Effects of anesthesia on the activity of pyramidal neurons

A previous study using extracellular recordings on pyramidal neurons in the primary visual cortex found that tuning properties in the awake animals were similar to those measured in the anesthetized animals (Niell et al., 2010). However, since this comparison was made from two neuronal populations in separate experiments, it is not clear whether this similarity can be confirmed in subsequent recordings performed under both conditions in the same cells. In this part, by using in vivo population calcium imaging, we compared the tuning properties of pyramidal neurons in the awake and the anesthetized states. We analyzed 86 cells, which showed visual responses in one or the other conditions and classified them into three groups based on their response patterns. In the first group, the cells didn’t change their preferred orientation or direction when the brain state switched from the awake to the anesthetized state. In the second group, the cells showed responses only in the anesthetized state. In the third group, the cells changed their preferred orientation or direction when the brain state switched from the awake to the anesthetized state.

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Figure 19. Effects of anesthesia on responses of the suppressed response interneuron.

(A) In vivo two-photon image of an EGFP-expressing 5HT3aR+ interneuron with the cell-attached pipette that was filled with 50 µM Alexa 488 dissolved in normal Ringer solution.

(B) Top: Responses of the interneuron to drifting grating at eight different directions in the awake state. Bottom: Responses of the interneuron to drifting grating at eight different

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directions in the anesthetized state. (C) Peri-stimulus time histogram of 8 repeats from recording in the awake (top panel) and anesthetized (bottom) state. Bin size: 1s. (D) Response curve of the interneuron to drifting grating at eight directions in the awake (red) and anesthetized (black) state.

Figure 20A shows an example cell with a tuning response pattern that is not affected by anesthesia. In the awake state, the cell reliably showed responses to one orientation (Figure 20B). In the anesthetized state, the preference for this orientation remained the same, even though response reliability and the amplitude of the response were smaller than in the awake state (Figure 20C). However, these alterations were transient and partially recovered when the mouse returned to wakefulness (Figure 20D). Figure 21A shows an experiment in which the cells’ responses to visual stimulation were present under anesthesia only. In the awake state, no responses of the cell to the presentation of the drifting grating were observed, irrespective of the directions of the drifting grating. The only activity observed occurred spontaneously (Figure 21B). However, in the anesthetized state, the cell showed a preferred response to one direction of the drifting grating (Figure 21C). After returning to the awake state the cell’s response disappeared again (Figure 21D). An experiment in which a cell changed tuning patterns with the two cortical states tested is shown in Figure 22A. In the awake state, this cell responded strongly and distinctively to one orientation of the drifting grating (Figure 22B, left panel and 22C). Interestingly, when the brain state switched from the awake state to the anesthetized state, the cell shifted its preference to a slightly different orientation (Figure 22B, middle panel and 22C). After returning to the awake state, the cell’s response also returned to the initial tuning (Figure 22B, right panel and 22C). To determine the cell type of this neuron, we injected OGB-1 and biocytin into the cell and did a DAB staining. We found that this cell had the typical appearance of a pyramidal cell (Figure 22D).

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Figure 20. Tuning unchanged response pattern in the anesthetized and awake states.

(A) In vivo two-photon image of cortical neurons in layer 2/3 of the V1 stained with Cal-520 AM dye. (B-C) Calcium transients evoked by drifting grating of the cell indicated by red dashed circle in (A) showed a decreased amplitude of the response to the preferred direction in anesthetized state (C) comparing to the response under awake state (B, D).

Single trials are represented with black lines and the average of all trials is shown in green.

The preferred orientation of the cell in the awake and anesthetized state are marked in red.

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Figure 21. Cell showed response to visual stimulus only in the anesthesia

(A) In vivo two-photon image of cortical neurons in layer 2/3 of the V1 stained with Cal-520 AM dye. (B-C) Calcium transients of the cell indicated by red dashed circle in (A) showed an evoked response only in the anesthetized state (C) but not in the awake state (B, D). Single trials are represented with black lines and the average of all trials is shown in green. The preferred direction of the cell in the anesthetized state is marked in red.

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Figure 22. Tuning changed response pattern in the anesthetized and awake states.

(A) In vivo two-photon image of cortical neurons in layer 2/3 of the V1 stained with Cal-520 AM dye. (B) Calcium transients evoked by drifting grating of the cell indicated by red dashed circle in (A) showed a change of the preferred orientation in the anesthetized state (Middle panel) comparing to the response in the awake state (left and right panel). Single trials are represented with black lines and the average of all trials is shown in green. The preferred orientation of the cell in the awake is marked in red. (C) Polar plots of the neuron’s response to drifting gratings at eight directions in the awake (red and green trace) and anesthetized state (black trace). (D) Left: The cell indicated by red dashed circle in (A) was electroporated with an OGB-1 and biocytin contained solution after recording.

Right: Biocytin-filled cell was visualized by DAB staining.

In summary, nearly 60% of the neurons in our study (51/86) had stable tuning patterns that were insensitive to the anesthetized state. In around one-quarter of the neurons

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(21/86), preferred orientation or direction of the drifting grating was affected by anesthesia.

Finally, around 16% of the neurons (14/86) were irresponsive to visual stimulation in the awake state but responded under anesthesia (Figure 23). It should be noted here that our assumption that these cells are pyramidal cells depends largely on their tuning properties.

We cannot exclude the possibility that some of them are interneurons unless more tests, like morphology reconstruction are done after the recording.

Figure 23: Relative proportions of neurons showing responses in anesthesia only, tuning sensitive to anesthesia and tuning insensitive to anesthesia response patterns.