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Optical imaging of respiratory burst activity in newborn rat medullary block preparations
NEUROSCIENCE RESERRCH Neuroscience Research 25 (1996) 183-190
ELSEVIER
Optical imaging of respiratory burst activity in newborn rat medullary block preparations Hiroshi Onimaru*, Arata Kanamaru, Ikuo Homma Department of Physiology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
Received 7 February 1996; accepted 11 April 1996
Abstract
We report on the optical imaging of excitation propagation induced by electrical stimulation of the nucleus tractus solitarius (NTS) area and subsequent inspiratory burst activity in the ventrolateral medulla (VLM) of a medullary block preparation. A medullary block preparation with a thickness of 1.0-1.4 mm was made from brainstems isolated from 0- to 4-day-old rats and stained with a fluorescent voltage-sensitive dye, RH795. Neuronal responses in the VLM evoked by electrical stimulation were recorded as a fluorescence change using an optical recording apparatus with a 128 x 128 photodiode array and a maximum time resolution of 0.6 ms. Motoneuronal activity was simultaneously recorded at the contralateral hypoglossal nerve roots. Neuronal excitation evoked by stimulation of the NTS area propagated to the VLM through the intermediate reticular zone (IRt). In contrast, caudal VLM stimulation induced excitation which propagated to the rostral VLM without any detectable excitation propagation in the IRt toward the NTS area from the VLM. NTS stimulation also induced an inspiratory burst activity in the hypoglossal nerve root activity with a 150-200 ms delay. Fluorescence changes corresponding to the inspiratory burst activity were observed in the VLM which coincided with the area in which the localization of many respiratory neurons had been demonstrated in previous studies using whole-brainstem preparations. The present results show the feasibility of using optical recordings for the analysis of respiratory neuron activity as well as for analysis of the transmission pathway of afferent and/or efferent information in the medulla. Keywords: Respiratory center; Optical imaging; Ventrolateral medulla; Nucleus tractus solitarius; Intermediate reticular zone; Newborn rat
I. Introduction
In order to elucidate the neural mechanisms of respiratory rhythm generation and modulation, it is important to understand the macroscopic behaviour of the respiratory neuron network as well as the membrane properties of individual neurons or synaptic connections a m o n g the neurons. Recent developments in optical recording methods of neuronal activity have facilitated the analysis of neural networks, to a certain extent, by visualizing their activity (Cohen and Lesher, 1986; Grinvald et al., 1988; Ichikawa et al., 1992; Sato et al., 1995; Tanifuji et al., 1994). Respiratory neurons located in the ventrolateral medulla (VLM) are important in the generation of respiratory rhythm and in* Corresponding author, Tel.: + 81 33784 8113; fax: + 81 33784 0200.
spiratory pattern, and thus, are thought to compose the central pattern generator of respiration (Bianchi et al., 1995; Euler, 1986; Ezure, 1990; Feldman, 1986; Onimaru, 1995; Richter et al., 1992). We report on optical imaging of excitation propagation and subsequent inspiratory burst activity in the VLM, caused by electrical stimulation of the nucleus tractus solitarius (NTS) area which mediates and integrates afferent inputs to the respiratory center and/or vasomotor center in the V L M (Ciriello et al., 1986; Feldman and Ellenberger, 1988).
2. Materials and methods
The brainstems of 0- to preparations) were isolated sia, according to methods maru et al., 1988; Suzue,
0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PH SO168-0102(96)01048-6
4-day-old Wistar rats (20 under deep ether anaesthepreviously described (Oni1984). The brainstem was
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transected rostrally at the level of the IX/Xth cranial nerve roots and caudally at the middle level of the hypoglossal nerve roots, to make a medullary block preparation with a thickness of 1.0-1.4 mm (Fig. 1A). The most rostral and caudal level of the block preparation corresponded to the level of the retrofacial nucleus and rostral part of the lateral reticular nucleus (i.e. Figs. 2(f) and (a) in Arata et al., 1990), respectively. Hence, the block preparation included the medullary region, which is essential for respiratory rhythm generation (Onimaru et al., 1987, 1988), and was shown to generate rhythmic inspiratory activity in hypoglossal nerves (Funk et al., 1993; Smith et al., 1991). The medullary block preparation was stained by incubation for 20 min in a modified Krebs solution (see below) containing 0.2-0.5 mg/ml of a fluorescent voltage-sensitive dye, RH795 (Molecular Probes; Grinvald et al., 1986). The block preparation was then transferred to a 1-ml perfusion chamber (Flexiperm, Heraeus Co.), which was mounted on an inverted microscope (IMT-2, Olympus). The block preparation was placed in the chamber with the caudal face up (Fig. 1B), and superfused continuously at a rate of 2-3 ml/min with the following modified Krebs solution (in mM): NaCI, 124; KC1, 5.0; KH2PO4, 1.2; CaC12, 2.4; MgCI2, 1.3; NaHCO3, 26; glucose, 30; equilibrated with 95% 02 and 5% COz; pH 7.4 at 26-27°C. In some experiments, the potassium concentration of the perfusate was increased to 8-11 mM for activation of