Author contributions: M.Y.C., L.E.F., and G.K.S. designed research; M.Y.C., E.H.W., W.J.W., S.W., G.S., A.G.L., and A.A. performed research; A.G.L. and K.D. contributed new reagents/analytic tools; M.Y.C. and G.K.S. analyzed data; and M.Y.C. and G.K.S. wrote the paper.

Edited by Anders Bjorklund, Lund University, Lund, Sweden, and approved July 17, 2014 (received for review March 3, 2014)

Significance

Stroke is the leading cause of disability in the United States and has very limited treatment options. Brain stimulation techniques that promote recovery after stroke are a promising area of research; however, current stimulation techniques nonspecifically activate/inhibit the target area, which not only leads to undesired side effects but also makes it difficult to understand which cell types and mechanisms drive recovery. We used the optogenetic technique to specifically stimulate only neurons after stroke and demonstrate that selective neuronal stimulations can activate beneficial mechanisms and promote recovery. Understanding the cell type and mechanisms driving recovery may identify potential drug targets for stroke treatment, as well as ultimately help develop precise brain stimulation techniques for stroke therapy.

Keywords: stroke recovery, channelrhodopsin

Significance

Abstract

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area’s excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery.

Abstract

Stroke is a major acute neurological insult that disrupts brain function and causes neuron death. Functional recovery after stroke has been observed and is currently attributed to both brain remodeling and plasticity (1 –4). Structural and functional remodeling of areas next to an infarct or remote regions can alter signaling within bilateral neuronal networks and thus contribute to functional recovery (3 –7). Rewiring of neural connections is mediated by electrical activity, which can activate a number of plasticity mechanisms, including the release of activity-dependent neurotrophins such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) (8 –10). Both BDNF and NGF have been shown to improve recovery by enhancing axonal and dendritic sprouting (10 –12).

Tremendous effort has been focused on promoting recovery after stroke, including pharmacological treatment, rehabilitation (e.g., constraint-induced therapy), stem cell transplantation, and brain stimulation (1, 4, 13). Brain stimulation is a promising area of research because it allows direct activation and manipulation of the target area’s excitability (14 –16). The primary motor cortex (M1) is a commonly stimulated area as it directly innervates the corticospinal tract to initiate movement (1, 7). Although electrical stimulation and transcranial magnetic stimulation show promise in promoting recovery (17, 18), these techniques are limited by imprecision and indiscriminate activation or inhibition of all cell types near the stimulated site; thus, they can produce undesired effects such as psychiatric and motor/speech problems (19 –21). In addition, it has been difficult to elucidate the cell type and mechanisms driving recovery, as multiple cell types such as neurons, astrocytes, and oligodendrocytes have been shown to contribute to remodeling and recovery processes after stroke (5, 22 –27).

To elucidate whether activation of neurons alone can promote recovery, we used optogenetics to selectively manipulate the excitability of specific cell groups with millisecond-scale temporal precision in a manner more similar to endogenous neuronal firing patterns (21, 28, 29). This technique uses light-activated microbial proteins such as Channelrhodopsin 2 (ChR2), which depolarizes neurons when illuminated with blue light, or Halorhodopsin (NpHR), which hyperpolarizes neurons (21, 28, 29). Optogenetic approaches have been used in rodents to probe neuronal circuits for several neurological/neurodegenerative diseases, including Parkinson disease (30) and epilepsy (31). Recent studies have also used optogenetics to map functional organization after stroke (32 –35). The safety and efficacy of using optogenetics in nonhuman primates has also been characterized (29, 36).

In this study, we used optogenetics to selectively stimulate neurons in layer V of the ipsilesional primary motor cortex (iM1) and examine the effects of repeated neuronal stimulations in normal and stroke mice. Sensory-motor behavior tests were used to evaluate functional recovery after stroke, and plasticity-associated mechanisms, such as cerebral blood flow (CBF)/neurovascular coupling responses and activity-dependent neurotrophin expression, were investigated.

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Acknowledgments

We thank Robin Lemmens, Paul Kalanithi, and Marion Buckwalter for helpful discussions; Cindy H. Samos for scientific editing of the manuscript; Charu Ramakrishnan and Maisie Lo for their helpful technical discussions on optogenetic-related techniques; and Corinne Bart, Alex Bautista, and Aatman Shah for their technical assistance in some of the experiments. This work was supported in part by National Institutes of Health National Institute of Neurological Disorders and Stroke Grant 1R21NS082894, Russell and Elizabeth Siegelman, and Bernard and Ronni Lacroute (G.K.S.). G.K.S. is a member of the Medtronic Neuroscience Strategic Advisory Board.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1404109111/-/DCSupplemental.

Footnotes

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