Functional imaging of neuronal activity of auditory cortex by using Cal-520 in anesthetized and awake mice

Abstract

Abstract: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 Ca 2+ 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 Ca 2+ 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. Therefore, Cal-520 is a reliable and useful Ca 2+ indicator for in vivo functional imaging of the Au1.

Functional Organization of Mouse Primary Auditory Cortex in adult C57BL/6 and F1 (CBAxC57) mice

“…Initial studies using synthetic dyes in anesthetized animals described a local heterogeneous frequency preference (Bandyopadhyay et al, 2010;Rothschild et al, 2010) in L2/3. These results in L2/3 have been confirmed using the sensitive synthetic dye Cal-520 (Li et al, 2017;Tischbirek et al, 2019;Zeng et al, 2019) in anesthetized and awake mice and rats, with the ultrasensitive genetically encoded calcium indicator (GECIs) GCaMP6s in awake mice (Meng et al, 2017;Liu et al, 2019), as well as by in vivo patch clamp studies (Maor et al, 2016). In addition to primary auditory cortex, L2/3 of ACX areas such as the anterior auditory field and secondary ACX were also shown to contain fractured tonotopic organization (Liu et al, 2019).…”

“…While on large scales A1 in rodents shows a tonotopic organization, nearby neurons can show different frequency preference (Bandyopadhyay et al, 2010;Rothschild et al, 2010;Winkowski and Kanold, 2013;Kanold et al, 2014;Li et al, 2017;Meng et al, 2017;Panniello et al, 2018;Liu et al, 2019;Tischbirek et al, 2019;Zeng et al, 2019). We thus investigated if this local heterogeneity varied between mouse strains.…”

“…A classic hallmark of A1 organization in all species has been the discovery of tonotopic maps which describe a smooth distribution of tone preference across the surface of the primary and secondary auditory fields when probed with low spatial resolution techniques (Merzenich et al, 1975;Reale and Imig, 1980;Stiebler et al, 1997;Wessinger et al, 1997;Nelken et al, 2004;Bizley et al, 2005;Woods et al, 2010;Striem-Amit et al, 2011;van Dijk and Langers, 2013;Issa et al, 2014;Moerel et al, 2014;Baba et al, 2016;Liu et al, 2019). However, the application of large-scale high-resolution optical techniques in mice or rats using synthetic dyes (OGB1,Fluo4, or sensitive genetically encoded Ca 2+ indicators (GCaMP6) has shown that on the cellular level the organization of tuning in A1 is more heterogeneous than previously appreciated (Bandyopadhyay et al, 2010;Rothschild et al, 2010;Winkowski and Kanold, 2013;Kanold et al, 2014;Li et al, 2017;Meng et al, 2017;Panniello et al, 2018;Liu et al, 2019;Tischbirek et al, 2019;Zeng et al, 2019) suggesting that at least in rodent layer 2/3 (L2/3) functional tonotopic maps might be fractured. The precise degree of heterogeneity varies slightly between these studies, and this difference might be due to differences in anesthetic condition, cell inclusion criteria, tonal stimuli used, and/or species.…”

“…We performed in vivo 2-photon imaging experiments in awake mice of C57 strain as well as CBA background (CBAxC57 F1) and quantified the functional responses across L2/3 and L4 of A1. We focused on young adult animals (~2-3 months old) which is the age frequently used in functional studies, especially those involving mouse behavior (Bandyopadhyay et al, 2010;Rothschild et al, 2010;Carcea et al, 2017;Guo et al, 2017;Kuchibhotla et al, 2017;Li et al, 2017;Meng et al, 2017;Resnik and Polley, 2017;Francis et al, 2018;Liu et al, 2019;Tischbirek et al, 2019). To compare strains we imaged the mid-frequency region of A1 (4-16kHz) which is also where mice are most sensitive to sound (Zheng et al, 1999).We find that A1 of CBA mice contains more neurons tuned to high frequencies and a higher number of significant tone-responses overall, including a higher proportion of off-responses (Liu et al, 2019).…”

“…The primary auditory cortex (A1) plays a key role for sound perception since it represents one of the first cortical processing stations for sounds. Recent studies have investigated the functional organization and circuitry of mouse auditory cortex (Bandyopadhyay et al, 2010;Rothschild et al, 2010;Li et al, 2017;Meng et al, 2017;Liu et al, 2019;Tischbirek et al, 2019). A classic hallmark of A1 organization in all species has been the discovery of tonotopic maps which describe a smooth distribution of tone preference across the surface of the primary and secondary auditory fields when probed with low spatial resolution techniques (Merzenich et al, 1975;Reale and Imig, 1980;Stiebler et al, 1997;Wessinger et al, 1997;Nelken et al, 2004;Bizley et al, 2005;Woods et al, 2010;Striem-Amit et al, 2011;van Dijk and Langers, 2013;Issa et al, 2014;Moerel et al, 2014;Baba et al, 2016;Liu et al, 2019).…”