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Heterogeneity of input activity to ventral midbrain neurons in vivo (#170)
1 UTSA, Neurosciences Institute, San Antonio, United States of America
The firing pattern of midbrain dopamine neurons is controlled by afferents to encode reward prediction error and drive reward-related behavior. However, the underlying synaptic mechanisms driving their activity is unknown. We obtained whole-cell recordings of identified midbrain dopamine neurons to measure subthreshold activity in vivo. Similar to their activity in vitro, dopamine neurons in vivo fire action potentials in a single spike pattern that is insensitive to synaptic noise, but can be driven by changes in synaptic activity state to fire bursts or pauses in firing. We identify two types of bursts: one driven by a depolarized state in membrane potential, and another driven by a rebound from a hyperpolarizated state. Rather than a single burst signature, dopamine neurons display different bursts elicited by separate classes of afferents.
Keywords: in vivo whole-cell
The diversity of dopamine signals in macaque monkeys (#395)
M. Matsumoto1, 2
1 University of Tsukuba, Faculty of Medicine, Tsukuba, Japan
Dopamine neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) are known to encode “reward prediction error”, and have been implicated in learning and motivation to obtain rewards. On the other hand, our recent studies in macaque monkeys have shown that a subgroup of dopamine neurons in the SNc transmits a signal related to the “salience” of external events rather than reward prediction error. These neurons are activated not only by rewarding stimuli but also by aversive stimuli and cognitively demanding stimuli. In this talk, I will introduce our recent data suggesting that these dopamine neurons regulate a cognitive ability, called “response inhibition”, to inhibit planned or ongoing motor actions that would lead to unwanted outcomes. We recorded single-unit activity from dopamine neurons in the SNc and VTA and neurons in the caudate nucleus while monkeys performed a saccadic countermanding task. In this task, after the monkey gazed a fixation point, the point disappeared and a saccadic target was presented. In 70% of the trials, the monkey was required to make a saccadic eye movement to the target. In the remaining 30%, the fixation point reappeared as a “stop signal” after the onset of the saccadic target. The monkey was required to cancel the planned saccadic eye movement. We found that dopamine neurons in the SNc, but not in the VTA, exhibited a significant excitation to the stop signal. This excitatory response decreased when the monkey failed to cancel planned saccadic eye movements. We also found that caudate neurons, which receive dopaminergic projections mainly from the SNc, exhibited a significant excitation to the stop signal as well. Furthermore, injecting haloperidol, D2 antagonist, into the caudate nucleus impaired the performance of canceling planned saccadic eye movements. These results suggest that the nigrostriatal dopamine pathway, mainly transmitting salience signal, regulates saccadic response inhibition.
Keywords: dopamine, monkey, response inhibition
Early dysfunctions of the striatonigral dopaminergic system in a rat model of Parkinson Disease (#435)
N. B. Mercuri1, 2, S. Kraschia1, 3, O. Riess4, N. Casadei4, A. Cordella1, V. Chirchiu1, 3, M. D'Amelio1, 3
1 Fondazione S Lucia , Rome, Italy
Using a rat model of Parkinson Disease (Nuber S. et al., Brain 2013) in which human α-synuclein (Syn) has been overexpressed in the brain, we have found, by using electrophysiological in vitro techniques that electrophysiological and pharmacological properties of dopaminergic neurons are altered. There is a reduction in firing activity, increase of AHP currents and rise of cytoplasmic calcium in substantia nigra dopaminergic cells of Syn animals. Interestingly, there is also an impairment of the stimulus-evoked DA release in the striatum as shown by in vitro amperometric experiments. Noteworthy, these changes were not detected in 2 months old Syn animals but were present in 4-5-month-old rats. Behavioral tests demonstrated altered motor performances in 4 months old animals.
These early changes are coupled with microglia activation and perturbations of inflammatory and pro-resolving mediators, namely IFN-y and resolvin D1 (RvD1).
Our results demonstrate early changes in the dopaminergic system associated with an imbalance of neuroinflammatory processes in PD animals.
