Segregation and Crosstalk of D1 Receptor-Mediated Activation of ERK in Striatal Medium Spiny Neurons upon Acute Administration of Psychostimulants
[['The interplay between dopamine and glutamate in the striatum is considered to mediate the role of this basal ganglia structure in reinforcement learning and action selection [1] .', 'Glutamatergic projections arising from the cortex carry environmental information ( a context , a cue or an action ) into the striatum where dopamine is released by afferents from the midbrain .', 'This dopamine signal occurs in response to salient events such as unexpected rewards [2] , [3] .', 'The glutamatergic and dopaminergic inputs converge on striatal medium spiny neurons ( MSN ) where they trigger neuronal plasticity mechanisms that allow the animal to associate the salient event and the environment that preceded it .', 'This system is hijacked by psychostimulants like cocaine and amphetamines , which acting directly on the dopaminergic terminals generate an increase in the striatal dopamine levels [4] so that the context paired with the administration of the drug is actively sought by the animal even after just a single trial [5] .', 'This behavior is considered to be a correlate of addiction in humans .', 'The elucidation of the receptor-induced signaling cascades taking place in the neurons of the circuitry integrating dopaminergic and glutamatergic inputs is considered a natural requirement for the design of effective pharmacological treatments for preventing/curing addiction .', 'In this regard , it has been found that acute psychostimulant administration ( APA ) to naïve animals produces conspicuous molecular phenotypes in dopamine type 1 receptor ( D1R ) expressing MSNs ( D1R+MSN ) which constitute half of striatal MSNs [6]–[8] ( throughout this work we use a broad definition of phenotype [9]–[11] that comprises not only macroscopic observables in mutant and wild type living animals upon some treatment like APA , but also traits in samples from these animals like immunoblot bands from striatal slices , microscopy images and membrane currents ) .', 'The co-stimulation with dopamine and glutamate has been found to be required for the activation of the mitogen activated protein kinase ERK2 ( hereafter just ERK ) [12] .', 'Classically , this convergence is also required for the mobilization of GluR1-containing AMPAR ( 2-Amino-3- ( 3-hydroxy-5-Methyl-isoxazol-4-yl ) Propanoic Acid Receptor ) to the postsynaptic density through the dual phosphorylation of GluR1 by PKA and PKC/CaMKII [13] , [14] but just the PKA site has been found to be modified in the psychostimulant paradigms [13] , [15] .', 'The phosphorylation of ERK and GluR1 are part of plasticity mechanisms which result in measurable behaviors [16] , [17] .', 'The effects of APA in D1R+MSNs are mediated through two signaling cascades , the AC5 ( Adenylyl Cyclase type 5 ) /cAMP/PKA axis ( AC5 axis ) and the NMDAR ( N-Methyl-D-Aspartate Receptor ) /Ca2+/RAS ( NMDAR axis ) .', 'These are both activated by dopamine signaling via D1R coupled to Golf .', 'The AC5 axis is activated by the Golf α subunit [18] and the NMDAR axis is sensitized to glutamatergic input by the Golf βγ dimer [19] ( Fig . 1 ) .', 'In order to study this system , mice with single copies of the genes encoding D1R ( Drd1a+/− ) and Gαolf ( GnaI+/− ) have been used .', 'In both cases these animals have shown clear signs of haploinsufficiency ( reduced amount of the gene product ) , with 20% and 40% of the wild type ( WT ) striatal levels of D1R and Gαolf ( and thereafter the Golf heterotrimer ) [20] , respectively .', 'APA experiments with these mutant mice have shown that the phosphorylation of GluR1 and ERK are associated to different sets of behaviors and that both the phosphorylation and the associated behaviors are segregated as explained in more detail below .', 'The AC5 axis comprises the classical generation of cAMP by adenylyl cyclase ( AC5 ) with the consequent activation of PKA and inhibition of its counteracting PP1 via DARPP32 ( D32 ) ( Fig . 