The vast majority of ascending sensory information reaches the cortex via the thalamus. To understand the functional organization of cortical circuits, it is therefore crucial to uncover the rules of thalamocortical connectivity (Jones, 2007; Sherman and Guillery, 2013; Winer et al., 2005). Most of the brain's auditory neurons, including those in the medial geniculate body (MGB) of the thalamus, are tuned to sound frequency and their spatial arrangement reflects the tonotopic organization established by the biomechanical properties of the cochlea. Tonotopy is preserved across species and at every lemniscal stage of the ascending auditory pathway up to the cortex (Kaas, 2011; Schreiner and Winer, 2007; Woolsey and Walzl, 1942). Like the receptor surface maps that are also hallmarks of the visual and somatosensory pathways, the presence of sound frequency gradients within each of these brain regions is therefore the most well characterized feature of the auditory system.

While the existence of cortical tonotopy is universally accepted, how precise this organization really is has recently been debated (Guo et al., 2012; Kanold et al., 2014; Rothschild and Mizrahi, 2015). In particular, the opportunity to image the activity of large populations of neurons at single-cell resolution in the mouse auditory cortex (Bandyopadhyay et al., 2010; Issa et al., 2014; Rothschild et al., 2010; Winkowski and Kanold, 2013) has questioned the smooth tonotopic organization revealed with microelectrode recordings (Guo et al., 2012; Hackett, 2011; Stiebler et al., 1997) or low-resolution imaging methods (Horie et al., 2013; Moczulska et al., 2013; Tsukano et al., 2016). The current view holds that neurons in the main thalamorecipient layers 4 and 3b, which tend to be most commonly sampled by microelectrode recordings, exhibit precise tonotopy that transitions into a coarse and more heterogeneous frequency organization in the supragranular layers (Kanold et al., 2014).

One implication of this arrangement is that the homogenous tonotopy of the middle cortical layers is inherited from thalamic input which is itself precisely tonotopically ordered. However, it is unclear how tightly organized this projection actually is. Although retrograde tracing of thalamocortical inputs (Brandner and Redies, 1990; Hackett et al., 2011) suggests strict topography, anterograde tracing (Huang and Winer, 2000) and reconstruction of single thalamic axons (Cetas et al., 1999) indicate considerable divergence in the auditory thalamocortical pathway. Indeed the frequency tuning of thalamic inputs that converge onto individual auditory cortical neurons can span several octaves (Liu et al., 2007), suggesting a need for integration across differently tuned afferent terminals. Furthermore, while most thalamocortical projections target the middle cortical layers, axons from the MGB can also be found in other layers (Frost and Caviness, 1980; Huang and Winer, 2000; Ji et al., 2016; Kimura et al., 2003; Llano and Sherman, 2008), but nothing is currently known about the relative specificity or precision of these inputs.

Our current understanding of the functional organization of the auditory thalamocortical pathway is limited by the relatively poor spatial resolution of the methods that have so far been used to investigate it. In this study, we employed in vivo two-photon (Denk et al., 1990) axonal calcium imaging (Glickfeld et al., 2013; Petreanu et al., 2012; Roth et al., 2016) to measure for the first time the frequency selectivity of individual boutons on auditory thalamocortical axons. Across different anesthetic states and different strains of mice, we found that the tuning of neighboring boutons is surprisingly heterogeneous, that frequency gradients are apparent at a large spatial scale only, and that thalamic inputs to cortical layers 1 and 3b/4 share a similarly coarse tonotopic organization. Furthermore, we demonstrate that this organization, which provides a potential basis for the broad spectral integration and experience-dependent plasticity that are characteristic features of the tuning properties of auditory cortical neurons, reflects almost exclusively the properties of the lemniscal thalamocortical projection originating in the ventral division of the medial geniculate body.

We initially expressed GCaMP6m (Chen et al., 2013) throughout the auditory thalamus (Figure 1A) in order to functionally characterize its input to the auditory cortex. Most of the thalamic axons were found in the middle layers, L3b/4, but substantial input was also observed in L1. We measured calcium transients, which correlate with somatic spiking activity (Petreanu et al., 2012), in individual putative synaptic boutons of thalamocortical axons (Figure 1B) in L1 and L3b/4 of anesthetized mice during presentation of pure tones and assessed their frequency sensitivity (Figure 1C).

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For each 90 × 100 µm region of auditory cortex we recorded from dozens to hundreds of tone-responsive (L1: 90.5 ± 64 (median ± interquartile range), n = 36 imaged regions; L3b/4: 87 ± 84, n = 36) and mostly well-tuned boutons (Figure 1—figure supplement 1). Given that the auditory cortex is tonotopically organized and that this organization must be inherited from the thalamus, the cortex's sole source of ascending auditory information, we expected the thalamic input to be tightly tonotopically ordered. Consequently, when sampling from a small patch of cortex, the boutons found therein ought to be tuned to similar frequencies. To our surprise, even neighboring boutons could be tuned to frequencies several octaves apart both in L1 and L3b/4 (Figure 1D–G). In order to quantify the variation in frequency selectivity among a population of nearby thalamocortical boutons, we determined each bouton's best frequency (BF), defined as the frequency at which the strongest response occurred in the level-averaged tuning curve (Guo et al., 2012) (Figure 1F–I), measured the co-tuning (the standard deviation of the BF distribution) for each imaged region (Figure 1J) and compared regions recorded at depths corresponding to L1 (55 ± 39 µm) with those recorded at the same x-y coordinates but at depths corresponding to L3b/4 (311 ± 43.5 µm) (Figure 1K). While the average co-tuning of thalamic boutons in a 90 × 100 µm region of auditory cortex was about one octave, there was a slight, but statistically significant, difference between the inputs to the different layers. Input to L3b/4 shows stronger co-tuning (0.93 ± 0.25 octaves) and is, thus, more homogeneous than the input to L1 (1.15 ± 0.37 octaves, p<0.001, effect size: r = 0.43, n = 36, Wilcoxon signed-rank test, Figure 1J). Within L3b/4 there was no relationship between depth and co-tuning (R = 0.14, p=0.41, n = 36, Spearman's correlation), which suggests that layer 4 is no more homogeneous than lower layer 3 (Figure 1—figure supplement 2).

Given the size of the imaged regions relative to the size of the auditory cortex and its subfields, the variation in frequency tuning appeared unexpectedly large so, for comparison, we pooled all boutons across all imaged regions and animals to obtain overall BF distributions for L1 and L3b/4. Mice are sensitive to frequencies between about 1 and 100 kHz (Willott, 2001), but their brains do not represent all frequencies within that range equally. Most auditory nerve fibers (Ehret, 1979) and most neurons in the inferior colliculus (Stiebler and Ehret, 1985) and thalamus (Anderson and Linden, 2011) are tuned to frequencies in the middle one to two octaves of the mouse's hearing range. Consistent with the frequency distributions reported in the inferior colliculus and thalamus, we found that the overall BF distribution of thalamocortical inputs had a pronounced bias towards frequencies near the center of the mouse's hearing range (Figure 1H,I).

