Somatic vs. dendritic responses to hypercapnia in chemosensitive locus coeruleus neurons from neonatal rats

Nick A. Ritucci, Jay B. Dean, and Robert W. Putnam

Department of Neuroscience, Cell Biology and Physiology, Wright State University School of Medicine, Dayton, Ohio

Submitted 8 July 2004 ; accepted in final form 8 July 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Cardiorespiratory control is mediated in part by central chemosensitive neurons that respond to increased CO2 (hypercapnia). Activation of these neurons is thought to involve hypercapnia-induced decreases in intracellular pH (pHi). All previous measurements of hypercapnia-induced pHi changes in chemosensitive neurons have been obtained from the soma, but chemosensitive signaling could be initiated in the dendrites of these neurons. In this study, membrane potential (Vm) and pHi were measured simultaneously in chemosensitive locus coeruleus (LC) neurons from neonatal rat brain stem slices using whole cell pipettes and the pH-sensitive fluorescent dye pyranine. We measured pHi from the soma as well as from primary dendrites to a distance 160 µm from the edge of the soma. Hypercapnia [15% CO2, external pH (pHo) 7.00; control, 5% CO2, pHo 7.45] resulted in an acidification of similar magnitude in dendrites and soma (~0.26 pH unit), but acidification was faster in the more distal regions of the dendrites. Neither the dendrites nor the soma exhibited pHi recovery during hypercapnia-induced acidification; but both regions contained pH-regulating transporters, because they exhibited pHi recovery from an NH4Cl prepulse-induced acidification (at constant pHo 7.45). Exposure of a portion of the dendrites to hypercapnic solution did not increase the firing rate, but exposing the soma to hypercapnic solution resulted in a near-maximal increase in firing rate. These data show that while the pHi response to hypercapnia is similar in the dendrites and soma, somatic exposure to hypercapnia plays a major role in the activation of chemosensitive LC neurons from neonatal rats.

acid; brain stem; intracellular pH; pyranine; respiratory control; whole cell


INCREASED CO2 (HYPERCAPNIA) stimulates ventilation. This ventilatory response to hypercapnia is largely mediated by CO2-sensitive neurons in the brain stem, referred to as central chemosensitive neurons (for recent reviews, see Refs. 26 and 34). Chemosensitive neurons are located in a variety of sites in the brain stem, and their response to hypercapnia is thought to be mediated in part by a decrease in intracellular pH (pHi) (12, 26, 41, 42). Cell acidification can alter excitability by inhibiting a variety of ion channels (13). However, all of the studies of the role of pHi changes in chemosensitive neurons have measured pHi of the soma only (12, 20, 24, 26, 32, 33, 39, 42). It may be that the chemosensitive signal arises not in the soma but in the dendrites (14, 22, 34). If this is the case, then the neuron would be responding to a signal from a specified area, not to one from a larger region of the neuron. Furthermore, dendrites from chemosensitive neurons in a variety of brain stem regions project to the surface or associate with blood vessels (1, 6, 15, 22), suggesting that they can respond more easily to systemic changes in CO2/H+. This possibility is difficult to assess because changes in pHi in the dendrites of chemosensitive neurons in response to hypercapnia have not been measured. The responses of chemosensitive neuron firing rate to local acidification within the vicinity of the soma or of the dendrites also have not been measured.

We used a technique involving whole cell pipettes to load neuronal soma and dendrites reliably with a pH-sensitive dye and thereby measure pHi and membrane potential (Vm) simultaneously (35, 43). We used neonatal rat brain stem slices and studied neurons from the locus coeruleus (LC) because >80% of the neurons from this region respond to hypercapnia with an increased firing rate (12, 21, 23). Using this technique, we found that the pHi responses of LC dendrites to hypercapnia are similar to the pHi responses of the soma, consistent with chemosensitive signaling arising in the soma, the dendrites, or both. We further studied the firing rate response of LC neurons to local exposure of hypercapnia. We found that exposing only a dendrite to hypercapnic acidotic solution did not increase LC neuron firing rate but that somatic exposure elicited a near-maximal firing rate response.

Preliminary reports of this work have been published previously (27, 28).


