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Three forebrain structures directly inform the auditory midbrain of echolocating bats
Neuroscience Letters 712 (2019) 134481
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Research article
Three forebrain structures directly inform the auditory midbrain of echolocating bats
T
⁎
Tetsufumi Itoa, , Ryo Yamamotob, Takafumi Furuyamab,c, Kazuma Hasec, Kohta I Kobayasic, Shizuko Hiryuc, Satoru Honmaa a
Department of Anatomy, Kanazawa Medical University, Uchinada, Ishikawa, 920-0293, Japan Department of Physiology, Kanazawa Medical University, Uchinada, Ishikawa, 920-0293, Japan c Neuroethology and Bioengineering Laboratory, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, 610-0394, Japan b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Medial prefrontal cortex Basolateral amygdala Auditory cortex Inferior colliculus Descending projection
Echolocating bats emit various types of vocalizations for navigation and communication, and need to pay attention to vocal sounds. Projections from forebrain centers to auditory centers are involved in the attention to vocalizations, with the inferior colliculus (IC) being the main target of the projections. Here, using a retrograde tracer, we demonstrate that three forebrain structures, namely, the medial prefrontal cortex (mPFC), amygdala, and auditory cortex (AC), send direct descending projections to the central nucleus of IC. We found that all three structures projected to the bilateral IC. A comparison of the patterns of retrogradely labeled cells across animals suggests that the ipsilateral AC-IC projection is topographically organized, whereas mPFC-IC or amygdala-IC projections did not show clear topographic organization. Together with evidence from previous studies, these
Abbreviations: AC, Auditory cortex; ACC, Anterior cingulate cortex; A1, Primary auditory cortex; Bmg, Magnocellular part of the basal amygdala; Bpc, Parvocellular part of the basal amygdala; CE, Central amygdala; COp, Posterior cortical amygdaloid nucleus; DP, Dorsal peduncular cortex; Ect, Ectorhinal cortex; FG, Fluorogold; FM, Frequency modulation; GAD67, Glutamic acid decarboxylase 67; In, Intercalated nucleus of the amygdala; IC, Inferior colliculus; ICC, Central nucleus of the IC; IL, Infralimbic cortex; La, Lateral amygdala; Ma, Medial amygdala; MD, Mediodorsal thalamic nucleus; MGB, Medial geniculate body; mPFC, Medial prefrontal cortex; M2, Secondary motor cortex; ORB, Orbitofrontal cortex; PAC, Periamygdaloid cortex; PAG, Periaqueductal gray; PrL, Prelimbic cortex; S1, Primary somatosensory cortex; S2, Secondary somatosensory cortex; TEA, Temporal association area; TTd, Dorsal part of taenia tecta; +, Positive ⁎ Corresponding author. E-mail address: [email protected] (T. Ito). https://doi.org/10.1016/j.neulet.2019.134481 Received 2 April 2019; Received in revised form 2 September 2019; Accepted 4 September 2019 Available online 05 September 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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results suggest that three descending projections to the IC form loops between the forebrain and IC to make attention to various vocal sounds.
1. Introduction
relationships between centers for navigation, communication, and hearing in echolocating bats. The medial prefrontal cortex (mPFC) plays important roles in navigation [4–6]. Because the mPFC is essentially multimodal [7], attention to navigation by mPFC is likely to modify the activity of auditory centers in bats. Bats behaviorally show selective attention to communication calls, which convey emotional value [1]. The basolateral amygdala integrates multimodal sensory information and links emotional value to information [8], and is important for communication call processing in bats [9–11]. These studies demonstrate both navigatory and emotive attention for hearing and suggest connections between forebrain centers and
Vocalizations are critical for both navigation and communication in echolocating bats. Both functions require individuals to carefully attend to the incoming sound signals, which means that their auditory system need to be "tuned" to these sounds to extract the maximum information from them. For navigation, bats listen to the echoes returning from biosonar pulses and for communication they listen to the social calls of conspecifics [1]. Behavioral evidence of attention for biosonar pulses is jamming avoidance [2]. To concentrate on their own pulses, which may change dynamically during navigation [3], bats need to optimize their hearing system quickly since echoes of pulses arrive few milliseconds after the vocalization. Therefore, there are likely to be strong
Fig. 1. Nissl cytoarchitecture of mPFC of the Japanese house bat. (A-C) Micrographs of coronal sections of 160-μm-intervals. (D-F) Micrographs of sagittal sections of 160-μm-intervals. Scale bars: 250 μm. 2
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3.1. Medial prefrontal cortex (mPFC)
auditory nuclei in bats. The inferior colliculus (IC) is a good candidate auditory nucleus according to the neuroethological theory of IC operation [12]. In the theory, IC transforms the rapid inputs of biologically important sounds to slowed output to match the rhythm of cortical activity, which is related to the timing of specific behaviors. In bats, communication calls and biosonar pulses are considered to be sign stimuli because they elicit fixed action patterns for hunting, escape, or vocal communication [3,9–11,13]. Accordingly, we hypothesize that the IC is involved as a part of conditional loops with other brain structures controlling different behaviors. Here, we demonstrate that three forebrain centers, namely the mPFC, basolateral amygdala, and AC make direct descending projections to the IC of Japanese house bats. Together with previous studies, the findings support the presence of hypothetical loops.
In the medial prefrontal regions, located dorsal and rostral to the corpus callosum and medial to the secondary motor cortex (M2), we identified the anterior cingulate, prelimbic, infralimbic, and dorsal peduncular cortices (ACC, PrL, IL, and DP), and the dorsal part of the taenia tecta (TTd) from dorsal to ventral, respectively, using Nissl staining and in accordance with a cytoarchitectonic study in the rat [15](Fig. 1, S1). Pyramidal cell layers (layers 2, 3, 5) were clearly identified based upon the presence of densely accumulated triangular cell bodies. All of these areas lacked accumulation of granule cells, and layer 4 was undistinguishable with Nissl stain. M2 had a distinct layer 2 and contained numerous large cells in layer 5 (Fig. S1A). ACC was characterized by a distinct layer 2 and a small cell size in layer 6 (Fig. S1B). PrL also had a distinct layer 2, although its cells were less aligned than those in the ACC (Fig. S1C). In IL, layers 2 and 3 were not distinguishable, and the cell density of layer 6 was higher than that in the PrL (Fig. S1D). In the DP, layer 2/3 was thin and the cell density of layer 2/3 was sparser than that in the IL (Fig. S1E). The cell density in layer 2/3 of TTd was higher than that in DP (Fig. S1F). Although the cytoarchitectonic features were distinct among areas, the delineation of areas may be inaccurate because it was based upon anatomy only. The same is true for the delineation of the amygdaloid complex and temporal cortical fields described below.
2. Materials and methods Japanese house bats (Pipistrellus abramus) were used in this study. All animals were also used for previous papers which focused on the cytoarchitecture and synaptic organization of brainstem auditory pathways [14], and methods were described in the previous paper [14] and supplemental methods. 3. Results We first describe the cytoarchitecture of forebrain where the retrogradely labeled cells were consistently found. Next, we present the distribution pattern of the retrogradely labeled cells in the forebrain structures.
