Adenosine A2A receptors modulate BDNF both in normal conditions and in experimental models of Huntington’s disease

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Functional interactions between adenosine A_2A receptors (A_2ARs) and BDNF have been recently reported. In this article, we report some recent findings from our group showing that A_2ARs regulate both BDNF functions and levels in the brain. Whereas BDNF (10 ng/ml) increased the slope of excitatory postsynaptic field potentials (fEPSPs) in hippocampal slices from wild-type (WT) mice, it was completely ineffective in slices taken from A_2AR knock-out (KO) mice. Furthermore, enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A_2AR KO vs. WT mice. Having found an even marked reduction in the striatum of A_2AR KO mice, and as both BDNF and A_2ARs have been implicated in the pathogenesis of Huntington’s disease (HD), an inherited striatal neurodegenerative disease, we then evaluated whether the pharmacological blockade of A_2ARs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration (quinolinic acid-lesioned rats) and in a transgenic mice model of HD (R6/2 mice). In both QA-lesioned rats and early symptomatic R6/2 mice (8 weeks), the systemic administration of the A_2AR antagonist SCH58261 significantly reduced striatal BDNF levels. These results indicate that the presence and the tonic activation of A_2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone. The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation.     

Cav1.3 L-type Ca2+ channels mediate long-term adaptation in dopamine D2L-mediated GluA1 trafficking in the dorsal striatum following cocaine exposure

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesostriatal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization in mice we find that repeated cocaine results in a basal reduction of Ser 845 GluA1 and cell surface GluA1 levels in the dorsal striatum (dStr) following a protracted withdrawal period, an adaptation that is dependent on Ca_v1.3 channels but not those expressed in the VTA. We find that the basally-induced decrease in this phosphoprotein is the result of recruitment of the striatal dopamine D2 pathway, as evidenced by enhanced levels of D2 receptor (D2R) mRNA expression and D2R function as examined using the D2R antagonist, eticlopride, as well as alterations in the phosphorylation status of several downstream molecular targets of D2R’s, including CREB, DARPP-32, Akt and GSK3β. Taken together with our recently published findings examining similar phenomena in the nucleus accumbens (NAc), these results underscore the utilization of divergent molecular mechanisms in the dStr, in mediating cocaine-induced persistent behavioral changes.     

Cav1.3 L-type Ca2+ channels mediate long-term adaptation in dopamine D2L-mediated GluA1 trafficking in the dorsal striatum following cocaine exposure

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesostriatal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization in mice we find that repeated cocaine results in a basal reduction of Ser 845 GluA1 and cell surface GluA1 levels in the dorsal striatum (dStr) following a protracted withdrawal period, an adaptation that is dependent on Ca_v1.3 channels but not those expressed in the VTA. We find that the basally-induced decrease in this phosphoprotein is the result of recruitment of the striatal dopamine D2 pathway, as evidenced by enhanced levels of D2 receptor (D2R) mRNA expression and D2R function as examined using the D2R antagonist, eticlopride, as well as alterations in the phosphorylation status of several downstream molecular targets of D2R’s, including CREB, DARPP-32, Akt and GSK3β. Taken together with our recently published findings examining similar phenomena in the nucleus accumbens (NAc), these results underscore the utilization of divergent molecular mechanisms in the dStr, in mediating cocaine-induced persistent behavioral changes.     

Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission

An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.     

BDNF prevents NMDA‐induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved

NMDA receptor‐mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain‐derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A_2ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA‐induced effects in HD models, and the possible involvement of A_2ARs. In corticostriatal slices from wild‐type mice and age‐matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild‐type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A_2AR blockade. The protective effect of BDNF against NMDA‐induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A_2AR ligands in HD.     

