In the last few years, several strategies for silencing neurons optogenetically have emerged. The most popular approaches make use of two types of microbial light-driven ion pumps. One type is an optimized version of halorhodopsin, an inward chloride pump isolated from the halophilic bacterium Natronomonas pharaonis. Over the past couple years, halorhodopsin was tweaked by adding a series of trafficking signals to improve its addressing to the cytoplasmic membrane (Gradinaru et al, 2010). The latest version is eNpHR3.0. The other type corresponds to archeorhodopsins, which are outward proton pumps isolated from halophilic archaebacteria of the genus Halorubrum. The two popular archeorhodopsins are Arch from Halorubrum sodomense and ArchT from Halorubrum genus (Chow et al., 2010, Han et al., 2011). The same optimization tricks used for halorhodopsin also worked for Arch and ArchT. Both types of pumps generate a hyperpolarizing current in mammalian neurons in response to 500-600 nm light and are effective optogenetic silencers of neuronal activity.
A recent head-to-head comparison performed in the lab of K. Deisseroth has shown that, under matched experiments conditions, archeorhodopsins seem to perform slightly better than the best available halorhodopsin mutant (Mattis et al., 2012). In particular eArchT3.0 (an enhanced version of ArchT) generates about twice as much photocurrent as eNpHR3.0. This property is probably due to a higher expression of eArchT3.0 at the cytoplasmic membrane since eNpHR3.0 displays the highest photocurrent/fluorescence ratio (all these tools are fused to YFP to assess their expression level and addressing). The kinetics of both types of pumps are somehow comparable (eArchT3.0 photocurrents kick in a bit faster than eNpHR3.0) and so is their light sensitivity. In terms of desensitization, eArch3.0 and eArchT3.0 exhibit a more severe dropoff than eNpHR3.0 during prolonged illumination with the same optical power. But when the power is adjusted to obtain matched photocurrent amplitudes, eArch3.0 photocurrents decay more slowly than eNpHR3.0 photocurrents.
Cell-attached recordings of CA3 pyramidal neurons expressing eNpHR3.0-EYFP (left) or Arch-GFP (right). The recordings show synaptically evoked spiking before and after activation of the silencer (15 s, 532 nm, 7.9 mW mm−2 for eNpHR3.0 and 76.1 mW mm−2 for Arch). Spike probability was set to approximately 0.4 before laser activation (measured over ten trials). The before stimulus was delivered 1,250 ms before laser onset and the after stimulus was delivered 250 ms after laser offset.
In an upcoming issue of Nature Neuroscience, the team of Colin Akerman in Oxford drops a little bombshell in the field of optogenetic silencers (Raimondo et al., 2012). They show that the reasons to prefer archeorhodopsins over halorhodopsins might go beyond considerations of photocurrent, kinetics etc. Using gene gun expression of eNpHR3.0 and Arch in rat hippocampal organotypic slices, they show that brief episodes of eNpHR3.0 activation tend to increase the probability of spiking in response to a volley of presynaptic action potentials, while Arch activation do not. The experiment was performed as follows: eNpHR3.0- or Arch-expressing CA1 and CA3 pyramidal neurons were recorded using the cell-attached configuration of the patch clamp technique. Postsynaptic action potentials were evoked 12.50 s before or 0.25 s after a 15 s light pulse (532 nm, 20 mW/mm2), using a brief electrical stimulation of the Schaffer collateral pathway. The electrical stimulation was set in order to achieve an average postsynaptic spike probability of 0.4 before the light pulse. In these conditions, the spike probability after the light pulse was around 0.8 for eNpHR3.0-expressing cells and was unchanged for Arch-expressing cells. The amount of hyperpolarizing photocurrent evoked by the light pulse was comparable for both pumps (235 and 237 pA respectively) and can therefore not be held responsible for the difference in this after-pulse increased excitability.
The main difference between eNpHR3.0 and Arch is that the first one translocates chloride into the cytoplasm while the other one extrudes protons in the extracellular space. In other words, eNpHR3.0 accumulates chloride intracellularly and Arch tends to alkalinize the cytoplasm. Raimondo et al. showed that the post-hyperpolarization effect observed with eNpHR3.0 results most probably from a collapse in the Cl- transmembrane gradient which causes a depolarizing shift of the reversal potential of Cl- (ECl-). In other words, inhibitory post-synaptic currents evoked by the Schaffer collateral stimulation through feedforward and/or feedback inhibition mechanisms (and carried by the Cl- conducting GABA-A receptors) would be less effective at hyperpolarizing the neurons in the after-pulse condition compared to the pre-pulse condition. Using the perforated patch technique (in order to preserve the ECl- of the cell), the authors showed that a 15 s activation of eNpHR3.0 producing an hyperpolarization comprised between 90 and 400 pA induced a 20 mV depolarizing shift of ECl- (from around -70 mV to around -50 mV). This was sufficient to turn hyperpolarizing GABA puffs into depolarizing ones at resting potential. The rate of recovery of ECl- after eNpHR3.0 activation had a time constant of around 15 s.
In summary, the use of eNpHR3.0 can significantly affect GABAergic synaptic transmission during and after prolonged tissue illumination. So take into account this parameter when using this silencer in your experiments! Let’s wait and see if the inward proton flux in the case of archeorhodopsins also cause unwanted side effects… Stay tuned.
Read also: When the electricity (and the lights) go out: transient changes in excitability, by Emily Ferenczi & Karl Deisseroth.