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Optical mapping of pontine chemosensitive regions of neonatal rat
Neuroscience Letters 366 (2004) 103–106
Optical mapping of pontine chemosensitive regions of neonatal rat Yoko Ito a,1 , Yoshitaka Oyamada a,∗ , Yasumasa Okada b,2 , Haruhiko Hakuno a,1 , Ryoma Aoyama c,3 , Kazuhiro Yamaguchi a,1 a
b
Department of Medicine, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Medicine, Keio University Tsukigase Rehabilitation Center, Tsukigase 380-2, Izu City, Shizuoka-ken 410-3215, Japan c Department of Orthopedics, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Received 15 March 2004; received in revised form 22 April 2004; accepted 10 May 2004
Abstract We analyzed the neuronal response to hypercapnic acidosis, using an optical recording technique with a fluorescent voltage-sensitive dye (di-4-ANEPPS), in pontine slice preparations of neonatal rats, containing the locus coeruleus (LC), which has been electrophysiologically demonstrated to be chemosensitive. The dye-stained preparation was continuously superfused with artificial cerebrospinal fluid. Epifluorescence of the slice was detected using a high-sensitivity optical recording system. Changes in the intensity of fluorescence were serially analyzed while switching artificial cerebrospinal fluid from control to hypercapnic acidosis, or vice versa. The optical recording method revealed that the LC, as reported in previous studies, reversibly showed a depolarizing response to hypercapnic acidosis in 56% of the examined preparations. The A5 area (56%) also exhibited a reversible, depolarizing response to hypercapnic acidosis. The response was preserved under conditions in which chemical synaptic transmission was blocked by low Ca2+ -high Mg2+ solution. These results suggest that the optical recording method is applicable to identification of potentially chemosensitive areas, which deserve further electrophysiological analysis, and that the A5 area could be chemosensitive. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Chemosensitivity; Optical recording; Pons; Locus coeruleus; A5 area
It is now believed that neurons sensitive to extracellular pH/PCO2 (i.e., chemosensitive neurons) are widely distributed in the mammalian brainstem. These neurons could be responsible for central chemosensitivity (i.e., the ability of the central nervous system to sense extracellular pH/PCO2 in the absence of peripheral afferent input) for respiration. Chemosensitivity of neurons is electrophysiologically defined as an ability of the neuron to change membrane potential and, eventually, its burst frequency, in response to a shift of extracellular pH/PCO2 , independent of chemical synaptic input. Such neuronal chemosensitivity has been investigated mainly in the medulla [2,8,14]. Meanwhile, in the pons, neuronal chemosensitivity has been electrophyiologically proved only in the locus coeruleus ∗ Corresponding author. Tel.: +81 3 3353 1211x62889; fax: +81 3 3353 2502. E-mail address: [email protected] (Y. Oyamada). 1 Tel.: +81 3 3353 1211x62889; fax: +81 3 3353 2502. 2 Tel.: +81 558 85 1701; fax: +81 558 85 1810. 3 Tel.: +81 3 3353 1211x62344; fax: +81 3 3353 6597.
(LC) [11,12], though Haxhiu et al. [4] suggested that the A5 area could be chemosensitive by demonstrating its expression of Fos-protein—a marker for neuronal excitation—in rats exposed to hypercapnic conditions. Optical recording of neuronal activity using voltagesensitive dyes is a useful technique in the analysis of brain function [9,15]. This technique makes it possible to simultaneously analyze multi-point neuronal activities in the area of concern. The objective of the present study was to identify possible chemosensitive regions in the pons deserving further electrophysiological investigation, using an optical recording technique. For this purpose, we used a slice preparation of the pons containing the LC, which has been demonstrated to be chemosensitive, as a positive control. This study was approved by the Laboratory Animal Care and Use Committee of the School of Medicine, Keio University. The brainstem was isolated en bloc from neonatal rats (Wistar, 1–4 days old) under deep anesthesia with ether, according to a previously described procedure [6,11,12]. Serial transverse slices (300–500 m) of the brainstem were made, using a brain-slicer (DTK-1000, DSK, Kyoto, Japan)
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.05.035
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in a chamber filled with control artificial cerebrospinal fluid (ACSF) containing (mM): 125 NaCl, 4 KCl, 2 CaCl2 , 1 MgSO4 , 0.5 NaH2 PO4 , 26 NaHCO3 , and 30 d-glucose equilibrated with a gas mixture consisting of 2% CO2 and 98% O2 (pH 7.8). Slices containing the LC were selected under a dissection microscope with reference to cytoarchitectonic landmarks, especially the shape of the floor of the fourth ventricle [13]. Details of the optical recording method using voltage-sensitive dyes are described elsewhere [9,15,16]. Briefly, slice preparations were stained with a fluorescent voltage-sensitive
dye (di-4-ANEPPS, Molecular Probe, OR, USA; concentration 0.1 mg/ml) for 15 min. After the preparation was rinsed with control ACSF, it was fixed in a recording chamber (2 ml) placed under an epifluorescent microscope (Optiphoto 2UD, Nikon, Tokyo, Japan) with an objective lens (Plan Apo 4X, NA: 0.20). The preparation was continuously superfused with control ACSF at a constant rate of 6–7 ml/min, at 25–26 ◦ C. The neural structures in the preparations were identified under a microscope prior to the following measurement (later histologically identified in some preparations), with reference to a rat brain atlas [13].