3.2. Amygdaloid complex Like other bat species, the Japanese house bat has a hypertrophied amygdaloid complex [16,17]. The amygdaloid complex is composed of
Fig. 2. Cytoarchitectonic features of the amygdala defined with Nissl stain and GAD67 immunoreactivity. (A) A low-power micrograph of a Nissl-stained coronal section containing the central amygdala (CE). (B) A low-power micrograph of a GAD67-immunostained section located next to the sections shown in A. (C-N) Highpower micrographs showing the morphology of cell bodies (upper) and pattern of GAD67 immunoreactivity (lower) in subdivisions of the amygdaloid complex. The images were taken from the central part of each region. Scale bars: 500 μm (A, B), and 25 μm (C-N). 3
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higher than that in the Bpc, AB, and medio-ventral part of La (Fig. 2F). Bpc was located lateral to Bmg and was composed of irregularly shaped, intensely stained small cells (Fig. 2K). AB was located ventral to Bmg and Bpc and consisted of medium-sized, sparsely distributed cells (Fig. 2M). The density of GAD67+ fibers was intermediate to that of Bmg and Bpc. La was further subdivided into medio-ventral and dorsolateral parts: the medio-ventral part was composed of weakly stained medium-sized neurons, while the dorso-lateral part was composed of intensely stained small neurons (Fig. 2G, I). The density of GAD67+ fibers was lowest in the medio-ventral part and highest in the dorsolateral part of the amygdaloid complex (Fig. 2H, J).
six areas, namely the lateral (La), basal, accessory basal (AB), intercalated (In), and central (CE) nuclei, as well as the cortical amygdaloid areas (Fig. 2). The basal nucleus was further divided into magnocellular and parvocellular (Bmg and Bpc) areas based on somatic size. The cortical areas were distinguished as the medial nucleus (Ma), the posterior cortical nucleus, and the periamygdaloid cortex from medial to lateral, respectively. The cortical areas were easily discriminated from other amygdaloid nuclei by their laminar formation. The other nuclei were distinguishable based on Nissl cytoarchitecture [16,17] and distribution of GABAergic structures [monkey,18](Fig. 2). La, Bmg, Bpc, and AB formed a large mass located lateral to the CE, and In was located between them. The In was composed of densely packed small neurons and was intensely immunoreactive for GAD67. CE was composed of densely packed medium-sized neurons (Fig. 2A, C). The density of GAD67+ fibers was high, especially in the center of the nucleus (Fig. 2B, D). Bmg stood out due to the presence of intensely stained large cells (Fig. 2E), and the density of GAD67+ fibers in the Bmg was
3.3. Temporal cortical regions In the temporal and parietal cortical areas, layer 2 is clearly identified with a high packing density. Conversely, there was no sharp border between layer 3 and 4. In the dorsal part of the temporal cortex,
Fig. 3. Nissl cytoarchitecture of the auditory cortex (AC) and surrounding cortical structures. (A) Low-magnification micrographs of coronal (left) and horizontal (right) sections containing the AC. (B-F) High-power micrographs showing laminar organization of cortical areas. Panels B–D were acquired from coronal sections, while panels E and F were from horizontal sections. Scale bars: 500 μm (A), and 100 μm (B-F). 4
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surrounding areas (Fig. 3A, C). In addition to the properties shared with the horseshoe bat, layer 6 of AC in Japanese house bat is thicker than surrounding areas, and inside layer 6, cells were evenly distributed. The cell density in layer 2/3 was higher in A1 than in the surrounding areas. In TEA, the cell density was lower and the somatic size was smaller than in A1 (Fig. 3D). In Ect, layer 4 was undistinguishable from other layers, and the cell density of layer 2/3 was lower than that in A1 (Fig. 3E). S2 was characterized by a lower organization of layer 2/3 and a thinner layer 4 compared with A1 (Fig. 3F). From serial Nissl-stained sections cut coronally, we were able to reconstruct the three-dimensional (3D) organization of the IL, A1, and Bmg (Fig. S2). For the following analyses, we used the cytoarchitectonic
density of cells in layer 4 increased, and we identified the region as AC (Fig. 3A). AC was located ventral to the primary somatosensory cortex (S1) and dorsal to the temporal association area (TEA) in coronal sections. In horizontal sections, the AC was rostral to the ectorhinal cortex (Ect), and caudal to the secondary somatosensory cortex (S2) and Ect in dorsal and ventral sections, respectively (Fig. 3A). The AC itself is likely subdivided into several regions: in the central part of the AC, the cytoarchitecture was clearly different from the surrounding areas, and we assumed the central part to be the primary AC (A1). A1 was distinguished from surrounding cortical areas by the thickness of the cortical layers [horseshoebat,19] and the cell density [rat,20]: Layer 3/ 4 was markedly thicker than layer 5, and layer 1 was thinner than
Fig. 4. Images of horizontal sections containing retrogradely labeled neurons (C, G, J) and the adjacent sections stained with cresyl violet (A, B, E, F, I) or for GAD67 antibody (D, H). Case of Pa128. (A-D) Ipsilateral mPFC. Retrogradely labeled cell bodies were located in layer 6 of the IL. A box in A indicates the approximate location of panels B–D. (E-H) Ipsilateral amygdala. Retrogradely labeled cell bodies were located in Bmg. A box in E indicates the approximate location of panels F-H. (I, J) Ipsilateral auditory cortex. Retrogradely labeled cell bodies were located in layer 5. Scale bars: 500 μm (A, E), and 200 μm (others). 5
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small leakage of the tracer in the periaqueductal gray (PAG) and two other cases due to very weak labeling in the forebrain structures. It was noted that the case with leakage showed a similar pattern of retrograde labeling in the forebrain. From the distribution of labeled cells around the injection site (red dots in Fig. S3E-H), we conclude that the injection sites of four cases covered most parts of the ICC and retrogradely labeled cells were likely to target the ICC. We normalized dorso-ventral and medio-lateral position of the injection centers (Fig. S3J-L; see also Fig. 1K, L of Ito et al., 2018) [14].