1 ) .', 'The PKA phosphorylation of GluR1 results in the exocytosis of GluR1-containing AMPA to the membrane and increases in single channel currents [14] .', 'While no changes in the membrane levels of GluR1 has been found in the APA paradigm [15] , the PKA phosphorylation of GluR1 correlates with the locomotor activation observed upon APA .', 'The phosphorylation of GluR1 upon APA and the behavior associated to it are affected in GnaI+/− mice but neither in Drd1a+/− mice [11] , [15] nor in mice treated with a MEK inhibitor that prevents the activation of ERK [21] ( APA-phenotypes in Table 1 ) .', 'In the NMDAR axis dopamine enhances NMDAR-mediated Ca2+ entry which results in the downstream activation of ERK through the RAS/RAF/MEK/ERK cascade .', 'The activation of ERK , which triggers the expression of several genes , has been found to be essential for the development of conditioned place preference and locomotor sensitization which are considered behavioral signatures of addiction to psychostimulants [5] , [21] .', 'Furthermore , the activation of ERK has been found to mediate APA-induced LTP in D1R+MSN [22] .', 'The activation of ERK upon APA and the behaviors associated to it are affected in Drd1a+/− mice but not in GnaI+/− mice ( APA-phenotypes in Table 1 ) [11] , [14] .', 'The AC5 and the NMDAR signaling axes have several crosstalking edges ( Fig . 1 ) .', 'The Ca2+ entering through NMDAR activates serine/threonine phosphatases regulating the phosphorylation of D32 and inhibits AC5 activity [23] .', 'On the other hand , the broadcast from the AC5 axis to the NMDAR axis is mediated by the tyrosine phosphatase STEP ( Striatal Enriched tyrosine Phosphatase ) which exists in two forms and is claimed to be inactivated by PKA-mediated phosphorylation which is counteracted by PP1 [24]–[26] .', 'The phosphatase activity of STEP counteract the activation of Fyn , NMDAR and ERK in the NMDAR axis [26] .', 'The significantly lower activation of not only GluR1 but also ERK upon APA in D32KO mice , where PP1 is not inhibited and the STEP phosphorylation by PKA is affected , has been interpreted as an evidence of the PKA-sensitive crosstalk mediated by STEP [12] .', 'The segregation of the effects in GnaI+/− and Drd1a+/− mice into the AC5 and the NMDAR axes is puzzling for at least two reasons .', 'One is that as the D1R-catalyzed Golf activation monotonically increases with dopamine levels , thus the effectors that are changed in one mutant should be the same or a subset of the effectors that are changed in the other mutant .', 'The other is that the crosstalk between the axes is expected to have a homogenizing effect by transferring the sensitivities from one axis to the other [27] .', 'How can the segregation arise in the first place ?', 'Then , how is it maintained in the face of crosstalk ?', 'In this work , we have developed a quantitative signaling model which explains this data pattern .', 'In this model the segregation arises from the distribution of a common pool of D1R and Golf in two D1R/Golf signaling compartments according to the affinity and capacity of compartment anchors .', 'Each D1R/Golf compartment is dedicated to only one signaling axis .', 'The segregation is kept in the face of crosstalk if the latter involves just a single pool of STEP whose activity is affected by PKA phosphorylation in a substrate dependent manner .', 'These assumptions are backed by an increasing body of experimental support [24] , [25] , [28]–[32] .'], ['The model developed in this work integrates a substantial portion of the intracellular signaling triggered in MSNs by dopamine acting on D1R and glutamate on NMDAR .', 'Two main signaling axes with several crosstalking edges are considered both of which are modulated upstream by dopamine via D1R coupled to Golf .', 'One is the classical generation of cAMP by AC5 with the consequent activation of PKA and inhibition of its counteracting PP1 via D32 .', 'The other is the enhancement of NMDAR-mediated Ca2+ entry which results in the downstream activation of ERK .', 'A detailed description follows ( Fig . 1 ) .', 'This signaling cascade is triggered in dendritic spines and most of the processes occur there .', 'The entire reaction network is modeled in a single well-stirred volume using ordinary differential equations ( ODEs ) that describe species concentration changes over time .', 'However , the model includes the representation ( through the indexing of species ) of a few reaction compartments which stand for segregated reaction sites located in the spine such as the post-synaptic density ( PSD ) , an internal NMDAR reservoir and two D1R/Golf signaling compartments .', 'A few others processes which are part of the DUSP ( dual specificity phosphatases ) -mediated negative feedback loop inactivating ERK occur in other locations like the dendritic shaft , the soma and the nucleus and but these locations were not considered explicitly .', 'The model was constrained with several quantitative molecular