Functional Imaging In Primary Auditory Cortex Of Mice By Using Cal-520AM In Vivo

Object:The organization in the primary auditory cortex(Au1)is critical to the basic function of auditory information processing and integration.However,recent mapping experiments using in vivo two-photon imaging with different Ca2+ indicators have reached controversial conclusions on this topic,possibly because of the different sensitivities and properties of the indicators used.Therefore,it is essential to identify a reliable Ca2+ indicator for use in in vivo functional imaging of the Au1,to understand its functional organization.Here,we demonstrate that a previously reported indicator,Cal-520,performs well in both anesthetized and awake conditions.Cal-520 shows a sufficient sensitivity for the detection of single action potentials,and a high signal-to-noise ratio.Cal-520 reliably reported on both spontaneous and sound-evoked neuronal activity in anesthetized and awake mice.After testing with pure tones at a range of frequencies,we confirmed the local heterogeneity of the functional organization of the mouse Au1.We also make sure that the neurons in primary auditory cortex will generate predictive signal following rhythmic sound stimuli with the help of Cal-520 calcium imaging.Therefore,Cal-520 is a reliable and useful Ca2+ indicator for in vivo functional imaging of the Au1.Method:We exposed the right Au1 of each mouse to two-photon imaging under anesthesia.Each mouse was anesthetized with 1.5% isoflurane in pure oxygen and kept on a warm plate(37.5–38 °C).After local lidocaine injection,the skin and muscles over the Au1 were removed.We then glued a custom-made plastic chamber to the skull with UHU glue and performed a small craniotomy(2 mm × 2 mm)(the center point: Bregma-2.5 mm,4.5 mm lateral to midline).Afterward,we transferred the mouse to the imaging setup under a lower level of anesthesia(0.4-0.8%)(Breathing rates 90-130 breaths per minute).The craniotomy was filled with 1.5% low-melting-point agarose.The recording chamber was perfused with normal ACSF(p H 7.4 when bubbled with 95% oxygen and 5% CO2).We used the highly sensitive fluorescent Ca2+ indicator Cal-520 AM for multicell bolus loading in the Au1.Cal-520 AM was dissolved in DMSO with 20% Pluronic F-127 to a final concentration of 567 ?M with ACSF for bolus loading.The loading procedure was performed according to previous studies.We performed the Ca2+ imaging 1 hour after dye injection,and the imaging lasted for up to 8 hours.The body temperature of the mouse was kept between 36.5–37.5 °C throughout the experiments.In two-photon imaging experiments for awake mice with head fixation,we used a custom-made plastic chamber designed for experiments on head-fixed mice.After surgery as described above for gluing the chamber to the skull of the mouse,the mouse was allowed to recover for 5 days.During this period,the mouse underwent head-fixation training for 3-5 days(from 1 to 4 hours per day).The head and the body of the mouse were rotated approximately 70° on the basis of the craniocaudal axis to keep the surface of the Au1 perpendicular to the microscope objective.Mice gradually adapted to this posture and were able to sit comfortably for 4 hours after these head-fixation training sessions.A small craniotomy(2 mm × 2 mm)was performed on the recording day under local anesthesia,and this was followed by bolus loading of Cal-520 AM.A camera(frame rate 30 Hz)under infrared illumination was used to monitor the status of the mice.Result:1.Ca2+ imaging of Au1 neurons in vivo using Cal-520 AM:The Cal-520 signals reliably reported single action potentials at a rate of nearly 100%.2.Spontaneous and sound-evoked responses of Au1 neurons in anesthetized mice: We randomly sampled several neurons and their adjacent neuropils' signals,the signals detected in neurons were not observed in their nearby neuropils.In addition,we also selected small regions of neuropils and compared their signals with those of the adjacent neurons.Similarly,we found no contamination of neuronal signals by nearby neuropils.We observed both spontaneous activity and sound-evoked activation in Au1 L2/3 neurons in anesthetized mice.The frequency of the spontaneous activity of Au1 L2/3 neurons was rather low,less than 0.1 Hz.The response of Au1 L2/3 neurons to broadband noise stimulation had success rates varying from 0 to 100%(mean 43%;1263 neurons from 12 mice);45% of the recorded neurons showed responses with success rates higher than 50%,43% had success rates lower than 50%,and 12% were silent.3.Spontaneous and broadband noise-induced responses of Au1 neurons in awake mice: Similarly to the data under anesthetized conditions,the spontaneous activity of neurons in awake mice exhibited a low frequency in a range of 0 to 0.3 Hz.During broadband noise stimulation,the average response rate of Au1 L2/3 neurons was 34%,with a range of 0 to 100%.Here,30% of the recorded neurons showed responses with success rates higher than 50%,59% had success rates lower than 50%,and 11% were silent.4.Pure tone-induced responses of Au1 neurons in anesthetized and awake mice: Across populations in both anesthetized and awake mice,Au1 neurons showed strong responses to pure tones,with single-peaked tuning curves and there was no spatial clustering of neurons with a similar BF.Moreover,the maximal distance of the neurons with ?BF of 0 to 1 octave reached up to 200 ?m in both anesthetized and awake mice.5.In anesthetized and awake mice,the neurons in primary cortex will generate predictive signals after rhythmic sound stimuli.We call this predictive signal “echo response”.The echo response has frequency selectivity.Similarly,in the correspondent behavioral experiment,the mouse will have predictive licking after ten times of sound stimuli.Conclusion:1.Cal-520 AM in the Au1 performs well under both anesthetized and awake conditions.It has a high SNR and detection sensitivity for single action potentials.2.Spontaneous and sound-evoked responses of Au1 neurons in anesthetized mice: Neuronal signals will not be contaminated by adjacent neuropil in population Ca2+ imaging.The frequency of the spontaneous activity of Au1 L2/3 neurons is rather low.The response of Au1 L2/3 neurons to broadband noise stimulation has success rates varying from 0 to 100%.3.The spontaneous activity of neurons in awake mice exhibits a low frequency.During broadband noise stimulation,the average response rate of Au1 L2/3 neurons is 34%,with a range of 0 to 100%.4.Pure tone-induced responses of Au1 neurons in anesthetized and awake mice: The results suggest that there is no smooth tonotopy but instead suggest a heterogeneous organization of local circuits in the Au1.5.The neurons in primary auditory cortex will generate predictive signal following rhythmic sound stimuli.Similarly,the correspondent behavioral experiment will show the predictive behavior.