inspiratory burst activity (Smith et al., 1991). For electrical stimulation (200 /zs, 10-30 /~A), a tungsten electrode (tip diameter, 50 pm) with a resistance of 2 Mf~ was inserted into the NTS area or VLM through the surface of the caudal cross section of the block preparation. Motoneuronal activity was monitored at the contralateral hypoglossal nerve roots using a glass capillary suction electrode and a high pass filter with a 0.3-s time constant, and was recorded simultaneously in digital memory together with optical imaging data (see below). The neuronal responses in the VLM evoked by electrical stimulation were recorded as a fluorescence change in the voltage-sensitive dye by an optical recording apparatus (HR Deltaron 1700, Fuji Photo Film Co.) through a 510-550 nm excitation filter, a 570 nm dichroic mirror and a 590 nm long pass barrier filter (U-MWG mirror unit, Olympus), using a tungstenhalogen lamp (300 W) as the light source. The recording system consisted of a camera head with a 128 × 128 photodiode array (16 384 pixels) and a processing unit, which included digital frame memories, having a maximum time resolution of 0.6 ms (Ichikawa et al., 1992). Magnification of the microscope was adjusted so that an area of 1.1 x 1.1 mm 2 was covered by the image sensor using the 10 × objective. In each pixel of the sensor, therefore, changes in fluorescence in 8.6 x 8.6
~m 2 of the block preparation were detected. The microscope objective was focused on the surface plane of the rostral cross section of the block preparation. To determine the structural details of the recording area, the surface view of the cross section was also monitored using a CCD camera (CCD-72 Series, Dage-MTI Inc.) and stored on video tape. The stimulation point and the level of block preparation were confirmed in serial 70 p m sections made after the experiments. In each trial, a real-time image was taken about 500 ms before the stimulation and stored in a frame memory (reference image). The differences between the reference image and subsequent frames taken every 1.2 or 2.4 ms were amplified 1000 times, then stored in another frame memory (difference images). The difference image was averaged for 4-8 trials. All optical signal data were stored on an MO disk for subsequent off-line analysis using a computer (Sun4/50, Sun Microsystems Inc.). Fractional changes in fluorescence were estimated to be less than 0.05%, while the noise level was less than 0.01%. To consider the variability in pixel values, relative changes in the optical signal intensities were determined by relating the amplitude of fluorescence changes V ~
A
o
I,~p b n ~ t
Fig. 1. Schema of the block preparation and recording apparatus. (A) Ventral view of the brainstem-spinal cord preparation. To make a medullary block preparation with a thickness of 1.0-1.4 m m , the brainstem was transected rostrally at the level of the IX/Xth cranial nerve roots and caudally at the middle level of the hypoglossal nerve roots. V-XII, cranial nerves; C I - C 4 , cervical ventral nerves. (B) The block preparation was stained with the fluorescent voltage-sensitive dye, RH795 (0.2-0.5 mg/ml, 20 min) in modified Krebs solution. The block preparation was transferred to a 1-ml perfusion chamber mounted on an inverted microscope, with the caudal face up. The NTS area was stimulated electrically by a tungsten electrode. Motoneuronal activity was monitored at the contralateral hypoglossal nerve roots. F1, 510-550 n m excitation filter; D M , 570 nm dichroic mirror; F2, 590 n m long pass barrier filter; MOS IS, MOS image sensor which covers 1.1 × 1.1 m m 2 of the cross section. (C) The block preparation was positioned so that the NTS area was in the upper right direction and the VLM was at the lower left of the optical imaging area. A M B / R F N , the nucleus ambiguus or retrofacial nucleus.
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A NTS stimulation 0 ms
NTS
2.4 ms
4.8 ms
9.6 ms
168 ms
V L M stimulation 0 ms
1.2 ms
3.6 ms
"VS 6.0 ms
8.4 ms
20.4 ms
"~'VS 7.2 ms ~ ~ +>
B
,
~,
,
y
i I
i
xSD <-2
0
+4
+8<
Fig. 2. Short-term propagation of excitation evoked by electrical stimulation in the NTS area (A) or caudal VLM (B). Representative optical images were selected from image data of a total of 512 frames sampled every 1.2 ms. An average of eight trials. The numbers on each frame image show the time before or after stimulation. The NTS area is in the upper right direction and VLM is at the lower left of the optical imaging area (see Fig. IC). VS, ventral surface. See the legend in Fig. 3 for the white square in the first frame of (A) and (B).
to the average baseline noise levels, i.e. standard deviations (S.D.) of prestimulus baseline activity (Cinelli and Kauer, 1995). First, the mean and S.D. of prestimulus baseline activity were calculated in each pixel of 16 frames before stimulation as controls. The control
mean value was subtracted from values in each pixel after stimulation, and the ratios of the differences to the control S.D. value were used to evaluate changes in the optical signal intensity in each pixel. The ratios were represented as different color components using Adobe
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PHOTOSHOP 2.5 on a Macintosh computer. In following optical imaging figures, the block preparations were positioned so that the NTS area was in the upper right direction and VLM was at the lower left of the optical imaging area (see Figs. 1 and 4).