If the thalamic input exhibits a tight tonotopic organization, the BF distributions of individual imaged regions should be much more narrow, that is they should show stronger co-tuning, than the overall BF distribution. If there is no relationship between spatial position and frequency, the BF distributions of individual regions (Figure 1L,M, bars) should resemble the overall BF distribution (Figure 1L,M, lines). We found that the overall BF distributions for L1 (1.29 octaves) and L3b/4 (1.05 octaves) exhibited slightly but significantly weaker co-tuning than individual imaged regions (L1: p=0.003, effect size: r = 0.50, n = 36; L3b/4: p=0.014, effect size: r = 0.42, n = 36, Wilcoxon signed-rank test), indicating some selectivity in the BFs represented within imaged regions. Furthermore, the difference in co-tuning between pairs of individual L1 and L3b/4 regions (Figure 1J) could be accounted for by the difference between the overall BF distributions for L1 and L3b/4 (co-tuning of boutons within imaged regions / co-tuning of overall BF distribution: for L1 = 89.4 ± 29.1%; for L3b/4 = 88.9 ± 24.3%, p=0.56, n = 36, Wilcoxon signed-rank test).

If the difference in co-tuning between individual regions and the overall BF distribution is the result of tonotopic organization, then neighboring boutons —even within a small patch of cortex— should be more similar in their tuning than topographically distant ones. Indeed, we observed a relationship between topographic distance and the difference in BF (Figure 2A). Interestingly, this very small but statistically significant correlation was present not only in the main thalamic input to L3b/4 (R = 0.034, p<10⁻⁴⁵, for all possible pairs of boutons, n = 167521, Spearman’s correlation), but also in L1 (R = 0.035, p<10⁻⁶³, n = 237107, Spearman’s correlation), suggesting that input to L1 has a similar degree of topographic order. The relationship between distance and frequency selectivity was not simply the result of a topographic clustering of boutons from the same axon because the correlation remained even when pairs with the same BF were excluded from the analysis (L3b/4: R = 0.027, p<10⁻²⁴, n = 142108; L1: R = 0.024, p<10⁻²⁸, n = 210001, Spearman’s correlation).

Figure 2

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Next we asked whether the inputs to L1 and L3b/4 are in register. We found that there is a close correspondence between the mean BF of a region imaged in L3b/4 and the mean BF of one imaged in L1 immediately above, suggesting that the two input channels are matched tonotopically (Figure 2B, R = 0.59, p=0.0002, n = 36, Pearson correlation). Finally, we examined whether, on a more global scale spanning several hundred micrometers of cortex and several imaged regions, tonotopic gradients might become apparent. Figure 2C illustrates the results from an experiment in which gaps in the vasculature allowed us to image several regions close together. The caudo-rostral low-to-high tonotopic gradient indicative of mouse A1 now emerged both in the inputs to L3b/4 and the inputs to L1. Furthermore, the co-tuning in these regions (green dots in Figure 1J) was representative of the co-tuning of the entire sample, suggesting either that most of the data were collected in A1 or that the co-tuning of the thalamic input is similar across cortical fields.

The C57BL/6 strain employed in the above experiments is the most popular laboratory mouse strain, and is used as genetic background for the overwhelming majority of genetically modified mouse strains, the availability of which make this species such a useful model system for neuroscience research. C57BL/6 mice are not normally considered to suffer from impaired hearing at the age used here (Ison et al., 2007), but there have been some reports that a decline in the number of neurons tuned to high frequencies can be detected as early as 1–2 months after birth, especially at higher levels of the auditory pathway such as the cortex (Willott et al., 1993). We therefore carried out additional experiments on a novel C57BL/6 strain in which the Cdh23^ahl allele that otherwise predisposes this strain to age-related high frequency hearing loss has been corrected (Mianné et al., 2016). Furthermore, and in order to rule out that any of the above reported results are dependent on the effects of anesthesia, we carried out these experiments in awake, passively listening animals.

While the C57BL/6NTac.Cdh23^753A>G mice also showed a bias for frequencies near the middle of their hearing range, the proportion of high frequency BFs was greater than in the C57BL/6 mice and the overall BF distribution, thus, broader (Figure 3A,B). Overall, the median number of tone-responsive boutons obtained per imaging region was lower (29.5 ± 23), which could be partly due to the effects of anesthesia vs wakefulness. However, this might also reflect strain differences or other differences in methodology, such as the fact that we tended to image these animals slightly sooner after the virus injections (3–4 weeks), but over several days rather than in a single session immediately after the window implantation. Otherwise, the results were remarkably similar. Thus, the average co-tuning per imaged region of auditory cortex was just above one octave both near the cortical surface and in the middle layers (Figure 3C) with slightly stronger co-tuning in L3b/4 (1.21 ± 0.64 octaves) than in L1 (1.42 ± 0.50 octaves, p=0.011, effect size: r = 0.40, n = 20, Wilcoxon signed-rank test, Figure 3D). Moreover, as in the preceding experiments, the inputs to L1 and L3b/4 were matched tonotopically (Figure 3E, R = 0.67, p=0.0013, n = 20, Pearson correlation). Where it was possible to image several regions over a large enough area, the caudo-rostral, low-to-high and high-to-low tonotopic gradients that are respectively indicative of A1 and the anterior auditory field (AAF), the primary cortical areas of the mouse, emerged (Figure 3F). We followed up these experiments with microelectrode recordings to obtain cortical multi-unit frequency maps that helped us to attribute individual imaging regions to particular cortical fields even in those cases when the thalamic input frequency maps were inconclusive (Figure 3G). These recordings demonstrated that the vast majority (18/20) of imaging regions were located in the primary cortical areas.

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The auditory thalamus consists of several subnuclei. Besides the ventral division of the MGB (MGBv), which is the largest subnucleus and part of the lemniscal pathway, these are the non-lemniscal dorsal division of the MGB (MGBd) and the paralaminar nuclei —the medial division of the MGB (MGBm), the posterior intralaminar nucleus (PIN), the suprageniculate nucleus (SG) and peripeduncular nucleus (PP). Our imaging experiments were designed to characterize the full extent of the auditory thalamic input available to auditory cortex. To better understand the contributions of the lemniscal, non-lemniscal and paralaminar subnuclei to the thalamocortical projection we next carried out a number of mostly anatomical experiments.