Fig. 1. Response of LC to hypercapnic acidosis (a) Image of dorsal pons in control ACSF. (b–d) Hypercapnic acidosis elicited a depolarizing response in LC. (e) F/F in LC (upper trace) started to change with a delay of 8.3 s from the shift in bath pH (lower trace). Arrows indicate when images (a–d) were obtained (from left to right, a–d). (f) Schematic drawing of slice preparation. Open square indicates the investigated area shown in images (a–d). LC: locus coeruleus. 4V: fourth ventricle; RPn: pontine raphe nucleus.
Y. Ito et al. / Neuroscience Letters 366 (2004) 103–106
The dye-stained preparation was illuminated through an excitation filter (λ = 535 ± 10 nm) and a shutter using a high power tungsten halogen lamp (250 W; Oriel, CT, USA), driven by a stable DC current supplier (PD 35-20, Kenwood, Tokyo, Japan). Epifluorescence of the slice was detected through an absorption filter (long pass λ = 610 nm) using a high-sensitivity optical recording system, MiCAM01 (Brain Vision, Tokyo, Japan), that provides high spatial (60 × 90 pixels) and temporal (20 ms) resolution. Changes in the intensity of fluorescence (F) in each pixel over the area of interest (i.e., the unilateral half of the slice) relative to the initial intensity of fluorescence (F) were serially determined at a rate of 20 ms/frame while switching ACSF from the control to hypercapnic acidosis (the same composition as control ACSF, but equilibrated with 8% CO2 and 92% O2 : pH 7.2), or vice versa. The fractional change in fluorescent intensity (F/F) was used to normalize the difference in the amount of dye within the slice. A negative F/F corresponds to depolarization of the neuronal membrane. The temporal and spatial profiles of F/F over the area of interest were displayed as pseudocolor images, using temporal and spatial filters. The maximum length of each recording session was 1 min 50 sec. This rendered us to perform on-phase (i.e., from control to hypercapnic acidosis) and off-phase (i.e., from hypercapnic acidosis to control) analyses, separately. In some preparations, chemical synaptic transmission was blocked, using low Ca2+ -high Mg2+ ACSF (125 NaCl, 4 KCl, 0.2 CaCl2 , 5 MgSO4 , 0.5 NaH2 PO4 , 26 NaHCO3 , and 30 d-glucose: all in mM). Bath pH was recorded with a conventional glass pH electrode.