3. Results
Neuronal excitation evoked by stimulation of the NTS area (n = 11) propagated mainly to the VLM through the intermediate reticular zone (IRt; Paxinos and Watson, 1986), while part of the excitation propagated laterally in the NTS region. Fig. 2A shows an example of such optical imaging of the excitation, averaging the results of eight trials recorded with a 1.2-ms time resolution. The excitation reached the VLM in 4.8-6.0 ms, and maximum responses in the VLM appeared 9.6-12 ms after stimulation. Excitation in the VLM continued for 100-150 ms with gradual reduction (Fig. 3A, see also Fig. 4). In contrast to the NTS stimulation, caudal VLM stimulation (n = 9) induced excitation which propagated to the rostral VLM in 1.2 ms, and maximum responses appeared 3.6-4.8 ms after stimulation (Fig. 2B). The fast spikelike reA N T S stimuidlm x~l~ 6 4
lOOm
2
0
-2
sponse was followed by small-amplitude excitation, which decreased slowly (Fig. 3B). No detectable excitation toward the NTS area from the VLM was observed in the IRt. To detect optical signals corresponding to the inspiratory burst activity induced by NTS stimulation, fluorescence changes were measured every 2.4 ms for 1024 frames (i.e. about 2.5 s in total). In such long-term measurement, gradual increases in background fluorescence changes were not negligible in the present study (Fig. 4A, red trace). To cancel background fluorescence changes, the mean value in a 20 × 20 pixel (172 /~m square) area, in which the changes in optical signals correlating to stimulation were minimum during measurement, was subtracted from each pixel value of the optical recording data. Fig. 4 shows the result of such calculations from the optical data of responses evoked by NTS stimulation. The time course of the subtracted signal intensity changes in 20 x 20 pixels in the VLM indicated strong correlation with that of inspiratory hypoglossal motoneuronal activity which appeared with a 150-200 ms delay and was of about 600 ms duration (Fig. 4A). Fig. 4B shows several optical images in which background fluorescence changes were canceled. After fast propagation of excitation from the NTS to the VLM by stimulation of the NTS (Fig. 4B, 12.0 ms frame) as shown in Fig. 2, fluorescence changes in the VLM were observed for 210-850 ms, corresponding to the inspiratory burst activity of the hypoglossal motoneurons. Fig. 5 shows an optical image averaged temporally between 400 and 500 ms after NTS stimulation, in which the hypoglossal nerve recording indicated almost maximum inspiratory burst activity. The active VLM region was found to be ventral to the nucleus ambiguus or retrofacial nucleus and close to the ventral surface. In contrast to this, no significant fluorescence change was detected in the VLM after fast propagation of excitation from the NTS to VLM when no hypoglossal inspiratory burst activity was induced by subthreshold NTS stimulation (data not shown).
8
't -2
Fig. 3. Time course of change in the. optical signal in the VLM (the same experiment as outlined in Fig. 2, i.e. electrical stimulation in the NTS (A) or caudal VLM (B)). The average optical signal was calculated in 20 x 20 pixels in the VLM indicated by the white square in the first frame of Fig. 2A or B.
4. Discussion
The excitation by NTS stimulation propagated to the VLM while activating IRt neurons and continued for more than 100 ms. Although the fast components of the excitation might include antidormic activation of the IRt and VLM neurons, the excitation was considered to reflect mainly stimulus-induced EPSP or postsynaptic action potentials in neurons in these areas, including secondary-induced activation of the local network of neurons in these areas. This possibility should be confirmed by elimination of the response under synaptic blockade conditions (Sato et al., 1995). On the other hand, caudal VLM stimulation induced a fast spikelike
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H. Onimaru et al./ Neuroscience Research 25 (1996) 183-190
A
.J-L
x SD +5' +4' +3' +2 +1 0 HUI-DaCK
B -4.8 ms
410 ms
12.0 ms
200 ms
550 ms
1130 ms
xSD >+6 +3
0 <-3
Fig. 4. Long-term optical recording in the VLM following electrical stimulation in the NTS. Fluorescence changes were sampled every 2.4 ms for a 1024-frame optical recording. An average of four trials. (A) Time courses of fluorescence changes. ROI (region of interest, blue trace), change in the mean optical signal in 20 x 20 pixels in the VLM indicated by the white square in the figure at the left side; back (red trace), change in the mean optical signal in 20 × 20 pixels in the mid-lateral medulla indicated by the red square in the figure at the left side, used for background fluorescence change. ROl-back (black trace), result of subtraction of back from ROI. XII, hypoglossal motoneuronal activity recorded simultaneously; AMB/RFN, the nucleus ambiguus or retrofacial nucleus, An inspiratory burst activity was induced with a delay of about 200 ms. Note the good correlation in the time course between the subtracted optical signal (ROI-back) and motoneuronal activity (XII). (B) Changes in the optical images induced by a single-shot NTS stimulation in which background fluorescence changes were canceled. The numbers on each frame image show the time before or after stimulation, corresponding to the time indicated by the arrowheads in the record of hypoglossal motoneuronal activity (A, XII). See (A) (left side) for the position of the preparation; the NTS area is in the upper right direction and VLM is at the lower left (see also Fig. 5). Note the optical signals in the VLM (410, 550 ms frames) corresponding to the inspiratory hypoglossal motoneuronal burst.
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Fig. 5. A temporally averaged optical image corresponding to the inspiratory burst activity. The frame memory data of Fig. 4 were averaged between 400 and 500 ms after NTS stimulation, in which hypoglossal inspiratory burst activity was maximum. The active region was found to be ventral to the nucleus ambiguus or retrofaeial nucleus (AMB/RFN). VS, ventral surface.