phenotypes gathered from the literature ( Table 1 ) .', 'These phenotypes can be classified as APA and non-APA phenotypes depending on whether they were obtained with the APA protocol .', 'There are 10 phenotypes several of them with more than one phenotypic variable .', 'The names of the phenotypic variables result from the merge of the phenotype name and the marker name .', 'Some of these phenotypic variables are measurements of protein markers by immunohistochemistry and/or immunoblotting .', 'Whenever possible , the measured values of protein levels by immunoblotting were corrected if ,', 'i ) the samples have a relevant cellular heterogeneity such as those from the striatum , but', 'ii ) the effect is cell specific and', 'iii ) other cells types in the sample contribute to the basal levels of the protein of interest .', 'The correction performed was , ( 4 ) FC is the cell-specific fold change over the basal , W is the fold change quantified with Western blots in the heterogeneous sample and fc and ft are the fraction of positive cells for the marker as measured by immunohistochemistry for the control and the treatment , respectively .', 'The derivation of this equation is presented in Text S1 ( Supporting Information ) .', 'Time series and dose-response phenotypic data was compressed by parameterization with the fitting of simple parametric models .', 'For example , the dose-response data of phenotypes activeRAS and sensitizedNMDAR was parameterized with a Hill model .', '( 5 ) where Cmin and Cmax are the minimal and maximal levels of the response ( Cx ) , respectively .', 'K and h are the dosage for half of the total response change and the Hill coefficient .', 'In the case of sensitizedNMDAR , where two dose-response data series were fitted , it is reported the ratio between the half-activating concentrations without and with dopamine .', 'The time series of trafficNMDAR was parameterized with a single exponential .', '( 6 ) where k is the rate constant .', 'Thus , the quality of the fit of the ODE model to the experimental data was represented and evaluated in these cases through the comparison of optimal parameters for the experimental data versus those of the simulated model .', 'No simple parametric model was found to fit the time series of APAib and they were represented independently .', 'After these transformations and excluding the phenotype ‘sensitizedNMDAR’ which was matched just qualitatively , there are a total of 17 phenotypic variables to constrain the model .', 'Considerable system insights can be retrieved by analyzing how the output ( corresponding to different phenotypic variables ) depends on the model parameters [92] .', 'Both when it comes to how robust the model is in terms of parameter variations [93] , [94] , as well as mapping out which parameters that have the largest influence on the model output [95] .', 'It has for example been observed that biological models tend to have a “sloppy” spectra of parameter sensitivities [96] , meaning that there are many parameters that has minor or no effect on the model output when perturbed slightly .', 'Other studies have shown that different model outputs can have different groups of parameters that are the most influential to the behavior [95] , [97] .', 'In order to investigate these features in our model we performed a local sensitivity analysis , calculating normalized sensitivities Sij .', 'This corresponds to investigating what effect minor perturbations of the parameter values have on the different model outputs .', 'The local sensitivity of the output oi with respect to the parameter pj , was given by ( 10 ) which ( for small enough values of Δpj ) is an approximation of the normalized partial derivative calculated at pj .', 'Here we used a relative perturbation size of 1% , i . e . The retrieved sensitivities was used to consider a number of model-features;', 'i ) the sensitivity profile of the output ( the phenotypic variables ) , e .', 'g .', 'which phenotypic variables are in general most sensitive to perturbations ,', 'ii ) the sensitivity profile of the parameters , e . g . how influential are the different parameters on the different outputs ,', 'iii ) the similarity ( or dissimilarity ) of the subgroups , consisting of the most sensitive parameters , for each phenotypic variable .', 'The comparison between subgroups of sensitive parameters was done by considering all pairs of phenotypic variables .', 'For each pair we calculated the overlap between the respective sensitive parameters , i . e . the number of sensitive parameters that the two phenotypic variables have in common .', 'This was next divided by the number of sensitive parameters in the larger of the two subgroups to get a relative measure .', 'This means that totally overlapping groups have an overlapping measure of one , whereas non-overlapping groups get zero .', 'Even though during the modelling process the enzymatic reactions was modelled directly with mass action kinetics with no steady-state assumption ( using forward and backward rate constants kf , kr and catalytic efficiency kcat ) for the sensitivity analysis only kcat and Michaelis constant KM were considered .', 'This was motivated from the realization that for enzymatic reactions , kf and kr could be changed considerably , but as long as the relation was kept so that KM ( = ( kr+kcat ) /kf ) was not changed this had no effect on the model fitting ( Supporting Information , Fig . S2 ) .', 'KM was perturbed through kf ( i . e . a perturbation of kf with the factor ( 1±0 . 005 ) −1 ) and when kcat was perturbed we introduced a balancing change in kf and kr to keep KM constant so that the relevance of kcat were probed independent of its contribution to KM ( i . e . when perturbing kcat with a factor 1±0 . 005 , kf and kr were also both perturbed with the same factor ) .', 'Overall , each of the 316 parameters ( all 276 kinetic rate constants , as well as the total amounts of all 40 species ) was varied one-at-a-time and each of the 17 different phenotypic variables was recorded .'], ['The final version of the model developed in this work contains 235 reactions involving 184 species and 358 reaction rate parameters ( Fig . 1 and Fig . S1 ) .', 'This constitutes a near 3 fold expansion with respect to a previous version [23] .', 'The model was manually constrained to a relative large collection of molecular phenotypic data obtained from the literature .', 'These data come from in vivo , slices and cell culture experiments and most of them correspond to D1R+MSN phenotypes but there is also some from unspecified MSNs , prefrontal cortex and hippocampal neurons .', 'The data from striatal slices either after in vivo treatment ( such as the APA protocol ) or treatment of the slices themselves were corrected to account for cell type-specific response ( Equation 4 , Table 2 ) .', 'The validity of this simple correction is illustrated through the correspondence between the estimated fold-change for D32p34 in the DAsliceD32 phenotype ( 11× ) and the value measured experimentally with a novel cell-type specific technique ( 12× ) [6] .', 'This is relevant because the capacity to sequester PP1 is doubled , thus generating a larger boost to PKA-mediated phosphorylation .', 'However , while this procedure alleviates the underestimation of fold changes characteristic of immunoblot measurements of cell-type specific events in heterogeneous samples , the reported data seldom have all the elements required for the estimation .', 'For example , an important part of the experiments with mutant mice lacks immunohistochemistry analysis so that estimations are not possible in a simple way .', 'Thus , when comparing the activation of relevant effectors upon APA in mutant versus wild type mice , the ratio of changes in the APA relative to saline was used .', 'The quality of fit of the model to non-APA phenotypes for both single time/dose measurements ( Fig . 2A and 2B ) and time-series/dose-response ( Fig . 2C and 2D ) was very high with divergences below 20% in most cases ( Fig . 2E ) .', 'The parameterization of time-series and dose-response sets with a monoexponential ( Eq . 6 ) and the Hill equation ( Eq . 5 ) , respectively , was successful for both experimental and simulated data ( r2>0 . 95 ) ( Fig . 2C and 2D ) .', 'The remaining non-APA phenotype , sensitizedNMDAR [19] , was reproduced qualitatively by the model ( Fig . 3 ) .', 'The model shows that a fast rise in dopamine concentration does produce a sudden sensitization of the NMDAR response as measured by the increase of the scaling factor of the Ca2+ level .', 'This results in the left-shift of the dose-response curve for ERK activation ( Fig . 3 ) .', 'In the model , this increase in the scaling factor operates on a simulated Ca2+ elevation that reproduces the one described in experiments ( Fig . 3A1 ) [19] making it more effective activating ERK .', 'Comparing the three mechanisms of DA/D1R triggered enhancement of NMDAR-Ca2+ currents , the sensitization was seen for single channel enhancement triggered by tyrosine phosphorylation and not for PKA-mediated single channel or Fyn-mediated traffic-based enhancements as these two were too slow to match the Ca2+ pulse ( Fig . 