Differential regulation of dopamine D1 and D2 signaling by nicotine in neostriatal neurons

Nicotine, acting on nicotinic acetylcholine receptors (nAChRs) expressed at pre-synaptic dopaminergic terminals, has been shown to stimulate the release of dopamine in the neostriatum. However, the molecular consequences of pre-synaptic nAChR activation in post-synaptic neostriatal neurons are not clearly understood. Here, we investigated the effect of nAChR activation on dopaminergic signaling in medium spiny neurons by measuring phosphorylated DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (the PKA-site) in mouse neostriatal slices. Nicotine produced dose-dependent responses, with a low concentration (1 microm) causing a sustained decrease in DARPP-32 Thr34 phosphorylation and a high concentration (100 microm) causing a transient increase in DARPP-32 Thr34 phosphorylation. Depending on the concentration of nicotine, either dopamine D2 or D1 receptor signaling was predominantly activated. Nicotine at a low concentration (1 microm) activated dopamine D2 receptor signaling in striatopallidal/indirect pathway neurons, likely by activating alpha4beta2* nAChRs at dopaminergic terminals. Nicotine at a high concentration (100 microm) activated dopamine D1 receptor signaling in striatonigral/direct pathway neurons, likely by activating (i) alpha4beta2* nAChRs at dopaminergic terminals and (ii) alpha7 nAChRs at glutamatergic terminals, which, by stimulating the release of glutamate, activated NMDA/AMPA receptors at dopaminergic terminals. The differential effects of low and high nicotine concentrations on D2- and D1-dependent signaling pathways in striatal neurons may contribute to dose-dependent actions of this drug of abuse.     

The Cdk5 inhibitor Roscovitine increases LTP induction in corticostriatal synapses

In corticostriatal synapses, LTD (long-term depression) and LTP (long-term potentiation) are modulated by the activation of DA (dopamine) receptors, with LTD being the most common type of long-term plasticity induced using the standard stimulation protocols. In particular, activation of the D1 signaling pathway increases cAMP/PKA (protein kinase A) phosphorylation activity and promotes an increase in the amplitude of glutamatergic corticostriatal synapses. However, if the Cdk5 (cyclin-dependent kinase 5) phosphorylates the DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) at Thr⁷⁵, DARPP-32 becomes a strong inhibitor of PKA activity. Roscovitine is a potent Cdk5 inhibitor; it has been previously shown that acute application of Roscovitine increases striatal transmission via Cdk5/DARPP-32. Since DARPP-32 controls long-term plasticity in the striatum, we wondered whether switching off CdK5 activity with Roscovitine contributes to the induction of LTP in corticostriatal synapses. For this purpose, excitatory population spikes and whole cell EPSC (excitatory postsynaptic currents) were recorded in striatal slices from C57/BL6 mice. Experiments were carried out in the presence of Roscovitine (20 μM) in the recording bath. Roscovitine increased the amplitude of excitatory population spikes and the percentage of population spikes that exhibited LTP after HFS (high-frequency stimulation; 100Hz). Results obtained showed that the mechanisms responsible for LTP induction after Cdk5 inhibition involved the PKA pathway, DA and NMDA (N-methyl-D-aspartate) receptor activation, L-type calcium channels activation and the presynaptic modulation of neurotransmitter release.     

Is the functional interaction between adenosine A2A receptors and metabotropic glutamate 5 receptors a general mechanism in the brain? Differences and similarities between the striatum and the hippocampus

The aim of the present paper was to examine, in a comparative way, the occurrence and the mechanisms of the interactions between adenosine A_2A receptors (A_2ARs) and metabotropic glutamate 5 receptors (mGlu5Rs) in the hippocampus and the striatum. In rat hippocampal and corticostriatal slices, combined ineffective doses of the mGlu5R agonist 2-chloro-5-hydroxyphenylglycine (CHPG) and the A_2AR agonist CGS 21680 synergistically reduced the slope of excitatory postsynaptic field potentials (fEPSPs) recorded in CA1 and the amplitude of field potentials (FPs) recorded in the dorsomedial striatum. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway appeared to be involved in the effects of CGS 21680 in corticostriatal but not in hippocampal slices. In both areas, a postsynaptic locus of interaction appeared more likely. N-methyl-D-aspartate (NMDA) reduced the fEPSP slope and FP amplitude in hippocampal and corticostriatal slices, respectively. Such an effect was significantly potentiated by CHPG in both areas. Interestingly, the A_2AR antagonist ZM 241385 significantly reduced the NMDA-potentiating effect of CHPG. In primary cultures of rat hippocampal and striatal neurons (ED 17, DIV 14), CHPG significantly potentiated NMDA-induced lactate dehydrogenase (LDH) release. Again, such an effect was prevented by ZM 241385. Our results show that A_2A and mGlu5 receptors functionally interact both in the hippocampus and in the striatum, even though different mechanisms seem to be involved in the two areas. The ability of A_2ARs to control mGlu5R-dependent effects may thus be a general feature of A_2ARs in different brain regions (irrespective of their density) and may represent an additional target for the development of therapeutic strategies against neurological disorders.     