atlases and 3D reconstructions for reference. 3.4. Injection sites of fluorogold (FG) In all seven cases which are described in a previous paper [14], FG was injected inside the IC. Of these, four cases (Pa119, Pa125, Pa128, and Pa131) showed no leakage of FG outside the IC and used for analysis (Fig. S3A–H; see also Fig. 1 of [14]) and intensely labeled cells in the forebrain structures. We excluded one case (Pa114) because of
Fig. 5. (A-D) Side views of 3D models of the forebrain and borders of AC, IL, and Bmg, and spatial distribution of retrogradely labeled cells (dots), reconstructed from horizontal serial sections of the four IC injection cases. Upper panels indicate the ipsilateral hemisphere, and lower panels indicate the contralateral side. (E) Schematic diagrams showing the distribution of labeled cells of the four cases. Blue, green, red, and orange borders correspond to ventro-lateral (Pa119), dorsolateral (Pa125), dorso-intermediate (Pa131), and dorso-medial (Pa128) injection, respectively. Gray grids: 100 μm. (F) Patterns of retrogradely labeling in IL, AC, and Bmg along one axis (dorso-ventral axis for IL, and rostro-caudal axis for others). Percentages to total retrogradely labeled forebrain cells are shown in the vertical axis. Blue, green, red, and orange lines and symbols correspond to ventro-lateral (Pa119), dorso-lateral (Pa125), dorso-intermediate (Pa131), and dorso-medial (Pa128) injection, respectively. 6
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3.5. Distribution of retrogradely labeled cells
4.1. Technical considerations
In the horizontal forebrain sections of all four cases analyzed, retrogradely labeled cells were consistently found in three regions, namely the mPFC, the temporal cortex, and the amygdala. In the bilateral mPFC (Fig. 4A), retrogradely labeled cells were found rostral to the corpus callosum. Based on neighboring sections stained with cresyl violet (Fig. 4B) and GAD67 antibody (Fig. 4D), we found that the retrogradely labeled cells were restricted to layer 6 of the IL (Fig. 4C). In the amygdala (Fig. 4E), the labeled cells were located rostral to the lateral ventricle and formed a cluster that was elongated rostro-caudally (Fig. 4G). Based on neighboring sections stained with cresyl violet (Fig. 4F) and GAD67 antibody (Fig. 4H), we found that the retrogradely labeled cells were restricted in the Bmg of the amygdaloid complex. In the temporal cortex, the labeled cells were found in layer 5 of the AC (Fig. 4I, J). In the thalamus, although we found labeled cells in shell nuclei of auditory thalamus, but did not analyze because brains were transected around the medial geniculate body (MGB) and therefore it was impossible to reconstruct auditory thalamus. Next, we made a 3D reconstruction of the forebrain, AC, IL, and Bmg based on Nissl cytoarchitecture and distribution of retrogradely labeled cells in the forebrain (Fig. 5A–D). Although the retrogradely labeled cells were found bilaterally, the number of cells was higher in the ipsilateral side than in the contralateral side. This tendency was more obvious in the AC. In the ipsilateral AC of Pa119, which received a large injection in the ventro-lateral IC (the highest frequency injection; Fig. S3A, E, J), the retrogradely labeled cells were found throughout the AC and the density was lower in the dorsal part (Fig. 5A). In case of Pa125, which received an injection in dorso-lateral IC (the second highest frequency injection; Fig. S3B, F, J), the labeled cells were sparsely distributed and the cell density was higher in the ventral part. In the rostro-caudal axis, the cell density was higher in the midpoint (Fig. 5B). In case of Pa131, which received an injection in the dorso-intermediate part of the IC (the lowest frequency injection; Fig. S3C, G, J), the density of the retrogradely labeled cells was higher in the dorsal part, specifically in the midpoint of the dorso-ventral axis (Fig. 5C). In case of Pa128, which received an injection in dorso-medial IC (the second lowest frequency injection; Fig. S3D, H, J), the labeled cells were denser in the caudal and dorsal parts. Except for some labeled cells in ventral parts, the rostral part of the AC was devoid of retrogradely labeled cells (Fig. 5D). The rostro-caudal distribution of retrogradely labeled cells revealed that the highest percentage occurred in the more rostral part of ipsilateral AC in Pa119 (highest frequency injection) and the more caudal part in Pa128 and Pa131 (low frequency injection) (Fig. 5F). Such topographic organization was not obvious in the contralateral side, which contained few labeled cells. Taken together, these findings suggest that caudal part of AC projects to dorsal, low frequency part of the IC, while rostral part projects to ventral, high frequency part of the IC. The medio-lateral axis of the ICC is likely related to the dorso-ventral axis of the AC. In the IL and Bmg, there was no obvious difference in the distribution of labeled cells among cases. Due to the small sample size and variable distributions in the ipsilateral side, no strong conclusions can be drawn about topographic organization. The distribution patterns of labeled cells in each case are summarized in Fig. 5E and Table S1.
As the direct projection from the prefrontal cortex to the IC has not been previously described, alternative interpretations of results require discussion. Nonspecific labeling is unlikely as the labeling pattern in other structures (i.e., amygdala, AC, and brainstem nuclei) was consistent with previous studies [17,21]. Trans-synaptic transport of FG is unlikely because the projection from the brainstem nuclei to the IC, which was described in another study [14], was very similar to that in other bat species [21]. FG uptake by the fibers of the passage is unlikely because this can only occur if IL fibers pass evenly inside the IC without termination. In other mammalian species, although IL fibers terminate in the PAG, they do not enter the IC [22]. As none of the four cases reported here showed leakage of FG from the IC (Fig. S3), it is unlikely that IL fibers that terminated or passed through the PAG took up FG. Furthermore, PAG-projecting IL neurons are located in layer 5 [23], while IC-projecting IL neurons were located in layer 6 (Fig. 4C). Taken together, the results of the current study strongly support the presence of direct projections from the IL to IC in Japanese house bats.
4.2. Comparative views of forebrain–IC loops In non-bat mammals, only IC-AC loop is present (dotted lines in Fig. 6): the IC projects to the MGB, the auditory thalamic structure, the MGB then projects to the AC, and the AC in turn projects back to the IC [24]. The pathway to A1 is tonotopically organized. Vespertilionidae bats include Japanese house bats and use downward frequency-modulated (FM) sweeps with a terminal phase showing a pure-tone-like constant frequency (quasi-CF) [13]. In several species of Vespertilionidae bats, the AC is composed of a smaller rostral field not sensitive to a delay of sound, and a larger caudal field sensitive to the delay [25]. Both fields showed tonotopicity and the caudal field exhibited a caudalto-rostral frequency gradient. Other bat species with FM calls (Carollia perspicillata) also have a large caudal field with a caudal-to-rostral frequency gradient [26]. The distribution of retrogradely labeled cells in the ipsilateral AC (Fig. 5) is likely to reflect the caudal-to-rostral frequency gradient in the caudal auditory field, and this suggests that the tonotopic organization of Japanese house bat AC is similar to other FM bat species. The amygdala receives auditory information from both the MGB
4. Discussion In this study, we found that three different forebrain regions, the IL, AC, and Bmg, directly innervate the bilateral IC in the Japanese house bat. Combined with previous studies on other bat and non-bat mammalian species, we suggest the presence of three loops between the IC and the forebrain regions of the bat brain (Fig. 6).
Fig. 6. Three forebrain structures directly inform the IC. Dotted arrows indicate connections commonly found in mammalian species. Thick arrows indicate connections found in echolocation bats. 7
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Acknowledgments
[27] and AC [28]. In non-bat mammals, the amygdala is interconnected with the mPFC [7] and AC [29]. In echolocating bats, the Bmg projects directly to the IC [currentstudy,17]; No clear topographic organization was found in the projection, and activation of the pathway may elicit a change of sensitivity of the whole IC. Since frequency spectra of bat communication calls are broad [11], the organization of projections may be effective for the attention and reaction to communication calls. In non-bat mammals, the relationship between the AC and mPFC is indirect [7]. In bats, reciprocal connection with PFC was found between AC [30] and suprageniculate nucleus, a shell region of MGB [31]. Consequently, auditory responses are evoked in the frontal auditory field of the PFC of bats [32], although the correspondence between frontal auditory field and IL is not clear. We thus estimate that there are at least three loops between the IC and forebrain structures of echolocating bats.
