Monday, November 5, 2012

Annals of underwhelming papers: Channelrhodopsinning

While I am no Boyden-level expert in channelrhodopsin, I have photostimulated neurons in the olfactory bulb, and now am trying to use the delightfully named PINP technique to record from specific layers of cortex. Given this rudimentary experience, I am increasingly frustrated by the sloppiness of optogenetic research in good journals. Today I would like to highlight four articles that used channelrhodopsin sub-optimally. (To be 100% clear, I am not commenting on the quality of these papers as a whole, or the researchers who conducted the experiments. I am merely saying that I think the optogenetic experiments could have been done better).

Two Pillars of Channelrhodopsin Photostimulation

In the channelrhodopsin experiments I have done so far, I have identified two keys to channelrhodopsin use, which I have dubbed the Two Pillars of Channelrhodopsin Photostimulation.*

The first pillar is to always perform negative controls. Light is energy, which means light can cause heat. Light can heat tissue, but Michale Fee even thinks that metal electrodes can heat up if stimulated long enough. For example, in a set of experiments I did last fall, I photostimulated neuromodulatory neurons while recording from the olfactory bulb, and saw some interesting changes in activity. When I performed the histology, I found out that the infection had not taken, and what I was witnessing was a heat-induced artifact. To control for heat during photostimulaiton, you must do two things: first, reduce your stimulation power and time as much as possible to minimize the risk of heat artifacts; and second, always, ALWAYS perform negative controls in non-channelrhodopsin animals.

* Back in my two-photon days, it was easy to put your slice under the microscope, turn to the computer, and not see an image. 90% of the time this was due to a few routine mistakes, so whenever I taught people how to use the microscope, I invented a Buddhist-style "Four Pillars of Two-photon Imaging." (Move the filter wheel, close the fluorescence shutter, turn down the fluorescence lamp, and switch the imaging mirror.)

The second pillar is to pulse your light. Channelrhodopsin is a non-specific cation channel. This means that when the channel initially opens, the increase in conductance causes a large depolarization. However, if you keep the channel open, you have no idea what's going on. Some neurons will continue to happily fire action potentials, while others will go silent. You could even, theoretically, get shunting inhibition from the increased conductance. To avoid these problems, and have a better idea about what your stimulated neurons are doing, you should pulse your photostimulation for 1-10 ms, and stimulate at 1-50Hz (or even higher if you have a new fancy channelrhodopsin). Pulsed light stimulation also helps avoid heat artifacts.

Given these pillars, I'd like to highlight four papers in high profile journals from big name labs that did not use them. Hopefully I don't stick my foot in my mouth.

Poulet et. al., 2012


From the Petersen lab at EPFL, this paper followed neither pillar. They recorded intracellulary from neurons in barrel cortex while photostimulating thalamic neurons expressing ChR2. When they stimulated the thalamus, the used a 5 second ramped light pulse.

Intracellular recording from barrel cortex during photostimulation of the thalamus. Photostimulation caused a change in the frequency of membrane fluctuations.
From Figure 3, Poulet et. al., 2012.
In the supplementary information, they tried to justify... something by showing that a stepped light pulse caused whisker deflections, while ramped light pulses did not cause whisker deflections (see below). To me, this is just another argument in favour of short, pulsed stimulation. With the step pulse, the whisker activity was at the beginning of the pulse, when you know the photostimulation is causing spikes; the lack of whisking afterwards shows you don't know what's happening during the last four seconds of the pulse. Furthermore, the ramped pulse elicits no whisking activity, showing you don't know what the heck that protocol is doing.
(left) Square pulse photostimulation causes whisker deflections (individual trials, green traces), primarily at stimulus onset. (right) Ramped photostimulation causes no whisker deflections.
From Supplemental Figure 6, Poulet et. al., 2012.
I don't know squat about barrel cortex or the thalamus, but this is a flabbergasting experiment: their light stimulation is too long, and may cause heating; they don't have any stimulus control; and they never performed photostimulation in a non-ChR2 animal.

Tan et. al., 2012

From the Luscher lab next door, this paper has a couple problems. They recorded in vivo from the VTA of GAD-cre mice infected with floxed-ChR2 (viz. GABAergic ChR2 neurons). To identify whether neurons were GABAergic, they used 1 second light pulses. This is a minor sin, since the neurons are fast spiking, and they infected GABAergic neurons, making non-specific excitatory activity unlikely. However, they really should have PINPed it.