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Solutions and materials. Control solution contained (in mM) 124 NaCl, 3 KCl, 1.24 NaH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, and 10 glucose and was equilibrated with 5% CO2-95% O2 (control, pH ~7.45 at 37°C). Hypercapnic acidosis solution was equilibrated with 10% CO2-90% O2 (pH ~7.15 at 37°C), 15% CO2-85% O2 (pH ~7.00 at 37°C) or with 20% CO2-80% O2 (pH ~6.85 at 37°C). During NH4Cl prepulse experiments (5), 30 mM NaCl in control solution was isosmotically replaced with 30 mM NH4Cl. The pHi calibration solution contained (in mM) 129 KCl, 1.24 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 17 K+-HEPES, 10 glucose, and 0.004 nigericin and was titrated with either KOH or HCl to pH values ranging from 6.2 to 8.6. Nigericin was purchased from Sigma and was added from a 16.7 mM stock solution in DMSO. Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) was purchased from Molecular Probes (Eugene, OR) and was added from a 4 mM stock solution in water.

Preparation of pontine brain slices. Transverse pontine slices (200–300 µm) were prepared from preweanling Sprague-Dawley rats (postnatal days P0P12) beginning at the level of cranial nerve VII and extending rostrally for ~1–1.5 mm. All animal procedures were approved by the Wright State University Institutional Animal Care and Use Committee. Wright State University is accredited by American Association for Accreditation of Laboratory Animal Care and is covered by National Institutes of Health assurance no. A3632-01. Slices were cut into ice-cold control solution using a vibratome (Pelco 101 series 1000) and stored at room temperature. Experiments began at least 1 h and no longer than 6 h after slicing. Individual slices were then placed into a superfusion chamber (1.0-ml volume), which was mounted on the stage of an upright Nikon Eclipse E600 microscope, immobilized with a grid of nylon fibers, and superfused at ~4 ml/min with control solution (37°C).

Visualization of brain slices. Individual LC neurons were visualized using infrared videomicroscopy. A x60 water-immersion lens objective (working distance, 3 mm; numerical aperture, 0.8) was used during these experiments and contained Hoffman Modulated Contrast optics. Light was directed to a Nikon multi-image port module equipped with a 505-nm dichroic mirror, which allowed 100% of the infrared image and 100% of the fluorescence image (see below) to each port simultaneously. The infrared image was then directed to a Sony charge-coupled device (CCD)-IRIS video camera and was displayed on a Sony video monitor.

Imaging of pyranine-loaded slices. The pH-sensitive dye pyranine was loaded into individual LC neurons with either whole cell pipettes or sharp electrodes (see below). Pyranine-loaded LC neurons were excited (with light from a xenon arc lamp) alternately at 450 ± 10 nm (pH sensitive) and 415 ± 10 nm (pH insensitive) using a Sutter Lambda 10-2 filter wheel. Image acquisition was achieved within ~2 s and was repeated at 10- to 60-s intervals. There was no excitation light between acquisitions. Emitted fluorescence at 515 ± 10 nm (all filters were obtained from Omega Optical) was directed to the Nikon multi-image port module and was then directed to a GenIISys image intensifier and a CCD 100 camera (both obtained from Dage-MTI). The subsequent fluorescence images were acquired using a Gateway 2000 E-3100 computer and were collected and processed using Metafluor 4.6r5 software (Universal Imaging, Downingtown, PA), and the 450/415 fluorescence ratios (Rfl) were determined. To convert Rfl to pHi, we calibrated pyranine using the high-K+/nigericin technique (37) to obtain Rfl values at known pH values ranging from 6.2 to 8.6. These Rfl values were then normalized to the Rfl value at pH 7.4, yielding normalized Rfl (Nfl). A calibration of Nfl vs. pHi was created, which produced a calibration curve according to the following equation:

This allowed us to perform a one-point calibration at pH 7.4 at the end of each experiment. The Rfl values measured during the experiment were divided by the calibration Rfl value at pH 7.4, yielding Nfl, and Nfl was converted to pHi using the above equation.

Sharp electrode recordings. Sharp electrodes (125–150 M{Omega}) were fabricated from borosilicate glass (1B100F-4; World Precision Instruments, Sarasota, FL) using a Flaming/Brown P-87 electrode puller and contained 3 M K+-acetate and 10 mM pyranine. The electrode holder contained a Ag-AgCl wire, and the circuit was completed with a Ag-AgCl wire placed into the superfusion bath downstream from the brain slice. Impalement was obtained by placing the electrode tip onto the cell membrane and inducing a ~4-ms pulse using the remote buzz button from a Dagan BVC-700 amplifier. Once an LC neuron had been impaled, pyranine was iontophoresed into the neuron with hyperpolarizing current (–0.1 nA, 500 ms, 1 Hz) for ~5 min. Impalements were administered only on the outer edge of the soma (as opposed to at the center) to ensure that the fluorescence from pyranine in the electrode would not interfere with the fluorescence of pyranine in the soma. Stable Rfl was achieved within ~10 min. Vm was measured using the Dagan BVC-700 amplifier.