This work was supported by grants from the Japan Society for the Promotion of Science (KAKENHI Grant Number 19H04212 for TI; 18K07572 and 18KK0468 for RY; 16H06542 for SH; 17H01769 for KIK), Okawa Foundation for Information and Telecommunications, Suzuken Memorial Foundation, and Daiwa Securities Health Foundation to TI. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.neulet.2019.134481. References [1] G.S. Wright, et al., Social calls of flying big brown bats (Eptesicus fuscus), Front. Physiol. 4 (2013) 214. [2] K. Hase, et al., Rapid frequency control of sonar sounds by the FM bat, Miniopterus fuliginosus, in response to spectral overlap, Behav. Processes 128 (2016) 126–133. [3] E. Fujioka, et al., Echolocating bats use future-target information for optimal foraging, Proc. Natl. Acad. Sci. U. S. A. 113 (17) (2016) 4848–4852. [4] B. Delatour, P. Gisquet-Verrier, Functional role of rat prelimbic-infralimbic cortices in spatial memory: evidence for their involvement in attention and behavioural flexibility, Behav. Brain Res. 109 (1) (2000) 113–128. [5] V. Hok, et al., Coding for spatial goals in the prelimbic/infralimbic area of the rat frontal cortex, Proc. Natl. Acad. Sci. U. S. A. 102 (12) (2005) 4602–4607. [6] H.T. Ito, E.I. Moser, M.B. Moser, Supramammillary nucleus modulates spike-time coordination in the prefrontal-thalamo-hippocampal circuit during navigation, Neuron 99 (3) (2018) p. 576-587 e5. [7] W.B. Hoover, R.P. Vertes, Anatomical analysis of afferent projections to the medial prefrontal cortex in the rat, Brain Struct. Funct. 212 (2) (2007) 149–179. [8] S. Duvarci, D. Pare, Amygdala microcircuits controlling learned fear, Neuron 82 (5) (2014) 966–980. [9] J. Ma, J.S. Kanwal, Stimulation of the basal and central amygdala in the mustached bat triggers echolocation and agonistic vocalizations within multimodal output, Front. Physiol. 5 (2014) 55. [10] R.T. Naumann, J.S. Kanwal, Basolateral amygdala responds robustly to social calls: spiking characteristics of single unit activity, J. Neurophysiol. 105 (5) (2011) 2389–2404. [11] M.A. Gadziola, S.J. Shanbhag, J.J. Wenstrup, Two distinct representations of social vocalizations in the basolateral amygdala, J. Neurophysiol. 115 (2) (2016) 868–886. [12] J.H. Casseday, E. Covey, A neuroethological theory of the operation of the inferior colliculus, Brain Behav. Evol. 47 (6) (1996) 311–336. [13] K. Goto, S. Hiryu, H. Riquimaroux, Frequency tuning and latency organization of responses in the inferior colliculus of Japanese house bat, Pipistrellus abramus, J. Acoust. Soc. Am. 128 (3) (2010) 1452–1459. [14] T. Ito, et al., Organization of projection from brainstem auditory nuclei to the inferior colliculus of Japanese house bat (Pipistrellus abramus), Brain Behav. 8 (8) (2018) p. e01059. [15] C.G. Van Eden, H.B. Uylings, Cytoarchitectonic development of the prefrontal cortex in the rat, J. Comp. Neurol. 241 (3) (1985) 253–267. [16] T. Yokoyama, Comparative anatomical studies on the amygdaloid nuclear complex, especially in Chiroptera, Acta Anat. Niigata Sect. Anat. Univ. Niigata 35 (1955) 47–68. [17] R.A. Marsh, et al., Projection to the inferior colliculus from the basal nucleus of the amygdala, J. Neurosci. 22 (23) (2002) 10449–10460. [18] A. Pitkanen, D.G. Amaral, The distribution of GABAergic cells, fibers, and terminals in the monkey amygdaloid complex: an immunohistochemical and in situ hybridization study, J. Neurosci. 14 (4) (1994) 2200–2224. [19] S. Radtke-Schuller, Cytoarchitecture of the medial geniculate body and thalamic projections to the auditory cortex in the rufous horseshoe bat (Rhinolophus rouxi). I. Temporal fields, Anat. Embryol. (Berl.) 209 (1) (2004) 59–76. [20] N. Palomero-Gallagher, K. Zilles, Isocortex, in: G. Paxinos (Ed.), The Rat Nervous System, Elsevier, London, 2015, pp. 601–625. [21] J.M. Zook, J.H. Casseday, Origin of ascending projections to inferior colliculus in the mustache bat, Pteronotus parnellii, J. Comp. Neurol. 207 (1) (1982) 14–28. [22] S.L. Buchanan, et al., Efferent connections of the medial prefrontal cortex in the rabbit, Exp. Brain Res. 100 (3) (1994) 469–483. [23] A.N. Ferreira, et al., Highly differentiated cellular and circuit properties of infralimbic pyramidal neurons projecting to the periaqueductal gray and amygdala, Front. Cell. Neurosci. 9 (2015) 161. [24] T. Ito, M.S. Malmierca, Neurons, connections, and microcircuits of the inferior colliculus, in: D.L. Oliver (Ed.), The Mammalian Auditory Pathways, Springer, New York, 2018, pp. 127–167. [25] W.E. O’Neill, Bat auditory cortex, in: A.N. Popper, R.R. Fay (Eds.), Hearing by Bats, Springer-Verlag, New York, 1995, pp. 416–480. [26] K.H. Esser, A. Eiermann, Tonotopic organization and parcellation of auditory cortex in the FM-bat Carollia perspicillata, Eur. J. Neurosci. 11 (10) (1999) 3669–3682. [27] J.E. LeDoux, D.A. Ruggiero, D.J. Reis, Projections to the subcortical forebrain from
4.3. Functional considerations According to the neuroethological theory of the IC operation [12], a slowed signal processing in the IC is suitable for tuning to sign stimuli, which have biological importance and trigger fixed action patterns. In this aspect, descending projections from the three forebrain centers to the IC will control the sensitivity to different sign stimuli, and in turn change the balance of activity in the forebrain centers responsible for specific behaviors related with sign stimuli. The sign stimuli related to the AC-IC projection corresponds likely to biosonar pulses: The tonotopic organization of the descending pathway from the AC enables control of the sensitivity of IC neurons to a specific and optimal frequency. After tones associated with the fearful stimulus (foot shock), frequency tuning of IC neurons shifts toward the frequency of tones, and AC is responsive for the plastic change [33]. This type of plasticity may underlie attention to shifted quasi-CF of biosonar pulses occurring when conspecific bats are present [2]. Therefore, it is likely that the AC-IC projection is related to echolocation behaviors, and plays a role in biasing “cognitive attention” to relevant sounds via experience-dependent plasticity. The sign stimuli related to the Bmg-IC projection is likely to be communication calls: It has been shown that the basal amygdala of bats is related with expression and reception of conspecific vocalization conveying emotional values [9–11]. The amygdalocollicular projection may produce emotional attention by modifying activity of whole IC immediately after calls with a particular emotional value. We speculate that the function of the IL-IC projection is based upon anatomical organization of the IL, which has been extensively studied in rodents [23,34]. Layer 6 of IL projects to the mediodorsal thalamic (MD) nucleus [34], whereas other layers project to amygdala and PAG [23,34]. These observations suggest that descending commands to the IC are closely related to those to the MD. Since synchronization of the MD and PFC is known to be important for working memory tasks [35], the IL-IC projection, also originated from layer 6, may be important for cognitive tasks, i.e., echolocation-based navigation. Considering the reciprocal connections among the three forebrain regions in bats, we speculate that the three forebrain regions cooperate and send output commands to the IC to modify the sensitivity to sign stimuli based on the behavior that the animal is engaged in. Loops between these three forebrain regions and IC may play an important attentional role in modulating, selecting and/or amplifying inputs, possibly via egocentric selection. By informing or providing feedback to the IC of executive or cognitive functions or emotional state, the IC can transmit the most relevant information to the frontal and auditory cortices and the amygdala for navigational, cognitive and emotive processing, respectively. In this way, as predicted earlier [12], the IC may participate in slowing outputs for the most effective and timely control of behavior.