excitation, presumed to be antidromic activation of the rostral VLM neurons, and slowly decrementing excitation of small amplitude, possibly corresponding to postsynaptic activation of the neurons. Excitation propagation toward the NTS from the VLM was not detected in the present study in spite of the presence of many axonal projections to the NTS from the VLM via the IRt (Ellenberger and Feldman, 1990). This may be due to an insufficient membrane area for the detection of axonal excitation even if it occurs (see e.g. Grinvald et al., 1988 or Sato et al., 1995 for a detailed discussion of the amplitude of the optical signals). The IRt has been delineated as a distinct nuclear zone in the rat (Paxinos and Watson, 1986). The IRt
receives afferents from, and sends projections to, many other brainstem areas, and plays an important role in various autonomic functions (Huang and Paxinos, 1995). The results of many neuro-anatomical studies have implied that neuronal projections from the NTS to VLM pass through the IRt (e.g. EUenberger and Feldman, 1990; Ross et al., 1985). The present results suggest that the IRt can function as a specific transmission pathway of afferent information from the NTS area to the VLM which is an important region in the central vasomotor control and/or respiratory control. NTS stimulation induced a hypoglossal inspiratory burst lasting about 600 ms, after a delay of about 200 ms. The time course of optical signals appearing after a
H. Onimaru et al. / Neuroscience Research 25 (1996) 183-190
similar delay in the VLM correlated well with that of the hypoglossal inspiratory burst activity (Fig. 4). Furthermore, such optical signals could not be detected when a hypoglossal inspiratory burst activity was not induced by NTS stimulation. It has been suggested that inspiratory activity in hypoglossal motoneurons is driven by inspiratory neurons in the VLM (Funk et al., 1993). Therefore, we concluded that the optical signals corresponding to the inspiratory burst probably reflected depolarizing driving potentials of inspiratory neurons whose amplitude was 5-15 mV (Onimaru and Homma, 1992). Respiratory neurons are distributed in the rostro-caudal column of the VLM in the present block preparation (Arata et al., 1990). It is presumed that a photodiode could detect fluorescence signals from two or three hundred microns deep (London et al., 1989). In the present study, the optical signals might have originated mainly from the rostral part of the VLM column in which both the dye concentration and the light intensity were greatest. Respiratory neurons in the VLM are located among many non-respiratory neurons without any distinct layer structure. This suggests that analysis of the respiratory neuron network would be difficult using optical measurement. Nevertheless, the present results demonstrate the feasibility of using optical recordings for the analysis of respiratory neuron activity. The delay (150-200 ms) of inspiratory burst initiation from NTS stimulation was not due to limitation of the propagation speed, because excitation in the NTS area reaches the VLM in several milli seconds. A similar delay was observed after contralateral VLM stimulation in brainstem-spinal cord preparations, in which stimulation first induced premature pre-inspiratory neuron firings before an inspiratory burst activity (Onimaru et al., 1987; Onimaru et al., 1988). In the present experiments, therefore, a certain neuronal activity that precedes and triggers the inspiratory burst may be induced by the stimulation, although optical signals indicating such neuronal activity are not evident. Discrimination of respiratory neuron activity with a different burst pattern may be the next significant step in studies using optical imaging techniques. The area in which optical signals corresponding to inspiratory burst activity were detected (Fig. 5) coincided with the area in which the localization of many respiratory neurons had been demonstrated, in previous studies, using whole-brainstem preparations: ventral to the nucleus ambiguus or retrofacial nucleus and close to the ventral surface (Arata et al., 1990; Kashiwagi et al., 1993; Onimaru et al., 1987). These distributions of respiratory neurons in the newborn rat preparation resembled those in the adult rat (Ezure et al., 1988; Pilowsky et al., 1990).
189
Acknowledgements We are grateful to H. Ayukawa and N. Yaginuma in the Bio-imaging Systems Division of Fuji Photo Film Co. for their technical support.
References Arata, A., Onimaru, H. and Homma, I. (1990) Respiration-related neurons in the ventral medulla of newborn rats in vitro. Brain Res. Bull., 24: 599-604. Bianchi, A.L., Denavit-Saubie, M. and Champagnat, J. (1995) Central control of breathing in mammals: Neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev., 75: 1-45. Cinelli, A.R. and Kauer, J.S. (1995) Salamander olfactory bulb neuronal activity observed by video rate, voltage-sensitive dye imaging. II. Spatial and temporal properties of responses evoked by electric stimulation. J. Neurophysiol., 73: 2033-2052. Ciriello, J., Caverson, M.M. and Polosa, C. (1986) Function of the ventrolateral medulla in the control of the circulation. Brain Res., 11: 359-391. Cohen, L.B. and Lesher, S. (1986) Optical monitoring of membrane potential: methods of multisite optical measurement. In: P. De Weer and B.M. Salzberg (Eds), Optical Methods in Cell Physiology, Wiley, New York, pp. 71-99. Ellenberger, H.H. and Feldman (1990) Brainstem connections of the rostral ventral respiratory group of the rat. Brain Res., 513: 35-42. Euler, C. yon (1986) Brainstem mechanisms for generation and control of breathing pattern. In: A.P. Fishman (Ed.), The Respiratory System, Handbook of Physiology, Section 3, Waverly, MD, pp. 1-67. Ezure, K. (1990) Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog. Neurobiol., 35:429-450: Ezure, K., Manabe, M. and Yamada, H. (1988) Distribution of medullary respiratory neurons in the rat. Brain Res., 455: 262270. Funk, G.D., Smith, J.C. and Feldman, J.L. (1993). Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids. J. Neurophysiol., 70:1497 1515. Feldman, J.L. (1986) Neurophysiology of breathing in mammals. In: F.E. Bloom (Ed.), The Nervous System, Volume IV, Intrinsic Regulatory Systems of the Brain, Handbook of Physiology, Section 1, American Physiological Society, Bethesda, MD, pp. 463524. Feldman, J.L. and Ellenberger, H.H. (1988) Central coordination of respiratory and cardiovascular control in mammals. Annu. Rev. Physiol., 50: 593-606. Grinvald, A., Lieke, E., Frostig, R.D., Gilbert, C.D. and Wiesel, T.N. (1986) Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature, 342: 361-364. Grinvald, A., Frostig, R.D., Lieke, E. and Hildesheim, R. (1988) Optical imaging of neuronal activity. Physiol. Rev., 68:1285 1366. Huang, X.-F. and Paxinos, G. (1995) Human intermediate reticular zone: A cyto- and chemoarchitectonic study. J. Comp. Neurol., 360: 571-588. Ichikawa, M., lijima, T. and Matsumoto, G. (1992) Real-time optical recording of neuronal activities in the brain. In: T. Ono, L.R. Squire, M.E. Raichle, D.I. Perrett and M. Fukuda (Eds), Brain Mechanisms of Perception and Memory, Oxford University Press, New York, pp. 639-647.