Whole cell recordings. The whole cell recording technique, which was used to measure Vm and pHi simultaneously, was modified from the technique described by Schwiening and Willoughby (35, 43). Whole cell pipettes (5 M{Omega}) were fabricated from borosilicate glass (TW150F-4; World Precision Instruments) using a Narishige PP-830 dual-stage pipette puller and contained (in mM) 130 K+-gluconate, 1 MgCl2, 10 HEPES, 0.4 EGTA, 2 Na2ATP, 0.3 Na2GTP, and 0.2–0.4 pyranine [external pH (pHo) ~7.3; 37°C] (13). The pipette holder contained a Ag-AgCl wire, and the circuit was completed with a Ag-AgCl wire placed into the superfusion bath downstream from the brain slice. Positive pressure was applied to the whole cell recording pipette as soon as it was put into the bath solution. To achieve a tight seal, the pipette was initially manipulated to touch the membrane of the soma. Tight seals were attempted solely on the outer edge of the soma so that the fluorescence from pyranine in the pipette would not interfere with the fluorescence of pyranine in the soma. Once the pipette touched the outer edge of the soma, negative pressure was applied to the pipette to form a tight (G{Omega}) seal with the cell membrane. The tight seal was then ruptured, and a whole cell patch was created. Pyranine diffused from the whole cell pipette into the neuron, and stable Rfl values were achieved within ~15 min (17). Vm was measured using the Dagan BVC-700 amplifier. We also performed experiments in which the neurons were loaded with pyranine and then the whole cell pipette was rapidly but carefully detached from the neuron (detached neuron). We then loaded a second neuron adjacent to the pyranine-loaded detached neuron, but this time, the whole cell pipette remained attached to the neuron (attached neuron). Rfl in the detached neuron was stable, and the neuron remained unchanged morphologically for >2 h, both of which indicated that the neuron remained healthy.

Determination of effect of firing rate on pHi. Constant current was injected into a whole cell patched LC neuron via a Dagan BVC-700 amplifier for 5–10 min to depolarize the neuron and increase its firing rate. We simultaneously measured pHi (as described above) during increased firing rate. The amount of current was varied to increase the firing rate by 25%, 100%, 200%, 300%, or 400% and was performed in a stepwise manner. The neuron was allowed to return to resting firing rate and pHi between each step. The change in pHi during each step was determined in the soma and in various regions along the dendrite (0–25 µm, 25–50 µm, and 50–100 µm from the edge of the soma).

Microinjection of hypercapnic solution over the soma or dendrites. The same pipettes used for whole cell recordings were used as microinjection pipettes, filled with control solution + 200 µM pyranine (equilibrated with 100% CO2), and placed into the holder of a Picospritzer II D microinjection system (General Valve). Hypercapnic solution was injected for 2 min over discrete regions of a whole cell-patched LC neuron while Vm was measured. The injected hypercapnic solution was visualized from the pyranine fluorescence within the solution and encompassed a region that was ~100 µm in diameter. Upon cessation of the injection, it took ~2 min for the hypercapnic solution to be superfused away completely. Pipettes were initially positioned over the distal region of a dendrite, which allowed just the dendrite to be exposed to the injected hypercapnic solution. Pipettes were then positioned over the proximal region of the same dendrite, which allowed both the dendrite and the soma to be exposed to the injected hypercapnic solution. Finally, pipettes were positioned next to the soma, which allowed the soma (and, to a lesser extent, the proximal regions of dendrites) to be exposed to the injected hypercapnic solution. Between each of the above-described injections, the neurons were allowed to return to resting Vm conditions. Control experiments were performed identically to those described above, except that the microinjection pipette was filled with control solution (equilibrated with 5% CO2, pHo 7.45).