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The on-phase analysis was performed in 18 preparations, without synaptic blockade. In 7 preparations, a negative F/F was elicited by hypercapnic acidosis in the LC (Fig. 1). Meanwhile, hypercapnic acidosis induced a negative F/F in the A5 area in seven preparations. In another set of three preparations, a negative F/F in response to hypercapnic acidosis was observed both in the LC and the A5 area. That is, the LC and the A5 area exhibited depolarizing responses to hypercapnic acidosis in 10 preparations for each. The peak F/F of the A5 area was −0.41 ± 0.15% (mean ± standard error), comparable to the hypercapnic response of the LC (−0.36 ± 0.08%, P = 0.79, unpaired t-test). The reversibility of the depolarizing response was confirmed in three preparations in which the off-phase analysis was performed (Fig. 2a and b). There were also some excitatory regions that could not be assigned to specific anatomical regions. A neuronal response to hypercapnic acidosis and its reversibility were examined in the absence of chemical synaptic transmission in three preparations. The preparation was superfused with low Ca2+ -high Mg2+ ACSF for more than 15 min. The depolarizing response to hypercapnic acidosis was reversibly evoked also in the low Ca2+ -high Mg2+ ACSF (Fig. 2c and d). Previous studies have demonstrated that LC neurons exhibit an intrinsic, depolarizing response to hypercapnic acidosis [11,12]. Since the LC is the only pontine nucleus that has been electrophysiologically proven to be chemosensitive, we used the LC as a positive control for the present study. We were able to detect its depolarizing response, consistent with the findings obtained in previous studies [11,12],
Fig. 2. Reversibility of response to hypercapnic acidosis: (a) hypercapnic acidosis elicited a depolarizing response in the LC and the A5 area. This image was obtained at 68 s after switching control ACSF to hypercapnic acidosis. (b) Image of the same preparation as in (a) obtained at 31 s after switching ACSF back to control. The LC and the A5 area showed a hyperpolarizing response. In this preparation, an undefined area (arrow head) also showed a reversible, depolarizing response to hypercapnic acidosis. (c) A depolarizing response of the A5 area to hypercapnic acidosis in low Ca2+ -high Mg2+ ACSF. (d) Image of the same preparation as in (c). The off-phase analysis revealed that the depolarizing response was reversible. In this preparation, the LC exhibited no response to hypercapnic acidosis. LC: locus coeruleus; A5: A5 area; 4V: fourth ventricle.
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with this newly-applied optical recording method. This justifies application of the optical recording method for identification of chemosensitive regions. We revealed that the A5 area was possibly intrinsically chemosensitive. The A5 area is noradrenergic and is located ventrolaterally to the LC. This area is considered to be involved in the determination of expiratory time and respiratory frequency [1,3,5,7,10]. In the isolated brainstem-spinal cord preparation of the neonatal rat, this area exerts an inhibitory effect on the frequency of respiratory activity [3,5]. This is also true in in vivo rats; i.e., stimulation of the A5 area elicited prolongation of the expiratory time and an eventual decrease in respiratory frequency in anesthetized rats [7]. The A5 area is also considered to play an important role in the post-hypoxic ventilatory decline [1,10]. Haxhiu et al. [4] demonstrated that the A5 area expressed Fos-protein—a marker for neuronal excitation—in rats exposed to hypercapnic conditions, suggesting that the area could be chemosensitive. Our study provided more direct evidence for the chemosensitivity of this area. In summary, we demonstrated that several pontine regions in the slice preparation of the neonatal rat exhibited a depolarizing response to hypercapnic acidosis, using an optical recording method with a voltage-sensitive dye. The present study suggests that the method is applicable to identification of potentially chemosensitive regions, which deserve further electrophysiological analysis. Acknowledgements This study was supported by Research Grants for Life Sciences and Medicine from Keio University Medical Science Fund, a Research Grant for Specific Diseases from the Japanese Ministry of Health and Welfare, and a Grant-in-Aid for Exploratory Research from the Japan Society for the Promotion of Science. References [1] S.K. Coles, T.E. Dick, Neurons in the ventrolateral pons are required for post-hypoxic frequency decline in rats, J. Physiol. 497 (1996) 79–94.
