Luz M. Suárez

L-DOPA Oppositely Regulates Synaptic Strength and Spine Morphology in D1 and D2 Striatal Projection Neurons in Dyskinesia

Dopamine depletion in Parkinsons disease (PD) produces dendritic spine loss in striatal medium spiny neurons (MSNs) and increases their excitability. However, the synaptic changes that occur in MSNs in PD, in particular those induced by chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, are still poorly understood. We exposed BAC-transgenic D1-tomato and D2-eGFP mice to PD and dyskinesia model paradigms, enabling cell type-specific assessment of changes in synaptic physiology and morphology. The distinct fluorescence markers allowed us to identify D1 and D2 MSNs for analysis using intracellular sharp electrode recordings, electron microscopy, and 3D reconstructions with single-cell Lucifer Yellow injections. Dopamine depletion induced spine pruning in both types of MSNs, affecting mushroom and thin spines equally. Dopamine depletion also increased firing rate in both D1- and D2-MSNs, but reduced evoked-EPSP amplitude selectively in D2-MSNs. L-DOPA treatment that produced dyskinesia differentially affected synaptic properties in D1- and D2-MSNs. In D1-MSNs, spine density remained reduced but the remaining spines were enlarged, with bigger heads and larger postsynaptic densities. These morphological changes were accompanied by facilitation of action potential firing triggered by synaptic inputs. In contrast, although L-DOPA restored the number of spines in D2-MSNs, it resulted in shortened postsynaptic densities. These changes in D2-MSNs correlated with a decrease in synaptic transmission. Our findings indicate that L-DOPA-induced dyskinesia is associated with abnormal spine morphology, modified synaptic transmission, and altered EPSP-spike coupling, with distinct effects in D1- and D2-MSNs.

Taurine potentiates presynaptic NMDA receptors in hippocampal Schaffer collateral axons

We have previously shown that activation of presynaptic N‐methyl‐d‐aspartate (NMDA) receptors (NMDAR) enhances the amplitude of the presynaptic fibre volley (FV) evoked in Schaffer collateral axons of rat hippocampal slices, by a mechanism independent of extracellular Ca2+. Here we compared the pharmacological characteristics of presynaptic NMDARs affecting axon excitability (activated by 10–300 µm NMDA for 10 min), with those mediating field excitatory postsynaptic potentials (NMDA‐fEPSP). We found that NMDA‐induced potentiation was completely inhibited by NVP‐AAM077, an antagonist of NR2A‐containing NMDAR, but not by ifenprodil, an NR2B‐selective antagonist. The inhibitor of the glycine‐binding site in NMDARs, 7‐clorokynurenic acid (7‐CK), was more potent against NMDA‐fEPSP (IC50 = 6.3 ± 1.3 µm) than against the NMDA‐induced FV potentiation (IC50 = 26.5 ± 1.3 µm). Moreover, both post‐ and presynaptic NMDAR‐mediated phenomena were enhanced by glycine and d‐serine, but taurine, an endogenous analogue of glycine, only enhanced the latter (EC50 = 19 µm). Taurine was able to block the inhibitory effect of low doses of 7‐CK on NMDA‐induced FV potentiation, while glycine and d‐serine only reduced the effects of higher concentrations of this drug. Surprisingly, the enhancing effect of taurine on NMDA‐induced FV potentiation was blocked when it was co‐applied with glycine. Furthermore, the glutamate released synaptically with a train of stimuli also increased FV amplitude by a mechanism dependent on NMDARs; this was potentiated by taurine but not by co‐application of taurine and glycine. These results reveal that presynaptic NMDARs have unique properties that mediate the facilitation of axon excitability.