Data analysis. Vm was saved with both a digital videocassette recorder (model 400; Vetter) and Axoscope software (version 8.0) for later analysis. Firing rates were determined (10-s bins) using a window discriminator/integrator (Winston Electronics). Curve fitting for the pHi calibration curve was performed using the NFIT software program (Island Products, Galveston, TX). The rate of acidification upon exposure to hypercapnia was measured from the point just before the beginning of acidification to the point at which the initial fast drop in pHi was complete (~2 min, which corresponds to ~12 points). The rate of pHi recovery in response to hypercapnia-induced acidification in the presence of maintained hypercapnia was measured from the point at which the initial fast drop in pHi was complete until the point at which hypercapnia was removed. This period was at least 5 min in duration and included from 5–15 points. The rate of pHi recovery from an NH4Cl prepulse-induced acidification was measured beginning at the point of minimum pHi and followed for ~3 min, corresponding to ~18 points. All of these rates were determined as the slope of the best fit and least squares linear regression line fit to the appropriate points as described above. Significant differences between two means were determined by Student’s t-tests. Comparisons of more than two means were conducted using one-way ANOVA. Multiple paired comparisons were performed using the Student-Newman-Keuls test. For the statistical comparison of a mean difference with 0, a 95% confidence interval was constructed. For all tests, P < 0.05 was the criterion for statistical significance. All values shown are means ± SE.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Whole cell vs. sharp electrode recordings. We wanted to use whole cell pipettes to load the dendrites as well as the soma of LC neurons with pyranine as had been performed previously with nonchemosensitive neurons (35, 43). However, we were concerned that there might be some clamping of pHi or washout of the electrical response to hypercapnia with the use of these pipettes (8, 15, 17, 31). Although we used a filling solution that had low EGTA concentration and no added Ca2+, which has been shown previously to reduce whole cell washout (11), we wanted to compare simultaneous measurements of pHi and Vm using whole cell pipettes with measurements obtained using sharp electrodes, which have limited diffusional exchange with the cytoplasm.

The Vm response to hypercapnia was similar when using whole cell pipettes vs. sharp electrodes (Figs. 1, A and B). The initial Vm was –45 ± 1 mV (n = 15) and –45 ± 2 mV (n = 5), depolarized by 4 and 3 mV, respectively, in response to hypercapnia (10% CO2), and returned to normal Vm of –44 ± 1 and –45 ± 2 mV, respectively, upon return to normocapnia (5% CO2) with whole cell vs. sharp electrodes. Hypercapnia also induced a similar reversible increase in firing rate from 0.8 ± 0.03 to 1.7 ± 0.03 (115 ± 19% increase) back to 1.0 ± 0.03 Hz measured with whole cell pipettes (Figs. 1, A and D) and 1.2 ± 0.2 to 2.3 ± 0.3 (107 ± 29% increase) back to 1.0 ± 0.3 Hz measured with sharp electrodes.



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Fig. 1. Simultaneous measurements of intracellular pH (pHi) and firing rate in response to hypercapnia [10% CO2; external pH (pHo) 7.15] of locus coeruleus (LC) neurons measured with either whole cell pipettes or sharp electrodes. A: pHi (top) and integrated firing rate (middle) as a function of time in response to hypercapnia as measured with a whole cell pipette. Note the reversible and maintained acidification and increased firing rate in response to hypercapnia. Brief segments (13) of individual action potentials are shown in the bottom trace. The segments were taken from the numbered regions as indicated in the middle trace. Time and voltage calibrations for these traces are indicated at right. B: pHi (top) and integrated firing rate (middle) as a function of time in response to hypercapnia as measured with a sharp electrode. Brief segments (13) of individual action potentials are shown in the bottom trace. The segments were taken from the numbered regions as indicated in the middle trace. Time and voltage calibrations for these traces are indicated at right. C: means ± SE of the hypercapnia-induced change of pHi measured with whole cell pipettes (closed bars) or sharp electrodes (open bars). Hypercapnia-induced acidification (~0.12 pH unit) was the same when measured with either technique. D: means ± 1 SE of the hypercapnia-induced %increase in firing rate measured with whole cell pipettes (closed bars) or sharp electrodes (open bars). Hypercapnia-induced increase firing rate (~100%) was the same when measured with either technique.

 
The pHi response to hypercapnia was also similar when using whole cell pipettes vs. sharp electrodes (Fig. 1, A and B). When measured in neurons patched with whole cell pipettes, hypercapnia (10% CO2) caused a maintained 0.13 ± 0.01 pH unit intracellular acidification from an initial pHi of 7.34 ± 0.01 pH, which returned to 7.32 ± 0.03 pH upon return to normocapnia (5% CO2) (Fig. 1, A and C). When measured in neurons impaled with sharp electrodes, hypercapnia induced a similar maintained intracellular acidification of 0.11 ± 0.01 pH from an initial pHi of 7.32 ± 0.02 pH, which returned to 7.30 ± 0.04 pH upon return to normocapnia (Figs. 1, B and C). Thus we observed no significant differences in hypercapnia-induced changes in pHi (Fig. 1C) or firing rate (Fig. 1D) between neurons studied with whole cell pipettes vs. sharp electrodes. This indicates that whole cell pipettes can reliably be used to study the responses of LC neurons to hypercapnia, and we used whole cell pipettes in the remainder of the experiments performed in the present study.