Extracellular recording in vivo in GAD-ChR2 mice. Photostimulation excites GABAergic neurons.
From Figure 1, Tan et. al., 2012.
More troubling, they performed a conditioning paradigm, where "the laser was continuously activated when mice entered the conditioned chamber for a maximum duration of 30 seconds to avoid any overheating of the brain structures." In my opinion, a better way to avoid heat artifacts would have been pulsing the laser. They did perform negative controls in non-expressing animals.

Atallah et. al., 2012


This paper from the HHMI Scanziani lab looked at how inhibitory neuron activity shapes pyramidal neuron tuning properties. They expressed ChR2 in parvalbumin cells, and recorded in vivo using loose-patch. They then stimulated the cells using one second light pulses (blue bar), in combination with visual stimulation (grey area). Like Tan above, this is a minor sin, since they recorded from the cells they photostimulated, and know what activity they are inducing. In the supplement, they also provide negative controls.

(Left) Image of a tdTomato-ChR2 expressing parvalbumin cell, recorded via loose-patch. (Right) Raster plot and PSTH from the cell in response to visual stimulation (grey area). The activity was higher for a combination of light (blue bar) and visual stimulation.
From Figure 2, Atallah et. al., 2012.
Kravitz et. al., 2012

From Young Investigator Kreitzer's lab, this paper looks at how the direct and indirect pathways in the striatum influenced learning.** To ensure that they were stimulating the cell type they intended, they used a chronic in vivo optrode to record spikes in response to light stimulation. For a veneer of control, they used different light intensities, ranging from 0.1-3mW, but used constant 1 second light pulses.
PSTH of a neuron in response to constant 1 second light pulse.
From Supplemental Figure 1, Kravitz et. al., 2012.
In their words, "Neurons were classified as ChR2 expressing if they exhibited, within 40 ms of the laser onset, a firing rate more than threefold greater than the s.d. of the 1 s preceding the laser pulse." This strikes me as a crap criterion, since if there is any excitatory feedback in the network, it would be active within 40 ms. For example, the gradual increase in firing rate for this neuron looks odd. I asked the Luscher lab about connectivity in the striatum, and they said that the direct and indirect pathways inhibit each other, so it is unlikely in this case that Kravitz is recording the wrong neuron type. In any case, this is sloppy.

** Which Cre mice did they use? "Bacterial artificial chromosome (BAC) transgenic mouse lines that express Cre recombinase under control of the dopamine D1 receptor and A2A receptor regulatory elements were obtained from GENSAT." Is it too much to ask them to name which of the nine D1A or three A2a lines they used? There can be variability between lines which nominally target the same populations.

Maybe I'm just being a pedantic curmudgeon. Maybe it's my background in cellular neuroscience where you always have to perform negative controls. But I think we should have higher standards for channelrhodopsin experiments. If these papers in high tier journals from respected labs (including Deisseroth on one paper!) can get away with sloppy channelrhodopsin work, what's getting through in other journals? If people simply followed the Two Pillars of Channelrhodopsin Photostimulation, their results would be cleaner and more reproducible, and get less guff from reviewers like me.

References

Atallah BV, Bruns W, Carandini M, & Scanziani M (2012). Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli. Neuron, 73 (1), 159-70 PMID: 22243754

Kravitz AV, Tye LD, & Kreitzer AC (2012). Distinct roles for direct and indirect pathway striatal neurons in reinforcement. Nature neuroscience, 15 (6), 816-8 PMID: 22544310

Poulet, J., Fernandez, L., Crochet, S., & Petersen, C. (2012). Thalamic control of cortical states Nature Neuroscience, 15 (3), 370-372 DOI: 10.1038/nn.3035

Tan KR, Yvon C, Turiault M, Mirzabekov JJ, Doehner J, Labouèbe G, Deisseroth K, Tye KM, & Lüscher C (2012). GABA neurons of the VTA drive conditioned place aversion. Neuron, 73 (6), 1173-83 PMID: 22445344

Thursday, November 1, 2012

PINPing ain't easy

With my olfaction research ending at Zeno speed, I've started pilot experiments for everyone's favourite sensory modality, taste! The idea is to record from each layer of taste (insular) cortex, and compare the "taste receptive fields" of neurons from each layer. To identify the layer I record from, I'm using channelrhodopsin-aided cell identification: I record extracellulary from random neurons in cortex; the mouse I'm recording expresses ChR2 in a specific cortical layer; and then I identify which layer of cortex I'm recording from by photostimulating and recording evoked spikes. To get ChR2 expression in specific cortical layers, I'm using transgenic mouse lines that express Cre in specific cortical layers, and crossing those Cre mice with floxed-Channelrhodopsin (all lines are from MMRRC, and the names are available on request).