3A1 and A2 ) .', 'Similar to what is seen in the experimental data , the dose-response curves for measurements at 8 minutes show a non-monotonic behavior ( Fig . 3C ) [19] .', 'The maximum ERK activation at different Ca2+ amplitudes shows a typical monotonic behavior , but the higher the level of the maximum the faster the decay and then the time courses intersect and produce the non-monotonic at longer times ( Fig . 3B and 3C ) .', 'The removal of the negative-feedback loop ( DUSP-mediated ) partially uncouples maximum ERK activation levels from faster decays thus displacing the intersection of time courses curved toward longer times ( Fig . 3B ) .', 'The model also qualitatively reproduced another result obtained in cultures of striatal neurons treated with high concentration of glutamate ( 100 uM ) , this is , the inhibition of PP2B with Cyclosporin A increased the level of phosphorylated STEP and reduced the activation of ERK ( Fig . S3 ) [83] .', 'The psychostimulant induced dopamine increase in the striatum was modeled according to experimental measurements .', 'While most of the measurements of dopamine in this and other paradigms have been performed from the extracellular fluid recovered in a microdialysis cannula , this technique underestimates both amplitude and rate [90] , [98] .', 'From a collection of several measurements made with faster and less invasive techniques , such as PET and FSCV which were fitted to a sum of exponentials ( Eq . 7 ) , a consensus psychostimulant induced DA transient with k1\u200a=\u200a0 . 15 min−1 and k2\u200a=\u200a0 . 055 min−1 was used ( Fig . 4B ) .', 'The Ca2+ input was modeled as a random Poisson train of transients with a frequency of 0 . 1 Hz ( Fig . 4A , inset ) but in most simulations a train of regularly spaced Ca2+ spikes with the same frequency was used in order to reduce the computational cost associated with several replicate runs .', 'Each Ca2+ transient resembles the form of synaptically evoked Ca2+ elevation measured in hippocampal neurons ( Fig . 4A ) [88] .', 'Then , the model was challenged with the DA increase generated by APA together with the Ca2+ spike train whose basal amplitude of 500 nM was scaled up with each of the three modeled mechanisms of DA-triggered NMDAR function enhancement: single channel enhancement through serine/threonine phosphorylation by PKA or tyrosine phosphorylation by Src-like kinases , and Fyn-mediated traffic based enhancement ( Fig . 4C ) .', 'The traffic-based mechanism matches very closely what has been measured experimentally in immunoblots from APA-treated mice ( r2\u200a=\u200a0 . 87 ) ( Fig . 4D ) but the two single channel mechanisms reach maximum ERK activation far earlier than what has been reported experimentally with this technique [12] .', 'Importantly , both dopamine increase and NMDAR Ca2+ entry are required for the activation of ERK ( Fig . 4E ) , reproducing previous findings of ERK as an AND gate which is opened ( activated ) upon APA by the convergence of both inputs [12] .', 'In the case of GluR1 phosphorylation there were no differences between the three mechanisms in the model , but the goodness of fit to the experimental data varies ( Fig . 4F ) .', 'For both effectors , the experimental data was transformed by removing its reference to the baseline levels and scaling the result so just the kinetics of the process is considered in the comparison with the simulation .', 'Almost all the phenotypes challenging the model so far were also reproduced by a model with a single D1R/Golf signaling compartment and a single pool PKA-sensitive crosstalk via STEP at the three nodes in the NMDAR axis ( Fig . 1 ) .', 'However , this model failed to reproduce the activation pattern of GluR1 and ERK upon APA in D32KO , Drd1a+/− and GnaI+/− mice as described below ( Fig . S4 ) .', 'The haploinsufficiency of D1R and Gαolf and the effects of D32 knock-out on the psychostimulant induced phosphorylation of GluR1 and ERK has been studied experimentally with quantitative detail [12] , [19] , [20] .', 'In order to explain the opposing patterns of activation of GluR1 and ERK in haploinsufficient mice for D1R and Gαolf , we have assumed that the total pool of these elements is distributed in two compartments for D1R/Golf mediated signaling ( Fig . 