[2] J.B. Dean, D.A. Bayliss, J.T. Erickson, W.L. Lawing, D.E. Millhorn, Depolarization and stimulation of neurons in nucleus tractus solitarii by carbon dioxide does not require chemical synaptic input, Neuroscience 36 (1990) 207–216. [3] S. Errchidi, G. Hilaire, R. Monteau, Permanent release of noradrenaline modulates respiratory frequency in the newborn rat: an in vitro study, J. Physiol. 429 (1990) 497–510. [4] M.A. Haxhiu, K. Yung, B. Erokwu, N.S. Cherniack, CO2 -induced c-fos expression in the CNS catecholaminergic neurons, Respir. Physiol. 105 (1996) 35–45. [5] G. Hilaire, R. Monteau, S. Errchidi, Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area: an in vitro study in the newborn rat, Brain Res. 485 (1989) 325– 332. [6] Y. Ito, Y. Oyamada, K. Yamaguchi, Age-dependent chemosensitive pontine inhibition of medullary respiratory rhythm generation in the isolated brainstem of the neonatal rat, Brain Res. 887 (2000) 418– 420. [7] J.S. Jodokowski, S.K. Coles, T.E. Dick, Prolongation in expiration evoked from ventrolateral pons of adult rats, J. Appl. Physiol. 82 (1997) 377–381. [8] A. Kawai, D. Ballantyne, K. Mückenhoff, P. Scheid, Chemosensitive medullary neurons in the brainstem–spinal cord preparation of the neonatal rat, J. Physiol. 492 (1996) 277–292. [9] Y. Okada, Z. Chen, H. Yoshida, S. Kuwana, W. Jiang, H. Maruiwa, Optical recording of the neuronal activity in the brainstem–spinal cord, Adv. Exp. Med. Biol. 499 (2001) 113–118. [10] Y. Okada, A. Kawai, K. Mückenhoff, P. Scheid, Roles of the pons in hypoxic respiratory depression in the neonatal rat, Respir. Physiol. 111 (1998) 55–63. [11] Y. Oyamada, K. Andrzejewski, K. Mückenhoff, P. Scheid, D. Ballantyne, Locus coeruleus neurones in vitro: pH-sensitive oscillations of membrane potential in an electrically coupled network, Respir. Physiol. 118 (1999) 131–147. [12] Y. Oyamada, D. Ballantyne, K. Mückenhoff, P. Scheid, Respirationmodulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem–spinal cord of the neonatal rat, J. Physiol. 513 (1998) 381–398. [13] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. [14] G.B. Richerson, Response to CO2 of neurons in the rostral ventral medulla in vitro, J. Neurophysiol. 73 (1995) 933– 944. [15] T. Tominaga, Y. Tominaga, H. Yamada, G. Matsumoto, M. Ichikawa, Quantification of optical signals with electrophysiological signals in neural activities of di-4-ANEPPS stained rat hippocampal slices, J. Neurosci. Methods 102 (2000) 11–23. [16] H. Yoshida, Y. Okada, H. Maruiwa, K. Fukuda, M. Nakamura, K. Chiba, Y. Toyama, Synaptic blockade plays a major role in the neural disturbance of experimental spinal cord compression, J. Neurotrauma 20 (2003) 1365–1376.
Optical mapping of pontine chemosensitive regions of neonatal rat Yoko Ito a,1 , Yoshitaka Oyamada a,∗ , Yasumasa Okada b,2 , Haruhiko Hakuno a,1 , Ryoma Aoyama c,3 , Kazuhiro Yamaguchi a,1 a
b
Department of Medicine, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Medicine, Keio University Tsukigase Rehabilitation Center, Tsukigase 380-2, Izu City, Shizuoka-ken 410-3215, Japan c Department of Orthopedics, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Received 15 March 2004; received in revised form 22 April 2004; accepted 10 May 2004
Abstract We analyzed the neuronal response to hypercapnic acidosis, using an optical recording technique with a fluorescent voltage-sensitive dye (di-4-ANEPPS), in pontine slice preparations of neonatal rats, containing the locus coeruleus (LC), which has been electrophysiologically demonstrated to be chemosensitive. The dye-stained preparation was continuously superfused with artificial cerebrospinal fluid. Epifluorescence of the slice was detected using a high-sensitivity optical recording system. Changes in the intensity of fluorescence were serially analyzed while switching artificial cerebrospinal fluid from control to hypercapnic acidosis, or vice versa. The optical recording method revealed that the LC, as reported in previous studies, reversibly showed a depolarizing response to hypercapnic acidosis in 56% of the examined preparations. The A5 area (56%) also exhibited a reversible, depolarizing response to hypercapnic acidosis. The response was preserved under conditions in which chemical synaptic transmission was blocked by low Ca2+ -high Mg2+ solution. These results suggest that the optical recording method is applicable to identification of potentially chemosensitive areas, which deserve further electrophysiological analysis, and that the A5 area could be chemosensitive. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Chemosensitivity; Optical recording; Pons; Locus coeruleus; A5 area
It is now believed that neurons sensitive to extracellular pH/PCO2 (i.e., chemosensitive neurons) are widely distributed in the mammalian brainstem. These neurons could be responsible for central chemosensitivity (i.e., the ability of the central nervous system to sense extracellular pH/PCO2 in the absence of peripheral afferent input) for respiration. Chemosensitivity of neurons is electrophysiologically defined as an ability of the neuron to change membrane potential and, eventually, its burst frequency, in response to a shift of extracellular pH/PCO2 , independent of chemical synaptic input. Such neuronal chemosensitivity has been investigated mainly in the medulla [2,8,14]. Meanwhile, in the pons, neuronal chemosensitivity has been electrophyiologically proved only in the locus coeruleus ∗ Corresponding author. Tel.: +81 3 3353 1211x62889; fax: +81 3 3353 2502. E-mail address: [email protected] (Y. Oyamada). 1 Tel.: +81 3 3353 1211x62889; fax: +81 3 3353 2502. 2 Tel.: +81 558 85 1701; fax: +81 558 85 1810. 3 Tel.: +81 3 3353 1211x62344; fax: +81 3 3353 6597.
