- Email: [email protected]
Intrinsic photosensitivity of a deep brain photoreceptor
Current Biology Vol 24 No 13 R596
Correspondences
Intrinsic photosensitivity of a deep brain photoreceptor Yusuke Nakane1, Tsuyoshi Shimmura1,5, Hideki Abe2, and Takashi Yoshimura1,3,4,5,* In addition to having a photoreceptive retina and pineal organ, nonmammalian vertebrates are known to have photoreceptors in the deeper regions of the brain that mediate seasonal changes in physiology and behavior [1]. Cerebrospinal fluid (CSF)contacting neurons extend knob-like dendrites into the ventricular cavity, where they form ciliated terminals [2]. The dendritic structures of photoreceptor cells in the developing retina and the pineal organ resemble those of CSF-contacting neurons. Thus, CSF-contacting neurons have been suggested to function as deep brain photoreceptors. Although the localization of a novel shortwavelength-sensitive photopigment (OPN5) that detects violet and ultraviolet (UV) light has been recently demonstrated in CSF-contacting neurons [3,4], there has been no direct evidence of photosensitivity in deep brain CSF-contacting neurons. Here, we report that the OPN5-positive CSF-contacting neurons in the paraventricular organ (PVO) of the quail mediobasal hypothalamus (MBH) are intrinsically photosensitive and are involved in the regulation of seasonal reproduction. In order to adapt to seasonal changes in their environment, organisms use changes in the photoperiod as a calendar. Recent studies demonstrated that longday induction of the -subunit of thyrotropin (thyroid-stimulating hormone, TSH) in the pars tuberalis (PT) of the pituitary gland triggers photoperiodic responses of gonads [5]. Non-mammalian vertebrates are known to have deep brain photoreceptors that mediate seasonal changes in physiology and behavior such as seasonal reproduction and migration. Benoit demonstrated that blind ducks show normal testicular
growth in response to a long-day stimulus, whereas a black cap placed on the duck’s head abolished this response [1]. Menaker et al. demonstrated that injection of India ink under the scalp of a sparrow similarly abolished testicular recrudescence, whereas pinealectomy did not interfere with this response [6]. Removing the eyes and/or the pineal organ from quail also did not influence the photoperiodic response. Conversely, local illumination of the MBH or septal region of the telencephalon leads to gonadal growth in quail, suggesting the existence of deep brain photoreceptors in these regions [7]. Photoreceptor proteins have been immunohistochemically identified in the CSF-contacting neurons of various non-mammalian vertebrates. Silver et al. first demonstrated rhodopsin-like immunoreactivity in CSF-contacting neurons in the septal and MBH areas of the ring dove brain [8]. Subsequently, colocalization of rhodopsin and the -subunit of rodtype retinal G protein (transducin, Gt1) in septal CSF-contacting neurons was reported in the pigeon. In addition to rhodopsin, localization of the non-visual photopigment OPN5 (Opsin 5) [3,4] has been reported in the CSF-contacting neurons in the PVO of birds. Since CSF-contacting neurons bear a solitary 9×2+0 cilium and resemble developing photoreceptor cells cytologically [2], CSF-contacting neurons could be likely candidates for deep brain photoreceptors. However, retinal photoreceptors have outer segment disk membranes; it is not known whether CSF-contacting neurons, which lack outer segments, can function as photoreceptors [2]. Therefore, direct evidence for the intrinsic photosensitivity of deep brain CSF-contacting neurons and their physiological functions has not yet been reported. To determine whether these neurons are capable of phototransduction, we performed whole-cell patchclamp recordings from the PVO CSF-contacting neurons (Figure 1A–C). A 1-klx white light stimulus that contains short-wavelength light induced strong depolarization and fast action potentials in most of these neurons (n = 33/44; Figure 1D). To confirm the presence of OPN5 in these photosensitive CSF-contacting neurons, we labeled recorded neurons intracellularly with biocytin, and
