A method for combining multiple-units readout of optogenetic control with natural stimulation-evoked eyeblink conditioning in freely-moving mice

Abstract

A growing pool of transgenic mice expressing Cre-recombinases, together with Cre-dependent opsin viruses, provide good tools to manipulate specific neural circuits related to eyeblink conditioning (EBC). However, currently available methods do not enable to get fast and precise readout of optogenetic control when the freely-moving mice are receiving EBC training. In the current study, we describe a laser diode (LD)-optical fiber (OF)-Tetrode assembly that allows for simultaneous multiple units recording and optical stimulation. Since the numbers of various cables that require to be connected are minimized, the LD-OF-Tetrode assembly can be combined with CS-US delivery apparatus for revealing the effects of optical stimulation on EBC in freely- moving mice. Moreover, this combination of techniques can be utilized to optogenetically intervene in hippocampal neuronal activities during the post-conditioning sleep in a closed-loop manner. This novel device thus enhances our ability to explore how specific neuronal assembly contributes to associative motor memory in vivo.

Figure 1

LD-OF-tetrode assembly for optogenetic control in freely-moving mice. Left: Schematic drawing of a homemade microdrive equipped with the LD-OF-tetrode assembly. Each LD-OF-tetrode assembly includes: ① 4 tungsten wire tetrodes, ② 4 polymicro pipes, ③ 1 optical fiber, ④ 1 green laser diode, ⑤ 16 gold pins, ⑥ 1 PCB board, and ⑦ 1 microdrive. The tip of optical fiber was attached ~500 um above the tips of tetrodes. Right: Picture of the LD-OF -tetrode assembly depicted in the left panel. Care should be taken to keep a 5-degree (or less) angle between the optical fiber and the tetrodes.

Figure 2

Combination of MUR and optical implants with CS-US delivery in freely-moving mice. (A) Top and (B) side view of the schematic diagram illustrating the copper mesh wall. A total of 3 SIP socket connectors were tethered to this wall. (C) Schematic diagram of various assembled components within the headstage. The description of various parts is listed in Table 1. (D) Front (upper) and side (bottom) view photograph of the assembly headstage in a freely-moving mouse. (E) Top view photograph of the assembled headstage including various SIP socket connectors and LD-OF-Tetrode assembly.

Figure 3

Acquisition of TEBC in freely-moving mice. (A) Schematic diagram of TEBC training in freely-moving mice. (B) Example of responses from a mouse in a non-CR (B1, blue) and CR (B2, red) trial, respectively. The CS was a 150-ms blue LED light (470 nm in wave length), while the US was a 100-ms airpuff to the cornea. A 250-ms time interval was inserted between the CS offset and the US onset. The top trace of each panel shows the raw EMG signal, whereas the bottom trace shows the rectified and integrated EMG signal. (C) Averaged eyelid responses of the valid CS-US presentation trials across five training days for each freely-moving mouse. (D) CR incidence and (E) CR peak amplitude measured from freely-moving mice (n = 4) across 5 conditioning training days. (F) Incidence of spontaneous eyeblink responses during the 300-ms baseline period. (G) Incidence of startle eyeblink responses (SR) in the 50-ms period after the CS onset. Data are shown as mean ± S.E.M. The CRs and URs were indicated by the arrows.

Figure 4

Multiple units recording in freely-moving mice during TEBC. (A) Representative hippocampal CA1 neuronal activities during TEBC in a freely-moving mouse. Upper: Firing activities of 3 representative isolated single units in a CS-US paired presentation trial. Lower: The rectified and integrated EMG signal for the same CS-US trial. (B) Spike waveforms (Left) and autocorrelograms (Right) of three single isolated units shown in (A). The spike waveforms on each recording channel of a tetrode are illustrated. Same isolated units and colors in (A,B). (C) Peri-stimulus histogram (PSTH, left) and raster plot (right) revealed the firing patterns of three isolated units illustrated in (A).

Figure 5

Readout of optogenetic intervention during TEBC. (A) Optogenetic inhibition of hippocampal PYR units during TEBC. Upper: (A) CS-US presentation trial without green light stimulation and a CS-US trial with 400-ms green light stimulation after CS onset. 2 representative isolated units are shown. They were obviously inhibited by the green light. Lower: Two rectified and integrated EMG signals for the same CS-US trials in the upper panel. (B) The recorded hippocampal units were classified into 2 groups: putative pyramidal cells (PYR, n = 47, triangles) and putative interneurons (n = 2, circles) based on their firing rates and spike waveform width. (C) Representative recording site in the hippocampus of CamkIIa-cre::ArchT- EGFP mouse. Schematic drawings of the valid recording sites in 4 mice are exhibited. (D) Upper: Normalized gain of spike activities across putative PYR units in the CS-US trials without and CS-US trials with green-light stimulation (n = 47). Shaded area represents S.E.M. Lower: The peri-CS firing activities were illustrated for each putative PYR unit before (left) and after (right) the optogenetic intervention. (E) Efficiency of the optogenetic inhibition at the population level (n = 47). 87% of the recorded putative PYR units were inhibited by the 400-ms green light pulses.

Figure 6

Real-time optogenetic silencing of putative PYR contingent upon SWR detection. (A) The threshold for real-time SWR detection was set to 5× s.d. above the mean power. Top, raw LFP traces recorded from 2 tetrodes in the dorsal hippocampal CA1 area. Middle, a trace filtered in a SWR-frequency (150–250 Hz) band; Bottom, a trace representing the LFP envelope amplitude. The asterisks indicate the detected SWR onsets. (B) Schematic diagram of real-time optogenetic experiments during the post-TEBC sleep. (C) Representative abolishment of the ongoing SWR by the optogenetic inhibition. (D) Power of LFP oscillations 100-ms before (black traces) and 100-ms after (red trace) the onset of green lights. It was revealed that LFP power in high-frequency band (150–250 Hz) oscillation was significantly decreased after the green light onset. (E) Population responses of putative PYRs (n = 18) before (black) and after (red) the green light onset.