Novel AAV-based rat model of forebrain synucleinopathy shows extensive pathologies and progressive loss of cholinergic interneurons.

Synucleinopathies, characterized by intracellular aggregation of α-synuclein protein, share a number of features in pathology and disease progression. However, the vulnerable cell population differs significantly between the disorders, despite being caused by the same protein. While the vulnerability of dopamine cells in the substantia nigra to α-synuclein over-expression, and its link to Parkinson's disease, is well studied, animal models recapitulating the cortical degeneration in dementia with Lewy-bodies (DLB) are much less mature. The aim of this study was to develop a first rat model of widespread progressive synucleinopathy throughout the forebrain using adeno-associated viral (AAV) vector mediated gene delivery. Through bilateral injection of an AAV6 vector expressing human wild-type α-synuclein into the forebrain of neonatal rats, we were able to achieve widespread, robust α-synuclein expression with preferential expression in the frontal cortex. These animals displayed a progressive emergence of hyper-locomotion and dysregulated response to the dopaminergic agonist apomorphine. The animals receiving the α-synuclein vector displayed significant α-synuclein pathology including intra-cellular inclusion bodies, axonal pathology and elevated levels of phosphorylated α-synuclein, accompanied by significant loss of cortical neurons and a progressive reduction in both cortical and striatal ChAT positive interneurons. Furthermore, we found evidence of α-synuclein sequestered by IBA-1 positive microglia, which was coupled with a distinct change in morphology. In areas of most prominent pathology, the total α-synuclein levels were increased to, on average, two-fold, which is similar to the levels observed in patients with SNCA gene triplication, associated with cortical Lewy body pathology. This study provides a novel rat model of progressive cortical synucleinopathy, showing for the first time that cholinergic interneurons are vulnerable to α-synuclein over-expression. This animal model provides a powerful new tool for studies of neuronal degeneration in conditions of widespread cortical α-synuclein pathology, such as DLB, as well an attractive model for the exploration of novel biomarkers

Thalamic inputs to dorsomedial striatum are involved in inhibitory control: evidence from the five-choice serial reaction time task in rats

Rationale Corticostriatal circuits are widely implicated in the top-down control of attention including inhibitory control and behavioural flexibility. However, recent neurophysiological evidence also suggests a role for thalamic inputs to striatum in behaviours related to salient, reward-paired cues. Objectives Here, we used designer receptors exclusively activated by designer drugs (DREADDs) to investigate the role of parafascicular (Pf) thalamic inputs to the dorsomedial striatum (DMS) using the five-choice serial reaction time task (5CSRTT) in rats. Methods The 5CSRTT requires sustained attention in order to detect spatially and temporally distributed visual cues and provides measures of inhibitory control related to impulsivity (premature responses) and compulsivity (perseverative responses). Rats underwent bilateral Pf injections of the DREADD vector, AAV2-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine. The DREADD agonist, clozapine N-oxide (CNO; 1 μl bilateral; 3 μM) or vehicle, was injected into DMS 1 h before behavioural testing. Task parameters were manipulated to increase attention load or reduce stimulus predictability respectively. Results We found that inhibition of the Pf-DMS projection significantly increased perseverative responses when stimulus predictability was reduced but had no effect on premature responses or response accuracy, even under increased attentional load. Control experiments showed no effects on locomotor activity in an open field. Conclusions These results complement previous lesion work in which the DMS and orbitofrontal cortex were similarly implicated in perseverative responses and suggest a specific role for thalamostriatal inputs in inhibitory control

Beyond equilibrium climate sensitivity

ISSN:1752-0908ISSN:1752-089

Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum

The intricate balance between dopaminergic and cholinergic neurotransmission in the striatum has been thoroughly difficult to characterize. It was initially described as a seesaw with a competing function of dopamine versus acetylcholine. Recent technical advances however, have brought this view into question suggesting that the two systems work rather in concert with the cholinergic interneurons (ChIs) driving dopamine release. In this study, we have utilized two transgenic Cre-driver rat lines, a choline acetyl transferase ChAT-Cre transgenic rat and a novel double-transgenic tyrosine hydroxylase TH-Cre/ChAT-Cre rat to further elucidate the role of striatal ChIs in normal motor function and in Parkinson's disease. Here we show that selective and reversible activation of ChIs using chemogenetic (DREADD) receptors increases locomotor function in intact rats and potentiate the therapeutic effect of L-DOPA in the rats with lesions of the nigral dopamine system. However, the potentiation of the L-DOPA effect is accompanied by an aggravation of L-DOPA induced dyskinesias (LIDs). These LIDs appear to be driven primarily through the indirect striato-pallidal pathway since the same effect can be induced by the D2 agonist Quinpirole. Taken together, the results highlight the intricate regulation of balance between the two output pathways from the striatum orchestrated by the ChIs

DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor

Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson's disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still unknown. Here we have used a new model where the activity of the transplanted DA neurons can be selectively modulated using a bimodal chemogenetic (DREADD) approach, allowing either enhancement or reduction of the therapeutic effect. We show that exclusive activation of a cAMP-linked (Gs-coupled) DREADD or serotonin 5-HT6 receptor, located on the grafted DA neurons, is sufficient to induce GIDs. These findings establish a mechanistic link between the 5-HT6 receptor, intracellular cAMP, and GIDs in transplanted PD patients. This effect is thought to be mediated through counteraction of the D2 autoreceptor feedback inhibition, resulting in a dysplastic DA release from the transplant

High-power histological analysis of α-synuclein expression.