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Kashiwagi, M., Onimaru, H. and Homma, I. (1993) Correlation analysis of respiratory neuron activity in ventrolateral medulla of brainstem-spinal cord preparation isolated from newborn rat. Exp. Brain Res., 95: 277-290. London, J.A., Cohen, L.B. and Wu, J.-Y. (1989) Optical recordings of the cortical response to whisker stimulation before and after the addition of an epileptogenic agent. J. Neurosci., 9:2182 2190. Onimarn, H. (1995) Studies of respiratory center using isolated brainstem-spinal cord preparations. Neurosci. Res., 21:183 190. Onimaru, H., Arata, A., and Homma, I. (1987) Localization of respiratory rhythm-generating neurons in the medulla of brainstem-spinal cord preparations from newborn rats. Neurosci. Lett., 78: 151-155. Onimaru, H., Arata, A. and Homma, I. (1988) Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat. Brain Res., 445: 314-324. Onimaru, H. and Homma, I. (1992) Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats. Pfliigers Arch., 420: 399-406. Paxinos, G. and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Sydney. Pilowsky, P.M., Jiang, C. and Lipski, J. (1990) An intracellular study of respiratory neurons in the rostral ventrolateral medulla of rat
and their relationship to catecholamine-containing neurons. J. Comp. Neurol., 301: 604-617. Richter, D.W., Ballanyi, K. and Schwarzacher, S. (1992) Mechanisms of respiratory rhythm generation. Curr. Opin. Neurobiol., 2: 788-793. Ross, C.A., Ruggiero, D.A. and Reis, D.J. (1985) Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla. J. Comp. Neurol., 242:511 534. Sato, K., Momose-Sato, Y., Sakai, T., Hirota, A. and Kamino, K. (1995) Responses to glossopharyngeal stimulus in the early embryonic chick brainstem: Spatiotemporal patterns in three dimensions from repeated multiple-site optical recording of electrical activity. J. Neurosci., 15:2123 2140. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W. and Feldman, J.L. (1991) Pre-B6tzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science, 254: 726-729. Suzue, T. (1984) Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J. Physiol., 354: 173-183. Tanifuji, M., Sugiyama, T. and Murase, K. (1994) Horizontal propagation of excitation in rat visual cortical slices revealed by optical imaging. Science, 266: 1057-1059.
ELSEVIER
Optical imaging of respiratory burst activity in newborn rat medullary block preparations Hiroshi Onimaru*, Arata Kanamaru, Ikuo Homma Department of Physiology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan
Received 7 February 1996; accepted 11 April 1996
Abstract
We report on the optical imaging of excitation propagation induced by electrical stimulation of the nucleus tractus solitarius (NTS) area and subsequent inspiratory burst activity in the ventrolateral medulla (VLM) of a medullary block preparation. A medullary block preparation with a thickness of 1.0-1.4 mm was made from brainstems isolated from 0- to 4-day-old rats and stained with a fluorescent voltage-sensitive dye, RH795. Neuronal responses in the VLM evoked by electrical stimulation were recorded as a fluorescence change using an optical recording apparatus with a 128 x 128 photodiode array and a maximum time resolution of 0.6 ms. Motoneuronal activity was simultaneously recorded at the contralateral hypoglossal nerve roots. Neuronal excitation evoked by stimulation of the NTS area propagated to the VLM through the intermediate reticular zone (IRt). In contrast, caudal VLM stimulation induced excitation which propagated to the rostral VLM without any detectable excitation propagation in the IRt toward the NTS area from the VLM. NTS stimulation also induced an inspiratory burst activity in the hypoglossal nerve root activity with a 150-200 ms delay. Fluorescence changes corresponding to the inspiratory burst activity were observed in the VLM which coincided with the area in which the localization of many respiratory neurons had been demonstrated in previous studies using whole-brainstem preparations. The present results show the feasibility of using optical recordings for the analysis of respiratory neuron activity as well as for analysis of the transmission pathway of afferent and/or efferent information in the medulla. Keywords: Respiratory center; Optical imaging; Ventrolateral medulla; Nucleus tractus solitarius; Intermediate reticular zone; Newborn rat
I. Introduction
In order to elucidate the neural mechanisms of respiratory rhythm generation and modulation, it is important to understand the macroscopic behaviour of the respiratory neuron network as well as the membrane properties of individual neurons or synaptic connections a m o n g the neurons. Recent developments in optical recording methods of neuronal activity have facilitated the analysis of neural networks, to a certain extent, by visualizing their activity (Cohen and Lesher, 1986; Grinvald et al., 1988; Ichikawa et al., 1992; Sato et al., 1995; Tanifuji et al., 1994). Respiratory neurons located in the ventrolateral medulla (VLM) are important in the generation of respiratory rhythm and in* Corresponding author, Tel.: + 81 33784 8113; fax: + 81 33784 0200.
spiratory pattern, and thus, are thought to compose the central pattern generator of respiration (Bianchi et al., 1995; Euler, 1986; Ezure, 1990; Feldman, 1986; Onimaru, 1995; Richter et al., 1992). We report on optical imaging of excitation propagation and subsequent inspiratory burst activity in the VLM, caused by electrical stimulation of the nucleus tractus solitarius (NTS) area which mediates and integrates afferent inputs to the respiratory center and/or vasomotor center in the V L M (Ciriello et al., 1986; Feldman and Ellenberger, 1988).