In the mouse, calretinin (CR) has been identified as a useful marker for distinguishing between different parts of the thalamus. Among the auditory subnuclei, only neurons in the MGBd, MGBm, SG, PIN and PP, but not the MGBv, contain CR (Lu et al., 2009), so we injected a mixture of viruses driving the cre-dependent expression of a red fluorescent protein and the non-cre dependent expression of a green fluorescent protein into the auditory thalamus of a CR-IRES-cre (Taniguchi et al., 2011) mouse, which expresses cre recombinase only in CR+ neurons. While green labelled neurons were found throughout the auditory thalamus, red labelled neurons were found exclusively outside of the MGBv (Figure 4A,B), which confirmed that the CR-IRES-cre line is suitable for targeting the non-lemniscal and paralaminar nuclei of the auditory thalamus. We found that input to the cortex from the neurons in these nuclei is restricted mostly to secondary auditory areas, particularly the ventrally located area A2. The few axons found in primary auditory cortical areas were restricted mostly to layer 1 and the even fewer axons found in the middle layers were located primarily below the main thalamic input (Figure 4C). Projections to regions outside the auditory cortex were found mostly in the amygdala and striatum.

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To better resolve the organization of thalamic axons in the auditory cortex we performed minute injections of a mixture of highly diluted cre-expressing and cre-dependent eGFP-expressing viruses in different parts of the auditory thalamus of C57BL/6 mice. Using this approach (Chen et al., 2013; Xu et al., 2012), we were able to transfect very small numbers of neurons (12-53) and could reveal that projections from the medial part of the auditory thalamus (MGBm/PIN) provide only extremely sparse input to auditory cortex (Figure 5A). This input primarily terminates in L1, and otherwise is located below the middle layer(s) where input from the MGBv is densest. Projections from the PP do not enter the auditory cortex and instead remain subcortical where they target amygdala, striatum and midbrain (Figure 5B). Projections from the MGBv to primary auditory cortical areas are several orders of magnitudes more extensive than the projections from other thalamic nuclei, both in L1 and in the middle layers (Figure 5C,D). Closer inspection of the MGBv axons revealed that they tend to travel from the middle layers to L1 in columnar fashion, that is, in an almost straight line (Figure 5C, inset), an arrangement which provides an anatomical substrate for our finding that L1 and L3b/4 thalamic input are in register tonotopically and exhibit a very similar organization. Furthermore, by partially reconstructing the axon from one MGBv neuron, we were able to confirm previous work in the rabbit (Cetas et al., 1999) showing how extraordinarily wide the arbors of MGBv axons tend to be, and that the same MGBv neurons provide input to different layers of the auditory cortex (Figure 5D).

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Finally, we employed a cre-dependent virus in the CR-IRES-cre mice to express GCaMP6m exclusively in CR+ neurons outside the MGBv and performed two-photon calcium imaging of their axonal boutons in the auditory cortex of awake mice. These boutons typically responded very poorly to acoustic stimulation and, consequently, only very few FRAs were obtained that passed our inclusion criterion. In these experiments, we first identified A2 by its ventral location and the particularly dense thalamocortical axon labelling. Areas slightly dorsal of A2 were deemed to be in primary auditory areas. Five out of six imaged areas in primary auditory areas produced no FRAs at all, and one produced three FRAs. Even in A2, where the labelling was typically very dense (Figure 4—figure supplement 1), these numbers were very low. Here, the median number of FRAs obtained per imaged area was 1 ± 3.5 (range: 0–9, n = 9 areas) suggesting that axon boutons with clearly defined FRAs are predominantly a feature of the projection from MGBv to the primary auditory cortical areas.

We have shown that while auditory cortical layers 1 and 3b/4 receive tonotopically matching thalamic input, the frequency selectivity of neighboring axon boutons is highly heterogeneous. That the thalamocortical projection is topographically arranged has been known for a long time (Clark, 1936; Walker, 1937). Until recently, however, it has not been possible to characterize the receptive fields of individual thalamic boutons (Roth et al., 2016), preventing any physiological assessment of how precisely organized this projection is.

We observed very similar patterns of results across two strains of C57BL/6 mice, one of which had the Cdh23^ahl allele that otherwise predisposes this strain to age-related high frequency hearing loss corrected, and across both anesthetized and awake animals. The proportion of neurons with high BFs was greater in the C57BL/6NTac.Cdh23^753A>G mice, potentially indicative of the beginning of high-frequency loss in the other animals. While the study was designed to capture the full extent of the thalamic input available to the auditory cortex, our own anatomical data together with the work of others (Hackett et al., 2011; Llano and Sherman, 2008), show that the organization we describe reflects almost exclusively the properties of the lemniscal thalamocortical projection from the MGBv to the primary cortical areas.

The presence of a well-defined tonotopic organization in the main thalamorecipient middle layers of auditory cortex (Guo et al., 2012; Hackett et al., 2011; Winkowski and Kanold, 2013) implies that the thalamic input should also be precisely arranged. Although the diffuse organization observed with axon bouton imaging contrasts with that expectation, it does provide an explanation for other findings. For instance, the observation that focal electrical stimulation of the MGB causes widespread activation of the cortex over several hundred micrometers (Hackett et al., 2011; Kaur et al., 2005) is easier to reconcile with a diffuse thalamacortical connectivity pattern in which similarly tuned thalamic axons, or even the same axon (Cetas et al., 1999), can connect with neurons located far apart in the auditory cortex. Similarly, the demonstration that thalamic inputs determine the bandwidth of the broadly tuned excitatory synaptic FRAs of auditory cortical neurons (Liu et al., 2007) can be explained more readily by our finding that most auditory cortical neurons have, within the boundaries of their dendritic trees (Richardson et al., 2009), access to thalamic terminals tuned to frequencies that collectively span several octaves.

Our results show that the cortical frequency map is built from a thalamic input map which is itself poorly organized. Thalamic projections synapse preferentially on spines within 100 µm of the soma of L3 and L4 neurons (Richardson et al., 2009), but how exactly these neurons integrate the available thalamic input to produce a more precisely ordered cortical frequency representation is unclear. Several mechanisms could contribute to this transformation. First, recent work in the visual cortex has shown that dendritic nonlinearities can affect the tuning of neurons (Wilson et al., 2016). Second, recurrent connections between cortical neurons, comprising over half of their inputs (Lübke et al., 2000), can amplify (Happel et al., 2010; Li et al., 2013) and may potentially homogenise (Liu et al., 2007) local tuning, especially if they are biased (Cossell et al., 2015). Finally, auditory cortical neurons may sample their thalamic inputs in a biased manner, similar to what has been proposed in the visual system (Reid and Alonso, 1995). However, given the broad synaptic tuning reported for thalamic inputs onto individual auditory cortical neurons — in rats the range of frequencies covered by the thalamic inputs onto a single L4 neuron lies between 3 and 5 octaves (Liu et al., 2007)— such biased connectivity seems less likely in the auditory thalamocortical system.

