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. 2016 Jan 1;115(1):470-85.
doi: 10.1152/jn.00858.2015. Epub 2015 Nov 4.

Anatomical localization of Cav3.1 calcium channels and electrophysiological effects of T-type calcium channel blockade in the motor thalamus of MPTP-treated monkeys

Annaelle Devergnas  1 , Erdong Chen  2 , Yuxian Ma  2 , Ikuma Hamada  2 , Damien Pittard  2 , Stefan Kammermeier  3 , Ariana P Mullin  4 , Victor Faundez  5 , Craig W Lindsley  6 , Carrie Jones  6 , Yoland Smith  7 , Thomas Wichmann  7
Affiliations

Anatomical localization of Cav3.1 calcium channels and electrophysiological effects of T-type calcium channel blockade in the motor thalamus of MPTP-treated monkeys

Annaelle Devergnas et al. J Neurophysiol. .

Abstract

Conventional anti-Parkinsonian dopamine replacement therapy is often complicated by side effects that limit the use of these medications. There is a continuing need to develop nondopaminergic approaches to treat Parkinsonism. One such approach is to use medications that normalize dopamine depletion-related firing abnormalities in the basal ganglia-thalamocortical circuitry. In this study, we assessed the potential of a specific T-type calcium channel blocker (ML218) to eliminate pathologic burst patterns of firing in the basal ganglia-receiving territory of the motor thalamus in Parkinsonian monkeys. We also carried out an anatomical study, demonstrating that the immunoreactivity for T-type calcium channels is strongly expressed in the motor thalamus in normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. At the electron microscopic level, dendrites accounted for >90% of all tissue elements that were immunoreactive for voltage-gated calcium channel, type 3.2-containing T-type calcium channels in normal and Parkinsonian monkeys. Subsequent in vivo electrophysiologic studies in awake MPTP-treated Parkinsonian monkeys demonstrated that intrathalamic microinjections of ML218 (0.5 μl of a 2.5-mM solution, injected at 0.1-0.2 μl/min) partially normalized the thalamic activity by reducing the proportion of rebound bursts and increasing the proportion of spikes in non-rebound bursts. The drug also attenuated oscillatory activity in the 3-13-Hz frequency range and increased gamma frequency oscillations. However, ML218 did not normalize Parkinsonism-related changes in firing rates and oscillatory activity in the beta frequency range. Whereas the described changes are promising, a more complete assessment of the cellular and behavioral effects of ML218 (or similar drugs) is needed for a full appraisal of their anti-Parkinsonian potential.

Keywords: Parkinsonism; T-type calcium channel; nondopaminergic therapy; rebound burst.