then stained them with anti-OPN5 antibody. Thus, we determined that the CSF-contacting neurons showing light-evoked responses possessed OPN5-positive immunoreactivity (Figure 1E–G). Synaptic inputs to CSF-contacting neurons are reported in the PVO [9]. However, persistence of the response to light was observed even if we blocked action potential activity with a bath application of 1 µM TTX for 10 min (Figure S1 in Supplemental Information, published with this article online). Furthermore, we blocked major synaptic inputs with a 10 min application of a cocktail containing antagonists of the major neurotransmitters (NMDA receptors, DL-2-amino-5-phosphonovaleric acid (APV); AMPA/kainate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); GABAA receptors, Pictoroxin). As a result, robust light-evoked firing of CSF-contacting neurons was observed even in the presence of this antagonist cocktail (Figure 1H). These data strongly indicate that the CSF-contacting neurons in the PVO are intrinsically photosensitive. We observed changes in the latency of the response to light in the presence of the antagonist cocktail (Figure 1H). It is possible that some synaptic inputs are modulating the activity of OPN5-positive CSF-contacting neurons. Japanese quail show rapid and robust photoperiodic responses. A single long-day stimulus induces elevation of plasma gonadotropin concentration by the end of the first long-day [5]. Recent studies demonstrated that long-day-induced TSH in the PT of the pituitary gland is the primary factor regulating seasonal reproduction in birds and mammals [5]. In addition, projection of OPN5positive CSF-contacting neurons to the quail PT has been reported [3]. To assess whether OPN5 is involved in the seasonal regulation of reproduction, we examined the effect of siRNA-mediated OPN5 knockdown on long-day induction of TSHB in eye-patched, pinealectomized quail. Since other opsins such as rhodopsin, melanopsin, and VA-opsin that detect green and blue light are also present in the avian brain [8], we used UV light to specifically stimulate OPN5. Intracerebroventricular injection of OPN5 siRNA significantly reduced the number of OPN5-positive cells in the PVO (Figure 1I,J). Induction
Magazine R597
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on tro kn O l oc PN kd 5 ow n
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Number of OPN5 +ve cells/area
5 sec
Current Biology
Figure 1. Intrinsic photosensitivity of OPN5-positive CSF-contacting neurons in the paraventricular organ (PVO). (A) Schematic drawing of the quail mediobasal hypothalamus. PVO, paraventricular organ; PT, pars tuberalis of the pituitary gland; OT, optic tract; 3V, third ventricle. (B) OPN5-positive CSF-contacting neurons in the PVO of 1-day-old quail. (C) Schematic drawing of representative OPN5-positive CSF-contacting neurons in panel (B). S, subependymal layer; E, ependymal layer. (D) Light (hv, indicated by a bar below) -evoked strong depolarization and fast action potentials in a CSF-contacting neuron. (E–G) The recorded cell filled with biocytin showed OPN5-immunoreactivity. (H) Light response apparent in normal ACSF (black trace) persisted during bath application of an antagonist cocktail (50 µM APV, 50 µM CNQX, 50 µM Picrotoxin) that blocks conventional neurotransmission (red trace). (I) Effect of ICV injection of OPN5 siRNA on OPN5 immunoreactivity in the PVO of eye-patched, pinealectomized quail. (J) The number of OPN5-positive neurons was significantly reduced by ICV injection of siRNA. (K) Long-day-induced expression of TSHB mRNA was significantly suppressed by siRNA-mediated OPN5 knockdown in eye-patched, pinealectomized quail. TSHB mRNA levels were determined by in situ hybridization (also see Figure S2). *P < 0.05 (Student’s t-test). The number in parentheses indicates the number of animals used. Values are mean + SEM. Control, Scrambled control; OPN5 knockdown, OPN5 siRNA. Scale bars: (B) 20 µm; (E) 10 µm; (I) 50 µm.