Attached vs. detached whole cell recordings. We were surprised to find that the soma remained loaded with pyranine even when the whole cell pipette became detached. We studied this phenomenon directly by loading a neuron with pyranine and then deliberately detaching the patch electrode (detached neuron). A second neuron adjacent to the pyranine-loaded detached neuron was then patched and loaded, with the whole cell pipette remaining attached to this neuron (attached neuron) (Fig. 2A). Rfl in the detached neuron was stable for >2 h, and morphologically, the neuron remained unchanged, both of which indicate that it successfully resealed. We then compared the changes of pHi in response to hypercapnic acidosis in both the detached and attached neurons. Brain slices were superfused with control solution until an initial stable pHi baseline was established for both detached and attached neurons (7.31 ± 0.02 and 7.35 ± 0.02, respectively; n = 5). Upon superfusion with hypercapnic acidotic solution equilibrated with 15% CO2-85% O2 (we used 15% CO2 instead of 10% CO2 to produce a larger pHi response so that we could better compare changes between the two somata), there was an intracellular acidification of 0.25 pH in both detached and attached neurons that was maintained for the duration of hypercapnic exposure (Fig. 2, B and C). Upon return to control solution equilibrated with 5% CO2-95% O2, pHi values in detached and attached neurons returned to their initial levels of 7.30 ± 0.02 and 7.33 ± 0.02, respectively. The rate of acidification upon exposure to hypercapnic acidosis was also similar in the detached and attached neurons: –0.061 ± 0.010 pH unit/min and –0.058 ± 0.007 pH unit/min, respectively (Fig. 2D). Thus it appears that the whole cell pipette can be detached from an LC neuron after loading with pyranine, and the neuron will still produce normal pHi responses to hypercapnic acidosis.



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Fig. 2. Comparison of the pHi response to hypercapnia in detached vs. attached LC neurons. A: pseudocolor image of the somata of two pyranine-loaded LC neurons. One was previously loaded with a whole cell pipette that was then detached from the cell body (green), while the other was loaded and has the whole cell pipette attached (blue). B: pHi response of the two neurons to hypercapnia (15% CO2, pHo 7.00). C: magnitude of pHi change in the soma induced by hypercapnia for attached (blue) and detached (green) LC neurons. D: rate of the acidification induced by hypercapnia for attached (blue) and detached (green) LC neurons. Note that the change of pHi and the rate of acidification are the same in both attached and detached somata. The height of a bar represents the mean, and the error bar represents SE.

 
Effects of increasing firing rate on pHi. Because increased firing rate has previously been shown to acidify neurons (3, 35, 43), we wanted to determine to what extent the increase in firing rate observed with hypercapnic acidosis contributed to the decrease in pHi. Once stable Vm and pHi baseline recordings were established, various levels of constant positive direct current were injected into the neuron via the amplifier to increase the neuronal firing rate (Fig. 3, A and B). The initial current injection caused an increase in firing rate similar to that of hypercapnic acidosis (10% CO2). Subsequent current injections caused 100%, 200%, 300%, and 400% increases in firing rate (Fig. 3A, top). Between each current injection, the firing rate was allowed to return to baseline levels. The change in pHi was also recorded during current injections in the soma and at various distances down the dendrites (Fig. 3A, bottom). The firing rate-induced changes in pHi did not differ between the soma and the dendrites and amounted to at most ~0.06 pH unit for a 400% increase in firing rate (Fig. 3B).



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Fig. 3. Determination of the effect of firing rate on pHi. A: constant depolarizing current pulses (5- to 10-min duration) were injected into a whole cell patched LC neuron to increase firing rate (top) while pHi was measured (bottom). The current injected was varied to increase firing rate by 25% (similar to increase observed with 10% CO2), 100%, 200%, 300%, or 400% and was performed in a stepwise manner. The neuron was allowed to return to resting firing rate and pHi between each step. The change of pHi during each step was determined in the soma and at various regions along the dendrite (0–25 µm, 25–50 µm, and 50–100 µm from the edge of the soma). B: means ± SE of the change of pHi as a function of the %change in firing rate. Note the linear relationship between the two, the similar pHi changes in the soma and dendrites, and the small magnitude of the change of pHi for even large increases in firing rate.

 
Comparing hypercapnia-induced pHi changes in somata vs. dendrites. We next wanted to measure directly the response of pHi to hypercapnic acidosis in the dendrites vs. the soma of LC neurons. We loaded neurons using whole cell pipettes and increased the pyranine concentration from 200 to 400 µM. This was done to get good loading of the dendrites and somata. By increasing excitation light and imaging system gain, dendrites could be visualized a considerable distance from the soma; but in these cases, somatic fluorescence was saturated. An example of such a pyranine-loaded neuron is shown in Fig. 4. This neuron is similar in morphology to multipolar LC neurons previously described (9), with a pyramidal soma ~20–40 µm across and four or five major dendrites that taper to ~2 µm in diameter. Often at least one of the dendrites extended a considerable distance within the same focal plane as the soma (Fig. 4).