Keywords: Dopaminergi neurons, Parkinson's Disease, neuroinflammation
A novel mouse model of CACNA1D-associated autism spectrum disorder (#393)
N. J. Ortner1, N. T. Hofer1, M. Kharitonova1, E. Paradiso2, P. Tuluc1, L. Guarina3, A. Sah1, L. Schwankler1, N. Stefanova4, F. Ferraguti2, N. Singewald1, E. Carbone3, J. Striessnig1
1 University of Innsbruck, Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, Innsbruck, Tirol, Austria
Voltage-gated Cav1.3 L-type Ca2+ channels (LTCCs) convert electrical activity into Ca2+-influx and comprise ~10% of total LTCCs in the brain where they shape cellular excitability, support pacemaker function and regulate Ca2+-dependent gene expression. Heterozygous Cav1.3-deficiency does not induce a detectable brain pathology in human or mice. In contrast, eight patients harboring germline de novo mutations in the CACNA1D gene (coding for the Cav1.3 α1-subunit) have been identified with neurodevelopmental disease including autism spectrum disorder (ASD). All of them induced pronounced channel gating changes with gain-of-function features when expressed in HEK293 cells. Therefore we predict a disease-causing enhanced Ca2+-influx through Cav1.3 mutant channels in vivo which raises the exciting opportunity of symptomatic treatment with clinically approved LTCC-inhibiting drugs.
We have successfully introduced the ASD-associated A749G CACNA1D mutation into C57Bl/6N mice. Electrophysiological recordings in adrenal chromaffin cells isolated from heterozygous mutant mice revealed the expected changes of Cav1.3 channel gating, which altered spontaneous cellular activity patterns. Using Western blot analysis we found similar expression levels of the Cav1.3 α1-subunit protein in the brain of wildtype and mutant mice. Male and female mutants showed a delayed gain of body weight – compatible with a developmental delay. In adult male mice we found highly reproducible and gene dose-dependent behavioral alterations such as a challenge-induced hyperlocomotion, reduced social interaction (three chamber social test), a mild anxiety-like phenotype (light-dark test) and reduced marble burying. However, gross brain morphology, the number of dopamine midbrain neurons, the density of cerebellar Purkinje cells, striatal volume and cortical layering were not altered in adult male mutant mice.
In summary, we confirm for the first time that the human ASD A749G CACNA1D mutation is sufficient to induce a neurodevelopmental phenotype including disease-relevant behavioral alterations. Our new mouse model is particularly attractive as the disease phenotype was already present in the heterozygous state without gross structural alterations in the brain – both reflecting the human patient situation. Therefore, it represents a feasible tool to identify Cav1.3-associated disease mechanisms and to test potential treatments with clinically available LTCC-inhibiting drugs such as isradipine.
Keywords: Cav1.3, autism spectrum disorder, calcium
Using human iPSC derived dopamine neurons to investigate physiological changes in Parkinson Disease (#438)
D. Beccano-Kelly1, Y. Mousba2, M. Cherubini1, S. Vingill1, P. Rai1, K. Cramb1, J. Vowles2, S. Cowley2, R. Wade-Martins1
1 University of Oxford, Department of Physiology, Anatomy and Genetics, Oxford, United Kingdom
Understanding the pathophysiological timeline of disease is paramount to developing cures and preventative measures for PD. Specifically, identifying when in the disease course the various phenotypes begin and, how they interact with one another, will allow the scientific community to ascertain which mechanisms can be targeted and critically, when. Many useful therapeutics could currently have their validity missed due to administration at the wrong time. Thus generation of a timeline from models mimicking this human disease is of the utmost importance.
With the advent of induced pluripotent stem cell (iPSC) technology, it is now possible to work from a known genetic ontology (using autosomal dominant genetic forms of PD as a model for the mechanisms of late onset PD) and interrogate pathological phenotypes and stress over time in human neurons. Thus using iPSC-derived neurons from carriers of disease-associated mutations in GBA genes, we have investigated phenotypes at several time points in order to map the pathophysiological disease timeline.
We have found that these mutations cause perturbation in calcium handling within intracellular neuronal stores; specifically in GBA lines, decreased calcium release from mitochondria. This reduced calcium would make generation of ATP harder from an early age (35 DIV). Using transcriptomic and western blot analysis, it was possible to identify changes in the level of key modulators of this calcium-signalling pathway (namely phospholipase D- 1; PLD-1 and calcium independent phospholipase A2; iPLA2) in GBA lines.
Concurrently, we have identified that iPSC neurons develop increased and sustained mature neuronal excitability and neurotransmission function by 70 DIV. The evolution and persistent nature of these functions will provide increased demand for ATP. We hypothesize that this unbalanced supply and demand will result in significant mitochondrial stress.
In line with this, we have found progressive alterations in mitochondrial membrane potential (MMP) over time in GBA lines, which only becomes significantly different at 70DIV and above. This change in MMP indicates perturbation of mitochondrial function. Similar investigations are ongoing in LRRK2 neurons.
These data present that altered mitochondrial calcium handling is a target for disease modulation specifically at early stages of the disease process.
Keywords: Electrophysiology, Intracellular calcium release, iPSC derived neurons