of TSHB by long-day stimulus of UV light was suppressed by the knockdown of OPN5 (Figure 1K, Figure S2), suggesting that OPN5 is involved in the regulation of seasonal reproduction. To our knowledge, this is the first demonstration of the intrinsic photosensitivity of deep brain CSFcontacting neurons. This result is particularly plausible because photoreceptive organs such as the lateral eyes and the pineal organ arise developmentally as evaginations from the diencephalon, and hypothalamic CSF-contacting neurons have long been considered to be ancestral photoreceptors [2,10]. In conclusion, we demonstrated that OPN5-positive CSF-contacting neurons function as deep brain photoreceptors, and regulate seasonal reproduction. Our findings will shed further light on the evolution and development of photoreceptors.
Supplemental Information Supplemental Information includes two figures and experimental procedures, and can be found with this article online at http:// dx.doi.org/10.1016/j.cub.2014.05.038.
7.
8.
References 1. Benoit, J. (1935). Le role des yeux dans l’action stimulante de la lumiere sure le developpement testiulaire chez le canard. CR Soc. Biol. 118, 669–671. 2. Vigh, B., and Vigh-Teichmann, I. (1998). I. Actual problems of the cerebrospinal fluid-contacting neurons. Microsc. Res. Tech. 41, 57–83. 3. Nakane, Y., Ikegami, K., Ono, H., Yamamoto, N., Yoshida, S., Hirunagi, K., Ebihara, S., Kubo, Y., and Yoshimura, T. (2010). A novel mammalian neural tissue opsin (Opsin5) is a deep brain photoreceptor in birds. Proc. Natl. Acad. Sci. USA 107, 15264–15268. 4. Yamashita, T., Ohuchi, H., Tomonari, S., Ikeda, K., Sakai, K., and Shichida, Y. (2010). Opn5 is a UV-sensitive bistable pigment that couples with Gi subtype of G protein. Proc. Natl. Acad. Sci. USA 107, 22084–22089. 5. Nakao, N., Ono, H., Yamamura, T., Anraku, T., Takagi, T., Higashi, K., Yasuo, S., Katou, Y., Kageyama, S., Uno, Y., et al. (2008). Thyrotrophin in the pars tuberalis triggers photoperiodic response. Nature 452, 317–322. 6. Menaker, M., Roberts, R., Elliott, J., and Underwood, H. (1970). Extraretinal light
9.
10.
perception in the sparrow, III. The eyes do not participate in photoperiodic photoreception. Proc. Natl. Acad. Sci. USA 67, 320–325. Homma, K., Ohta, M., and Sakakibara, Y. (1979). Biological Rhythms and Their Central Mechanism, eds Suda, M., Hayaishi, O., and Nakagawa, H. (Amsterdam: Elsevier), pp. 85–94. Silver, R., Witkovsky, P., Horvath, P., Alones, V., Barnstable, C.J., and Lehman, M.N. (1988). Coexpression of opsin- and VIP-likeimmunoreactivity in CSF-contacting neurons of the avian brain. Cell Tissue Res. 253, 189–198. Vígh, B., Manzano e Silva, M.J., Frank, C.L., Vincze, C., Czirok, S.J., Szabó, A., Lukáts, A., and Szél, A. (2004). The system of cerebrospinal fluid-contacting neurons. Its supposed role in the nonsynaptic signal transmission of the brain. Histol. Histopathol. 19, 607–628. Lamb, T.D., Collin, S.P., and Pugh, E.N., Jr. (2007). Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nat. Rev. Neurosci. 8, 960–976.