Although thalamic inputs primarily target the middle cortical layers, they innervate all cortical layers and particularly L1 (Huang and Winer, 2000; Kimura et al., 2003; Kondo and Ohki, 2016; Roth et al., 2016; Rubio-Garrido et al., 2009; Sun et al., 2016). Thalamic axons in L1 have different neuronal targets, mostly L1 inhibitory neurons (Cruikshank et al., 2007; Ji et al., 2016) and the apical dendrites of supra- and infragranular excitatory neurons (Harris and Shepherd, 2015; Petreanu et al., 2009), from those terminating in the middle layers, but whether the content of the information transmitted to different cortical layers also differs is not well understood. We found that L1 and L3b/4 inputs are fairly well matched tonotopically and show only minor differences in the degree of BF heterogeneity. This is consistent with two other studies which also found only minor differences between the responses to oriented gratings of thalamic axons in L1 and L4 of visual cortex (Kondo and Ohki, 2016; Sun et al., 2016). Furthermore, our anatomical work revealed that many lemniscal thalamic axons travel from the middle layers up to L1 in a columnar fashion, a feature that helps explain why the properties of the thalamic input to L1 and to the middle layers are so similar. Traditionally, thalamic inputs to L1 and L3b/4 have been classified as belonging to separate channels, with L1 inputs described as matrix-type and L3b/4 input as core-type (Clascá et al., 2012; Harris and Shepherd, 2015; Jones, 2001). Yet, a number of single axon tracing studies in various species and cortical regions have described thalamic axons that form dense plexuses in L4 and project collaterals to L1 (Cetas et al., 1999; Hashikawa et al., 1995; Kuramoto et al., 2009; Oda et al., 2004). These and our current findings suggest that the laminar separation into matrix- and core-type inputs may not be so clear-cut. Nevertheless, L1 does receive a larger proportion of input from higher-order thalamic nuclei than L3b/4 (Frost and Caviness, 1980; Linke, 1999; Linke and Schwegler, 2000; Llano and Sherman, 2008; Ryugo and Killackey, 1974; Smith et al., 2010). Given that, in other sensory systems (Roth et al., 2016), input from higher-order thalamic nuclei has been shown to carry more motor and contextual sensory signals than the input from the first order nucleus, and that we observed generally poor responses to tone stimulation in higher-order thalamic axons, it is likely that recordings in behaving animals will reveal more pronounced differences between L1 and L3b/4 input.

A key question arising from our findings is why auditory thalamocortical projections are so imprecise. Precisely-organized tonotopic maps have been identified subcortically in the lemniscal part of the mouse inferior colliculus (Barnstedt et al., 2015; Portfors et al., 2011; Stiebler and Ehret, 1985), and anatomical and electrophysiological data indicate that the lemniscal thalamus is likely to be similarly organized (Hackett et al., 2011; Lee and Sherman, 2010; Wenstrup, 2005). Input from the dorsolateral geniculate nucleus to the visual cortex tends to be highly retinotopically ordered (Roth et al., 2016), so the mouse brain is capable of establishing and maintaining very precise connections between thalamus and cortex. This suggests that the diffuse topographic arrangement we observed in the auditory system may be functionally relevant. Broad spectral integration enables auditory cortical neurons to form representations of behaviorally-relevant sound sources (Bar-Yosef et al., 2002; Las et al., 2005). Furthermore, studies in different species have shown that auditory cortical frequency representations are highly plastic over multiple timescales (Dahmen and King, 2007), and individual neurons can rapidly change their stimulus selectivity with the behavioral context (Fritz et al., 2003). Such dynamic modulation of sound frequency processing can only be possible if cortical neurons have access to spectrally broad inputs (Chen et al., 2011; Intskirveli et al., 2016; Metherate et al., 2005; Miller et al., 2001; Winer et al., 2005). The organization of the thalamocortical projection revealed here is likely to be one part of the neural architecture underpinning this rapid plasticity and the cognitive flexibility it enables.

All experiments were approved by the local ethical review committee at the University of Oxford and licensed by the UK Home Office. Nine female C57BL/6 (Harlan Laboratories, UK) mice, five female C57BL/6NTac.Cdh23^753A>G (MRC Harwell Institute, UK) mice and two female as well as one male B6(Cg)-Calb2^tm1(cre)Zjh/J (‘CR-IRES-cre’, Jackson Laboratories, CA, USA, Stock No: 010774) mice were used for calcium imaging. A further four female C57BL/6 (Envigo, UK) mice and one female B6(Cg)-Calb2^tm1(cre)Zjh/J were used for anatomical experiments.

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Thank you for submitting your article "Thalamic input to auditory cortex is locally heterogeneous but globally tonotopic" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Shihab Shamma (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The two reviewers highlight the major interest of your results. However, they point out that the results of several experiments require clarification (see reviewers' comments below). We therefore strongly encourage you to respond to the major concerns of the reviewers. They both agree on one particular experimental requirement, the labeling of lemniscal pathways, and suggest that you address this issue, in the revised form of your manuscript, in a mouse strain not affected by hearing loss at the time at which the central auditory pathway is studied. A mouse strain is now available, C57BL/6N genetically corrected for the auditory phenotype which should allow you to solve this problem (Genome Med. 2016 Feb 15;8(1):16. doi: 10.1186/s13073-016-0273-4).

Reviewer #2:

This interesting and important manuscript investigates the functional responses of thalamocortical axons in auditory cortex. The authors perform Ca²⁺ imaging from MGB terminals in auditory cortex and show that neighboring terminals can show very heterogeneous frequency tuning. They also describe the tonotopic organization in layer 1, which is new. Despite methodological shortcomings, these results are novel and this paper adds significantly to our understanding of auditory processing. The shortcomings center on the non-specificity of the experiments which make the interpretation of the experiments difficult. Multiple subnuclei of MGB are labelled and unspecified regions of auditory cortex are imaged.

Major points:

1) Methodological issues: a) The authors use C57BL/6 mice that at the time of the experiment are 9-12 weeks old. These mice are known to show hearing deficits at these ages which can alter the results.

b) The authors label both lemniscal and non-lemniscal nuclei in the MGB as well as areas outside the MGB (see Figure 1B). Thus, the tuning heterogeneity observed could be due to intermingled non-lemniscal projections on a background of tonotopically organized lemniscal projections. This should be disambiguated or at least discussed in much more detail to highlight these serious limitations. The authors should also include photomicrographs of the injection sites of more animals. How consistent and how even is their labelling of the MGB? How did they determine the borders of the MGB in the fluorescence images?

c) The authors state that the image in "auditory cortex". The auditory cortex consists of many subfields but no detail is given about which cortical subfield the images were acquired. Given the functional diversity of auditory cortical fields (see Issa et al. 2014, 2017, Baba et al. 2016) the organization of thalamic terminals in the subfields might vary. To strengthen their case the authors should identify cortical subfields (e.g. by intrinsic or flavoprotein imaging) and then image terminals in A1 which would be expected to have the highest degree of tonotopic organization.

d) The authors only use female mice. This seems to be a curious choice given the general thought of higher variability in females due to the estrus cycle. Is the cortical organization different in males?

e) How many times are sound stimuli presented? How reliable are the responses? Are there differences between layer 1 and layer 4? Could varying reliability across terminals lead to uncertain BF determination?