<p>Histological analysis of the cerebral cortices revealed high expression of human α-synuclein (syn211) within the soma and axonal projections of the cortical neurons. These neurons displayed a high incidence of α-synuclein punctates within their soma (arrows), while phosphorylated α-synuclein (P-ser129) was highly increased within the neuronal soma, and to a lesser extent within the axonal projections (A–D). Striatal analysis revealed high incidence of neurons with prominent expression of human α-synuclein, with Lewy neurite's being a common find (arrow). Phosphorylation of α-synuclein within the striatal neurons was also upregulated (E–H) Projections within the thalamic nuclei were found to contain high levels of human α-synuclein with large α-synuclein positive structures, believed to be large axonal swellings, were present throughout the thalamus (arrows) (I–L). Large axonal swellings were also present to a large extent in the SNpR (arrows) while phosphorylation of α-synuclein appeared as only moderately increased (M–P). The neonatal injection of the AAV6 serotype displays an interesting ability to also infect neuronal progenitors within the rostral migratory stream (RMS) or the sub-ventricular zone. This is seen as mature neurons within the olfactory bulb expressing high levels of human α-synuclein (R–S). These neurons appear with healthy morphology with α-synuclein distributed throw-out the neuropil (S). Dotted lines within the cerebral cortex denote the position of the tissue punch taken for TEM analysis.</p

Modified isotropic fractionation for neuronal quantification reveals broad-spread cortical degeneration.

<p>Principals of modified isotropic fractionation for neuronal quantification (A). PFA fixed cerebral cortices were dissected and dissociated using the GentleMacs tissue dissociator. Following centrifugation and washing only a nuclear fraction remained. The nuclear fraction was stained for DAPI and the NeuN neuronal marker and counted in a haemocytometer, using florescence microscopy (B). Quantification revealed a significant neuronal degeneration within the cerebral cortices of animals expressing α-synuclein, relative to controls (C). There was however, no significant neuronal loss in the cerebellum, a region not expressing human α-synuclein, using our modified isotropic fractionation protocol (AAV6|a-Syn 463.1±24.9×10³, AAV6|GFP 475.1±15.1×10³, Intact ctrl 506.7±12.1×10³. (* = p<0.05, one-way ANOVA with Newman-Keuls post hoc test). Data is expressed as mean ± SEM.</p

Morphological changes and evidence of α-synuclein uptake in IBA-1 positive microglia.

<p>Immunohistochemical staining of IBA-1 using DAB precipitation coloring in the cerebral cortex revealed a substantial change in the microglial morphology in the cortex of AAV6|a-Syn animals compared to AAV6|GFP and Intact ctrl animals at 10 months post AAV injection (A–D). In the regions overlapping with the most intense human α-synuclein over-expression, most microglial cells appeared with very few visible processes and majority of the IBA-1 positive staining localized to the nucleus (A), this stood in stark contrast to the normal IBA-1 staining of microglia in the corresponding cortical regions of AAV6|GFP animals (B). In surrounding cortical regions, the microglial morphology in AAV6|a-Syn animals showed a second distinct morphology, with elongated cell body and fewer processer, suggesting a migratory profile (C). Using double-labeled, fluorescence with IBA-1 and human α-synuclein/GFP antibodies in a scanning confocal microscope, the presence of α-synuclein in microglia was investigated (E–F″). At the core of the human α-synuclein over-expression in the AAV6|a-Syn animals, microglia were found with significantly changes morphology and intra-cellular α-synuclein (E–E″). Interestingly, two localization patterns were observed, both in the cell soma in circular, vesicular structures and as small accumulations in distal processes of the microglia. No such patterns were observed in AAV6|GFP (F–F″) or intact controls.</p

Transmission electron microscopy assessing α-synuclein localization and associated pathologies.

<p>Analysis of the cerebral cortices through electron microscopy revealed a large number of apoptotic neurons. These neurons were identified by morphological features, such as large, swollen mitochondria, vacuole formation and disruption of the nuclear envelope (<i>e.g.</i>, * in A). Immunogold staining for human α-synuclein (Syn211) revealed a high presence of nuclear associated α-synculein in apoptotic neurons (black 10 nm dots marked by arrows in B) in addition to that of the soma (A–D). Large axonal swellings were also a common pathological finding throughout the cerebral cortices (* in C). These were found to have large accumulations of human α-synuclein (arrows in F) and also appeared to associate with the cellular membrane (E–H). Large and swollen mitochondria present in neuronal cells, as well as within the axons (* in I) appeared associated with large amounts of human α-synuclein (I–L). Human α-synuclein was also found to be present to a large extent in synapses of the cerebral cortices (M–P), However, these synapses appeared largely of normal morphology. The antibody staining in B–C, F–G, J–K and N–O is visualized using immunogold particles of 10 nm in diameter.</p