(LC) [11,12], though Haxhiu et al. [4] suggested that the A5 area could be chemosensitive by demonstrating its expression of Fos-protein—a marker for neuronal excitation—in rats exposed to hypercapnic conditions. Optical recording of neuronal activity using voltagesensitive dyes is a useful technique in the analysis of brain function [9,15]. This technique makes it possible to simultaneously analyze multi-point neuronal activities in the area of concern. The objective of the present study was to identify possible chemosensitive regions in the pons deserving further electrophysiological investigation, using an optical recording technique. For this purpose, we used a slice preparation of the pons containing the LC, which has been demonstrated to be chemosensitive, as a positive control. This study was approved by the Laboratory Animal Care and Use Committee of the School of Medicine, Keio University. The brainstem was isolated en bloc from neonatal rats (Wistar, 1–4 days old) under deep anesthesia with ether, according to a previously described procedure [6,11,12]. Serial transverse slices (300–500 m) of the brainstem were made, using a brain-slicer (DTK-1000, DSK, Kyoto, Japan)
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.05.035
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in a chamber filled with control artificial cerebrospinal fluid (ACSF) containing (mM): 125 NaCl, 4 KCl, 2 CaCl2 , 1 MgSO4 , 0.5 NaH2 PO4 , 26 NaHCO3 , and 30 d-glucose equilibrated with a gas mixture consisting of 2% CO2 and 98% O2 (pH 7.8). Slices containing the LC were selected under a dissection microscope with reference to cytoarchitectonic landmarks, especially the shape of the floor of the fourth ventricle [13]. Details of the optical recording method using voltage-sensitive dyes are described elsewhere [9,15,16]. Briefly, slice preparations were stained with a fluorescent voltage-sensitive
dye (di-4-ANEPPS, Molecular Probe, OR, USA; concentration 0.1 mg/ml) for 15 min. After the preparation was rinsed with control ACSF, it was fixed in a recording chamber (2 ml) placed under an epifluorescent microscope (Optiphoto 2UD, Nikon, Tokyo, Japan) with an objective lens (Plan Apo 4X, NA: 0.20). The preparation was continuously superfused with control ACSF at a constant rate of 6–7 ml/min, at 25–26 ◦ C. The neural structures in the preparations were identified under a microscope prior to the following measurement (later histologically identified in some preparations), with reference to a rat brain atlas [13].
Fig. 1. Response of LC to hypercapnic acidosis (a) Image of dorsal pons in control ACSF. (b–d) Hypercapnic acidosis elicited a depolarizing response in LC. (e) F/F in LC (upper trace) started to change with a delay of 8.3 s from the shift in bath pH (lower trace). Arrows indicate when images (a–d) were obtained (from left to right, a–d). (f) Schematic drawing of slice preparation. Open square indicates the investigated area shown in images (a–d). LC: locus coeruleus. 4V: fourth ventricle; RPn: pontine raphe nucleus.