Effect of Dopaminergic D1 Receptors on Plasticity Is Dependent of Serotoninergic 5-HT1A Receptors in L5-Pyramidal Neurons of the Prefrontal Cortex

Major depression and schizophrenia are associated with dysfunctions of serotoninergic and dopaminergic systems mainly in the prefrontal cortex (PFC). Both serotonin and dopamine are known to modulate synaptic plasticity. 5-HT_1A receptors (5-HT_1ARs) and dopaminergic type D1 receptors are highly represented on dendritic spines of layer 5 pyramidal neurons (L5PyNs) in PFC. How these receptors interact to tune plasticity is poorly understood. Here we show that D1-like receptors (D1Rs) activation requires functional 5HT_1ARs to facilitate LTP induction at the expense of LTD. Using 129/Sv and 5-HT_1AR-KO mice, we recorded post-synaptic currents evoked by electrical stimulation in layer 2/3 after activation or inhibition of D1Rs. High frequency stimulation resulted in the induction of LTP, LTD or no plasticity. The D1 agonist markedly enhanced the NMDA current in 129/Sv mice and the percentage of L5PyNs displaying LTP was enhanced whereas LTD was reduced. In 5-HT_1AR-KO mice, the D1 agonist failed to increase the NMDA current and orientated the plasticity towards L5PyNs displaying LTD, thus revealing a prominent role of 5-HT_1ARs in dopamine-induced modulation of plasticity. Our data suggest that in pathological situation where 5-HT_1ARs expression varies, dopaminergic treatment used for its ability to increase LTP could turn to be less and less effective.     

Neuron‐type specific cannabinoid‐mediated G protein signalling in mouse hippocampus

Type 1 cannabinoid receptor (CB1) is expressed in different neuronal populations in the mammalian brain. In particular, CB1 on GABAergic or glutamatergic neurons exerts different functions and display different pharmacological properties in vivo. This suggests the existence of neuron‐type specific signalling pathways activated by different subpopulations of CB1. In this study, we analysed CB1 expression, binding and signalling in the hippocampus of conditional mutant mice, bearing CB1 deletion in GABAergic (GABA‐CB1‐KO mice) or cortical glutamatergic neurons (Glu‐CB1‐KO mice). Compared to their wild‐type littermates, Glu‐CB1‐KO displayed a small decrease of CB1 mRNA amount, immunoreactivity and [³H]CP55,940 binding. Conversely, GABA‐CB1‐KO mice showed a drastic reduction of these parameters, confirming that CB1 is present at much higher density on hippocampal GABAergic interneurons than glutamatergic neurons. Surprisingly, however, saturation analysis of HU210‐stimulated [³⁵S]GTPγS binding demonstrated that ‘glutamatergic’ CB1 is more efficiently coupled to G protein signalling than ‘GABAergic’ CB1. Thus, the minority of CB1 on glutamatergic neurons is paradoxically several fold more strongly coupled to G protein signalling than ‘GABAergic’ CB1. This selective signalling mechanism raises the possibility of designing novel cannabinoid ligands that differentially activate only a subset of physiological effects of CB1 stimulation, thereby optimizing therapeutic action.     

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M (r) 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca(2+) -dependent activation of the Ca(2+) -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca(2+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.     

Neuronal adenosine A<Subscript>2A</Subscript> receptor overexpression is neuroprotective towards 3-nitropropionic acid-induced striatal toxicity: a rat model of Huntington’s disease