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[28]
[29] [30] [31]
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anatomically defined regions of the medial geniculate body in the rat, J. Comp. Neurol. 242 (2) (1985) 182–213. F. Mascagni, A.J. McDonald, J.R. Coleman, Corticoamygdaloid and corticocortical projections of the rat temporal cortex: a Phaseolus vulgaris leucoagglutinin study, Neuroscience 57 (3) (1993) 697–715. Y. Yang, et al., Selective synaptic remodeling of amygdalocortical connections associated with fear memory, Nat. Neurosci. 19 (10) (2016) 1348–1355. D.C. Fitzpatrick, J.F. Olsen, N. Suga, Connections among functional areas in the mustached bat auditory cortex, J. Comp. Neurol. 391 (3) (1998) 366–396. J.B. Kobler, S.F. Isbey, J.H. Casseday, Auditory pathways to the frontal cortex of the
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Three forebrain structures directly inform the auditory midbrain of echolocating bats
T
⁎
Tetsufumi Itoa, , Ryo Yamamotob, Takafumi Furuyamab,c, Kazuma Hasec, Kohta I Kobayasic, Shizuko Hiryuc, Satoru Honmaa a
Department of Anatomy, Kanazawa Medical University, Uchinada, Ishikawa, 920-0293, Japan Department of Physiology, Kanazawa Medical University, Uchinada, Ishikawa, 920-0293, Japan c Neuroethology and Bioengineering Laboratory, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto, 610-0394, Japan b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Medial prefrontal cortex Basolateral amygdala Auditory cortex Inferior colliculus Descending projection
Echolocating bats emit various types of vocalizations for navigation and communication, and need to pay attention to vocal sounds. Projections from forebrain centers to auditory centers are involved in the attention to vocalizations, with the inferior colliculus (IC) being the main target of the projections. Here, using a retrograde tracer, we demonstrate that three forebrain structures, namely, the medial prefrontal cortex (mPFC), amygdala, and auditory cortex (AC), send direct descending projections to the central nucleus of IC. We found that all three structures projected to the bilateral IC. A comparison of the patterns of retrogradely labeled cells across animals suggests that the ipsilateral AC-IC projection is topographically organized, whereas mPFC-IC or amygdala-IC projections did not show clear topographic organization. Together with evidence from previous studies, these
Abbreviations: AC, Auditory cortex; ACC, Anterior cingulate cortex; A1, Primary auditory cortex; Bmg, Magnocellular part of the basal amygdala; Bpc, Parvocellular part of the basal amygdala; CE, Central amygdala; COp, Posterior cortical amygdaloid nucleus; DP, Dorsal peduncular cortex; Ect, Ectorhinal cortex; FG, Fluorogold; FM, Frequency modulation; GAD67, Glutamic acid decarboxylase 67; In, Intercalated nucleus of the amygdala; IC, Inferior colliculus; ICC, Central nucleus of the IC; IL, Infralimbic cortex; La, Lateral amygdala; Ma, Medial amygdala; MD, Mediodorsal thalamic nucleus; MGB, Medial geniculate body; mPFC, Medial prefrontal cortex; M2, Secondary motor cortex; ORB, Orbitofrontal cortex; PAC, Periamygdaloid cortex; PAG, Periaqueductal gray; PrL, Prelimbic cortex; S1, Primary somatosensory cortex; S2, Secondary somatosensory cortex; TEA, Temporal association area; TTd, Dorsal part of taenia tecta; +, Positive ⁎ Corresponding author. E-mail address: [email protected] (T. Ito). https://doi.org/10.1016/j.neulet.2019.134481 Received 2 April 2019; Received in revised form 2 September 2019; Accepted 4 September 2019 Available online 05 September 2019 0304-3940/ © 2019 Elsevier B.V. All rights reserved.
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results suggest that three descending projections to the IC form loops between the forebrain and IC to make attention to various vocal sounds.
1. Introduction
relationships between centers for navigation, communication, and hearing in echolocating bats. The medial prefrontal cortex (mPFC) plays important roles in navigation [4–6]. Because the mPFC is essentially multimodal [7], attention to navigation by mPFC is likely to modify the activity of auditory centers in bats. Bats behaviorally show selective attention to communication calls, which convey emotional value [1]. The basolateral amygdala integrates multimodal sensory information and links emotional value to information [8], and is important for communication call processing in bats [9–11]. These studies demonstrate both navigatory and emotive attention for hearing and suggest connections between forebrain centers and
Vocalizations are critical for both navigation and communication in echolocating bats. Both functions require individuals to carefully attend to the incoming sound signals, which means that their auditory system need to be "tuned" to these sounds to extract the maximum information from them. For navigation, bats listen to the echoes returning from biosonar pulses and for communication they listen to the social calls of conspecifics [1]. Behavioral evidence of attention for biosonar pulses is jamming avoidance [2]. To concentrate on their own pulses, which may change dynamically during navigation [3], bats need to optimize their hearing system quickly since echoes of pulses arrive few milliseconds after the vocalization. Therefore, there are likely to be strong
Fig. 1. Nissl cytoarchitecture of mPFC of the Japanese house bat. (A-C) Micrographs of coronal sections of 160-μm-intervals. (D-F) Micrographs of sagittal sections of 160-μm-intervals. Scale bars: 250 μm. 2
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3.1. Medial prefrontal cortex (mPFC)
auditory nuclei in bats. The inferior colliculus (IC) is a good candidate auditory nucleus according to the neuroethological theory of IC operation [12]. In the theory, IC transforms the rapid inputs of biologically important sounds to slowed output to match the rhythm of cortical activity, which is related to the timing of specific behaviors. In bats, communication calls and biosonar pulses are considered to be sign stimuli because they elicit fixed action patterns for hunting, escape, or vocal communication [3,9–11,13]. Accordingly, we hypothesize that the IC is involved as a part of conditional loops with other brain structures controlling different behaviors. Here, we demonstrate that three forebrain centers, namely the mPFC, basolateral amygdala, and AC make direct descending projections to the IC of Japanese house bats. Together with previous studies, the findings support the presence of hypothetical loops.