1Laboratory
of Animal Physiology, 2Laboratory of Fish Biology, 3Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences, 4Institute of Transformative Bio-molecules (WPI-ITbM), Nagoya University, Nagoya 464-8601, Japan. 5Division of Seasonal Biology, National Institute for Basic Biology, Okazaki 444–8585, Japan. *E-mail: [email protected]
Correspondences
Intrinsic photosensitivity of a deep brain photoreceptor Yusuke Nakane1, Tsuyoshi Shimmura1,5, Hideki Abe2, and Takashi Yoshimura1,3,4,5,* In addition to having a photoreceptive retina and pineal organ, nonmammalian vertebrates are known to have photoreceptors in the deeper regions of the brain that mediate seasonal changes in physiology and behavior [1]. Cerebrospinal fluid (CSF)contacting neurons extend knob-like dendrites into the ventricular cavity, where they form ciliated terminals [2]. The dendritic structures of photoreceptor cells in the developing retina and the pineal organ resemble those of CSF-contacting neurons. Thus, CSF-contacting neurons have been suggested to function as deep brain photoreceptors. Although the localization of a novel shortwavelength-sensitive photopigment (OPN5) that detects violet and ultraviolet (UV) light has been recently demonstrated in CSF-contacting neurons [3,4], there has been no direct evidence of photosensitivity in deep brain CSF-contacting neurons. Here, we report that the OPN5-positive CSF-contacting neurons in the paraventricular organ (PVO) of the quail mediobasal hypothalamus (MBH) are intrinsically photosensitive and are involved in the regulation of seasonal reproduction. In order to adapt to seasonal changes in their environment, organisms use changes in the photoperiod as a calendar. Recent studies demonstrated that longday induction of the -subunit of thyrotropin (thyroid-stimulating hormone, TSH) in the pars tuberalis (PT) of the pituitary gland triggers photoperiodic responses of gonads [5]. Non-mammalian vertebrates are known to have deep brain photoreceptors that mediate seasonal changes in physiology and behavior such as seasonal reproduction and migration. Benoit demonstrated that blind ducks show normal testicular
growth in response to a long-day stimulus, whereas a black cap placed on the duck’s head abolished this response [1]. Menaker et al. demonstrated that injection of India ink under the scalp of a sparrow similarly abolished testicular recrudescence, whereas pinealectomy did not interfere with this response [6]. Removing the eyes and/or the pineal organ from quail also did not influence the photoperiodic response. Conversely, local illumination of the MBH or septal region of the telencephalon leads to gonadal growth in quail, suggesting the existence of deep brain photoreceptors in these regions [7]. Photoreceptor proteins have been immunohistochemically identified in the CSF-contacting neurons of various non-mammalian vertebrates. Silver et al. first demonstrated rhodopsin-like immunoreactivity in CSF-contacting neurons in the septal and MBH areas of the ring dove brain [8]. Subsequently, colocalization of rhodopsin and the -subunit of rodtype retinal G protein (transducin, Gt1) in septal CSF-contacting neurons was reported in the pigeon. In addition to rhodopsin, localization of the non-visual photopigment OPN5 (Opsin 5) [3,4] has been reported in the CSF-contacting neurons in the PVO of birds. Since CSF-contacting neurons bear a solitary 9×2+0 cilium and resemble developing photoreceptor cells cytologically [2], CSF-contacting neurons could be likely candidates for deep brain photoreceptors. However, retinal photoreceptors have outer segment disk membranes; it is not known whether CSF-contacting neurons, which lack outer segments, can function as photoreceptors [2]. Therefore, direct evidence for the intrinsic photosensitivity of deep brain CSF-contacting neurons and their physiological functions has not yet been reported. To determine whether these neurons are capable of phototransduction, we performed whole-cell patchclamp recordings from the PVO CSF-contacting neurons (Figure 1A–C). A 1-klx white light stimulus that contains short-wavelength light induced strong depolarization and fast action potentials in most of these neurons (n = 33/44; Figure 1D). To confirm the presence of OPN5 in these photosensitive CSF-contacting neurons, we labeled recorded neurons intracellularly with biocytin, and