The A_2A adenosine receptor (A_2AR) is widely distributed on different cellular types in the brain, where it exerts a broad spectrum of pathophysiological functions, and for which a role in different neurodegenerative diseases has been hypothesized or demonstrated. To investigate the role of neuronal A_2ARs in neurodegeneration, we evaluated in vitro and in vivo the effect of the neurotoxin 3-nitropropionic acid (3-NP) in a transgenic rat strain overexpressing A_2ARs under the control of the neural-specific enolase promoter (NSEA_2A rats). We recorded extracellular field potentials (FP) in corticostriatal slice and found that the synaptotoxic effect of 3-NP was significantly reduced in NSEA_2A rats compared with wild-type animals (WT). In addition, after exposing corticostriatal slices to 3-NP 10 mM for 2 h, we found that striatal cell viability was significantly higher in NSEA_2A rats compared to control rats. These in vitro results were confirmed by in vivo experiments: daily treatment of female rats with 3-NP 10 mg/kg for 8 days induced a selective bilateral lesion in the striatum, which was significantly reduced in NSEA_2A compared to WT rats. These results demonstrate that the overexpression of the A_2AR selectively at the neuronal level reduced 3-NP-induced neurodegeneration, and suggest an important function of the neuronal A_2AR in the modulation of neurodegeneration.     

DARPP-32 is a robust integrator of dopamine and glutamate signals

Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca²⁺ signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca²⁺ signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca²⁺ regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.     

Isoaspartate Accumulation in Mouse Brain Is Associated with Altered Patterns of Protein Phosphorylation and Acetylation,Some of Which Are Highly Sex-Dependent

Isoaspartate (isoAsp) formation is a major source of protein damage that is kept in check by the repair function of protein L-isoaspartyl methyltransferase (PIMT). Mice deficient in PIMT accumulate isoAsp-containing proteins, resulting in cognitive deficits, abnormal neuronal physiology and cytoarchitecture, and fatal epileptic seizures 30–60 days after birth. Synapsins I and II, dynamin-1, collapsin response mediator protein 2 (CRMP2), and α/β-tubulin are major targets of PIMT in brain. To investigate links between isoAsp accumulation and the neurological phenotype of the KO mice, we used Western blotting to compare patterns of in vivo phosphorylation or acetylation of the major PIMT targets listed above. Phosphorylations of synapsins I and II at Ser-9 were increased in female KO vs. WT mice, and acetylation of tubulin at Lys-40 was decreased in male KO vs. WT mice. Average levels of dynamin-1 phosphorylation at Ser-778 and Ser-795 were higher in male KO vs. WT mice, but the statistical significance (P>0.1) was low. No changes in phosphorylation were found in synapsins I and II at Ser-603, in CRMP2 at Ser-522 or Thr-514, in DARPP-32 at Thr-34, or in PDK1 at Ser-241. General levels of phosphorylation assessed with Pro-Q Diamond stain, or an anti-phosphotyrosine antibody, appeared similar in the WT and KO mice. We conclude that isoAsp accumulation is associated with altered functional status of several neuronal proteins that are highly susceptible to this type of damage. We also uncovered unexpected differences in how male and female mice respond to isoAsp accumulation in the brain.     

Altered Cortical GABAA Receptor Composition,Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABA_A receptors (GABA_ARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Het_α1KO) of the human epilepsy gene, the GABA_AR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Het_α1KO on the expression and physiology of GABA_ARs in the mouse cortex. We found that Het_α1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABA_ARs. Cortices partially compensated for Het_α1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Het_α1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Het_α1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Het_α1KO reduced base-line GABA_AR endocytosis, an effect that probably contributes to the observed changes in GABA_AR expression. These findings demonstrate that Het_α1KO exerts two principle disinhibitory effects on cortical GABA_AR-mediated inhibitory neurotransmission: 1) a modest reduction of GABA_AR number and 2) a partial compensation with GABA_AR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABA_ARs.     

Transient calcium and dopamine increase PKA activity and DARPP-32 phosphorylation

