2. Use of Optogenetics Techniques to Discover Pathway-specific Feedforward Circuits Between Thalamus and Neocortex

All brain functions involve coordinated neural activities in many brain regions. Therefore, it is important to understand how different brain regions interact with each other. One particularly important coupled system in the brain is the neocortex and the thalamus. The neocortex, the thalamus, and the axonal tracts that interconnect these structures comprise the vast majority of the mammalian brain and are crucial for sensation, perception and consciousness.  Thalamocortical (TC) pathways provide the major extrinsic input to neocortex, and corticothalamic (CT) pathways are a principal source of synaptic input to thalamus.  These pathways are entwined, making their study challenging by conventional electrical stimulation methods. We used cell- specific expression of channelrhodopsin-2 (ChR2), a light-sensitive cation channel, in either thalamocortical or corticothalamic projection cells to manipulate the activity on  these tracts and study their effects on their target locations in mouse brain slices.

Viral delivery of ChR2

Lentiviruses carrying fusion genes for ChR2 and fluorescent proteins (pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE) were injected into ventrobasal thalamic complex (VB) or the barrel cortex of ICR or GIN mice in vivo, between postnatal days 8 and 15. Typical viral titers were 1010 IU/ml. Injection volumes were between 0.3 and 2 µl. After allowing 1–3 weeks for ChR2 expression, acute somatosensory thalamocortical or horizontal brain slices (300 µm thick) were prepared for in vitro recording and stimulation (Figure 1).

Selective labeling of TC and CT pathways
The viral injections were made at two different locations to selectively express ChR2 in either TC or CT projections. The injections into ventrobasal thalamus produced ChR2/EYFP expression in TC relay cells, including their axonal projections within the cortex (Figure 1). As a result of ChR2 expression, the thalamic cells responded to laser stimulation by bursts of spikes (Figure 2).

On the other hand, injections into barrel cortex produced ChR2/EYFP expression in cortical neurons, including CT projection cells and their axons within ventrobasal thalamus and thalamic reticular nucleus (TRN) (Figure 3A and B). The optical stimulation of neurons in barrel cortex generated spike responses due to their ChR2 expression (Figure 3C and D).

Effects of TC activity on cortical cells
When cortical neurons were recorded in areas targeted by ChR2-expressing TC axons, synaptic responses could be evoked by local laser stimulation of those axons. We recorded from a variety of neurons in cortical layers 4 and 5/6, including regular-spiking (RS), fast-spiking (FS), and low threshold-spiking (LTS) cells. RS cells are glutamatergic excitatory neurons (mainly spiny stellate in layer 4 and pyramidal in other layers. FS and LTS cells are GABAergic inhibitory interneurons; LTS cells are sometimes called ‘‘regular spiking nonpyramidal’’. Each cell type has characteristic morphology, protein expression, synaptic connectivity, short-term synaptic dynamics, and intrinsic physiology which helps to identify them. In all of these cells, optical stimulation of ChR2 expressing TC arbors produced excitatory potentials (Figure 4).

These excitatory thalamic synapses onto cortical neurons can drive spiking in cortical inhibitory interneurons. Since these interneurons synapse make many local synapses, TC input is able to produce powerful feedforward inhibition in surrounding cells.  In accordance with this, we found that laser stimulation of ChR2-expressing TC arbors nearly always produced feedforward inhibition (inhibition was observed in 63/67 cortical cells tested; Figure 5).

Effects of CT activity on thalamic cells

The effects of ChR2 expressing CT arbors on VB and TRN consisted of fast excitation in VB and TRN followed by a feedforward inhibition only in VB (Figure 6). TRN projections to VB is known to be inhibitory. Therefore, the CT projections trigger an early excitation in both VB and TRN. As a result, the activity in TRN generate feedforward inhibition in VB.

Our results demonstrate the efficacy of the ChR2 optical method for selectively stimulating axonal pathways in thalamocortical systems.  Lentivirus delivery of ChR2/EYFP yielded strong expression in local neurons near injection sites, and in their extended axons, but did not lead to retrograde infection or expression.  This made it possible to selectively activate projection pathways with light.  We used this technique to analyze the specific neuronal targets engaged by TC and CT projections, including feedforward inhibitory circuits.  We observed pathway-specific and cell type-specific differences in processing mechanisms.  Our results indicated that, functionally, the TC system contains strong feedforward inhibition on inhibitory neurons whereas, in the CT system, feedforward inhibition on inhibitory neurons are weak.