5A ) with the amount of Golf ( heterotrimer ) equal to the amount of Gαolf .', 'One of these compartments is associated with the production of cAMP via AC5 and the other with the Fyn-mediated increase of NMDAR Ca2+ currents .', 'The distribution of the total amounts of D1R and Golf in each of these compartments depends on anchors that differ in affinity and capacity for D1R and Gαolf .', 'Haploinsufficiency was simulated by reducing the total amount of D1R or Gαolf to the fraction reported experimentally ( 0 . 2 and 0 . 4 respectively [20] ) , then equilibrating the system and finally running the simulation of the APA paradigm .', 'This was performed for each of the three mutants for 8 different PKA-sensitive STEP-mediated crosstalking schemes considered ( Fig . 6A and B ) in order to evaluate which of them can accommodate the existence of segregation .', 'All these crosstalking schemes were successful reproducing the non-APA phenotypes and the APA induced transient activation of ERK and GluR1 in the wild type .', 'They also reproduced changes in GluR1 phosphorylation for the mutants .', 'However , there were clear differences between them in the activation level of ERK in the mutants and just the scheme with a single STEP pool mediating the PKA-sensitive crosstalk just at the level of ERK ( 010 ) was able to reproduce all mutant phenotypes ( Fig . 6 ) .', 'The rest failed in one or more of the phenotypes and we did not find any parameter values to circumvent this .', 'In the case of Drd1a+/− , the amount of D1R in the AC5 compartment was at wild type levels but the amount in the NMDAR compartment was significantly reduced ( Fig . 5B ) .', 'Upon simulated APA , model versions with PKA-sensitive crosstalk via STEP at Fyn and NMDAR ( 1** ) did not show reductions in ERK activation ( Fig . 6D ) .', 'This crosstalking scheme is the one where just the non-phosphorylated form of STEP is active against Fyn and NMDAR ( i . e . edges 5 and 7 but not 6 and 8 in Fig . 6A ) .', 'In the case of GnaI+/− , the amount of Golf in the NMDAR compartment was at wild type levels but the amount in the AC5 compartment was significantly reduced ( Fig . 5C ) .', 'Upon simulated APA , there was a reduction in GluR1 phosphorylation in all schemes but this reduced flow in the AC5 axis affected the activation of ERK in all schemes with crosstalk at the level of ERK ( *1* ) ( i . e . edges 1 and/or 3 but not 2 and/or 4 in Fig . 6A ) except the one with a single STEP pool ( 010 ) ( i . e . edges 15678 in Fig . 6A ) .', 'The schemes with no crosstalk at all ( 00* ) ( i . e . all forms of STEP are equally active on each substrate ) and with crosstalk just at Fyn and NMDAR ( 10* ) did not show reduction in ERK activation ( Fig . 6E ) .', 'For the remaining mutant , D32KO , where the phosphorylation of both GluR1 and ERK has been reported to be significantly affected ( Fig . 6F ) the schemes with crosstalk at ERK ( *1* ) reproduced the reduction in ERK activation , while the rest were not affected ( Fig . 6F ) .', 'All the results presented so far can also be reproduced with an alternative version of the model where some assumptions were varied in order to meet some reported values ( Fig . S5 ) .', 'Considering that there are at least 25 GPCR in D1R+MSN which are annotated to signal via Gs , as estimated from the IUPHAR receptor database [99] and a D1R+MSN transcriptome [100] , it seems likely that Golf and possibly also D1R [101] are coupled to other signaling partners as well .', 'Thus , in this alternative version of the model it was explicitly introduced a third compartment .', 'Furthermore , the total amount of Golf that was set to 10 times the total amount of D1R [16] , [85] , [102] , [103] and a level of D1R in Drd1a+/− is 40% that of the WT [86] .', 'The rate constants and the total amount of model species ( conserved moieties ) in the updated model ( two D1R/Golf compartments and crosstalking scheme 010 ) were perturbed ±0 . 5% and for each perturbation the value of the 17 phenotypic variables was recorded .', 'The distribution of the maximum value of the sensitivity to each parameter across all phenotypic variables appeared multimodal ( Fig . 7A ) .', 'Considering the median of this distribution ( 0 . 26 ) as the demarcation between insensitive and sensitive parameters , it was clear that each phenotypic variable was sensitive to a varying number of parameters ( Fig . 7B ) .', 'The activation of ERK in Drd1a+/− mice upon APA was the most sensitive phenotypic variable with twice as many parameters with a large influence on the phenotype as the next in rank .', 'On the other hand , the phosphorylation of GluR1 in the same conditions was not sensitive to any parameter .', 'The phenotypic variables were compared pairwise ( Fig . 