2) The authors remark that inputs to layer 3b and 4 seem surprisingly similar. Given that layer 4 neurons can receive thalamic inputs on their soma but also at their proximal apical dendrites these labelled inputs could target the same layer 4 neurons. Thus, the similar organization of layers 3b and 4 does not seem to be surprising.

3) In general, the authors should elaborate more on the integrative properties of layer 4 neurons such as geometry and extent of their dendritic tree. These properties are crucial to transform the thalamic input map they see into a smooth functional map.

4) The Introduction posits that because one observes functional maps in the spiking output of layer 4 neurons the underlying thalamic input have to be organized.

5) Strawman since we know for decades that layer 4 integrates over thalamic input. For example in visual cortex graduated ocular dominance (e.g. binocular cells) emerge from monocular LGN inputs. Moreover, detailed in vivo path clamp recordings by the Zhang lab at USC have shown that layer 4 neurons receive broadly tuned thalamic input suggestive of integration over many differently tuned thalamic terminals. These issues should be flushed out more in the Introduction and Discussion (also see point above).

6) In the Introduction, the authors refer to anatomical evidence of precise tonotopic projections from MBG to A1. More detail about the spatial scale of these experiments (size and separation of injection sites) is required here to compare with the expectations and results of the current study.

Reviewer #3:

This is an excellently written report dealing with a very important question. The results are also striking in that they (apparently) contradict two previous cited (2-photon) studies that had shown an organized projection at least to the intermediate cortical layers. This work extends these results by looking directly at the tuning of the dendritic boutons in L1 and L3/4 which project from the thalamus. Clearly this study is technically superb despite its obvious limitations that the authors describe. So rather than going over the paper in detail, I will list below what I consider to be important questions that need to be clarified and discussed.

1) The two previous studies in mouse are consistent on the disorder of the upper layers, but were contradictory on the main tonotopic order in L3/4. Any issues with methodology that the authors can address to explain this disparity? Those studies reported that activity from the "neuropil" was tonotopically organized, which would argue that the axonal terminals are organized, and presumably also the tuning in the boutons.

2) As the authors discuss, the injections reflect all MGB nuclei, i.e., both Limniscal and non-limniscle (L and NL) pathways. I understand the present difficulties of labeling only the L pathway (which is tonotopically organized) so to what extent should this limitation affect the conclusions of the manuscript? That is, does it nullify the whole result? Can for example the L pathway be completely tonotopically organized, but that view be obscured by the influence of NL inputs? Is there a way to know the relative contributions of these 2 inputs so as to be certain that the results are at least indicative of the nontonotopicity of the L pathway?

3) One way to help disambiguate the two pathways might be to focus the analysis of the tuning correlations on the SHARPLY tuned boutons only. This assumes that the NL pathway is non-tuned and hence may help reveal a consistent (or clearer picture due to the L pathway alone). I think in all the analyses shown, simply labeling a bouton by one number (BF) without regard to how reliable this measurement is makes the analysis too noisy. After all, most of the time, the BF is unreliable to within 1/2 to 1 octave due to noise and broad tuning.

4) The measurements in Figure 1E reveal nicely isolated "spiking" or events. Why not exploit this to examine pairs of boutons that are highly spiking-correlated and hence presumably coming from the same axon? That would reveal if axons really spread their terminals across large distances or not. And then one can say something concrete about the topography? This would also apply to relating tonotopy to spatial spread.

5) Also one can ask and answer questions such as whether there is dependence in L1 versus L3/4 on either of the above that measures of tuning and inputs, and how they might correlate?

6) I did not get the sense that topography of the MGB projections to cortex can be (or is) addressed directly? For example, it really matters if the thalamic input is organized spatially in mouse? If it is organized, then one can explore the question of tonotopy independent of topography. For example, are we sure that the MGB in the mouse is strongly tonotopically organized? If not then perhaps the projections are organized but are not tonotopic because there is none in MGB. And so on.

7) Is this whole issue of a disordered mapping in cortex just a rodent issue? And is it possible that the tonotopy is "absent" in all areas up the auditory pathway in rodents?

Reviewer #2:

This interesting and important manuscript investigates the functional responses of thalamocortical axons in auditory cortex. The authors perform Ca²⁺ imaging from MGB terminals in auditory cortex and show that neighboring terminals can show very heterogeneous frequency tuning. They also describe the tonotopic organization in layer 1, which is new. Despite methodological shortcomings, these results are novel and this paper adds significantly to our understanding of auditory processing. The shortcomings center on the non-specificity of the experiments which make the interpretation of the experiments difficult. Multiple subnuclei of MGB are labelled and unspecified regions of auditory cortex are imaged.

Major points:

1) Methodological issues: a) The authors use C57BL/6 mice that at the time of the experiment are 9-12 weeks old. These mice are known to show hearing deficits at these ages which can alter the results.

C57BL/6 mice are not normally considered to suffer from impaired high frequency hearing at the age used here (e.g. Ison et al. 2007), but there have been some reports that a decline in the number of neurons tuned to high frequencies can already be detected 1-2 months after birth, especially at higher levels of the auditory pathway such as the cortex (Willott et al., 1993). We therefore obtained the mouse strain recommended by the Reviewing Editor and carried out additional experiments. While we did not expect any fundamental differences in our results, we predicted that a larger proportion of high-frequency thalamic neurons in the C57BL/6NTac.Cdh23^753A>G mice and, thus, a broader overall BF distribution would be reflected in even weaker co-tuning and, thus, higher frequency heterogeneity than in the young adult C57BL/6 mice we used previously. That is exactly what we found: Slightly broader overall BF distribution and even weaker co-tuning in individual imaged areas. And just as before, we found that thalamocortical inputs to cortical layer (L)1 and L3b/4 are in register tonotopically. Furthermore, in order to address another concern voiced by the reviewer, we carried out these additional experiments in awake rather than anesthetized animals and followed up these experiments with an electrophysiological cortical frequency mapping experiment to help us attribute individual imaging regions to particular cortical areas.

To describe these new results, we added a figure (new Figure 3) and the following paragraphs to the Results section:

“The C57BL/6 strain employed in the above experiments is the most popular laboratory mouse strain, and is used as genetic background for the overwhelming majority of genetically modified mouse strains, the availability of which make this species such a useful model system for neuroscience research. […] These recordings demonstrated that the vast majority (18/20) of imaging regions were located in the primary cortical areas.”

b) The authors label both lemniscal and non-lemniscal nuclei in the MGB as well as areas outside the MGB (see Figure 1B). Thus, the tuning heterogeneity observed could be due to intermingled non-lemniscal projections on a background of tonotopically organized lemniscal projections. This should be disambiguated or at least discussed in much more detail to highlight these serious limitations. The authors should also include photomicrographs of the injection sites of more animals. How consistent and how even is their labelling of the MGB? How did they determine the borders of the MGB in the fluorescence images?