Role of taurine uptake on the induction of long‐term synaptic potentiation

Taurine application in the CA1 area of rat hippocampal slices induces a long‐lasting potentiation of excitatory synaptic transmission that has some mechanistic similitude with the late phase of long‐term potentiation (L‐LTP). Previous indirect evidence such as temperature and sodium dependence indicated that taurine uptake is one of the primary steps leading to the taurine‐induced synaptic potentiation. We show that taurine‐induced potentiation is not related to the intracellular accumulation of taurine and is not impaired by 2‐guanidinoethanesulphonic acid, a taurine transport inhibitor that is a substrate of taurine transporter. We have found that taurine uptake in hippocampal synaptosomes was inhibited by SKF 89976A, a GABA uptake blocker that is not transportable by GABA transporters. SKF 89976A prevents the induction of synaptic potentiation by taurine application. This effect is neither mimicked by nipecotic acid, a broad inhibitor of GABA transporters that does not affect taurine uptake, nor by NO‐711, a specific and potent inhibitor of GABA transporter GAT‐1. In addition, L‐LTP induced by trains of high‐frequency stimulation is also inhibited by SKF 89976A, and taurine, at a concentration that does not change basal synaptic transmission, overcomes such inhibition. We conclude that taurine induces synaptic potentiation through the activation of a system transporting taurine and that taurine uptake is required for the induction of synaptic plasticity phenomena such as L‐LTP.

Systemic injection of kainic acid differently affects LTP magnitude depending on its epileptogenic efficiency.

Seizures have profound impact on synaptic function and plasticity. While kainic acid is a popular method to induce seizures and to potentially affect synaptic plasticity, it can also produce physiological-like oscillations and trigger some forms of long-term potentiation (LTP). Here, we examine whether induction of LTP is altered in hippocampal slices prepared from rats with different sensitivity to develop status epilepticus (SE) by systemic injection of kainic acid. Rats were treated with multiple low doses of kainic acid (5 mg/kg; i.p.) to develop SE in a majority of animals (72–85% rats). A group of rats were resistant to develop SE (15–28%) after several accumulated doses. Animals were subsequently tested using chronic recordings and object recognition tasks before brain slices were prepared for histological studies and to examine basic features of hippocampal synaptic function and plasticity, including input/output curves, paired-pulse facilitation and theta-burst induced LTP. Consistent with previous reports in kindling and pilocapine models, LTP was reduced in rats that developed SE after kainic acid injection. These animals exhibited signs of hippocampal sclerosis and developed spontaneous seizures. In contrast, resistant rats did not become epileptic and had no signs of cell loss and mossy fiber sprouting. In slices from resistant rats, theta-burst stimulation induced LTP of higher magnitude when compared with control and epileptic rats. Variations on LTP magnitude correlate with animals’ performance in a hippocampal-dependent spatial memory task. Our results suggest dissociable long-term effects of treatment with kainic acid on synaptic function and plasticity depending on its epileptogenic efficiency.

Differential Synaptic Remodeling by Dopamine in Direct and Indirect Striatal Projection Neurons in Pitx3−/− Mice, a Genetic Model of Parkinson's Disease