2. Materials and methods
The brainstems of 0- to preparations) were isolated sia, according to methods maru et al., 1988; Suzue,
0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PH SO168-0102(96)01048-6
4-day-old Wistar rats (20 under deep ether anaesthepreviously described (Oni1984). The brainstem was
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transected rostrally at the level of the IX/Xth cranial nerve roots and caudally at the middle level of the hypoglossal nerve roots, to make a medullary block preparation with a thickness of 1.0-1.4 mm (Fig. 1A). The most rostral and caudal level of the block preparation corresponded to the level of the retrofacial nucleus and rostral part of the lateral reticular nucleus (i.e. Figs. 2(f) and (a) in Arata et al., 1990), respectively. Hence, the block preparation included the medullary region, which is essential for respiratory rhythm generation (Onimaru et al., 1987, 1988), and was shown to generate rhythmic inspiratory activity in hypoglossal nerves (Funk et al., 1993; Smith et al., 1991). The medullary block preparation was stained by incubation for 20 min in a modified Krebs solution (see below) containing 0.2-0.5 mg/ml of a fluorescent voltage-sensitive dye, RH795 (Molecular Probes; Grinvald et al., 1986). The block preparation was then transferred to a 1-ml perfusion chamber (Flexiperm, Heraeus Co.), which was mounted on an inverted microscope (IMT-2, Olympus). The block preparation was placed in the chamber with the caudal face up (Fig. 1B), and superfused continuously at a rate of 2-3 ml/min with the following modified Krebs solution (in mM): NaCI, 124; KC1, 5.0; KH2PO4, 1.2; CaC12, 2.4; MgCI2, 1.3; NaHCO3, 26; glucose, 30; equilibrated with 95% 02 and 5% COz; pH 7.4 at 26-27°C. In some experiments, the potassium concentration of the perfusate was increased to 8-11 mM for activation of inspiratory burst activity (Smith et al., 1991). For electrical stimulation (200 /zs, 10-30 /~A), a tungsten electrode (tip diameter, 50 pm) with a resistance of 2 Mf~ was inserted into the NTS area or VLM through the surface of the caudal cross section of the block preparation. Motoneuronal activity was monitored at the contralateral hypoglossal nerve roots using a glass capillary suction electrode and a high pass filter with a 0.3-s time constant, and was recorded simultaneously in digital memory together with optical imaging data (see below). The neuronal responses in the VLM evoked by electrical stimulation were recorded as a fluorescence change in the voltage-sensitive dye by an optical recording apparatus (HR Deltaron 1700, Fuji Photo Film Co.) through a 510-550 nm excitation filter, a 570 nm dichroic mirror and a 590 nm long pass barrier filter (U-MWG mirror unit, Olympus), using a tungstenhalogen lamp (300 W) as the light source. The recording system consisted of a camera head with a 128 × 128 photodiode array (16 384 pixels) and a processing unit, which included digital frame memories, having a maximum time resolution of 0.6 ms (Ichikawa et al., 1992). Magnification of the microscope was adjusted so that an area of 1.1 x 1.1 mm 2 was covered by the image sensor using the 10 × objective. In each pixel of the sensor, therefore, changes in fluorescence in 8.6 x 8.6
~m 2 of the block preparation were detected. The microscope objective was focused on the surface plane of the rostral cross section of the block preparation. To determine the structural details of the recording area, the surface view of the cross section was also monitored using a CCD camera (CCD-72 Series, Dage-MTI Inc.) and stored on video tape. The stimulation point and the level of block preparation were confirmed in serial 70 p m sections made after the experiments. In each trial, a real-time image was taken about 500 ms before the stimulation and stored in a frame memory (reference image). The differences between the reference image and subsequent frames taken every 1.2 or 2.4 ms were amplified 1000 times, then stored in another frame memory (difference images). The difference image was averaged for 4-8 trials. All optical signal data were stored on an MO disk for subsequent off-line analysis using a computer (Sun4/50, Sun Microsystems Inc.). Fractional changes in fluorescence were estimated to be less than 0.05%, while the noise level was less than 0.01%. To consider the variability in pixel values, relative changes in the optical signal intensities were determined by relating the amplitude of fluorescence changes V ~
A
o
I,~p b n ~ t
Fig. 1. Schema of the block preparation and recording apparatus. (A) Ventral view of the brainstem-spinal cord preparation. To make a medullary block preparation with a thickness of 1.0-1.4 m m , the brainstem was transected rostrally at the level of the IX/Xth cranial nerve roots and caudally at the middle level of the hypoglossal nerve roots. V-XII, cranial nerves; C I - C 4 , cervical ventral nerves. (B) The block preparation was stained with the fluorescent voltage-sensitive dye, RH795 (0.2-0.5 mg/ml, 20 min) in modified Krebs solution. The block preparation was transferred to a 1-ml perfusion chamber mounted on an inverted microscope, with the caudal face up. The NTS area was stimulated electrically by a tungsten electrode. Motoneuronal activity was monitored at the contralateral hypoglossal nerve roots. F1, 510-550 n m excitation filter; D M , 570 nm dichroic mirror; F2, 590 n m long pass barrier filter; MOS IS, MOS image sensor which covers 1.1 × 1.1 m m 2 of the cross section. (C) The block preparation was positioned so that the NTS area was in the upper right direction and the VLM was at the lower left of the optical imaging area. A M B / R F N , the nucleus ambiguus or retrofacial nucleus.