This is an important point and we agree that it needs to be addressed properly. We therefore carried out a number of new experiments to better understand the contributions of lemniscal, non-lemniscal and paralaminar auditory thalamic nuclei to the thalamocortical projection.

First, we showed that the CR-IRES-cre mouse line is suitable for targeting exclusively neurons outside the lemniscal auditory thalamus, i.e. outside MGBv. Then we show that input to cortex from these neurons is restricted mostly to secondary auditory cortex (particularly area A2), that the few axons found in primary auditory cortical areas are restricted mostly to layer 1, and that the even fewer axons found in the middle layers are located primarily below the main thalamic input, i.e. in upper layer 5.

Second, we carried out a large number of experiments involving the transfection of very small numbers of neurons through minute (<5 nl) injections of a mixture of highly diluted (1:50000-100000 in PBS) cre-expressing AAV and cre-dependent eGFP-expressing AAV in different parts of the auditory thalamus. We have included the results from a subset of these experiments in which we managed to label a small number of neurons in a defined subsection of the auditory thalamus. These experiments confirm and extend the above results by showing that: a) Projections from the medial part of the auditory thalamus (MGBm/PIN) provide only extremely sparse input to auditory cortex, which primarily terminates in L1, and otherwise is located below the layer(s) in which input from the MGBv is densest. b) Projections from the peripeduncular nucleus (PP) do not enter the auditory cortex and instead remain subcortical. c) Projections from the MGBv to the primary auditory areas are several orders of magnitudes more extensive than the projections from other thalamic nuclei, both in L1 and in the middle layers. d) Many MGBv axons travel from the middle layers to L1 in an almost straight line, an arrangement that provides an anatomical substrate for our finding that L1 and L3b/4 thalamic input are in register tonotopically and exhibit a very similar organisation. By partially reconstructing the axon from one MGBv neuron, we confirm previous work in the rabbit (Cetas et al., 1999) showing how extraordinarily wide the arbours of MGBv axons tend to be and that the same MGBv neurons provide input to different layers of the auditory cortex.

We now show a total of 8 photomicrographs of 5 animals. The labelling was good throughout the thalamus, although there is some variability in the brightness of the fluorescence which correlates with cell density as is evident from the DAPI images in Figure 4A, B.

Finally, by employing the CR-IRES-cre line to perform functional imaging of thalamic boutons originating exclusively from outside the MGBv, we found that only a very small minority of these boutons respond to the stimuli we used and very few FRAs were obtained that pass our inclusion criterion: Less than one on average per imaging region in primary auditory areas.

To summarise, our new anatomical data, together with other work (e.g. Llano and Sherman, 2008; Hackett et al., 2011), show that the non-lemniscal and paralaminar auditory thalamic nuclei provide either no or very sparse input to primary auditory cortical areas, and particularly to the middle cortical layers. Furthermore, the functional imaging of boutons on axons originating from outside the MGBv revealed that very few of these boutons respond to the stimuli we employed. Consequently, we can conclude that the organization we describe reflects almost exclusively the properties of the lemniscal thalamocortical projection, particularly in those cases where we can demonstrate that the imaging was carried out in primary cortical areas.

We added two new figures (Figures 4 and 5) and the following text to the manuscript:

“The auditory thalamus consists of several subnuclei. Besides the ventral division of the MGB (MGBv), which is the largest subnucleus and part of the lemniscal pathway, these are the non-lemniscal dorsal division of the MGB (MGBd) and the paralaminar nuclei – the medial division of the MGB (MGBm), the posterior intralaminar nucleus (PIN), the suprageniculate nucleus (SG) and peripeduncular nucleus (PP). […] Even in A2, where the labelling was typically very dense (Figure 4—figure supplement 1), these numbers were very low. Here, the median number of FRAs obtained per imaged area was 1 + 3.5 (range: 0-9, n = 9 areas) suggesting that axon boutons with clearly defined FRAs are predominantly a feature of the projection from MGBv to the primary auditory cortical areas.”

c) The authors state that the image in "auditory cortex". The auditory cortex consists of many subfields but no detail is given about which cortical subfield the images were acquired. Given the functional diversity of auditory cortical fields (see Issa et al. 2014, 2017, Baba et al. 2016) the organization of thalamic terminals in the subfields might vary. To strengthen their case the authors should identify cortical subfields (e.g. by intrinsic or flavoprotein imaging) and then image terminals in A1 which would be expected to have the highest degree of tonotopic organization.

We already reported one case in the original manuscript in which we can show, based on the tonotopic gradient, that the imaging was done in A1. We have added further data from terminals imaged in the primary areas (A1 and AAF). To help with cases where it wasn’t clear from the imaging data alone which field we were in, we followed up the imaging with an electrophysiological mapping experiment.

There are several reasons why we are confident that the overwhelming majority of our data (including the original dataset, where we were not always able to verify the location of the imaging field) were obtained from primary auditory areas. First, we imaged only where we could see robust labelling in L3b/4. Given that thalamic input is far denser in the primary areas than outside of those areas (as shown, for example, in the new Figure 4A), the location of our imaging fields should have been strongly biased to those primary areas. Second, our imaging windows tended to be too dorsal to include much of A2, which is the largest of the mouse’s secondary auditory areas and lies at the ventral tip of auditory cortex (for imaging of the CR-IRES-cre line, we deliberately placed the windows slightly more ventral in order to target A2). Third, the imaging fields identified to be within A1 (based on the direction of the tonotopic gradient across the cortex) show co-tuning very similar to the rest of the dataset.

d) The authors only use female mice. This seems to be a curious choice given the general thought of higher variability in females due to the estrus cycle. Is the cortical organization different in males?

Given the smaller variability in body/brain size among female mice, stereotaxic injections are somewhat easier to perform in females than in males. Furthermore, other studies, including those exploring cortical tonotopy, have often used exclusively female mice (Hackett et al., 2011; Guo et al., 2012; Joachimsthaler et al., 2014), so we do not think that this is a curious choice. Finally, we have used both male and female mice in many other projects involving calcium imaging, electrophysiology and behavioural tests and have not come across any obvious differences.

e) How many times are sound stimuli presented? How reliable are the responses? Are there differences between layer 1 and layer 4? Could varying reliability across terminals lead to uncertain BF determination?

As stated in the Materials and methods section, each stimulus was presented 9 times. This is comparable to what other calcium imaging studies have used (Bandyopadhyay et al., 2010: 6-15 repeats; Rothschild et al., 2010: 8 repeats; Winkowski and Kanold, 2013: 10 repeats; Issa et al., 2014: 3-10 repeats). The frequency spacing (0.25 octaves) was identical to that used by most of the above studies. A further step we took to ensure a robust estimation of BFs was to present stimuli at several (4) different sound levels, something which was only done by two of the aforementioned studies, but has been shown to produce much more robust tonotopic maps (Guo et al., 2012). Moreover, we used a conservative inclusion criterion (p < 0.001) to avoid including unreliably responding boutons in the dataset. Finally, we confirmed that the level-averaged tuning curves from which we determined the BFs could generally be well approximated by a Gaussian function.

