±0.1 in vehicle vs. 1.4±0.2 in quinpirole). Paired-pulse depression was observed in the presence of bicuculline, which prevented the change in PPR (1.1±0.2) and the eEPSC amplitude increased (−95±13 pA). Bars: A and B, 30 mV, 20 ms; C–F, 30 mV, 2 min; G and H, 50 pA, 12.5 ms.

Wengang Wang, et al. Ann Neurol. 2013 March;73(3):355-369.

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