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A NTS stimulation 0 ms
NTS
2.4 ms
4.8 ms
9.6 ms
168 ms
V L M stimulation 0 ms
1.2 ms
3.6 ms
"VS 6.0 ms
8.4 ms
20.4 ms
"~'VS 7.2 ms ~ ~ +>
B
,
~,
,
y
i I
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0
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Fig. 2. Short-term propagation of excitation evoked by electrical stimulation in the NTS area (A) or caudal VLM (B). Representative optical images were selected from image data of a total of 512 frames sampled every 1.2 ms. An average of eight trials. The numbers on each frame image show the time before or after stimulation. The NTS area is in the upper right direction and VLM is at the lower left of the optical imaging area (see Fig. IC). VS, ventral surface. See the legend in Fig. 3 for the white square in the first frame of (A) and (B).
to the average baseline noise levels, i.e. standard deviations (S.D.) of prestimulus baseline activity (Cinelli and Kauer, 1995). First, the mean and S.D. of prestimulus baseline activity were calculated in each pixel of 16 frames before stimulation as controls. The control
mean value was subtracted from values in each pixel after stimulation, and the ratios of the differences to the control S.D. value were used to evaluate changes in the optical signal intensity in each pixel. The ratios were represented as different color components using Adobe
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PHOTOSHOP 2.5 on a Macintosh computer. In following optical imaging figures, the block preparations were positioned so that the NTS area was in the upper right direction and VLM was at the lower left of the optical imaging area (see Figs. 1 and 4).
3. Results
Neuronal excitation evoked by stimulation of the NTS area (n = 11) propagated mainly to the VLM through the intermediate reticular zone (IRt; Paxinos and Watson, 1986), while part of the excitation propagated laterally in the NTS region. Fig. 2A shows an example of such optical imaging of the excitation, averaging the results of eight trials recorded with a 1.2-ms time resolution. The excitation reached the VLM in 4.8-6.0 ms, and maximum responses in the VLM appeared 9.6-12 ms after stimulation. Excitation in the VLM continued for 100-150 ms with gradual reduction (Fig. 3A, see also Fig. 4). In contrast to the NTS stimulation, caudal VLM stimulation (n = 9) induced excitation which propagated to the rostral VLM in 1.2 ms, and maximum responses appeared 3.6-4.8 ms after stimulation (Fig. 2B). The fast spikelike reA N T S stimuidlm x~l~ 6 4
lOOm
2
0
-2
sponse was followed by small-amplitude excitation, which decreased slowly (Fig. 3B). No detectable excitation toward the NTS area from the VLM was observed in the IRt. To detect optical signals corresponding to the inspiratory burst activity induced by NTS stimulation, fluorescence changes were measured every 2.4 ms for 1024 frames (i.e. about 2.5 s in total). In such long-term measurement, gradual increases in background fluorescence changes were not negligible in the present study (Fig. 4A, red trace). To cancel background fluorescence changes, the mean value in a 20 × 20 pixel (172 /~m square) area, in which the changes in optical signals correlating to stimulation were minimum during measurement, was subtracted from each pixel value of the optical recording data. Fig. 4 shows the result of such calculations from the optical data of responses evoked by NTS stimulation. The time course of the subtracted signal intensity changes in 20 x 20 pixels in the VLM indicated strong correlation with that of inspiratory hypoglossal motoneuronal activity which appeared with a 150-200 ms delay and was of about 600 ms duration (Fig. 4A). Fig. 4B shows several optical images in which background fluorescence changes were canceled. After fast propagation of excitation from the NTS to the VLM by stimulation of the NTS (Fig. 4B, 12.0 ms frame) as shown in Fig. 2, fluorescence changes in the VLM were observed for 210-850 ms, corresponding to the inspiratory burst activity of the hypoglossal motoneurons. Fig. 5 shows an optical image averaged temporally between 400 and 500 ms after NTS stimulation, in which the hypoglossal nerve recording indicated almost maximum inspiratory burst activity. The active VLM region was found to be ventral to the nucleus ambiguus or retrofacial nucleus and close to the ventral surface. In contrast to this, no significant fluorescence change was detected in the VLM after fast propagation of excitation from the NTS to VLM when no hypoglossal inspiratory burst activity was induced by subthreshold NTS stimulation (data not shown).
8
't -2
Fig. 3. Time course of change in the. optical signal in the VLM (the same experiment as outlined in Fig. 2, i.e. electrical stimulation in the NTS (A) or caudal VLM (B)). The average optical signal was calculated in 20 x 20 pixels in the VLM indicated by the white square in the first frame of Fig. 2A or B.