In toxin-based models of Parkinsons disease (PD), striatal projection neurons (SPNs) exhibit dendritic atrophy and spine loss concurrent with an increase in excitability. Chronic l-DOPA treatment that induces dyskinesia selectively restores spine density and excitability in indirect pathway SPNs (iSPNs), whereas spine loss and hyperexcitability persist in direct pathway SPNs (dSPNs). These alterations have only been characterized in toxin-based models of PD, raising the possibility that they are an artifact of exposure to the toxin, which may engage compensatory mechanisms independent of the PD-like pathology or due to the loss of dopaminergic afferents. To test all these, we studied the synaptic remodeling in Pitx3−/− or aphakia mice, a genetic model of PD, in which most of the dopamine neurons in the substantia nigra fail to fully differentiate and to innervate the striatum. We made 3D reconstructions of the dendritic arbor and measured excitability in identified SPNs located in dorsal striatum of BAC-Pitx3−/− mice treated with saline or l-DOPA. Both dSPNs and iSPNs from BAC-Pitx3−/− mice had shorter dendritic trees, lower spine density, and more action potentials than their counterparts from WT mice. Chronic l-DOPA treatment restored spine density and firing rate in iSPNs. By contrast, in dSPNs, spine loss and hyperexcitability persisted following l-DOPA treatment, which is similar to what happens in 6-OHDA WT mice. This indicates that dopamine-mediated synaptic remodeling and plasticity is independent of dopamine innervation during SPN development and that Pitx3−/− mice are a good model because they develop the same pathology described in the toxins-based models and in human postmortem studies of advanced PD. SIGNIFICANCE STATEMENT As the only genetic model of Parkinsons disease (PD) that develops dyskinesia, Pitx3−/− mice reproduce the behavioral effects seen in humans and are a good system for studying dopamine-induced synaptic remodeling. The studies we present here establish that the structural and functional synaptic plasticity that occur in striatal projection neurons in PD and in l-DOPA-induced dyskinesia are specifically due to modulation of the neurotransmitter dopamine and are not artifacts of the use of chemical toxins in PD models. In addition, our findings provide evidence that synaptic plasticity in the Pitx3−/− mouse is similar to that seen in toxin models despite its lack of dopaminergic innervation of the striatum during development. Pitx3−/− mice reproduced the alterations described in patients with advanced PD and in well accepted toxin-based models of PD and dyskinesia. These results further consolidate the fidelity of the Pitx3−/− mouse as a PD model in which to study the morphological and physiological remodeling of striatal projection neurons by administration of l-DOPA and other drugs.

Taurine content in different brain structures during ageing: effect on hippocampal synaptic plasticity

A reduction in taurine content accompanies the ageing process in many tissues. In fact, the decline of brain taurine levels has been associated with cognitive deficits whereas chronic administration of taurine seems to ameliorate age-related deficits such as memory acquisition and retention. In the present study, using rats of three age groups (young, adult and aged) we determined whether the content of taurine and other amino acids (glutamate, serine, glutamine, glycine, alanine and GABA) was altered during ageing in different brain areas (cerebellum, cortex and hippocampus) as well non-brain tissues (heart, kidney, liver and plasma). Moreover, using hippocampal slices we tested whether ageing affects synaptic function and plasticity. These parameters were also determined in aged rats fed with either taurine-devoid or taurine-supplemented diets. With age, we found heterogeneous changes in amino acid content depending on the amino acid type and the tissue. In the case of taurine, its content was reduced in the cerebellum of adult and aged rats, but it remained unchanged in the hippocampus, cortex, heart and liver. The synaptic response amplitude decreased in aged rats, although the late phase of long-term synaptic potentiation (late-LTP), a taurine-dependent process, was not altered. Our study highlights the stability of taurine content in the hippocampus during ageing regardless of whether taurine was present in the diet, which is consistent with the lack of changes detected in late-LTP. These results indicate that the beneficial effects of taurine supplementation might be independent of the replenishment of taurine stores.

Morphological Plasticity in the Striatum Associated With Dopamine Dysfunction

The main entrance for information to the basal ganglia is the striatum. Striatal medium spiny neurons (MSNs) receive glutamate and dopamine inputs that are topographically organized on their dendritic spines. The configuration of the striatal synapses confers on dopamine a central role in regulating spine morphogenesis on MSNs. Dopamine can thus regulate the induction of long-term changes in the strength of corticostriatal synapses, which are implicated in learning and other behavioral plasticity. Indeed, changes in basal ganglia function in Parkinsons disease (PD) or drug addiction are associated with abnormal remodeling of the dendritic arbor of MSNs. In this chapter, we will summarize findings from patients and animal models of PD and drug addiction. We present new results obtained with state-of-the art technology, two-photon microscopy, and Lucifer yellow intracellular injections in identified striatal neurons, which allow the highest currently available resolution of single cell dendritic arbor reconstruction and spine morphology. Images reconstructed with this technique indicate a key role for dopamine in remodeling dendritic and spine morphology in MSNs.