Y. Ito et al. / Neuroscience Letters 366 (2004) 103–106
The dye-stained preparation was illuminated through an excitation filter (λ = 535 ± 10 nm) and a shutter using a high power tungsten halogen lamp (250 W; Oriel, CT, USA), driven by a stable DC current supplier (PD 35-20, Kenwood, Tokyo, Japan). Epifluorescence of the slice was detected through an absorption filter (long pass λ = 610 nm) using a high-sensitivity optical recording system, MiCAM01 (Brain Vision, Tokyo, Japan), that provides high spatial (60 × 90 pixels) and temporal (20 ms) resolution. Changes in the intensity of fluorescence (F) in each pixel over the area of interest (i.e., the unilateral half of the slice) relative to the initial intensity of fluorescence (F) were serially determined at a rate of 20 ms/frame while switching ACSF from the control to hypercapnic acidosis (the same composition as control ACSF, but equilibrated with 8% CO2 and 92% O2 : pH 7.2), or vice versa. The fractional change in fluorescent intensity (F/F) was used to normalize the difference in the amount of dye within the slice. A negative F/F corresponds to depolarization of the neuronal membrane. The temporal and spatial profiles of F/F over the area of interest were displayed as pseudocolor images, using temporal and spatial filters. The maximum length of each recording session was 1 min 50 sec. This rendered us to perform on-phase (i.e., from control to hypercapnic acidosis) and off-phase (i.e., from hypercapnic acidosis to control) analyses, separately. In some preparations, chemical synaptic transmission was blocked, using low Ca2+ -high Mg2+ ACSF (125 NaCl, 4 KCl, 0.2 CaCl2 , 5 MgSO4 , 0.5 NaH2 PO4 , 26 NaHCO3 , and 30 d-glucose: all in mM). Bath pH was recorded with a conventional glass pH electrode.
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The on-phase analysis was performed in 18 preparations, without synaptic blockade. In 7 preparations, a negative F/F was elicited by hypercapnic acidosis in the LC (Fig. 1). Meanwhile, hypercapnic acidosis induced a negative F/F in the A5 area in seven preparations. In another set of three preparations, a negative F/F in response to hypercapnic acidosis was observed both in the LC and the A5 area. That is, the LC and the A5 area exhibited depolarizing responses to hypercapnic acidosis in 10 preparations for each. The peak F/F of the A5 area was −0.41 ± 0.15% (mean ± standard error), comparable to the hypercapnic response of the LC (−0.36 ± 0.08%, P = 0.79, unpaired t-test). The reversibility of the depolarizing response was confirmed in three preparations in which the off-phase analysis was performed (Fig. 2a and b). There were also some excitatory regions that could not be assigned to specific anatomical regions. A neuronal response to hypercapnic acidosis and its reversibility were examined in the absence of chemical synaptic transmission in three preparations. The preparation was superfused with low Ca2+ -high Mg2+ ACSF for more than 15 min. The depolarizing response to hypercapnic acidosis was reversibly evoked also in the low Ca2+ -high Mg2+ ACSF (Fig. 2c and d). Previous studies have demonstrated that LC neurons exhibit an intrinsic, depolarizing response to hypercapnic acidosis [11,12]. Since the LC is the only pontine nucleus that has been electrophysiologically proven to be chemosensitive, we used the LC as a positive control for the present study. We were able to detect its depolarizing response, consistent with the findings obtained in previous studies [11,12],
Fig. 2. Reversibility of response to hypercapnic acidosis: (a) hypercapnic acidosis elicited a depolarizing response in the LC and the A5 area. This image was obtained at 68 s after switching control ACSF to hypercapnic acidosis. (b) Image of the same preparation as in (a) obtained at 31 s after switching ACSF back to control. The LC and the A5 area showed a hyperpolarizing response. In this preparation, an undefined area (arrow head) also showed a reversible, depolarizing response to hypercapnic acidosis. (c) A depolarizing response of the A5 area to hypercapnic acidosis in low Ca2+ -high Mg2+ ACSF. (d) Image of the same preparation as in (c). The off-phase analysis revealed that the depolarizing response was reversible. In this preparation, the LC exhibited no response to hypercapnic acidosis. LC: locus coeruleus; A5: A5 area; 4V: fourth ventricle.