OG PINP


This technique has been dubbed PINP (Photostimulation-assisted Identification of Neuronal Populations) by the Zador lab (you can blame them for the label). In the original PINP paper, to get cell-specific expression of ChR2, Lima and colleagues used parvalbumin-Cre mice, and injected floxed-ChR2 (AAV-LSL-ChR2-YFP) into A1 of auditory cortex. They then stuck a tungsten electrode into A1 (in vivo), and recorded spikes. Some units reliably responded to light stimulation (panel B, below), while other units did not (panel C, below); the ones that responded to light were 
presumably parvalbumin-expressing neurons.


From Lima et. al., 2009: "Figure 4. In vivo photostimulation of parvalbumin expressing auditory cortex neurons. (A) PV expressing neurons in the mouse auditory cortex, labeled with the binary Cre-AAV system, were tagged with ChR2 (green). (B) Spike rasters of a well isolated single unit that responded to light activation in the mouse auditory cortex. Light was on from 0 to 10 ms. (C) Reliability of light-evoked responses in all the cells recorded in the mouse auditory cortex. Reliability was computed as the fraction of trials in which the firing rate within the 40 ms after the start of the light pulse was greater than within the 40 ms immediately preceding the light pulse. (D) Action potentials originated from ChR2-expressing neurons were narrower than spikes originated from the rest of the population (green - ChR2 positive, gray – unlabeled cells)."
One thing that surprises me about this experiment is that the spikes were delayed by ~2.8 +/- 1 ms. In other channelrhodopsin papers, people are able to get strong channelrhodopsin effects with light pulses of only one or two milliseconds. For example, Smear et. al. expressed ChR2 in olfactory epithelial cells, and mice were able to detect fine changes in photostimulation latency using 1 ms pulses. For another example, Atasoy et. al. were able to detect strong IPSCs in brain slices following a 1 ms pulse (Fig. 3b). If Lima and Zador had used 1 ms pulses, they may not have been able to evoke spikes. Given the wide variability in cell sizes, conductances, and excitability, when performing PINP, it is probably important to calibrate the duration of the light pulses.

Other old school PINPs

Almost all of the papers that cite Lima are reviews about the potential of optogenetics (and there are a LOT of reviews about optogenetics), but I also found a few papers that actually used the PINP technique. The first is from the lab of Young Investigator Rui Costa. Xin and Costa recorded from the substantia nigra, and were interested in differentiating between dopaminergic and GABAergic neurons. In addition to well-established criteria like spike width and firing rate, they used PINP to identify TH-ChR2 neurons (generated by infecting AAV-ChR2 in TH-cre mice). They implanted tungsten microwires into the substantia nigra, and stimulated the area with 10 ms light pulses.


Raster plots and PSTHs from putative TH-ChR2 neurons following photostimulation at t=0 ms. Recorded using tungsten microwires, and photostimulation pulses of 10ms.
From supplemental figure 3 from Xin and Costa, 2010.
During photostimulation, they saw an increase in firing rate in the putative TH-ChR2 neurons. To be honest, I find these responses underwhelming due to the jitter. In the example above from Lima and Zador, the evoked spikes were delayed, but extremely precise. In comparison, this simply looks like an increase in firing rate. I know nothing about the connectivity of the substantia nigra, but if you told me that the above raster plots were recorded from a neuron downstream of TH-ChR2 neurons, I would not argue with you.

The other example PINP I found comes from the lab of Naoshige Uchida, a former olfaction researcher. Cohen and Uchida were interested in how dopaminergic and GABAergic neurons in VTA encoded reward. They targeted dopaminergic neurons using DAT-cre, and GABAergic neurons using Vgat-cre, and expressed ChR2 via AAV. They recorded extracellularly using twisted wire tetrodes, and stimulated using 5 ms laser pulses.


a. Voltage trace of light evoked activity. b. Photostimulation at 20 and 50 Hz evokes reliable and precise spikes. c. (left) Plot of photostimulat-ability ("light-evoked energy") versus the correlation between spontaneous and evoked waveforms for individual units. Only those cells that had light-evoked spikes, and well correlated waveforms were considered to be ChR2-expressing. (right) Example waveforms for different units shown on the left.
From Fig. 3, Cohen et. al., 2012
Following photostimulation, they were recorded precisely timed spikes (panels a & b, above), at up to 50Hz. To ensure the light-evoked units were the same as the natural units, they performed a control I quite liked: they calculated the correlation coeffecient between the waveform of the photostimulated spikes and the natural spikes (panel c, right). Only those units that were both light-activated and maintained spike shape were considered to be TH-ChR2 expressing (panel c, left, filled blue dots). The precision of these spikes casts further doubt in my mind on those observed by Xin and Costa.