In the medial prefrontal regions, located dorsal and rostral to the corpus callosum and medial to the secondary motor cortex (M2), we identified the anterior cingulate, prelimbic, infralimbic, and dorsal peduncular cortices (ACC, PrL, IL, and DP), and the dorsal part of the taenia tecta (TTd) from dorsal to ventral, respectively, using Nissl staining and in accordance with a cytoarchitectonic study in the rat [15](Fig. 1, S1). Pyramidal cell layers (layers 2, 3, 5) were clearly identified based upon the presence of densely accumulated triangular cell bodies. All of these areas lacked accumulation of granule cells, and layer 4 was undistinguishable with Nissl stain. M2 had a distinct layer 2 and contained numerous large cells in layer 5 (Fig. S1A). ACC was characterized by a distinct layer 2 and a small cell size in layer 6 (Fig. S1B). PrL also had a distinct layer 2, although its cells were less aligned than those in the ACC (Fig. S1C). In IL, layers 2 and 3 were not distinguishable, and the cell density of layer 6 was higher than that in the PrL (Fig. S1D). In the DP, layer 2/3 was thin and the cell density of layer 2/3 was sparser than that in the IL (Fig. S1E). The cell density in layer 2/3 of TTd was higher than that in DP (Fig. S1F). Although the cytoarchitectonic features were distinct among areas, the delineation of areas may be inaccurate because it was based upon anatomy only. The same is true for the delineation of the amygdaloid complex and temporal cortical fields described below.
2. Materials and methods Japanese house bats (Pipistrellus abramus) were used in this study. All animals were also used for previous papers which focused on the cytoarchitecture and synaptic organization of brainstem auditory pathways [14], and methods were described in the previous paper [14] and supplemental methods. 3. Results We first describe the cytoarchitecture of forebrain where the retrogradely labeled cells were consistently found. Next, we present the distribution pattern of the retrogradely labeled cells in the forebrain structures.
3.2. Amygdaloid complex Like other bat species, the Japanese house bat has a hypertrophied amygdaloid complex [16,17]. The amygdaloid complex is composed of
Fig. 2. Cytoarchitectonic features of the amygdala defined with Nissl stain and GAD67 immunoreactivity. (A) A low-power micrograph of a Nissl-stained coronal section containing the central amygdala (CE). (B) A low-power micrograph of a GAD67-immunostained section located next to the sections shown in A. (C-N) Highpower micrographs showing the morphology of cell bodies (upper) and pattern of GAD67 immunoreactivity (lower) in subdivisions of the amygdaloid complex. The images were taken from the central part of each region. Scale bars: 500 μm (A, B), and 25 μm (C-N). 3
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higher than that in the Bpc, AB, and medio-ventral part of La (Fig. 2F). Bpc was located lateral to Bmg and was composed of irregularly shaped, intensely stained small cells (Fig. 2K). AB was located ventral to Bmg and Bpc and consisted of medium-sized, sparsely distributed cells (Fig. 2M). The density of GAD67+ fibers was intermediate to that of Bmg and Bpc. La was further subdivided into medio-ventral and dorsolateral parts: the medio-ventral part was composed of weakly stained medium-sized neurons, while the dorso-lateral part was composed of intensely stained small neurons (Fig. 2G, I). The density of GAD67+ fibers was lowest in the medio-ventral part and highest in the dorsolateral part of the amygdaloid complex (Fig. 2H, J).
six areas, namely the lateral (La), basal, accessory basal (AB), intercalated (In), and central (CE) nuclei, as well as the cortical amygdaloid areas (Fig. 2). The basal nucleus was further divided into magnocellular and parvocellular (Bmg and Bpc) areas based on somatic size. The cortical areas were distinguished as the medial nucleus (Ma), the posterior cortical nucleus, and the periamygdaloid cortex from medial to lateral, respectively. The cortical areas were easily discriminated from other amygdaloid nuclei by their laminar formation. The other nuclei were distinguishable based on Nissl cytoarchitecture [16,17] and distribution of GABAergic structures [monkey,18](Fig. 2). La, Bmg, Bpc, and AB formed a large mass located lateral to the CE, and In was located between them. The In was composed of densely packed small neurons and was intensely immunoreactive for GAD67. CE was composed of densely packed medium-sized neurons (Fig. 2A, C). The density of GAD67+ fibers was high, especially in the center of the nucleus (Fig. 2B, D). Bmg stood out due to the presence of intensely stained large cells (Fig. 2E), and the density of GAD67+ fibers in the Bmg was
3.3. Temporal cortical regions In the temporal and parietal cortical areas, layer 2 is clearly identified with a high packing density. Conversely, there was no sharp border between layer 3 and 4. In the dorsal part of the temporal cortex,
Fig. 3. Nissl cytoarchitecture of the auditory cortex (AC) and surrounding cortical structures. (A) Low-magnification micrographs of coronal (left) and horizontal (right) sections containing the AC. (B-F) High-power micrographs showing laminar organization of cortical areas. Panels B–D were acquired from coronal sections, while panels E and F were from horizontal sections. Scale bars: 500 μm (A), and 100 μm (B-F). 4
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surrounding areas (Fig. 3A, C). In addition to the properties shared with the horseshoe bat, layer 6 of AC in Japanese house bat is thicker than surrounding areas, and inside layer 6, cells were evenly distributed. The cell density in layer 2/3 was higher in A1 than in the surrounding areas. In TEA, the cell density was lower and the somatic size was smaller than in A1 (Fig. 3D). In Ect, layer 4 was undistinguishable from other layers, and the cell density of layer 2/3 was lower than that in A1 (Fig. 3E). S2 was characterized by a lower organization of layer 2/3 and a thinner layer 4 compared with A1 (Fig. 3F). From serial Nissl-stained sections cut coronally, we were able to reconstruct the three-dimensional (3D) organization of the IL, A1, and Bmg (Fig. S2). For the following analyses, we used the cytoarchitectonic
density of cells in layer 4 increased, and we identified the region as AC (Fig. 3A). AC was located ventral to the primary somatosensory cortex (S1) and dorsal to the temporal association area (TEA) in coronal sections. In horizontal sections, the AC was rostral to the ectorhinal cortex (Ect), and caudal to the secondary somatosensory cortex (S2) and Ect in dorsal and ventral sections, respectively (Fig. 3A). The AC itself is likely subdivided into several regions: in the central part of the AC, the cytoarchitecture was clearly different from the surrounding areas, and we assumed the central part to be the primary AC (A1). A1 was distinguished from surrounding cortical areas by the thickness of the cortical layers [horseshoebat,19] and the cell density [rat,20]: Layer 3/ 4 was markedly thicker than layer 5, and layer 1 was thinner than
Fig. 4. Images of horizontal sections containing retrogradely labeled neurons (C, G, J) and the adjacent sections stained with cresyl violet (A, B, E, F, I) or for GAD67 antibody (D, H). Case of Pa128. (A-D) Ipsilateral mPFC. Retrogradely labeled cell bodies were located in layer 6 of the IL. A box in A indicates the approximate location of panels B–D. (E-H) Ipsilateral amygdala. Retrogradely labeled cell bodies were located in Bmg. A box in E indicates the approximate location of panels F-H. (I, J) Ipsilateral auditory cortex. Retrogradely labeled cell bodies were located in layer 5. Scale bars: 500 μm (A, E), and 200 μm (others). 5
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small leakage of the tracer in the periaqueductal gray (PAG) and two other cases due to very weak labeling in the forebrain structures. It was noted that the case with leakage showed a similar pattern of retrograde labeling in the forebrain. From the distribution of labeled cells around the injection site (red dots in Fig. S3E-H), we conclude that the injection sites of four cases covered most parts of the ICC and retrogradely labeled cells were likely to target the ICC. We normalized dorso-ventral and medio-lateral position of the injection centers (Fig. S3J-L; see also Fig. 1K, L of Ito et al., 2018) [14].