Reinforcement learning theorizes that strengthening of synaptic connections in medium spiny neurons of the striatum occurs when glutamatergic input (from cortex) and dopaminergic input (from substantia nigra) are received simultaneously. Subsequent to learning, medium spiny neurons with strengthened synapses are more likely to fire in response to cortical input alone. This synaptic plasticity is produced by phosphorylation of AMPA receptors, caused by phosphorylation of various signalling molecules. A key signalling molecule is the phosphoprotein DARPP-32, highly expressed in striatal medium spiny neurons. DARPP-32 is regulated by several neurotransmitters through a complex network of intracellular signalling pathways involving cAMP (increased through dopamine stimulation) and calcium (increased through glutamate stimulation). Since DARPP-32 controls several kinases and phosphatases involved in striatal synaptic plasticity, understanding the interactions between cAMP and calcium, in particular the effect of transient stimuli on DARPP-32 phosphorylation, has major implications for understanding reinforcement learning. We developed a computer model of the biochemical reaction pathways involved in the phosphorylation of DARPP-32 on Thr34 and Thr75. Ordinary differential equations describing the biochemical reactions were implemented in a single compartment model using the software XPPAUT. Reaction rate constants were obtained from the biochemical literature. The first set of simulations using sustained elevations of dopamine and calcium produced phosphorylation levels of DARPP-32 similar to that measured experimentally, thereby validating the model. The second set of simulations, using the validated model, showed that transient dopamine elevations increased the phosphorylation of Thr34 as expected, but transient calcium elevations also increased the phosphorylation of Thr34, contrary to what is believed. When transient calcium and dopamine stimuli were paired, PKA activation and Thr34 phosphorylation increased compared with dopamine alone. This result, which is robust to variation in model parameters, supports reinforcement learning theories in which activity-dependent long-term synaptic plasticity requires paired glutamate and dopamine inputs.     

Increase in Glutamatergic Terminals in the Striatum Following Dopamine Depletion in a Rat Model of Parkinson’s Disease

Dopaminergic neuron degeneration is known to give rise to dendrite injury and spine loss of striatal neurons, however, changes of intrastriatal glutamatergic terminals and their synapses after 6-hydroxydopamine (6OHDA)-induced dopamine (DA)-depletion remains controversial. To confirm the effect of striatal DA-depletion on the morphology and protein levels of corticostriatal and thalamostriatal glutamatergic terminals and synapses, immunohistochemistry, immuno-electron microscope (EM), western blotting techniques were performed on Parkinson’s disease rat models in this study. The experimental results of this study showed that: (1) 6OHDA-induced DA-depletion resulted in a remarkable increase of Vesicular glutamate transporter 1 (VGlut1) + and Vesicular glutamate transporter 2 (VGlut2)+ terminal densities at both the light microscope (LM) and EM levels, and VGlut1+ and VGlut2+ terminal sizes were shown to be enlarged by immuno-EM; (2) Striatal DA-depletion resulted in a decrease in both the total and axospinous terminal fractions of VGlut1+ terminals, but the axodendritic terminal fraction was not significantly different from the control group. However, total, axospinous and axodendritic terminal fractions for VGlut2+ terminals declined significantly after striatal DA-depletion. (3) Western blotting data showed that striatal DA-depletion up-regulated the expression levels of the VGlut1 and VGlut2 proteins. These results suggest that 6OHDA-induced DA-depletion affects corticostriatal and thalamostriatal glutamatergic synaptic inputs, which are involved in the pathological process of striatal neuron injury induced by DA-depletion.

     

DARPP-32 Expression in Rat Brain After an Inhibitory Avoidance Task

Step-down inhibitory avoidance (IA) is usually acquired in one single trial, which makes it ideal for studying processes initiated by training, uncontaminated by prior or further trials, rehearsals, or retrievals. Biochemical events in the hippocampus related to long-term memory (LTM) formation have been extensively studied in rats using a one trial step-down IA task. DARPP-32 (dopamine and cAMP regulated phosphoprotein of Mr 32 kDa) is a cytosolic protein that is selectively enriched in medium spiny neurons in the neostriatum. It has been shown that activation of DARPP-32 and the resultant inhibition of PP-1 activity is critical for the expression of two opposing forms of brain synaptic plasticity, striatal LTD and LTP. Both forms of plasticity are also critically linked to the activation of DA receptors. It has been shown with studies in DARPP-32 KO mice an important role of this protein in mediating the effects of DA on long term changes in neuronal excitability and to our knowledge, no studies have examined the effect of IA task on DARPP-32 expression. In order to demonstrate changes in the protein expression profile we analyzed DARPP-32 levels in the striatum, prefrontal cortex (PFC), hippocampus and entorhinal cortex of Wistar rats after step-down IA learning. Our results showed that IA induced changes on DARPP-32 expression in striatum and hippocampus. DARPP-32 expression changes corroborate with changes in expression and phosphorylation of CREB, NMDA, AMPA after IA that has been reported. These changes suggest that DARPP-32 might play a central role in the IA, as previously described as an integrator of the dopaminergic signal.     

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABA_A receptors (GABA_ARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABA_ARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABA_AR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABA_AR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABA_ARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.