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Fig. 4. Monochromatic image of an LC neuron loaded with pyranine and scanned at high gain. Note the three dendrites indicated by asterisks, one of which remains in focus for ~100 µm. Note also that a fourth dendrite has been pruned by slice preparation as is evident from the fluorescent bleb at its end (arrowhead).

 
To avoid saturation of somatic fluorescence while maintaining the ability to visualize dendritic fluorescence, we used neurons loaded with high levels of pyranine but without increasing system gain. Measurements of pHi were performed in the soma and only in dendrites within the same plane of focus as the soma. Measurements were obtained from three different regions: 0–25 µm, 25–50 µm, and 50–100 µm from the edge of the soma, where the soma clearly narrows to form the most proximal region of a dendrite (Fig. 5A). An example of the fluorescence of pyranine from different regions of the dendrite as well as the soma is shown in Fig. 5A. The magnitude of the pHi change before, during, and after hypercapnia in both the soma and the different regions of the dendrite, as well as the integrated firing rate during those same times, is shown in Fig. 5, B and C. This neuron was chemosensitive as shown by its reversible increase in firing rate in response to hypercapnic acidosis (15% CO2) (Fig. 5C). The magnitude and the rate of acidification appeared to be somewhat larger the farther down the dendrite the measurements were obtained (Fig. 5B).



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Fig. 5. Simultaneous measurement of the firing rate response and the pHi response to hypercapnia (15% CO2, pHo 7.00) in the soma and a dendrite of an LC neuron. A: pseudocolor image of an LC neuron with the dye-filled pipette attached. The four colored circles show the areas of interest that were studied. B: pHi response to hypercapnia in the soma and in three areas of a dendrite (0–25 µm, 25–50 µm, and 50–100 µm from edge of soma). C: integrated firing rate response to hypercapnia measured with the whole cell pipette.

 
We were able to obtain seven neurons with dendrites that loaded and remained in the same plane of focus as the soma at least 100 µm from the edge of the soma. To detect pHi changes in the dendrites farther from the soma, we loaded neurons as described above and then detached the whole cell pipette from the soma. This allowed us to follow a dye-loaded dendrite as far as possible by moving the stage. To further enhance our ability to visualize the most distal regions of the dendrites, we increased the gain of the intensifier. This allowed us to visualize dendrites as far as 160 µm from the edge of the soma. An additional five neurons were studied using this approach.

The summary of changes of pHi in the various regions in response to hypercapnia of these 12 neurons is shown in Fig. 6, A and B. In Fig. 6A, there was a trend for hypercapnia to induce a slightly greater acidification the more distal the region on the dendrite, but given the variation from neuron to neuron, this difference was not significantly different (P > 0.51; ANOVA). The overall mean hypercapnia-induced acidification was 0.26 ± 0.02 pH unit (n = 48). Similarly, the rate of acidification appeared to be slightly faster in the dendrites than in the soma (Fig. 6B), but these rates were not significantly different (P > 0.11; ANOVA), with an overall average rate of acidification of –0.070 ± 0.008 pH unit/min (n = 48). To eliminate the effect of interneuron variability, we were able to perform paired analysis of the somatic vs. dendritic effects for the seven neurons in which we had obtained simultaneous measurements of the pHi changes in both the soma and the dendrites. In this paired analysis, we observed a slightly larger acidification the farther down the dendrite we measured, which was significant just at 50–100 µm (Fig. 6C). Furthermore, the rate of acidification induced by hypercapnia was faster the farther down the dendrite we measured for all seven neurons, and the paired analysis showed that the rate was significantly faster at all distances down the dendrite compared with the soma. Finally, acidification in the dendrites was significantly faster at 50–100 µm from the edge of the soma than at regions of the dendrite closer to the soma (Fig. 6D).