7C ) to evaluate the extent of overlap of sensitive parameters .', 'The fraction of sensitive parameters that phenotypic variable pairs had in common covered all the range from 0 to 1 ( Fig . 7C ) .', 'There were several pairs of phenotypic variables with no sensitive parameters in common ( e . g . trafficNMDAR-k and DAslice-D32p34 ) and a few pairs where the sensitive parameters of one of the phenotypic variables could all be found among the sensitive parameters of the other phenotypic variable ( e . g . D32KO-GluR1 and D32KO-ERKpp ) .', 'Thus , as described for quantitative signaling models for other cell types and signaling pathways [95] , the parameter sensitivity profile depends on which phenotypic variable that are observed .', 'As an example of this , phenotypic variables referring to the same marker ( e . g . ERK ) measured for different mutants have significantly different number and identity of sensitive parameters .', 'In particular , we have yet to explore the origin of hypersensitivity of the activation of ERK in Drd1a+/− mice upon APA ( haploD1R-ERKpp ) .', 'Considering the detailed sensitivity profile of the parameters ( Fig . 7D ) , total amounts ( conserved moieties ) are significantly enriched in the first 60 most globally sensitive parameters ( 50% , p<10−10 ) .', 'The total amount of AC5 and the kcat of its Ca2+-free , Golf activated form are the two most sensitive parameters , being around 25% higher than the following in the rank ( Fig . 7D ) .', 'These two parameters as well as many others within the 60 most sensitive parameters are mostly related to generation and degradation of cAMP , in correspondence with the widespread effects of this second messenger in the modeled network .', 'In accordance with earlier studies of parameter sensitivities [96] we also find that a large part of the parameters have only a minute effect on the output .', 'Looking at all combinations of parameters and phenotypic variables , only 8 . 2% of these correspond to a parameter that have a sensitive effect on a phenotypic variable ( using the same threshold as earlier Sij\u200a=\u200a0 . 26 ) .', 'If the average effect that a parameter has on the phenotypic variables is considered then 4 . 4% of the parameters have an average sensitivity higher than the threshold ( i . e . ) .', 'Here we have performed a local sensitivity analysis , using small perturbations .', 'In studies of model robustness , larger parts of the parameter space are explored [94] , [97] , as well as the effect of perturbing combination of parameters .', 'This kind of study is out of the scope of this article .', 'We have however , within this study , tried to use a bit larger perturbations ( ±20% ) .', 'Interestingly enough , looking at the top 20 sensitive parameters and comparing ±20% to ±0 . 5% perturbations , the identity of the parameters are almost the same ( data not shown ) .'], ['Neither the Ca2+ spike train alone nor the psychostimulant induced dopamine in the absence of NMDAR Ca2+ spikes are able to activate ERK in the simulations .', 'This matches the experimental observations of ERK as an AND gate which is opened ( activated ) upon APA by the convergence of glutamatergic AND dopaminergic inputs [12] , [104] .', 'The D1R-triggered signaling operates on both ends of the MAPK cascade [12] , [19] .', 'Upstream , D1R activation triggers the enhancement of NMDAR mediated Ca2+ currents .', 'In the simulations , just the D1R-triggered traffic-based mechanism of NMDAR enhancement generates a time course of APA-induced ERK activation that matches the immunoblot-based observations .', 'The single-channel mechanisms operate too fast .', 'A faster APA-induced ERK activation has been measured by immunohistochemistry [8] which is more in line with the single-channel mechanism .', 'However , the activation is expressed as the percentage of positive cells and this will clearly produce a faster kinetics than immunoblots as the latter track total levels of active ERK .', 'On the other hand , in vivo measurements with Ca+2 indicators in D1R+MSNs upon APA have shown an increase in intracellular Ca2+ levels that matches the time scale of the slow traffic-based mechanism ( Fig . 