First PINP steps

I have started to try to perform PINP myself, and am using this post in part to structure my thinking about what is happening. Like all the previous papers, I'm using a Cre-lox system to get layer specific expression of ChR2. Unlike the previous papers, however, I'm using a transgenic floxed-ChR2 mouse to get expression.

Layer specific expression of YFP-ChR2 in somatosensory and taste cortex (blue box). Sagittal section taken at ~ -1.2mm from Bregma. Please pardon the overexposure.
As a pilot experiment, I anesthetized a mouse, and recorded from sensory cortex (I would have recorded from gustatory cortex, but had not stereotaxically head-posted the mouse, and decided not to risk an electrode recording by the edge of the skull). For recording, I used a Silicon optrode from NeuroNexusTech (A1x32-Poly3-5mm-25s-177-OA32). For my recordings in the olfactory bulb, I used electrodes that had an area of 312 um2. However, NeuroNexus does not make optrodes with electrode sizes that large (perhaps due to the photoelectric effect), and the electrode size for this experiment was 177 um2. In the olfactory bulb, using the 177 um2 electrodes, I recorded fewer spikes than with 312 um2 electrodes. Whether due to electrode size, the fact that the cortex is generally quiet, or due to the anesthesia, I didn't record any spikes in somatosensory cortex with the optrode.

Despite not recording any spontaneous spikes, I still tried photostimulating. The optrode consists of a single shank with electrodes forming two columns 200-300 um long; the optic fibre starts 200um above the top electrode. While doing this experiment, I neglected these measurements, and made a mistake. Somatosensory cortex is ~1mm deep, and the somas of the ChR2-expressing neurons are in the deepest layer. However, I only penetrated the optrode 500-600um, so I would not have been able to record evoked spikes.

Given the caveat that my electrodes were not in the layer of interest, I was able to record light-evoked LFPs. For the initial stimulations, I saw strong photoelectric artifacts at the beginning and end of the light pulses because the laser power was too high (panel A, below). The photoelectric artifact was easily removed by simply reducing the laser power.


LFPs recorded from light stimulation. A. (top) Light pulse. (bottom) LFP recorded. At high laser power, there is a photoelectric artifact at the beginning and end of light pulses. B. At medium laser power, there is no longer a photoelectric artifact. A 10 ms light pulse evokes a 2 ms LFP deflection. C.  A 1 ms light pulse evokes only a 1 ms LFP deflection.
With a more reasonable laser power, I was able to record LFP activity on every electrode I recorded. Even for longer light pulses, the LFP activity was fairly short. Whether this reflects a single evoked action potential in the deep layers, or something else is uncertain. To see if I could get the LFP activity with short laser pulses, I reduced the pulse duration to 1 ms, half the LFP duration. This in turn reduced the duration of the LFP activity. So there is some dynamic range in stimulation between 0 and 2 ms.

As the title of this post reflects, PINPing ain't easy. There are a few improvements I need to make before graduating to recording from taste cortex of (trained) awake mice. First, I need to penetrate the damn electrode to the layer of interest. This will be easy in somatosensory cortex, but may be complicated in insular cortex given that the layers are parallel to electrode penetration. Furthermore, I'm performing these experiments on mice head-posted 1-2 weeks earlier, which will complicate stereotaxy. Second, I need to be able to record spikes. All of the papers above recorded using tungsten wires, and got clean units. I'm concerned that the electrode sizes we are using are simply not large enough to record large number of clean units. Hopefully the difficulty of recording spontaneous spikes was due to the anesthesia and layers recorded. Finally, I am somewhat concerned that the transgenic expression is simply too high. If photostimulation activates all the neurons in a layer, will I be able to record single units? Or will the coactivation muddle any recordings? And will the strong LFP activity make isolating spikes difficult?

I have two priorities right now. First, I need to solidify the behavioural paradigm, and see how many training sessions it takes for mice to acquire the protocol, and measure how many trials I can expect out of a mouse before it is sated. And second, I need to refine PINP, and make sure that I can actually record from ChR2-identified neurons.

References


Cohen JY, Haesler S, Vong L, Lowell BB, & Uchida N (2012). Neuron-type-specific signals for reward and punishment in the ventral tegmental area. Nature, 482 (7383), 85-8 PMID: 22258508

Jin X, & Costa RM (2010). Start/stop signals emerge in nigrostriatal circuits during sequence learning. Nature, 466 (7305), 457-62 PMID: 20651684

Lima SQ, Hromádka T, Znamenskiy P, & Zador AM (2009). PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording. PloS one, 4 (7) PMID: 19584920