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with this newly-applied optical recording method. This justifies application of the optical recording method for identification of chemosensitive regions. We revealed that the A5 area was possibly intrinsically chemosensitive. The A5 area is noradrenergic and is located ventrolaterally to the LC. This area is considered to be involved in the determination of expiratory time and respiratory frequency [1,3,5,7,10]. In the isolated brainstem-spinal cord preparation of the neonatal rat, this area exerts an inhibitory effect on the frequency of respiratory activity [3,5]. This is also true in in vivo rats; i.e., stimulation of the A5 area elicited prolongation of the expiratory time and an eventual decrease in respiratory frequency in anesthetized rats [7]. The A5 area is also considered to play an important role in the post-hypoxic ventilatory decline [1,10]. Haxhiu et al. [4] demonstrated that the A5 area expressed Fos-protein—a marker for neuronal excitation—in rats exposed to hypercapnic conditions, suggesting that the area could be chemosensitive. Our study provided more direct evidence for the chemosensitivity of this area. In summary, we demonstrated that several pontine regions in the slice preparation of the neonatal rat exhibited a depolarizing response to hypercapnic acidosis, using an optical recording method with a voltage-sensitive dye. The present study suggests that the method is applicable to identification of potentially chemosensitive regions, which deserve further electrophysiological analysis. Acknowledgements This study was supported by Research Grants for Life Sciences and Medicine from Keio University Medical Science Fund, a Research Grant for Specific Diseases from the Japanese Ministry of Health and Welfare, and a Grant-in-Aid for Exploratory Research from the Japan Society for the Promotion of Science. References [1] S.K. Coles, T.E. Dick, Neurons in the ventrolateral pons are required for post-hypoxic frequency decline in rats, J. Physiol. 497 (1996) 79–94.
[2] J.B. Dean, D.A. Bayliss, J.T. Erickson, W.L. Lawing, D.E. Millhorn, Depolarization and stimulation of neurons in nucleus tractus solitarii by carbon dioxide does not require chemical synaptic input, Neuroscience 36 (1990) 207–216. [3] S. Errchidi, G. Hilaire, R. Monteau, Permanent release of noradrenaline modulates respiratory frequency in the newborn rat: an in vitro study, J. Physiol. 429 (1990) 497–510. [4] M.A. Haxhiu, K. Yung, B. Erokwu, N.S. Cherniack, CO2 -induced c-fos expression in the CNS catecholaminergic neurons, Respir. Physiol. 105 (1996) 35–45. [5] G. Hilaire, R. Monteau, S. Errchidi, Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area: an in vitro study in the newborn rat, Brain Res. 485 (1989) 325– 332. [6] Y. Ito, Y. Oyamada, K. Yamaguchi, Age-dependent chemosensitive pontine inhibition of medullary respiratory rhythm generation in the isolated brainstem of the neonatal rat, Brain Res. 887 (2000) 418– 420. [7] J.S. Jodokowski, S.K. Coles, T.E. Dick, Prolongation in expiration evoked from ventrolateral pons of adult rats, J. Appl. Physiol. 82 (1997) 377–381. [8] A. Kawai, D. Ballantyne, K. Mückenhoff, P. Scheid, Chemosensitive medullary neurons in the brainstem–spinal cord preparation of the neonatal rat, J. Physiol. 492 (1996) 277–292. [9] Y. Okada, Z. Chen, H. Yoshida, S. Kuwana, W. Jiang, H. Maruiwa, Optical recording of the neuronal activity in the brainstem–spinal cord, Adv. Exp. Med. Biol. 499 (2001) 113–118. [10] Y. Okada, A. Kawai, K. Mückenhoff, P. Scheid, Roles of the pons in hypoxic respiratory depression in the neonatal rat, Respir. Physiol. 111 (1998) 55–63. [11] Y. Oyamada, K. Andrzejewski, K. Mückenhoff, P. Scheid, D. Ballantyne, Locus coeruleus neurones in vitro: pH-sensitive oscillations of membrane potential in an electrically coupled network, Respir. Physiol. 118 (1999) 131–147. [12] Y. Oyamada, D. Ballantyne, K. Mückenhoff, P. Scheid, Respirationmodulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem–spinal cord of the neonatal rat, J. Physiol. 513 (1998) 381–398. [13] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. [14] G.B. Richerson, Response to CO2 of neurons in the rostral ventral medulla in vitro, J. Neurophysiol. 73 (1995) 933– 944. [15] T. Tominaga, Y. Tominaga, H. Yamada, G. Matsumoto, M. Ichikawa, Quantification of optical signals with electrophysiological signals in neural activities of di-4-ANEPPS stained rat hippocampal slices, J. Neurosci. Methods 102 (2000) 11–23. [16] H. Yoshida, Y. Okada, H. Maruiwa, K. Fukuda, M. Nakamura, K. Chiba, Y. Toyama, Synaptic blockade plays a major role in the neural disturbance of experimental spinal cord compression, J. Neurotrauma 20 (2003) 1365–1376.