atlases and 3D reconstructions for reference. 3.4. Injection sites of fluorogold (FG) In all seven cases which are described in a previous paper [14], FG was injected inside the IC. Of these, four cases (Pa119, Pa125, Pa128, and Pa131) showed no leakage of FG outside the IC and used for analysis (Fig. S3A–H; see also Fig. 1 of [14]) and intensely labeled cells in the forebrain structures. We excluded one case (Pa114) because of
Fig. 5. (A-D) Side views of 3D models of the forebrain and borders of AC, IL, and Bmg, and spatial distribution of retrogradely labeled cells (dots), reconstructed from horizontal serial sections of the four IC injection cases. Upper panels indicate the ipsilateral hemisphere, and lower panels indicate the contralateral side. (E) Schematic diagrams showing the distribution of labeled cells of the four cases. Blue, green, red, and orange borders correspond to ventro-lateral (Pa119), dorsolateral (Pa125), dorso-intermediate (Pa131), and dorso-medial (Pa128) injection, respectively. Gray grids: 100 μm. (F) Patterns of retrogradely labeling in IL, AC, and Bmg along one axis (dorso-ventral axis for IL, and rostro-caudal axis for others). Percentages to total retrogradely labeled forebrain cells are shown in the vertical axis. Blue, green, red, and orange lines and symbols correspond to ventro-lateral (Pa119), dorso-lateral (Pa125), dorso-intermediate (Pa131), and dorso-medial (Pa128) injection, respectively. 6
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3.5. Distribution of retrogradely labeled cells
4.1. Technical considerations
In the horizontal forebrain sections of all four cases analyzed, retrogradely labeled cells were consistently found in three regions, namely the mPFC, the temporal cortex, and the amygdala. In the bilateral mPFC (Fig. 4A), retrogradely labeled cells were found rostral to the corpus callosum. Based on neighboring sections stained with cresyl violet (Fig. 4B) and GAD67 antibody (Fig. 4D), we found that the retrogradely labeled cells were restricted to layer 6 of the IL (Fig. 4C). In the amygdala (Fig. 4E), the labeled cells were located rostral to the lateral ventricle and formed a cluster that was elongated rostro-caudally (Fig. 4G). Based on neighboring sections stained with cresyl violet (Fig. 4F) and GAD67 antibody (Fig. 4H), we found that the retrogradely labeled cells were restricted in the Bmg of the amygdaloid complex. In the temporal cortex, the labeled cells were found in layer 5 of the AC (Fig. 4I, J). In the thalamus, although we found labeled cells in shell nuclei of auditory thalamus, but did not analyze because brains were transected around the medial geniculate body (MGB) and therefore it was impossible to reconstruct auditory thalamus. Next, we made a 3D reconstruction of the forebrain, AC, IL, and Bmg based on Nissl cytoarchitecture and distribution of retrogradely labeled cells in the forebrain (Fig. 5A–D). Although the retrogradely labeled cells were found bilaterally, the number of cells was higher in the ipsilateral side than in the contralateral side. This tendency was more obvious in the AC. In the ipsilateral AC of Pa119, which received a large injection in the ventro-lateral IC (the highest frequency injection; Fig. S3A, E, J), the retrogradely labeled cells were found throughout the AC and the density was lower in the dorsal part (Fig. 5A). In case of Pa125, which received an injection in dorso-lateral IC (the second highest frequency injection; Fig. S3B, F, J), the labeled cells were sparsely distributed and the cell density was higher in the ventral part. In the rostro-caudal axis, the cell density was higher in the midpoint (Fig. 5B). In case of Pa131, which received an injection in the dorso-intermediate part of the IC (the lowest frequency injection; Fig. S3C, G, J), the density of the retrogradely labeled cells was higher in the dorsal part, specifically in the midpoint of the dorso-ventral axis (Fig. 5C). In case of Pa128, which received an injection in dorso-medial IC (the second lowest frequency injection; Fig. S3D, H, J), the labeled cells were denser in the caudal and dorsal parts. Except for some labeled cells in ventral parts, the rostral part of the AC was devoid of retrogradely labeled cells (Fig. 5D). The rostro-caudal distribution of retrogradely labeled cells revealed that the highest percentage occurred in the more rostral part of ipsilateral AC in Pa119 (highest frequency injection) and the more caudal part in Pa128 and Pa131 (low frequency injection) (Fig. 5F). Such topographic organization was not obvious in the contralateral side, which contained few labeled cells. Taken together, these findings suggest that caudal part of AC projects to dorsal, low frequency part of the IC, while rostral part projects to ventral, high frequency part of the IC. The medio-lateral axis of the ICC is likely related to the dorso-ventral axis of the AC. In the IL and Bmg, there was no obvious difference in the distribution of labeled cells among cases. Due to the small sample size and variable distributions in the ipsilateral side, no strong conclusions can be drawn about topographic organization. The distribution patterns of labeled cells in each case are summarized in Fig. 5E and Table S1.
As the direct projection from the prefrontal cortex to the IC has not been previously described, alternative interpretations of results require discussion. Nonspecific labeling is unlikely as the labeling pattern in other structures (i.e., amygdala, AC, and brainstem nuclei) was consistent with previous studies [17,21]. Trans-synaptic transport of FG is unlikely because the projection from the brainstem nuclei to the IC, which was described in another study [14], was very similar to that in other bat species [21]. FG uptake by the fibers of the passage is unlikely because this can only occur if IL fibers pass evenly inside the IC without termination. In other mammalian species, although IL fibers terminate in the PAG, they do not enter the IC [22]. As none of the four cases reported here showed leakage of FG from the IC (Fig. S3), it is unlikely that IL fibers that terminated or passed through the PAG took up FG. Furthermore, PAG-projecting IL neurons are located in layer 5 [23], while IC-projecting IL neurons were located in layer 6 (Fig. 4C). Taken together, the results of the current study strongly support the presence of direct projections from the IL to IC in Japanese house bats.