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Figures

Fig. 1.
Fig. 1.
Examples of immunolabeling to reveal tyrosine hydroxylase in the basal ganglia of a normal monkey (A) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkey (B; monkey C in this study). In both cases, a coronal section, corresponding to the anterior commissure (AC) 7.7-mm plane in the macaque atlas by Lanciego and Vazquez (2012), is shown. CN, caudate nucleus; Put, putamen; SN, substantia nigra.
Fig. 2.
Fig. 2.
A–E: light micrographs of coronal brain sections showing immunostaining for voltage-gated calcium channel, type 3.1 (Cav3.1) in the ventrolateral and ventroposterior thalamus of a normal (A) and MPTP-treated monkey (B and C). The overall intensity of labeling is similar across the ventral motor [ventral anterior nucleus magnocellularis (VAmc), ventral anterior nucleus parvocellularis (VApc), ventral lateral nucleus oral part (VLo)] and somatosensory [ventral posterolateral (VPL), ventral posteromedial (VPM)] thalamic nuclei. D and E: high-power views of Cav3.1 neutrophil immunostaining in VApc in a normal (D) and MPTP-treated (E) monkey. Original scale bars, 2 mm (A; also applies to B and C); 50 μm (D; also applies to E). F: Western blot showing the specificity of the NeuroMab Cav3.1 antibody when applied on monkey thalamic (T) and striatal (S) tissue. A single band corresponding to the predicted molecular weight of Cav3.1 protein (∼250 kD) is shown in this immunoblot. The lower band corresponds to beta-actin probing that was used as a loading control. Note the higher amount of Cav3.1 protein in the thalamic tissue compared with the striatal tissue. Rt, reticular nucleus; VL, ventral lateral nucleus; CM, centromedian nucleus.
Fig. 3.
Fig. 3.
Electron micrographs of Cav3.1-immunoreactive neuronal elements in the basal ganglia-receiving area of the thalamus of normal and MPTP-treated monkeys, as revealed with the pre-embedding immunoperoxidase (A–C) and immunogold methods (D). Note that the labeling is mostly expressed in dendrites of various sizes. Note also that the gold particles are associated with the plasma membrane (D). E: distribution of Cav3.1-labeled elements in the VApc of normal (black bars) and MPTP-treated (white bars) animals. F: distribution of Cav3.1-labeled dendritic profiles, separated by size. Values are means ± SE. There were no differences between the normal and Parkinsonian animals (tested with Mann-Whitney tests). Ax, axon; Den, dendrite. Original scale bars, 0.5 μm (A; also applies to B); 0.1 μm (C; also applies to D).
Fig. 4.
Fig. 4.
Location of thalamic neurons recorded in the normal (black circles) and post-MPTP states, after local microinjections of artificial cerebrospinal fluid (aCSF; white circles) or ML218 (gray circles). Delineation of nuclear boundaries and tracks in the thalamus are based on Nissl staining and the sagittal and coronal atlas of the macaque brain (Lanciego and Vazquez 2012). A: sagittal views of the location of neurons studied in monkeys A and B. A′: example of track reconstruction based on Nissl staining of a slice in the L1 plane (Lanciego and Vazquez 2012). B: coronal views of the location of neurons studied in monkey C. Lat, lateral; AM, anteromedian nucleus; Re, reticular nucleus. Original scale bars, 1 mm.
Fig. 5.
Fig. 5.
Effect of MPTP and ML218 on thalamic activity. Examples of 10 s of thalamic recordings and corresponding power spectra [normalized to the 1–100-Hz range; nonequispaced fast Fourier transform (nfft): 11; bin size: 0.49], before (A) and after (B) MPTP treatment. Same cell before (B1) and after (B2) ML218 injection. Horizontal lines above the recordings indicate events identified as burst. Horizontal lines with a black dot denote rebound bursts, and the 1 burst with an asterisk (*) was identified as a low-threshold spike (LTS)-like burst (see materials and methods). C: example of non-rebound burst (C1), rebound burst (C2), and LTS-like burst (C3). Original scale bars, 1 s (A; also applies to B); 0.1 s (C1; also applies to C2); 0.02 s (C3).
Fig. 6.
Fig. 6.
Effect of MPTP and ML218 on thalamic activity. A: firing rate in normal animals (black bars), Parkinsonian animals (white bars), and Parkinsonian animals after ML218 injection (at the random effect period; gray bars). B: averaged burst surprise value for all bursts. C: proportion of burst subtypes among all bursts. D: proportion of spikes in bursts for each burst subtype. E: average power spectra (normalized to the 1–100-Hz range; nfft: 11; bin size: 0.49) for the normal (black circles) and MPTP-treated (white circles) animals. Values are means ± SE compared with a Mann-Whitney rank sum test (*P < 0.05, **P < 0.01, and *** P < 0.001).
Fig. 7.
Fig. 7.
A: example trace of a thalamic neuron that increased its firing rate in response to ML218 injections. Green bar, 60-s control period; red bar, drug injection; black bar, maximal effect period; solid horizontal line, median firing rate at baseline; dashed lines, 90th and 10th percentiles; solid vertical line, maximal effect; vertical dashed line, moment at which the effect was considered to have started. B: delay of the onset of the ML218-induced effects and the timing of the maximal effect (both measured from the start of the injection), shown for cells with firing-rate decreases (black-striped bars) and cells with firing-rate increases (white-striped bars). Values are means ± SE compared with a Mann-Whitney rank sum test.
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