In order to measure response reliability, we took the same approach as in one of our previous studies (Barnstedt et al., 2015), in which we investigated the response properties of inferior colliculus neurons and corticocollicular axon boutons. Specifically, we measured the similarity between a bouton’s responses to each of the nine presentations of its best stimulus (the frequency-level combination that evoked the strongest average response) by calculating the average correlation coefficient for all pairwise combinations of the nine responses. Here, ‘response’ is defined as the entire 45-frame trace snippet post stimulus onset. As expected, the average reliability coefficient we measured for thalamic boutons (~0.15) was higher than that measured for cortical boutons (~0.08), but lower than that in the inferior colliculus (~0.34).

There was a small difference in response reliability between L1 and L3b/4, with L1 boutons being marginally but statistically significantly more reliable than L3b/4 boutons (0.155 vs. 0.138). If this difference were to lead to more uncertain BF estimates in L3b/4 than L1, we would expect to see more heterogeneous BF distributions in L3b/4 than in L1. However, this was not the case.

In summary, there is nothing to suggest that any aspect of our stimulation paradigm or the signals we measured may have caused an unusual degree of uncertainty in BF determination. These details are all in the Materials and methods section.

2) The authors remark that inputs to layer 3b and 4 seem surprisingly similar. Given that layer 4 neurons can receive thalamic inputs on their soma but also at their proximal apical dendrites these labelled inputs could target the same layer 4 neurons. Thus, the similar organization of layers 3b and 4 does not seem to be surprising.

We agree. We did not expect to see any difference between layer 3b and 4 inputs, but thought it would be informative to include this finding.

3) In general, the authors should elaborate more on the integrative properties of layer 4 neurons such as geometry and extent of their dendritic tree. These properties are crucial to transform the thalamic input map they see into a smooth functional map.

4) The Introduction posits that because one observes functional maps in the spiking output of layer 4 neurons the underlying thalamic input have to be organized.

5) Strawman since we know for decades that layer 4 integrates over thalamic input. For example in visual cortex graduated ocular dominance (e.g. binocular cells) emerge from monocular LGN inputs. Moreover, detailed in vivo path clamp recordings by the Zhang lab at USC have shown that layer 4 neurons receive broadly tuned thalamic input suggestive of integration over many differently tuned thalamic terminals. These issues should be flushed out more in the Introduction and Discussion (also see point above).

Points 3-5 are closely related so we address them together.

We already cite many of the studies relevant to the question how auditory cortical neurons integrate thalamic input (e.g. Richardson et al., 2009, as well as Liu et al., 2007 and Li et al., 2013 from the Zhang/Tao lab) and included a paragraph in the Discussion devoted to this topic. More specifically, we already made the very point about thalamocortical integration that the reviewer refers to: “given the broad synaptic tuning reported for thalamic inputs onto individual auditory cortical neurons – in rats the range of frequencies covered by the thalamic inputs onto a single L4 neuron lies between 3 and 5 octaves (Liu et al., 2007)…”).

Nevertheless, we have added an extra sentence to the Introduction to deal with this point:

“Indeed the frequency tuning of thalamic inputs that converge onto individual auditory cortical neurons can span several octaves (Liu et al., 2007), suggesting a need for integration across differently tuned afferent terminals.”

We have also added the following:

“Thalamic projections synapse preferentially on spines within 100 µm of the soma of L3 and L4 neurons (Richardson et al., 2009) but how exactly these neurons integrate the available thalamic input to produce…”

6) In the Introduction, the authors refer to anatomical evidence of precise tonotopic projections from MBG to A1. More detail about the spatial scale of these experiments (size and separation of injection sites) is required here to compare with the expectations and results of the current study.

The point we wished to make in the Introduction [“Although retrograde tracing of thalamocortical inputs (Brandner and Redies, 1990; Hackett et al., 2011) suggests strict topography, anterograde tracing (Huang and Winer, 2000) and reconstruction of single thalamic axons (Cetas et al., 1999) indicate considerable divergence in the auditory thalamocortical pathway.”] is that the anatomical evidence is conflicting, i.e. that retrograde tracing suggests precise topography whereas anterograde tracing implies the opposite. This is not a novel observation (see review by Winer et al., 2005, cited in the manuscript) and we don’t think the manuscript would benefit from including the methodological information requested by the reviewer. An interested reader will be able to find the information in the cited papers. In fact, the two retrograde tracing studies that we cited were quite different in their methodology, e.g. in the species and tracers used and the quantities injected (Brandner and Redies worked on cats and used several different retrograde tracers, some of which were ‘injected’ in crystalline form (HRP, Bisbenzimid) and others (nuclear yellow) in liquid form (0.05 µl to 0.1 µl), whereas Hackett et al. worked in mice, used CTB and injected 0.1-0.2 µl). Nevertheless, they came to very similar conclusions, suggesting that the precise methodological details are not particularly relevant. What matters is that anterograde tracing studies (and the in vivo patch clamp recordings referred to by the reviewer in the previous point) suggest very different conclusions about the organization of the thalamcortical projection, highlighting the need for the present study using in vivo calcium imaging of thalamocortical axon terminals.

Reviewer #3:

This is an excellently written report dealing with a very important question. The results are also striking in that they (apparently) contradict two previous cited (2-photon) studies that had shown an organized projection at least to the intermediate cortical layers. This work extends these results by looking directly at the tuning of the dendritic boutons in L1 and L3/4 which project from the thalamus. Clearly this study is technically superb despite its obvious limitations that the authors describe. So rather than going over the paper in detail, I will list below what I consider to be important questions that need to be clarified and discussed.

1) The two previous studies in mouse are consistent on the disorder of the upper layers, but were contradictory on the main tonotopic order in L3/4. Any issues with methodology that the authors can address to explain this disparity? Those studies reported that activity from the "neuropil" was tonotopically organized, which would argue that the axonal terminals are organized, and presumably also the tuning in the boutons.

In the manuscript we refer to four studies (Bandyopadhyay et al., 2010; Issa et al., 2014; Rothschild et al., 2010; Winkowski and Kanold, 2013) that investigated the tonotopic organisation of the mouse auditory cortex with two-photon calcium imaging. Three, including the one that shows the strongest evidence for tonotopic order in L4 (Winkowski and Kanold, 2013), used synthetic indicators that are injected into the cortex and could have been taken up by axons at the injection site including thalamocortical ones. However, the thalamic axons should be far outnumbered by axons and dendrites belonging to cortical neurons even in L3b/4, so the contribution made by thalamic axons to the neuropil signal is likely to be very minor in these studies. The fourth study (Issa et al., 2014), which found a particularly high degree of tonotopic order, used transgenic mice expressing calcium indicators in particular cell types. One of the transgenics used expressed GCaMP3 in EMX1 positive neurons. In these mice, no or very few thalamic neurons express GCaMP and so their axons would not have contributed to the neuropil signal picked up in the cortex. Nevertheless, different amounts of neuropil signal and different amounts of ‘contamination’ of the somatic signals by the neuropil signal might explain differences between some studies. However, thalamic axons are not likely to play a significant role.