then stained them with anti-OPN5 antibody. Thus, we determined that the CSF-contacting neurons showing light-evoked responses possessed OPN5-positive immunoreactivity (Figure 1E–G). Synaptic inputs to CSF-contacting neurons are reported in the PVO [9]. However, persistence of the response to light was observed even if we blocked action potential activity with a bath application of 1 µM TTX for 10 min (Figure S1 in Supplemental Information, published with this article online). Furthermore, we blocked major synaptic inputs with a 10 min application of a cocktail containing antagonists of the major neurotransmitters (NMDA receptors, DL-2-amino-5-phosphonovaleric acid (APV); AMPA/kainate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); GABAA receptors, Pictoroxin). As a result, robust light-evoked firing of CSF-contacting neurons was observed even in the presence of this antagonist cocktail (Figure 1H). These data strongly indicate that the CSF-contacting neurons in the PVO are intrinsically photosensitive. We observed changes in the latency of the response to light in the presence of the antagonist cocktail (Figure 1H). It is possible that some synaptic inputs are modulating the activity of OPN5-positive CSF-contacting neurons. Japanese quail show rapid and robust photoperiodic responses. A single long-day stimulus induces elevation of plasma gonadotropin concentration by the end of the first long-day [5]. Recent studies demonstrated that long-day-induced TSH in the PT of the pituitary gland is the primary factor regulating seasonal reproduction in birds and mammals [5]. In addition, projection of OPN5positive CSF-contacting neurons to the quail PT has been reported [3]. To assess whether OPN5 is involved in the seasonal regulation of reproduction, we examined the effect of siRNA-mediated OPN5 knockdown on long-day induction of TSHB in eye-patched, pinealectomized quail. Since other opsins such as rhodopsin, melanopsin, and VA-opsin that detect green and blue light are also present in the avian brain [8], we used UV light to specifically stimulate OPN5. Intracerebroventricular injection of OPN5 siRNA significantly reduced the number of OPN5-positive cells in the PVO (Figure 1I,J). Induction
Magazine R597
B
C
H
20 mV
A PVO OT
PT
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Control
-64 mV
OPN5 knockdown
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on tro kn O l oc PN kd 5 ow n
I
Number of OPN5 +ve cells/area
5 sec
Current Biology
Figure 1. Intrinsic photosensitivity of OPN5-positive CSF-contacting neurons in the paraventricular organ (PVO). (A) Schematic drawing of the quail mediobasal hypothalamus. PVO, paraventricular organ; PT, pars tuberalis of the pituitary gland; OT, optic tract; 3V, third ventricle. (B) OPN5-positive CSF-contacting neurons in the PVO of 1-day-old quail. (C) Schematic drawing of representative OPN5-positive CSF-contacting neurons in panel (B). S, subependymal layer; E, ependymal layer. (D) Light (hv, indicated by a bar below) -evoked strong depolarization and fast action potentials in a CSF-contacting neuron. (E–G) The recorded cell filled with biocytin showed OPN5-immunoreactivity. (H) Light response apparent in normal ACSF (black trace) persisted during bath application of an antagonist cocktail (50 µM APV, 50 µM CNQX, 50 µM Picrotoxin) that blocks conventional neurotransmission (red trace). (I) Effect of ICV injection of OPN5 siRNA on OPN5 immunoreactivity in the PVO of eye-patched, pinealectomized quail. (J) The number of OPN5-positive neurons was significantly reduced by ICV injection of siRNA. (K) Long-day-induced expression of TSHB mRNA was significantly suppressed by siRNA-mediated OPN5 knockdown in eye-patched, pinealectomized quail. TSHB mRNA levels were determined by in situ hybridization (also see Figure S2). *P < 0.05 (Student’s t-test). The number in parentheses indicates the number of animals used. Values are mean + SEM. Control, Scrambled control; OPN5 knockdown, OPN5 siRNA. Scale bars: (B) 20 µm; (E) 10 µm; (I) 50 µm.
