Membrane excitability of nucleus accumbens neurons gates the incubation of cocaine craving.

After drug withdrawal, a key factor triggering relapse is progressively intensified cue-associated drug craving, termed incubation of drug craving. After withdrawal from cocaine self-administration, incubation of cocaine craving develops more reliably in rats compared to mice. This species difference provides an opportunity to determine rat-specific cellular adaptations, which may constitute the critical mechanisms that contribute to incubated cocaine craving in humans. Expression of incubated cocaine seeking is mediated, in part, by cocaine-induced cellular adaptations in medium spiny neurons (MSNs) within the nucleus accumbens (NAc). In rats, decreased membrane excitability in NAc MSNs is a prominent cellular adaptation, which is induced after cocaine self-administration and lasts throughout prolonged drug withdrawal. Here, we show that, similar to rats, mice exhibit decreased membrane excitability of dopamine D1 receptor (D1)-, but not D2 (D2)-, expressing MSNs within the NAc shell (NAcSh) after 1 d withdrawal from cocaine self-administration. However, in contrast to rats, this membrane adaptation does not persist in mice, diminishing after 45-d withdrawal. We also find that restoring the membrane excitability of NAcSh MSNs after cocaine withdrawal decreases cocaine seeking in rats. This suggests that drug-induced membrane adaptations are essential for behavioral expression of incubated cocaine craving. In mice, however, experimentally inducing hypoactivity of D1 NAcSh MSNs after cocaine withdrawal does not alter cocaine seeking, suggesting that MSN hypo-excitability alone is insufficient to increase cocaine seeking. Together, our results demonstrate an overall permissive role of cocaine-induced hypoactivity of NAcSh MSNs in gating increased cocaine seeking after prolonged cocaine withdrawal.

Cocaine Triggers Astrocyte-Mediated Synaptogenesis.

BACKGROUND: Synaptogenesis is essential in forming new neurocircuits during development, and this is mediated in part by astrocyte-released thrombospondins (TSPs) and activation of their neuronal receptor, α2δ-1. Here, we show that this developmental synaptogenic mechanism is utilized during cocaine experience to induce spinogenesis and the generation of AMPA receptor-silent glutamatergic synapses in the adult nucleus accumbens shell (NAcSh). METHODS: Using multidisciplinary approaches including astrocyte Ca2+ imaging, genetic mouse lines, viral-mediated gene transfer, and operant behavioral procedures, we monitor the response of NAcSh astrocytes to cocaine administration and examine the role of astrocytic TSP-α2δ-1 signaling in cocaine-induced silent synapse generation as well as the behavioral impact of astrocyte-mediated synaptogenesis and silent synapse generation. RESULTS: Cocaine administration acutely increases Ca2+ events in NAcSh astrocytes, while decreasing astrocytic Ca2+ blocks cocaine-induced generation of silent synapses. Furthermore, knockout of TSP2, or pharmacological inhibition or viral-mediated knockdown of α2δ-1, prevents cocaine-induced generation of silent synapses. Moreover, disrupting TSP2-α2δ-1-mediated spinogenesis and synapse generation in NAcSh decreases cue-induced cocaine seeking after withdrawal from cocaine self-administration and cue-induced reinstatement of cocaine seeking after drug extinction. CONCLUSIONS: These results establish that silent synapses are generated by an astrocyte-mediated synaptogenic mechanism in response to cocaine experience and embed critical cue-associated memory traces that promote cocaine relapse.

Sarcomere length-dependent effects on Ca2+-troponin regulation in myocardium expressing compliant titin.

Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca2+-induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin-actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca2+-induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca2+-troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length-dependent enhancement of troponin regulation with both Ca2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length-dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length-dependent Ca2+-troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation of the myocardium.

Role of the C-terminus mobile domain of cardiac troponin I in the regulation of thin filament activation in skinned papillary muscle strips.

The C-terminus mobile domain of cTnI (cTnI-MD) is a highly conserved region which stabilizes the actin-cTnI interaction during the diastole. Upon Ca2+-binding to cTnC, cTnI-MD participates in a regulatory switching that involves cTnI to switch from interacting with actin toward interacting with the Ca2+-regulatory domain of cTnC. Despite many studies targeting the cTnI-MD, the role of this region in the length-dependent activation of cardiac contractility is yet to be determined. The present study investigated the functional consequences of losing the entire cTnI-MD in cTnI(1-167) truncation mutant, as it was exchanged for endogenous cTnI in skinned rat papillary muscle fibers. The influence of cTnI-MD truncation on the extent of the N-domain of cTnC hydrophobic cleft opening and the steady-state force as a function of sarcomere length (SL), cross-bridge state, and [Ca2+] was assessed using the simultaneous in situ time-resolved FRET and force measurements at short (1.8 µm) and long (2.2 µm) SLs. Our results show the significant role of cTnI-MD in the length dependent thin filament activation and the coupling between thin and thick filament regulations affected by SL. Our results also suggest that cTnI-MD transmits the effects of SL change to the core of troponin complex.

Nucleus accumbens feedforward inhibition circuit promotes cocaine self-administration.