2) As the authors discuss, the injections reflect all MGB nuclei, i.e., both Limniscal and non-limniscle (L and NL) pathways. I understand the present difficulties of labeling only the L pathway (which is tonotopically organized) so to what extent should this limitation affect the conclusions of the manuscript? That is, does it nullify the whole result? Can for example the L pathway be completely tonotopically organized, but that view be obscured by the influence of NL inputs? Is there a way to know the relative contributions of these 2 inputs so as to be certain that the results are at least indicative of the nontonotopicity of the L pathway?

3) One way to help disambiguate the two pathways might be to focus the analysis of the tuning correlations on the SHARPLY tuned boutons only. This assumes that the NL pathway is non-tuned and hence may help reveal a consistent (or clearer picture due to the L pathway alone). I think in all the analyses shown, simply labeling a bouton by one number (BF) without regard to how reliable this measurement is makes the analysis too noisy. After all, most of the time, the BF is unreliable to within 1/2 to 1 octave due to noise and broad tuning.

4) The measurements in Figure 1E reveal nicely isolated "spiking" or events. Why not exploit this to examine pairs of boutons that are highly spiking-correlated and hence presumably coming from the same axon? That would reveal if axons really spread their terminals across large distances or not. And then one can say something concrete about the topography? This would also apply to relating tonotopy to spatial spread.

5) Also one can ask and answer questions such as whether there is dependence in L1 versus L3/4 on either of the above that measures of tuning and inputs, and how they might correlate?

Points 2-5 all concern the question to which extent the organization described in our manuscript reflects that of the lemniscal thalamocortical projection or additional contributions from the input originating in other thalamic nuclei and are therefore addressed together. We appreciate the suggestion to use tuning properties to disambiguate between inputs from different thalamic sources. However, the existing evidence suggests that the tuning properties of neurons from different thalamic subnuclei (Anderson and Linden, 2011) can be very similar, making this approach unlikely to produce clear-cut results. Furthermore, we acknowledge the suggestion to use correlations between boutons to identify pairs belonging to the same axon and the distances between them. However, the problem with this approach is that two boutons may be highly correlated without belonging to the same axon and, even more importantly, that this approach would be limited to the size of the individual imaging regions, i.e. about 100 µm.

In order to properly address the extremely important question of to what extent the functional organization we observe reflects the properties of the lemniscal pathway or projections from other thalamic subnuclei, we have devoted a lot of time and effort to gathering additional anatomical and functional data. These data, which are shown in the new Figures 4 and 5 have largely resolved this question by allowing us to demonstrate the relative contributions of lemniscal, non-lemniscal and paralaminar auditory thalamic nuclei to the thalamocortical projection. As a result, we believe our manuscript has been greatly strengthened (The point was also raised by reviewer #2 so please see our response given to reviewer #2 point 1b.)

6) I did not get the sense that topography of the MGB projections to cortex can be (or is) addressed directly? For example, it really matters if the thalamic input is organized spatially in mouse? If it is organized, then one can explore the question of tonotopy independent of topography. For example, are we sure that the MGB in the mouse is strongly tonotopically organized? If not then perhaps the projections are organized but are not tonotopic because there is none in MGB. And so on.

While the anatomical and electrophysiological evidence certainly suggests that the mouse MGB is tonotopically organized (e.g. Horie et al., 2013; Hackett et al., 2011), we are not aware of any imaging data that could speak to the level of tonotopy in the mouse MGB at the fine sale. The only published two-photon imaging study of a subcortical auditory structure is one of our own studies (Barnstedt et al., 2015). Based on the strict tonotopic organization that we found to be present in the central nucleus of the mouse inferior colliculus (IC), it seems highly likely that the next stage of the lemniscal pathway, the MGBv, is also highly tonotopically organized even if the level of organisation may not be as strict as that in the IC. To answer the question of whether the thalamocortical projections are organized, we can look at published as well as our own new anatomical data presented in the revised manuscript. Cetas et al. (1999), for instance, reconstructed individual axons projecting from the MGBv to the auditory cortex in rabbits and showed that their arbors tend to be extraordinarily wide and project to parts of the cortex that are millimetres apart. Our own anatomical work suggests that the projection patterns of mouse MGBv axons, taking account of the smaller brain size, are very similar. So to summarise, the existing evidence favours an explanation in which the coarse tonotopic organization evident in the thalamic input is caused by the projection patterns of thalamocortical axons rather than a poorly organised MGBv.

In the revised manuscript we added the following sentence:

“Precisely-organized tonotopic maps have been identified subcortically in the lemniscal part of the mouse inferior colliculus (Barnstedt et al., 2015; Portfors et al., 2011; Stiebler and Ehret, 1985), and anatomical and electrophysiological data indicate that the lemniscal thalamus is likely to be similarly organized (Hackett et al., 2011; Lee and Sherman, 2010; Wenstrup, 2005).”

7) Is this whole issue of a disordered mapping in cortex just a rodent issue? And is it possible that the tonotopy is "absent" in all areas up the auditory pathway in rodents?

Our own two-photon calcium imaging experiments in ferret auditory cortex (Gaucher, Panniello, Dahmen, King, Walker 2017, ARO MWM abstract PS460) suggest that the fine-scale tonotopic organisation in rodents and carnivores is similar at the cortical level.

In Barnstedt et al., 2015 we performed two-photon calcium imaging in the central nucleus of the inferior colliculus of the mouse and found that the neurons there were very tightly tonotopically ordered, so strict tonotopy is certainly present up to at least the level of the inferior colliculus. This point is addressed for the thalamus in the previous comment. We therefore don’t think that this is a mouse issue. In proposing at the end of the Discussion that the imprecise organization of the thalamocortical projection could subserve spectral integration and task- or use-dependent plasticity in the cortex, we cited studies from different species (including cats and the reviewer’s own work in ferrets). To emphasize that point, we have added ‘in different species’ to the following sentence:

“Furthermore, studies in different species have shown that auditory cortical frequency representations are highly plastic over multiple timescales (Dahmen and King, 2007), and individual neurons can rapidly change their stimulus selectivity with the behavioral context (Fritz et al., 2003).”

Author details

1. Sebastian A Vasquez-Lopez

   Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4899-1221

2. Yves Weissenberger

   Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Michael Lohse

   Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   Software, Formal analysis, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Peter Keating

   1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
   2. Ear Institute, University College London, London, United Kingdom

   Contribution

   Resources, Software, Supervision, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0670-9075

5. Andrew J King

   Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   Resources, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   Senior Editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-5180-7179

6. Johannes C Dahmen

   Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   johannes.dahmen@dpag.ox.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9889-8303

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