4. Discussion
The excitation by NTS stimulation propagated to the VLM while activating IRt neurons and continued for more than 100 ms. Although the fast components of the excitation might include antidormic activation of the IRt and VLM neurons, the excitation was considered to reflect mainly stimulus-induced EPSP or postsynaptic action potentials in neurons in these areas, including secondary-induced activation of the local network of neurons in these areas. This possibility should be confirmed by elimination of the response under synaptic blockade conditions (Sato et al., 1995). On the other hand, caudal VLM stimulation induced a fast spikelike
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A
.J-L
x SD +5' +4' +3' +2 +1 0 HUI-DaCK
B -4.8 ms
410 ms
12.0 ms
200 ms
550 ms
1130 ms
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0 <-3
Fig. 4. Long-term optical recording in the VLM following electrical stimulation in the NTS. Fluorescence changes were sampled every 2.4 ms for a 1024-frame optical recording. An average of four trials. (A) Time courses of fluorescence changes. ROI (region of interest, blue trace), change in the mean optical signal in 20 x 20 pixels in the VLM indicated by the white square in the figure at the left side; back (red trace), change in the mean optical signal in 20 × 20 pixels in the mid-lateral medulla indicated by the red square in the figure at the left side, used for background fluorescence change. ROl-back (black trace), result of subtraction of back from ROI. XII, hypoglossal motoneuronal activity recorded simultaneously; AMB/RFN, the nucleus ambiguus or retrofacial nucleus, An inspiratory burst activity was induced with a delay of about 200 ms. Note the good correlation in the time course between the subtracted optical signal (ROI-back) and motoneuronal activity (XII). (B) Changes in the optical images induced by a single-shot NTS stimulation in which background fluorescence changes were canceled. The numbers on each frame image show the time before or after stimulation, corresponding to the time indicated by the arrowheads in the record of hypoglossal motoneuronal activity (A, XII). See (A) (left side) for the position of the preparation; the NTS area is in the upper right direction and VLM is at the lower left (see also Fig. 5). Note the optical signals in the VLM (410, 550 ms frames) corresponding to the inspiratory hypoglossal motoneuronal burst.
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Fig. 5. A temporally averaged optical image corresponding to the inspiratory burst activity. The frame memory data of Fig. 4 were averaged between 400 and 500 ms after NTS stimulation, in which hypoglossal inspiratory burst activity was maximum. The active region was found to be ventral to the nucleus ambiguus or retrofaeial nucleus (AMB/RFN). VS, ventral surface.
excitation, presumed to be antidromic activation of the rostral VLM neurons, and slowly decrementing excitation of small amplitude, possibly corresponding to postsynaptic activation of the neurons. Excitation propagation toward the NTS from the VLM was not detected in the present study in spite of the presence of many axonal projections to the NTS from the VLM via the IRt (Ellenberger and Feldman, 1990). This may be due to an insufficient membrane area for the detection of axonal excitation even if it occurs (see e.g. Grinvald et al., 1988 or Sato et al., 1995 for a detailed discussion of the amplitude of the optical signals). The IRt has been delineated as a distinct nuclear zone in the rat (Paxinos and Watson, 1986). The IRt
receives afferents from, and sends projections to, many other brainstem areas, and plays an important role in various autonomic functions (Huang and Paxinos, 1995). The results of many neuro-anatomical studies have implied that neuronal projections from the NTS to VLM pass through the IRt (e.g. EUenberger and Feldman, 1990; Ross et al., 1985). The present results suggest that the IRt can function as a specific transmission pathway of afferent information from the NTS area to the VLM which is an important region in the central vasomotor control and/or respiratory control. NTS stimulation induced a hypoglossal inspiratory burst lasting about 600 ms, after a delay of about 200 ms. The time course of optical signals appearing after a
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similar delay in the VLM correlated well with that of the hypoglossal inspiratory burst activity (Fig. 4). Furthermore, such optical signals could not be detected when a hypoglossal inspiratory burst activity was not induced by NTS stimulation. It has been suggested that inspiratory activity in hypoglossal motoneurons is driven by inspiratory neurons in the VLM (Funk et al., 1993). Therefore, we concluded that the optical signals corresponding to the inspiratory burst probably reflected depolarizing driving potentials of inspiratory neurons whose amplitude was 5-15 mV (Onimaru and Homma, 1992). Respiratory neurons are distributed in the rostro-caudal column of the VLM in the present block preparation (Arata et al., 1990). It is presumed that a photodiode could detect fluorescence signals from two or three hundred microns deep (London et al., 1989). In the present study, the optical signals might have originated mainly from the rostral part of the VLM column in which both the dye concentration and the light intensity were greatest. Respiratory neurons in the VLM are located among many non-respiratory neurons without any distinct layer structure. This suggests that analysis of the respiratory neuron network would be difficult using optical measurement. Nevertheless, the present results demonstrate the feasibility of using optical recordings for the analysis of respiratory neuron activity. The delay (150-200 ms) of inspiratory burst initiation from NTS stimulation was not due to limitation of the propagation speed, because excitation in the NTS area reaches the VLM in several milli seconds. A similar delay was observed after contralateral VLM stimulation in brainstem-spinal cord preparations, in which stimulation first induced premature pre-inspiratory neuron firings before an inspiratory burst activity (Onimaru et al., 1987; Onimaru et al., 1988). In the present experiments, therefore, a certain neuronal activity that precedes and triggers the inspiratory burst may be induced by the stimulation, although optical signals indicating such neuronal activity are not evident. Discrimination of respiratory neuron activity with a different burst pattern may be the next significant step in studies using optical imaging techniques. The area in which optical signals corresponding to inspiratory burst activity were detected (Fig. 5) coincided with the area in which the localization of many respiratory neurons had been demonstrated, in previous studies, using whole-brainstem preparations: ventral to the nucleus ambiguus or retrofacial nucleus and close to the ventral surface (Arata et al., 1990; Kashiwagi et al., 1993; Onimaru et al., 1987). These distributions of respiratory neurons in the newborn rat preparation resembled those in the adult rat (Ezure et al., 1988; Pilowsky et al., 1990).
189
Acknowledgements We are grateful to H. Ayukawa and N. Yaginuma in the Bio-imaging Systems Division of Fuji Photo Film Co. for their technical support.
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