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Fig. 6. Comparisons of the magnitude and the rate of hypercapnia-induced acidification in the soma and dendrites of LC neurons. A: comparison of the magnitude of hypercapnia-induced acidification of the soma and four dendritic regions. Means ± SE for the magnitude of hypercapnia-induced acidification in various regions of LC neurons that are shown do not differ. B: means ± SE for the rate of acidification induced by hypercapnia in various regions of LC neurons. The rates for the various regions are not significantly different. C: paired difference (means ± SE) between the magnitude of acidification for a particular region of the dendrite and the acidification of the soma for seven neurons (similar to the one shown in Fig. 5A) in which we obtained values for both the somata and the dendrites. Only for the most distant dendritic region is the magnitude of acidification significantly larger than the acidification of the soma. The values for the three dendritic regions do not differ significantly from one another. D: paired difference (means ± SE) between the rate of acidification for a particular region of the dendrite and the rate of acidification of the soma for seven neurons (similar to the one shown in Fig. 5A) for which we obtained values for both the somata and the dendrites. All three dendritic regions acidify significantly more rapidly than the soma, and the most distant dendritic region acidifies significantly more rapidly than the other dendritic regions. *P < 0.05 statistically significantly different from 0. {dagger}P < 0.05 statistically significant difference for a dendritic region from the values for the other two regions.

 
We also found that neither the soma nor any of the regions of the dendrite exhibited pHi recovery during exposure to hypercapnia (e.g., Figs. 1A, 2, and 5B). There was actually a slight additional acidification during the hypercapnic exposure that did not differ among the soma and the various dendritic regions (P > 0.84; ANOVA) and amounted to –0.007 ± 0.003 pH unit/min (n = 48).

As expected, the firing rate response to hypercapnia with 15% CO2 was somewhat larger than the response to hypercapnia with 10% CO2. In control solution (5% CO2), the firing rate was 0.90 ± 0.21 Hz (n = 7), which increased by 139 ± 33% (to 1.89 ± 0.32 Hz) in response to 15% CO2 and returned to control values (0.82 ± 0.19 Hz) when again exposed to the control solution.

Response to NH4Cl prepulse in dendrites vs. somata. Because there was no pHi recovery during hypercapnic acidosis in any region of LC neurons, we wanted to verify whether the dendrites have the ability to regulate pHi during an acid load. We previously showed that the soma exhibits pH recovery during an acid load if pHo is maintained constant, which occurs during an NH4Cl prepulse (33). We therefore exposed the slices to an NH4Cl prepulse and measured pHi recovery from acidification to pHi ~6.9. The soma as well as all regions of the dendrite exhibited pHi recovery (Fig. 7A). Furthermore, the rates of recovery in the soma and the three regions of the dendrite were all similar (Fig. 7B).



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Fig. 7. A: pHi response to an NH4Cl prepulse (30 mM) of the soma and of three dendritic regions (0–25 µm, 25–50 µm, and 50–100 µm from the soma) of LC neurons. B: means ± SE of the rate of pHi recovery from an NH4Cl prepulse-induced acidification in the soma and in the three dendritic regions. None of the recovery rates differed significantly among the various regions.

 
We used the change in pHi upon removal of NH4Cl to estimate the buffering power (4) in the dendrites and the soma (e.g., Fig. 7A). The buffering power determined in this way includes the intrinsic buffering power and the open buffering power due to intracellular HCO3 and is an overestimate because of the presence of pHi recovery (4). Nevertheless, our values can serve as an estimate for a comparison of buffering power in the dendrites vs. the somata. The overall buffering power (intrinsic plus HCO3 buffering) did not differ between the soma and the dendrites at 0–25 µm, 25–50 µm, and 50–100 µm from the soma (P > 0.39; ANOVA) and averaged 74.9 ± 3.5 mM/pH unit (n = 28). Estimating buffering power due to intracellular HCO3 yielded a value of ~30 mM/pH unit, which would mean that the intrinsic buffering power in the somata and dendrites of LC neurons is ~45 mM/pH unit, similar to values of intrinsic buffering power reported for medullary neurons (7, 32).

Firing rate response to acidification of dendrites or somata. Both the somata and the dendrites exhibit a similar pHi response to hypercapnia, but the basic question that remains is whether exposing dendrites, somata, or both is required for the activation of chemosensitive LC neurons. To address this question, we measured the firing rate of LC neurons while microinjecting hypercapnic solution over a dendrite or the soma. The response of a representative LC neuron is shown in Fig. 8. An LC neuron was patched and loaded with pyranine so that the soma and dendrites (2 are visible) could be observed (Fig. 8A). A separate microinjection pipette loaded with hypercapnic acidotic solution containing pyranine was placed such that solution could be microinjected on a dendrite only (Fig. 8B, arrowheads indicate superfused region). Upon cessation of microinjection, the hypercapnic solution washed off the dendrite (Fig. 8C). When the pipette was moved closer to the soma and the microinjection was repeated, solution covered the dendrite and the soma (Fig. 8D). Once again, the hypercapnic solution washed away upon cessation of microinjection (Fig. 8E). The microinjection pipette was then moved adjacent to the soma, and hypercapnic acidotic solution was microinjected on the soma and the proximal dendrites as well (Fig. 8F). Finally, cessation of microinjection resulted once again in the hypercapnic solution being washed away (Fig. 8G). This neuron had a spontaneous integrated firing rate of ~0.5 Hz that was largely unaffected by exposure of a dendrite to hypercapnia (Fig. 8, region B, bottom). However, when hypercapnic solution covered the soma as well (Fig. 8, D and F), the firing rate was markedly increased (Fig. 8, regions D and F, bottom). This hypercapnia-induced increased firing rate was reversible with the firing rate returning to initial values upon removal of the hypercapnic solution (Fig. 8, regions E and G, bottom).



