Phosphorylation of ribosomal protein S6 reflects neuronal activity in striatal cholinergic interneurons

<p>Cholinergic interneurons (CINs) provide the main source of acetylcholine to all striatal regions, and strongly modulate dopaminergic actions through complex regulation of pre- and post-synaptic acetylcholine receptors. Although striatal CINs have a well-defined electrophysiological profile, their biochemical properties are poorly understood, likely due to their low proportion within the striatum (2-3%). We report a strong and sustained phosphorylation of ribosomal protein S6 on its serine 240 and 244 residues (p-Ser240-244-S6rp), a protein integrant of the ribosomal machinery related to the mammalian target of the rapamycin complex 1 (mTORC1) pathway, which we found to be principally expressed in striatal CINs in basal conditions (A). We explored the functional relevance of this cellular event by pharmacologically inducing various sustained physiological activity states in CINs and assessing the effect on the levels of S6rp phosphorylation. The inhibitory effect of tetrodotoxin (TTX) on action potential firing was paralleled by a decrease in the p-Ser240-244-S6rp signal as detected by immunofluorescence after prolonged incubation (B-C). On the other hand, both the elevation in extracellular potassium concentration and the addition of apamin, which generate an increased firing rate and a burst-firing activity in CINs, respectively, significantly increased Ser240-244-S6rp phosphorylation above basal levels when incubated for one hour (B-C). Taken together, our results recently published in PLoS ONE (Bertran-Gonzalez & Chieng et al., 2012) demonstrate for the first time a link between the state of neuronal activity and a biochemical signaling event in striatal CINs, and suggest that immunofluorescence can be used to estimate the cellular activity of CINs under different pharmacological and/or behavioral conditions.</p

Study of Segregated Signaling Responses of Striatonigral and Striatopallidal Neurons in BAC Transgenic Mice

<p>The work compiled in the present thesis is the result of a project initially conceived to clarify a critical issue concerning the organization of the striatal neurons. The first question to be answered was what is the real distribution of the two major subtypes of dopamine receptors in striatal neurons. This issue is part of a classical debate concerning two contrasting views of striatal organization. On the one hand, some authors proposed decades ago that D1 receptors (D1Rs) and D2-receptors (D2Rs), the two main subtypes of dopamine receptors expressed in the striatum, were differentially distributed across the two subpopulations of projection neurons existing in the striatum, so that neurons projecting to the substantia nigra (striatonigral neurons) specifically expressed D1Rs, whereas those projecting to the lateral globus pallidus (striatopallidal neurons) expressed D2Rs. This proposal of striatal organization was subsequently challenged by a bulk of studies reporting different degrees of D1R and D2R overlapping expression in striatonigral and striatopallidal subpopulations. The final resolution of this concern is critical for the understanding of striatal and basal ganglia function, especially considering the opposing effect that D1Rs and D2Rs exert on intracellular signaling. In line with this, a second question to be addressed was what is the real degree of functional segregation existing between these two subpopulations of neurons. This question appeals to the completely different behavioral effects derived from two groups of drugs that exert their actions in striatal neurons: psychostimulants and antipsychotics. Although previous pharmachological data clearly showed that these drugs mediate their effects through the two different types of dopamine receptors, the specific (and possibly exclusive) involvement of striatonigral and striatopallidal neurons mediating psychostimulant and antipsychotic actions, respectively, had never been satisfactorily studied. A major problem responsible for the absence of conclusive studies to this respect was certainly the lack of technological approaches to convincingly distinguish between these two neuronal subpopulations, which are completely intermingled within the striatal tissue.</p> <p>In order to respond to these questions, our laboratory acquired BAC transgenic mice, a recently developed tool that could potentially solve the problem of the accurate distinction of striatal neuronal populations in vivo . Two different strains of BAC mice were bred: drd1a -EGFP mice, in which EGFP expression is targeted to D1R-expressing neurons; and drd2 -EGFP mice, in which EGFP is selectively expressed in D2R-expressing neurons. The goal was, first, to characterize these tools by performing in-depth neuroanatomical and histological studies. Second, once the model was properly validated, we aimed at studying the extent of segregation of the signaling pathways triggered by psychostimulants (mostly dependent on D1Rs) and typical antipsychotics (mostly dependent on D2Rs) in striatonigral and striatopallidal subpopulations. Beyond expectancies, our results pointed to highly segregated molecular responses in these neurons, and drove us to explore further signaling events exclusive of striatopallidal neurons, which appeared to have important different molecular regulations to those previously described in striatonigral neurons.</p> <p>Therefore, since the results obtained in BAC mice presented here not only address the molecular events occurring in striatal neurons but also deal with several aspects of the basal ganglia circuitry, the present thesis is introduced by an extensive review of the literature organized as follows. In Chapter I, a broad view of the anatomy, histology and function of the striatum provides a basis to understand how this structure supplies the inputs to the basal ganglia circuitry. In Chapter II, a profound review of the basal ganglia is performed, describing the connections existing between the different nuclei and exploring the functional circuits proposed in the literature. This chapter closes by pointing out the important role that dopamine plays in motor control and reinforcement learning. In Chapter III the scope is reduced to the intracellular events by which DA modulates the corticostrialal glutamatergic transmission in striatal neurons, especially pointing to the specific signaling pathways occurring in response to D1R and D2R activities. Finally, in Chapter IV the principles of the BAC transgenesis are explained, and a review of the numerous studies based on these tools that have appeared in the last years is also provided, describing some of the functional applications of these mice used so far.</p

Code for Manuscript "Adaptation of sequential action benefits from timing variability related to lateral basal ganglia circuitry"

Code used to generate and visualise the data for manuscript "Adaptation of sequential action benefits from timing variability related to lateral basal ganglia circuitry".</p