Cocaine experience causes cell-type specific synaptic changes on NAc core MSNs (Grueter et al, 2012; Kauer and Malenka, 2007). To study MSNs in a cell-type specific manner, we used WT and TLR4.KO mice carrying a bacterial artificial chromosome expressing the tdTomato fluorophore driven by the D1 dopamine receptor promotor as previously described (Grueter et al, 2013). Whole-cell voltage-clamp recordings were made in D1(+) and D1(-) MSNs corresponding to D1 and D2 MSNs, respectively
(Kashima and Grueter, 2017). We used male mice to perform voltage-clamp recordings following acquisition of cocaine locomotor sensitization (Figure 2A), a time point
associated with behavioral attenuations in both genders of TLR4.KO animals.
Non-contingent cocaine experience is associated with the generation of synapses containing only NMDAR on D1 MSNs (Graziane et al, 2016). These are termed “silent” synapses and play an important role in meta-plasticity (Graziane et al, 2016). We hypothesized that cocaine exposure causes differential changes in NAc core MSN synaptic properties in WT compared to TLR4.KO animals. To this end, we
assessed a series of pre- and post-synaptic properties the day following 5 days of saline or cocaine (15 mg/kg) injections (Figure 13A). We began by assessing the ratio of
1/CV2N:A, a measure of silent synapses (Grueter et al, 2013). In D1(+) MSNs, we found that cocaine significantly increased 1/CV2N:A in both WT and TLR4.KOs suggesting an increase in the proportion of silent synapses (Figure 13B-C, L-M). We also examined PPR, A/N ratio, dual-component decay kinetics, and sEPSC amplitude and frequency.
We found that cocaine experience did not alter PPR, an inverse measurement of
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Figure 13. Cocaine alters synaptic properties in TLR4.KO D1(+) MSNs.
(A) Experimental schematic. Recordings were made 24 h following 2 days saline habituation and 5 days of saline or cocaine injections. Similar to behavioral experiments, each injection was followed by 15 min. exposure to an open field chamber.
(B) Representative plot of WT coefficient of variance (CV) experiments. AMPAR (-70 mV) and NMDAR EPSCs (+40 mV) from D1(+) MSNs taken from saline (red) and cocaine (purple)-treated mice. (C) Summary ratio of 1/CV2N:A from WT D1(+) MSNs. (D) Representative traces from PPR experiments. (E) Summary plot of WT D1(+) PPR experiments. (F) Representative sEPSC traces. (G) Summary plot of sEPSC amplitude and (H) frequency. (I) Representative evoked current traces taken at +40 mV. (J) Summary plot of A/N ratio and (K) Time to half-peak (T1/2) of dual-component currents at +40 mV. (L-U) Example traces, experiments, and summary plots for 1/CV2N:A, PPR, sEPSC amplitude, sEPSC frequency, A/N ratio, and dual component T1/2 from D1(+) MSNs from TLR4.KO mice treated with either saline (black) or cocaine (purple). All recordings taken in the presence of picrotoxin (50 µM). n/N = 5-12 cells from 3-5 animals per group. Error bars denote SEM. *P < 0.05, unpaired t test.
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presynaptic release probability, in either WT or TLR4.KO D1(+) MSNs (Figure 13D-E, N-O). We also found no differences with cocaine in the A/N ratio and sEPSC amplitude;
measures of post-synaptic strength, or sEPSC frequency (Figure 13F-J). Interestingly, we found that cocaine exposure increased the time to half-peak (T1/2) of AMPAR and NMDAR dual component responses in D1(+) TLR4.KO but not WT MSNs (Figure 13K), suggestive of a change in NMDAR function.
In contrast to D1(+) MSNs, 1/CV2N:A was unchanged in D1(-) cells from cocaine- treated WT similar to what is seen in the NAc shell (Graziane et al, 2016) (Figure 14B- C). However, cocaine significantly increased 1/CV2N:A, decreased sEPSC frequency, and increased dual-component T1/2 in TLR4.KO animals (Figure 14L-M, R, U). In both WT and TLR4.KO D1(-) MSNs following cocaine, we observed no changes to PPR, A/N ratio, or sEPSC amplitude (Figure 14D-K, N-Q, S-T). The decrease in sEPSC
frequency observed in TLR4.KO D1(-) MSNs suggests one of: decreased presynaptic release probability, decreased number of synapses sampled from, or decreased network activity. Cocaine’s lack of effect on PPR argues against altered presynaptic release probability. In combination with the observed increase in 1/CV2N:A, the most parsimonious explanation for these findings is cocaine “silencing” previously active synapses on D1(-) MSNs. In addition, an increase in the dual-component T1/2 is consistent with decreased current flow through the AMPARs although it may also be due to a change in NMDAR function (Kashima and Grueter, 2017). Though we cannot rule out the possibility of de novo silent synapse generation paired with a reduction in network activity, this seems less likely given no differences in sEPSC frequencies were
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Figure 14. Cocaine alters synaptic properties in TLR4.KO D1(-) MSNs.
(A) Experimental schematic. (B) Representative plot of WT coefficient of variance (CV) experiments taken from saline (green) and cocaine (purple)-treated mice. (C) Summary of 1/CV2N:A from WT D1(-) MSNs. (D) Representative traces from PPR experiments. (E) Summary plot of WT D1(-) PPR experiments. (F) Representative sEPSC traces. (G) Summary plot of sEPSC amplitude and (H) frequency. (I) Representative evoked current traces taken at +40 mV. (J) Summary plot of A/N ratio and (K) T1/2 of dual- component currents at +40 mV. (L-U) Example traces, experiments, and summary plots for 1/CV2N:A, PPR, sEPSC amplitude, sEPSC frequency, A/N ratio, and dual component T1/2 from D1(-) MSNs from TLR4.KO mice treated with either saline (black) or cocaine (purple). All recordings taken in the presence of picrotoxin (50 µM). n/N = 6-13 cells from 4-6 animals per group. Error bars denote SEM. *P < 0.05; **P < 0.01, unpaired t test.
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seen in D1(+) MSNs. Regardless, we provide evidence for cocaine exposure differentially affecting WT and TLR4.KO MSNs in a cell-type specific manner.
III-2c. Few differences observed in synaptic properties following withdrawal from