Deciphering the Functional Connectome of the External Globus Pallidus with Electrical and Optogenetic Deep Brain Stimulation-fMRI
Daniel Albaugh1, Nathalie Van Den Berge2, Andrew Salzwedel3, Wei Gao3, Garret Stuber4, and Yen-Yu Ian Shih5

1Curriculum in Neurobiology, UNC-Chapel Hill, Chapel Hill, NC, United States, 2University of Ghent, Ghent, Belgium, 3Cedars-Sinai Medical Center, Los Angeles, CA, United States, 4Psychiatry, UNC-Chapel Hill, Chapel Hill, NC, United States, 5Biomedical Research Imaging Center, UNC-Chapel Hill, Chapel Hill, NC, United States

Synopsis

In this study, we unraveled the circuit and network connectivity of the rodent external globus pallidus (GPe), both in the healthy animals and a parkinson's disease model. We also employed multiple stimulation types (electrical and optogenetic), as well as fMRI modalities (evoked-fMRI and functional connectivity analyses) to provide an exhaustive analysis of this dynamic brain nucleus.

Purpose

The external globus pallidus (GPe) has been historically described as a simple relay nucleus through the basal ganglia, receiving striatal input and projecting output through the subthalamic nucleus1. However, recent anatomical studies have demonstrated newfound complexity in GPe connectivity, highlighting its potential role as far more than a relay nucleus2-3. In sum, such studies are providing a paradigm shift in our evaluation of the basal ganglia’s circuit architecture, necessitating closer evaluation of the functional connectivity patterns among its constituent nuclei. Complementary to anatomical studies, circuit stimulation approaches provide an ideal means for mapping the dynamic output patterns of target nuclei, including the directionality of downstream modulation (excitation or inhibition), and alterations in healthy vs. disease states. When combined with unbiased fMRI readouts, brain stimulation tools can be employed to map the global functional connectomes of brain nuclei with a high degree of circuit recruitment specificity. Here, we first used electrical stimulation of the GPe (at a range of stimulation frequencies) combined with evoked and functional connectivity (fc)-fMRI to map the functional connectome of the GPe in healthy rodents. Next, we employed optogenetic tools to more selectively stimulate GPe neurons, mapping the resultant downstream circuit modulation with evoked-fMRI. Finally, we compared the optogenetically-evoked modulation patterns in healthy rodents to those obtained by optogenetically stimulating the GPe in parkinsonian rats. Our results, detailed below, uncover a bewildering complexity in the GPe’s functional connectome, sensitive to stimulation type and frequency, as well as healthy vs. diseased state.

Methods

Wildtype Sprague-Dawley rats were either implanted with a tungsten microwire electrode unilaterally targeting the GPe for electrical stimulation studies (n=7), or prepared for optogenetic-fMRI studies (n=12) via microinjections with an adeno-associated virus (AAV) carrying the gene encoding channelrhodopsin-2 (ChR2), fused to an enhanced yellow fluorescent protein (EYFP) or only EYFP (synapsin promoter). A subset of animals also received ipsilateral microinjections of 6-hydroxydopamine targeting the medial forebrain bundle, the gold-standard approach for inducing experimental parkinsonism in rodents3-4. Electrical GPe stimulation was conducted using a current amplitude of 300uA, 500us pulse duration. Optogenetic GPe stimulation was conducted under the following parameters: 10 ms pulse width, 40Hz, 473-nm wavelength, 16-20mW light pulses. Each rat was endotracheally intubated and ventilated with 0.5% isoflurane and medical air. Dexmedetomidine (0.1 mg/ml) and pancuronium bromide (1.0 mg/ml) were infused intravenously for duration of scan. For CBV-weighted MRI, a tail-vein catheter was used to deliver monocrystalline iron oxide contrast agent at a dose of 30 mg Fe/kg. Single shot, single sampled GE-EPI sequences (BW= 300 kHz, TR= 1000 ms, TE= 8.107 ms, 80x80 matrix, FOV= 2.56 x 2.56 cm2, slice thickness= 1 mm) were acquired using a Bruker 9.4T MR scanner and home-made surface coil. Automatic co-registration using SPM codes were applied to realign time-series data within subjects and then again across subjects. Data were analyzed using our established pipelines detailed elsewhere5-7.