4D ) [105] .', 'Thus , the fast D1R-triggered sensitization of NMDAR to glutamate seen for MSNs in culture [19] may not be the one underlying the transient activation of ERK upon APA .', 'If a slow acting mechanism of ERK activation underlies the development of conditioned place preference , this may impose a minimum required residence time of the animal in the drug-paired site just after the injection for the correct association to be established .', 'In this regard , it is worth noting that previous models of signaling triggered by convergent glutamatergic and dopaminergic inputs on D1R+MSN have failed to reproduce the effective time windows between conditioning stimulus and reward in reinforcement learning paradigms [23] .', 'ERK has been reported to be activated in cultures of striatal neurons treated only with a D1R agonist ( SKF81297 ) [106] .', 'This effect was sensitive to PKA and Src inhibition and involves the tyrosine phosphatase Shp-2 which forms a complex with D1R .', 'However , the activation level of ERK achieved with D1R agonist alone is significantly lower than the level obtained in co-treatment with NMDAR agonists [19] in similar cultures which in turns
['Introduction', 'Methods', 'Results', 'Discussion']
['The convergence of corticostriatal glutamate and dopamine from the midbrain in the striatal medium spiny neurons ( MSN ) triggers synaptic plasticity that underlies reinforcement learning and pathological conditions such as psychostimulant addiction .', 'The increase in striatal dopamine produced by the acute administration of psychostimulants has been found to activate not only effectors of the AC5/cAMP/PKA signaling cascade such as GluR1 , but also effectors of the NMDAR/Ca2+/RAS cascade such as ERK .', 'The dopamine-triggered effects on both these cascades are mediated by D1R coupled to Golf but while the phosphorylation of GluR1 is affected by reductions in the available amount of Golf but not of D1R , the activation of ERK follows the opposite pattern .', 'This segregation is puzzling considering that D1R-induced Golf activation monotonically increases with DA and that there is crosstalk from the AC5/cAMP/PKA cascade to the NMDAR/Ca2+/RAS cascade via a STEP ( a tyrosine phosphatase ) .', 'In this work , we developed a signaling model which accounts for this segregation based on the assumption that a common pool of D1R and Golf is distributed in two D1R/Golf signaling compartments .', 'This model integrates a relatively large amount of experimental data for neurons in vivo and in vitro .', 'We used it to explore the crosstalk topologies under which the sensitivities of the AC5/cAMP/PKA signaling cascade to reductions in D1R or Golf are transferred or not to the activation of ERK .', 'We found that the sequestration of STEP by its substrate ERK together with the insensitivity of STEP activity on targets upstream of ERK ( i . e . Fyn and NR2B ) to PKA phosphorylation are able to explain the experimentally observed segregation .', 'This model provides a quantitative framework for simulation based experiments to study signaling required for long term potentiation in MSNs .']
['The molecular mechanisms by which a dog learns to associate a bell ring with incoming food and by which addiction to cocaine is developed have many things in common .', 'In both cases , glutamate-mediated inputs from the cortex converge on striatal neurons with the neuromodulator dopamine whose level increases upon food delivery or cocaine administration .', 'This convergence triggers intracellular reaction cascades that end up modifying the basal state of these neurons into what is currently considered the cellular correlate of learning and its overblown variant: addiction .', 'Understanding these signaling cascades is required to design interventions for the enhancement of cognitive capacities and the prevention/cure of addiction .', 'With this in mind , we developed a model of the dynamics of these reaction cascades upon the administration of cocaine .', "We did this by gathering the cascades' molecular components and interactions discovered by experimentalist so far and then setting the reaction parameters so that the model reproduces the timing of the molecular changes that lead to the discovery of those components and interactions .", 'With this integrationist endeavor we disclosed some inconsistencies and provided a rationale for experimental observations that were unexplained .', 'Our predictions are readily challengeable with future experiments .']
['systems biology', 'biochemistry', 'computer science', 'computer modeling', 'biology', 'computational biology', 'molecular cell biology', 'computerized simulations', 'neuroscience']