The basolateral amygdala (BLA) sends excitatory projections to the nucleus accumbens (NAc) and regulates motivated behaviors partially by activating NAc medium spiny neurons (MSNs). Here, we characterized a feedforward inhibition circuit, through which BLA-evoked activation of NAc shell (NAcSh) MSNs was fine-tuned by GABAergic monosynaptic innervation from adjacent fast-spiking interneurons (FSIs). Specifically, BLA-to-NAcSh projections predominantly innervated NAcSh FSIs compared with MSNs and triggered action potentials in FSIs preceding BLA-mediated activation of MSNs. Due to these anatomical and temporal properties, activation of the BLA-to-NAcSh projection resulted in a rapid FSI-mediated inhibition of MSNs, timing-contingently dictating BLA-evoked activation of MSNs. Cocaine self-administration selectively and persistently up-regulated the presynaptic release probability of BLA-to-FSI synapses, entailing enhanced FSI-mediated feedforward inhibition of MSNs upon BLA activation. Experimentally enhancing the BLA-to-FSI transmission in vivo expedited the acquisition of cocaine self-administration. These results reveal a previously unidentified role of an FSI-embedded circuit in regulating NAc-based drug seeking and taking.

Sarcomere length dependent effects on the interaction between cTnC and cTnI in skinned papillary muscle strips.

Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how the sarcomeres detect the length changes and convert them into altered activation of thin filament. In this study we investigated how the C-domain of cardiac troponin I (cTnI) functionally and structurally responds to the comprehensive effects of the Ca(2+), crossbridge, and sarcomere length of chemically skinned myocardial preparations. Using our in situ technique which allows for simultaneous measurements of time-resolved FRET and mechanical force of the skinned myocardial preparations, we measured changes in the FRET distance between cTnI(167C) and cTnC(89C), labeled with FRET donor and acceptor, respectively, as a function of [Ca(2+)], crossbridge state and sarcomere length of the skinned muscle preparations. Our results show that [Ca(2+)], cross-bridge feedback and sarcomere length have different effects on the structural transition of the C-domain cTnI. In particular, the interplay between crossbridges and sarcomere length has significant impacts on the functional structural change of the C-domain of cTnI in the relaxed state. These novel observations suggest the importance of the C-domain of cTnI and the dynamic and complex interplay between various components of myofilament in the LDA mechanism.

Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex.

Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context.

In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation.

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-µm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.

FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation.

FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca(2+) and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca(2+) and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca(2+) sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca(2+)-sensitivity. The rate of Ca(2+)-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca(2+)-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca(2+)-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca(2+)-sensitivity and likely play an important role in cardiac regulation.

Structural basis for the in situ Ca(2+) sensitization of cardiac troponin C by positive feedback from force-generating myosin cross-bridges.

The in situ structural coupling between the cardiac troponin (cTn) Ca(2+)-sensitive regulatory switch (CRS) and strong myosin cross-bridges was investigated using Förster resonance energy transfer (FRET). The double cysteine mutant cTnC(T13C/N51C) was fluorescently labeled with the FRET pair 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid (IAEDENS) and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and then incorporated into detergent skinned left ventricular papillary fiber bundles. Ca(2+) titrations of cTnC(T13C/N51C)AEDENS/DDPM-reconstituted fibers showed that the Ca(2+)-dependence of the opening of the N-domain of cTnC (N-cTnC) statistically matched the force-Ca(2+) relationship. N-cTnC opening still occurred steeply during Ca(2+) titrations in the presence of 1mM vanadate, but the maximal extent of ensemble-averaged N-cTnC opening and the Ca(2+)-sensitivity of the CRS were significantly reduced. At nanomolar, resting Ca(2+) levels, treatment with ADP·Mg in the absence of ATP caused a partial opening of N-cTnC. During subsequent Ca(2+) titrations in the presence of ADP·Mg and absence of ATP, further N-cTnC opening was stimulated as the CRS responded to Ca(2+) with increased Ca(2+)-sensitivity and reduced steepness. These findings supported our hypothesis here that strong cross-bridge interactions with the cardiac thin filament exert a Ca(2+)-sensitizing effect on the CRS by stabilizing the interaction between the exposed hydrophobic patch of N-cTnC and the switch region of cTnI.

Structural and kinetic effects of hypertrophic cardiomyopathy related mutations R146G/Q and R163W on the regulatory switching activity of rat cardiac troponin I.

Mutations in cardiac troponin I (cTnI) that cause hypertrophic cardiomyopathy (HCM) have been reported to change the contractility of cardiac myofilaments, but the underlying molecular mechanism remains elusive. In this study, Förster resonance energy transfer (FRET) was used to investigate the specific structural and kinetic effects that HCM related rat cTnI mutations R146G/Q and R163W exert on Ca(2+) and myosin S1 dependent conformational transitions in rat cTn structure. Ca(2+)-induced changes in interactions between cTnC and cTnI were individually monitored in reconstituted thin filaments using steady state and time resolved FRET, and kinetics were determined using stopped flow. R146G/Q and R163W all changed the FRET distances between cTnC and cTnI in unique and various ways. However, kinetic rates of conformational transitions induced by Ca(2+)-dissociation were universally slowed when R146G/Q and R163W were present. Interestingly, the kinetic rates of changes in the inhibitory region of cTnI were always slower than that of the regulatory region, suggesting that the fly casting mechanism that normally underlies deactivation is preserved in spite of mutation. In situ rat myocardial fiber studies also revealed that FRET distance changes indicating mutation specific disruption of the cTnIIR-actin interaction were consistent with increased passive tension.

Structural dynamics of C-domain of cardiac troponin I protein in reconstituted thin filament.

The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain (MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca(2+) binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca(2+)-bound to the Ca(2+)-free dynamic states were significantly faster (53.2-116.8 s(-1)) than that of the IR (14.1 s(-1)). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca(2+) dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca(2+) signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation.