Steinberg Lab Research: Optogenetics

Stroke is one of the major acute neurological insults that disrupts brain function and causes neuron death. Most stroke survivors suffer long-term deficits ranging from motor/sensory dysfunction to speech or memory loss, depending on where the stroke occurred in the brain. Functional recovery has been observed in animal and human studies of stroke, which is currently attributed to both brain remodeling and plasticity. The mechanisms that drive this recovery, however, are unclear. Elucidating the mechanisms of recovery is critical to identifying effective therapies for stroke.

One promising strategy is brain stimulation because it allows direct activation and manipulation of the target area’s excitability. Transcranial direct current stimulation (tDCS) and transcranial magnetic stimulation (TMS) in primary motor cortex (M1) have been shown to promote recovery after stroke in both human and animal models. However, these techniques are imprecise and activate/inhibit all cell types around the target area, thus can induce lingering and undesired side effects such as psychiatric and motor or speech problems. Furthermore, it is unclear what repair and plasticity mechanisms are being targeted by these techniques.

Optogenetics can overcome some of these shortcomings. This technique utilizes single-component microbial proteins such as Channelrhodopsin 2 (ChR2), which is a cation channel that depolarizes neurons when illuminated with blue light, or Halorhodopsin, which is a chloride pump that hyperpolarizes neurons when illuminated with yellow laser. By expressing these light-sensitive proteins in the cell type of interest, this allows selective stimulation and inhibition of specific cell types, as well as fast and precise millisecond manipulation of excitability.  Our research goals are (1) to study brain/circuit activation dynamics during stroke recovery (2) to investigate the effects of specific circuit manipulations on post-stroke recovery and (3) to determine the underlying repair/plasticity mechanisms that drive recovery.  We will use a combination of optogenetics, cutting edge imaging techniques and next generation sequencing to address these questions.

We recently demonstrated that selective neuronal stimulation in the ipsilesional primary motor cortex (iM1) can promote functional recovery after stroke (Cheng et al., 2014). We use Thy-1-ChR2-YFP transgenic mice that express high levels of ChR2 in layer V pyramidal neurons of M1 and an intraluminal suture model to generate infarct in the striatum and somatosensory cortex (Fig. 1). Stroke mice that received repeated iM stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response (Fig. 2), as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin 3 (NTF3) (Cheng et al., 2014).  Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of the plasticity-associated marker GAP43 (Cheng et al., 2014). Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests  (Fig. 3).  

Figure 1.  Optogenetic stimulation of the ipsilesional motor cortex

 (A) High expression of Thy1:ChR2-YFP in layer V pyramidal neurons of primary motor cortex (M1). Scale bar = 100 µm. (B) Diagram indicates infarct (striatum and somatosensory cortex) and fiber implant location (ipsilesional motor cortex). (C) Stimulation paradigm with includes 3 successive 1-min laser stimulations (blue bars) separated by 3-min rest intervals. 

Figure 2:  Repeated iM1 neuronal stimulations improved CBF and the neurovascular coupling response after stroke

Changes in CBF in response to a 1 min stimulation (blue bars) on either cM1 or iM1 were measured in stimulated and non-stimulated stroke mice at post-stroke day 15. At post-stroke day 15 both stimulated and non-stimulated stroke mice exhibited a similar neurovascular coupling response in the cM1, but only the stimulated stroke mice exhibited significant improvement of the neurovascular coupling response in the iM1. *P < 0.05, **P < 0.01, ***P < 0.001, Two-way ANOVA with Bonferroni’s posthoc test. n = 4–6 per group.

Figure 3.  Repeated iM1 neuronal stimulations improved functional recovery

(A) Stimulated stroke mice regained their body weight significantly faster than non-stimulated stroke mice at post-stroke day 14. Left:  Timecourse of body weight changes.  Right:  Average of percent body weight change during the stimulation period (*P < 0.05, Student’s t-test).  Stimulated mice performed significantly better in the rotating beam test, with a longer distance traveled (B) and a faster speed (C).  *P < 0.05, **P < 0.01, significant difference between stim and non-stim group, Two-way RM ANOVA with Fisher's LSD. Sham: n = 8, non-stim: n = 16, stim: n = 21.  Stimulation has no effect on distance traveled (D) or speed (E) in normal mice.  n = 6 per group.

In search of an optimal brain stimulation target, we have also stimulated a cerebellar target called the cerebellar dentate nucleus (cDN).  Post-stroke stimulations of cDN also promote recovery and the effect is permanent, as the mice continued to recover even after stimulations had stopped (Fig. 4).  Current studies focus on determining the underlying mechanisms of cDN-stimulation enhanced recovery using next generation sequencing.

Figure 4.  Optogenetic stimulations in the cerebellar dentate nucleus (cDN) promotes persistent post-stroke recovery

A) cDN stimulation can activate the dentato-thalamic-cortical pathway, resulting activation in the premotor, motor and sensory cortices.  B) Repeated cDN stimulations promotes functional recover.  cDN-stimulated mice exhibit persistent recovery (short stim) as mice continue to recover after stimulations had stopped.  

Results from our studies will advance the understanding of post-stroke neural circuit remodeling and molecular mechanisms driving recovery.  Our studies will also provide important insights into which brain/circuits may be important to target for designing brain stimulation strategies in future clinical studies.

Cheng MY, Woodson WJ, Wang EH, Wang S, Sun G, Lee AG, Arac A, Fenno L, Deisseroth K, Steinberg GK.  Optogenetic neuronal stimulation promotes functional recovery after stroke.  Proceedings of the National Academy of Sciences 2014 Sep 2;111(35):12913-8.