PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording

Abstract

Neural circuits are exquisitely organized, consisting of many different neuronal subpopulations. However, it is difficult to assess the functional roles of these subpopulations using conventional extracellular recording techniques because these techniques do not easily distinguish spikes from different neuronal populations. To overcome this limitation, we have developed PINP (Photostimulation-assisted Identification of Neuronal Populations), a method of tagging neuronal populations for identification during in vivo electrophysiological recording. The method is based on expressing the light-activated channel channelrhodopsin-2 (ChR2) to restricted neuronal subpopulations. ChR2-tagged neurons can be detected electrophysiologically in vivo since illumination of these neurons with a brief flash of blue light triggers a short latency reliable action potential. We demonstrate the feasibility of this technique by expressing ChR2 in distinct populations of cortical neurons using two different strategies. First, we labeled a subpopulation of cortical neurons-mainly fast-spiking interneurons-by using adeno-associated virus (AAV) to deliver ChR2 in a transgenic mouse line in which the expression of Cre recombinase was driven by the parvalbumin promoter. Second, we labeled subpopulations of excitatory neurons in the rat auditory cortex with ChR2 based on projection target by using herpes simplex virus 1 (HSV1), which is efficiently taken up by axons and transported retrogradely; we find that this latter population responds to acoustic stimulation differently from unlabeled neurons. Tagging neurons is a novel application of ChR2, used in this case to monitor activity instead of manipulating it. PINP can be readily extended to other populations of genetically identifiable neurons, and will provide a useful method for probing the functional role of different neuronal populations in vivo.

Figure 1. Identifying ChR2-tagged neurons in vivo.

Photostimulation-assisted identification of neuronal populations: general method for identifying neuronal populations during in vivo electrophysiological recordings. (A) ChR2 expression is restricted to distinct neuronal populations using methods that allow targeting of genetically identifiable populations of neurons (more detail in text). (B) Spikes from ChR2-positive (green) and ChR2-negative (black) single units are recorded extracellularly during a normal in vivo experiment, for example in response to sound stimulation. (C) ChR2-positive units are identified on the basis of their response to a flash of blue light.

Figure 2. Viral-mediated expression of ChR2-YFP into a class of inhibitory interneurons in the mouse auditory cortex.

(A) Neurons within the rodent auditory cortex can be excitatory or inhibitory. To express ChR2 in inhibitory parvalbumin expressing neurons of the mouse auditory cortex, we injected AAV carrying floxed ChR2-YFP in the left auditory cortex of PV-Cre mice, which express Cre recombinase only in fast-spiking interneurons. Although the virus can infect any cell, ChR2 is expressed only in PV-positive neurons. (B) Confocal micrograph of a section including the mouse primary auditory cortex shows fluorescence in cells expressing ChR2-YFP. (C) To test for PV specificity, the section was treated with an antibody against PV and counterstained with a red fluorescent dye. (D) Merging of the two channels shows that cells expressing YFP also counterstain for PV (merged cells show as yellow). Note that all ChR2+ (green) cells are also PV-positive (red) (i.e. there are only a few false positives), but that not all red PV-positive cells express ChR2.

Figure 3. Viral mediated retrograde labeling of neurons projecting to the primary auditory cortex.

(A) To tag neurons based on projection pattern, HSV1 expressing ChR2-YFP was injected into the right auditory cortex. Ten days later, coronal brain sections were made to assess infected cells (green); sections were counterstained with red fluorescent Nissl substance to stain neurons. (B) Coronal section showing the site of injection (asterisk) and ipsilateral secondary auditory cortex (above left box). Staining can also be seen in the auditory thalamus (middle box) and contralateral auditory cortex (right box). Retrogradely labeled areas include: (C) nearby ipsilateral secondary auditory cortex; (D) ipsilateral medial geniculate (auditory) thalamus; (E) neurons in layers 3 and 5 of the contralateral primary auditory cortex; (F) motor cortex; (G) somatosensory cortex; (H) visual cortex.

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).

Figure 5. In vivo photostimulation of callosally projecting auditory cortex neurons.

ChR2 expression in rat auditory cortex neurons can be used to tag and identify this neuronal population during in vivo recordings. (A) Callosally projecting neurons in the rat auditory cortex were labeled with ChR2 via retrogradely HSV1-mediated transfection; (B) Light-evoked activity of three well-isolated single units recorded simultaneously is shown as rasters. Each unit is color-coded (green, blue, red). After blocking fast glutamate (AMPA) receptors (left) with the selective antagonist NBQX, only the shortest latency unit (green, right) continued to show light-activated activity. This indicates that activity in the other two units was indirect, i.e. synaptic, and therefore blocked by NBQX. (C) Population histogram showing that ability to follow light flashes at higher repetition rate (5 Hz, ISI = 200 ms, 10 ms LED pulses) cleanly separated recordings into two classes, which we interpret as direct (ChR2-positive; dark green: single units responding after NBQX application) and synaptic (ChR2-negative; dark gray: single units not responding after NBQX simulation; light gray: multiunit recordings). The x-axis shows the spiking response reliability, computed as the fraction of trials in which the firing rate in 40 ms after the start of the second pulse of 5 Hz LED trains was greater than in the 40 ms immediately preceding the pulse. (D) Callosally projecting neurons (ChR2+) are non-responsive to white noise stimulation, showing different response reliability from the average population (Other). Reliability was computed as the fraction of trials in which the firing rate in 40 ms after sound onset was greater than in 40 ms immediately preceding the sound. Error bars show standard error of the mean.