of TSHB by long-day stimulus of UV light was suppressed by the knockdown of OPN5 (Figure 1K, Figure S2), suggesting that OPN5 is involved in the regulation of seasonal reproduction. To our knowledge, this is the first demonstration of the intrinsic photosensitivity of deep brain CSFcontacting neurons. This result is particularly plausible because photoreceptive organs such as the lateral eyes and the pineal organ arise developmentally as evaginations from the diencephalon, and hypothalamic CSF-contacting neurons have long been considered to be ancestral photoreceptors [2,10]. In conclusion, we demonstrated that OPN5-positive CSF-contacting neurons function as deep brain photoreceptors, and regulate seasonal reproduction. Our findings will shed further light on the evolution and development of photoreceptors.
Supplemental Information Supplemental Information includes two figures and experimental procedures, and can be found with this article online at http:// dx.doi.org/10.1016/j.cub.2014.05.038.
7.
8.
References 1. Benoit, J. (1935). Le role des yeux dans l’action stimulante de la lumiere sure le developpement testiulaire chez le canard. CR Soc. Biol. 118, 669–671. 2. Vigh, B., and Vigh-Teichmann, I. (1998). I. Actual problems of the cerebrospinal fluid-contacting neurons. Microsc. Res. Tech. 41, 57–83. 3. Nakane, Y., Ikegami, K., Ono, H., Yamamoto, N., Yoshida, S., Hirunagi, K., Ebihara, S., Kubo, Y., and Yoshimura, T. (2010). A novel mammalian neural tissue opsin (Opsin5) is a deep brain photoreceptor in birds. Proc. Natl. Acad. Sci. USA 107, 15264–15268. 4. Yamashita, T., Ohuchi, H., Tomonari, S., Ikeda, K., Sakai, K., and Shichida, Y. (2010). Opn5 is a UV-sensitive bistable pigment that couples with Gi subtype of G protein. Proc. Natl. Acad. Sci. USA 107, 22084–22089. 5. Nakao, N., Ono, H., Yamamura, T., Anraku, T., Takagi, T., Higashi, K., Yasuo, S., Katou, Y., Kageyama, S., Uno, Y., et al. (2008). Thyrotrophin in the pars tuberalis triggers photoperiodic response. Nature 452, 317–322. 6. Menaker, M., Roberts, R., Elliott, J., and Underwood, H. (1970). Extraretinal light
9.
10.
perception in the sparrow, III. The eyes do not participate in photoperiodic photoreception. Proc. Natl. Acad. Sci. USA 67, 320–325. Homma, K., Ohta, M., and Sakakibara, Y. (1979). Biological Rhythms and Their Central Mechanism, eds Suda, M., Hayaishi, O., and Nakagawa, H. (Amsterdam: Elsevier), pp. 85–94. Silver, R., Witkovsky, P., Horvath, P., Alones, V., Barnstable, C.J., and Lehman, M.N. (1988). Coexpression of opsin- and VIP-likeimmunoreactivity in CSF-contacting neurons of the avian brain. Cell Tissue Res. 253, 189–198. Vígh, B., Manzano e Silva, M.J., Frank, C.L., Vincze, C., Czirok, S.J., Szabó, A., Lukáts, A., and Szél, A. (2004). The system of cerebrospinal fluid-contacting neurons. Its supposed role in the nonsynaptic signal transmission of the brain. Histol. Histopathol. 19, 607–628. Lamb, T.D., Collin, S.P., and Pugh, E.N., Jr. (2007). Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nat. Rev. Neurosci. 8, 960–976.
1Laboratory
of Animal Physiology, 2Laboratory of Fish Biology, 3Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences, 4Institute of Transformative Bio-molecules (WPI-ITbM), Nagoya University, Nagoya 464-8601, Japan. 5Division of Seasonal Biology, National Institute for Basic Biology, Okazaki 444–8585, Japan. *E-mail: [email protected]