4.2. Comparative views of forebrain–IC loops In non-bat mammals, only IC-AC loop is present (dotted lines in Fig. 6): the IC projects to the MGB, the auditory thalamic structure, the MGB then projects to the AC, and the AC in turn projects back to the IC [24]. The pathway to A1 is tonotopically organized. Vespertilionidae bats include Japanese house bats and use downward frequency-modulated (FM) sweeps with a terminal phase showing a pure-tone-like constant frequency (quasi-CF) [13]. In several species of Vespertilionidae bats, the AC is composed of a smaller rostral field not sensitive to a delay of sound, and a larger caudal field sensitive to the delay [25]. Both fields showed tonotopicity and the caudal field exhibited a caudalto-rostral frequency gradient. Other bat species with FM calls (Carollia perspicillata) also have a large caudal field with a caudal-to-rostral frequency gradient [26]. The distribution of retrogradely labeled cells in the ipsilateral AC (Fig. 5) is likely to reflect the caudal-to-rostral frequency gradient in the caudal auditory field, and this suggests that the tonotopic organization of Japanese house bat AC is similar to other FM bat species. The amygdala receives auditory information from both the MGB
4. Discussion In this study, we found that three different forebrain regions, the IL, AC, and Bmg, directly innervate the bilateral IC in the Japanese house bat. Combined with previous studies on other bat and non-bat mammalian species, we suggest the presence of three loops between the IC and the forebrain regions of the bat brain (Fig. 6).
Fig. 6. Three forebrain structures directly inform the IC. Dotted arrows indicate connections commonly found in mammalian species. Thick arrows indicate connections found in echolocation bats. 7
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Acknowledgments
[27] and AC [28]. In non-bat mammals, the amygdala is interconnected with the mPFC [7] and AC [29]. In echolocating bats, the Bmg projects directly to the IC [currentstudy,17]; No clear topographic organization was found in the projection, and activation of the pathway may elicit a change of sensitivity of the whole IC. Since frequency spectra of bat communication calls are broad [11], the organization of projections may be effective for the attention and reaction to communication calls. In non-bat mammals, the relationship between the AC and mPFC is indirect [7]. In bats, reciprocal connection with PFC was found between AC [30] and suprageniculate nucleus, a shell region of MGB [31]. Consequently, auditory responses are evoked in the frontal auditory field of the PFC of bats [32], although the correspondence between frontal auditory field and IL is not clear. We thus estimate that there are at least three loops between the IC and forebrain structures of echolocating bats.
This work was supported by grants from the Japan Society for the Promotion of Science (KAKENHI Grant Number 19H04212 for TI; 18K07572 and 18KK0468 for RY; 16H06542 for SH; 17H01769 for KIK), Okawa Foundation for Information and Telecommunications, Suzuken Memorial Foundation, and Daiwa Securities Health Foundation to TI. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.neulet.2019.134481. References [1] G.S. Wright, et al., Social calls of flying big brown bats (Eptesicus fuscus), Front. Physiol. 4 (2013) 214. [2] K. Hase, et al., Rapid frequency control of sonar sounds by the FM bat, Miniopterus fuliginosus, in response to spectral overlap, Behav. Processes 128 (2016) 126–133. [3] E. Fujioka, et al., Echolocating bats use future-target information for optimal foraging, Proc. Natl. Acad. Sci. U. S. A. 113 (17) (2016) 4848–4852. [4] B. Delatour, P. 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4.3. Functional considerations According to the neuroethological theory of the IC operation [12], a slowed signal processing in the IC is suitable for tuning to sign stimuli, which have biological importance and trigger fixed action patterns. In this aspect, descending projections from the three forebrain centers to the IC will control the sensitivity to different sign stimuli, and in turn change the balance of activity in the forebrain centers responsible for specific behaviors related with sign stimuli. The sign stimuli related to the AC-IC projection corresponds likely to biosonar pulses: The tonotopic organization of the descending pathway from the AC enables control of the sensitivity of IC neurons to a specific and optimal frequency. After tones associated with the fearful stimulus (foot shock), frequency tuning of IC neurons shifts toward the frequency of tones, and AC is responsive for the plastic change [33]. This type of plasticity may underlie attention to shifted quasi-CF of biosonar pulses occurring when conspecific bats are present [2]. Therefore, it is likely that the AC-IC projection is related to echolocation behaviors, and plays a role in biasing “cognitive attention” to relevant sounds via experience-dependent plasticity. The sign stimuli related to the Bmg-IC projection is likely to be communication calls: It has been shown that the basal amygdala of bats is related with expression and reception of conspecific vocalization conveying emotional values [9–11]. The amygdalocollicular projection may produce emotional attention by modifying activity of whole IC immediately after calls with a particular emotional value. We speculate that the function of the IL-IC projection is based upon anatomical organization of the IL, which has been extensively studied in rodents [23,34]. Layer 6 of IL projects to the mediodorsal thalamic (MD) nucleus [34], whereas other layers project to amygdala and PAG [23,34]. These observations suggest that descending commands to the IC are closely related to those to the MD. Since synchronization of the MD and PFC is known to be important for working memory tasks [35], the IL-IC projection, also originated from layer 6, may be important for cognitive tasks, i.e., echolocation-based navigation. Considering the reciprocal connections among the three forebrain regions in bats, we speculate that the three forebrain regions cooperate and send output commands to the IC to modify the sensitivity to sign stimuli based on the behavior that the animal is engaged in. Loops between these three forebrain regions and IC may play an important attentional role in modulating, selecting and/or amplifying inputs, possibly via egocentric selection. By informing or providing feedback to the IC of executive or cognitive functions or emotional state, the IC can transmit the most relevant information to the frontal and auditory cortices and the amygdala for navigational, cognitive and emotive processing, respectively. In this way, as predicted earlier [12], the IC may participate in slowing outputs for the most effective and timely control of behavior.
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mustache bat, Pteronotus parnellii, Science 236 (4803) (1987) 824–826. [32] J.S. Kanwal, et al., Auditory responses from the frontal cortex in the mustached bat, Pteronotus parnellii, Neuroreport 11 (2) (2000) 367–372. [33] E. Gao, N. Suga, Experience-dependent corticofugal adjustment of midbrain frequency map in bat auditory system, Proc. Natl. Acad. Sci. U. S. A. 95 (21) (1998) 12663–12670. [34] P.L. Gabbott, et al., Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers, J. Comp. Neurol. 492 (2) (2005) 145–177. [35] S. Parnaudeau, et al., Inhibition of mediodorsal thalamus disrupts thalamofrontal connectivity and cognition, Neuron 77 (6) (2013) 1151–1162.
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