Results

The experimental setups for electrical and optogenetic stimulation of the GPe are shown in Fig 1a and 2a, respectively. Electrical stimulation of the GPe resulted in widespread modulation of evoked- and fcMRI signals (Fig 1b-d). Notable findings from our evoked-fMRI dataset include robust CBV increases of the ipsilateral prefrontal cortex, as well as bidirectional, stimulation frequency-dependent modulation of striatal CBV signals (negative at 10Hz, positive at 70Hz and above). fcMRI provided evidence of robust, frequency-dependent global network modulation by electrical GPe stimulation, with more robust network changes observed with 130Hz compared to 40Hz stimulation. Compared to electrical stimulation, optogenetic stimulation at 40Hz resulted in a more spatially restricted pattern of modulation, with prominent CBV increases locally within the GPe, and CBV decreases in the neighboring dorsal striatum (Fig 2b-c). Surprisingly, in Parkinsonian rats, optogenetically-evoked striatal vasoconstriction downstream was substantially reduced. Moreover, a unique prefrontal negative CBV signal emerged during GPe stimulation in these subjects.

Discussion

This study provides an in-depth characterization of the functional connectivity of the GPe, on circuit and network-levels, as well as in healthy and parkinsonian rodents. Our data clearly do not conform to the traditional model of the GPe as a straightforward relay nuclei; modulation of several noncanonical downstream circuits was observed, including prefrontal cortex and striatum. Our optogenetic experiments of GPe connectivity in the healthy and parkinsonian states was prompted by a number of studies demonstrating basal ganglia rewiring in the parkinsonian state3-4. Our finding of reduced striatal vasoconstriction during GPe stimulation in parkinsonian animals is strongly suggestive of pallidostriatal circuit rewiring in the dopamine-depleted state. Future studies will address the electrophyiological correlates of this disease-altered striatal fMRI signal.

Acknowledgements

We thank Jon Frank and Joseph Merill of theUNC Biomedical Research Imaging Center (BRIC) Small Animal Imaging (SAI) facility for technical assistance. We also thank members of the Shih laboratory for valuable discussions concerning the experiments described in this manuscript. D.L.A. was supported by the National Institute of Neurological Disorders and Stroke (NINDS)(NS087909). N.V.D.B. was supported by the Research Foundation Flanders. G.D.S. was supportedby the Klarman Family Foundation, the Brain and Behavior Research Foundation, the Foundation for Prader-Willi Research, the Foundation of Hope, the National Institute on Drug Abuse (DA032750 and DA038168), and the Department of Psychiatry at UNC Chapel Hill. W.G. was supported by the NINDS (NS088975) and Cedars-Sinai Medical Center. Y.Y.I.S. was supported by the NINDS(NS091236), the National Institute of Mental Health (MH106939), the National Institute on Alcohol Abuse and Alcoholism (AA020023), the National Institute of Health UL1TR001111 sub-awards 550KR81420 and 550KR91413, the Brain and Behavior Foundation Young Investigator Award and Ellen Schapiro & Gerald Axelbaum Investigator fund, the American Heart Association Scientist Development Award (15SDG23260025), and the Department of Neurology and the Biomedical Research Imaging Center at UNC Chapel Hill.

References

[1] Wichmann and Delong, Curr Opin Neurobiol 1996; 6(6):751-8.[2] Saunders et al., Nature 2015; 521(7550):85-9. [3] Mallet et al., Neuron 2012; 74(6):1075-86. [4] Gittis et al., Neuron 2011; 71(5):858-68. [5] Shih et al., J Neurosci 2009; 29(10):3036-44. [6] Shih et al., J Cereb Blood Flow Metab 2014; 34(9): 1483-92. [7] Elton and Gao, Cortex 2014; 51:56-66.

Figures

Fig.1. fMRI mapping of the GPe using an electrical stimulation approach. a. Electrode targeting. b. CBV functional activation maps at 40 vs. 130Hz GPe stimulation. c. Temporal dynamics and amplitudes of CBV modulation by GPe stimulation at varying frequencies. d. Cross-correlational functional connectivity matrices; network modulation by GPe stimulation

Fig.2. fMRI mapping of the GPe using an optogenetic stimulation approach: comparison of healthy and parkinsonian rats. a. Schematic of viral and 6-OHDA microinjections b. CBV functional activation maps of 40Hz GPe-ChR2 stimulation in healthy and parkinsonian rats. c. Temporal dynamics and amplitudes of CBV modulation by 40Hz optogenetic stimulation in ChR2 and EYFP control subjects.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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