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Fig. 8. Microinjection of hypercapnic acidotic solution on a dendrite or on the soma of an LC neuron and its effect on firing rate. A: LC neuron patched with a whole cell pipette and loaded with pyranine. The soma and two dendrites are evident. B: hypercapnic solution was microinjected for 2 min over one of the dendrites using a pipette filled with hypercapnic solution containing pyranine that was positioned over the distal region of a dendrite. The area covered by the hypercapnic solution was visualized by the extracellular pyranine fluorescence and encompassed a region ~100 µm in diameter (white arrowheads). C: microinjection was stopped, and the extracellular hypercapnic solution rapidly washed away. D: microinjection pipette was moved to a more proximal dendritic position, and the microinjection was repeated. In this case, the area encompassed by the hypercapnic solution included a proximal dendrite and the soma (white arrowheads). E: again the microinjection was stopped, and the external solution was allowed to return to normal. F: microinjection pipette was moved adjacent to the soma, and the microinjection was repeated. The hypercapnic solution covered the soma and the proximal dendrites. G: microinjection was stopped, and the hypercapnic solution was allowed to wash away. Bottom: integrated firing rate of the LC neuron depicted in AG. The firing rate corresponding to each image is indicated by the letter of that image above the firing rate trace. Note that the hypercapnic solution did not increase the firing rate when microinjected over a distal dendrite (point B) but increased the firing rate reversibly when microinjected over the proximal dendrites and soma (points D and F).

 
A summary of the response of five neurons exposed to hypercapnia on either the dendrites or the somata is shown in Fig. 9A. Compared with the control integrated firing rate of neurons in normocapnic solution, neurons exposed to hypercapnia only on a dendrite showed no significant increase in firing rate. However, neurons exposed on both a dendrite and the soma, or on the soma alone, showed a significant increase in firing rate of ~130% (Fig. 9A). This increase was compared with the increase in the firing rate of LC neurons induced by solutions equilibrated with CO2 of different levels superfused over the whole slice (Fig. 9C). The firing rate increased in LC neurons exposed to 10% CO2 and increased more in neurons exposed to 15% CO2. At higher levels of hypercapnia (20% CO2), LC neurons exhibited no further increased firing rate (Fig. 9C) and plateaued at an increased firing rate of ~150%. For comparison, our results for increased firing rate after microinjection of hypercapnic solution over the soma are shown in Fig. 9C. It can be observed that exposure of the soma to hypercapnic solution elicited a near-maximal firing rate response for LC neurons to hypercapnia.



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Fig. 9. Summary of the firing rate responses to focal microinjection of hypercapnic solutions in LC neurons. A: means ± SE (n = 5) of the firing rate when LC neurons were exposed to control solution and microinjection of hypercapnic solution over the distal dendrites alone, the proximal dendrites and soma, or the soma. The firing rate increased significantly (*) only when the area covered by the hypercapnic solution included the soma. B: means ± SE (n = 4) of the firing rate when LC neurons were exposed to microinjection of control solution (equilibrated with 5% CO2) over the distal dendrites, the proximal dendrites and soma, or the soma. There was no increase in firing rate when normocapnic solution was microinjected over any area of LC neurons. C: relationship of the %increase in the firing rate of LC neurons when the whole slice was superfused ({circ}) with solution equilibrated with different levels of hypercapnia (from 10% to 20% CO2). The %increase in firing rate increased with the degree of hypercapnia up to 15% CO2 and then plateaued. Each point represents %increase in firing rate (means ± SE; n = 9). Mean ± SE ({blacksquare}; n = 5) %increase in firing rate with microinjection of hypercapnic solution over the soma of LC neurons. Note that this microinjection induced a near-maximal increase in firing rate.

 
LC neuron firing rate responses to hypercapnia have been shown to be an intrinsic response (21), but we do not know whether the entire response reported herein reflects intrinsic responses or whether it is mediated in part by synaptic events.

We were concerned that our increase in firing rate was due to mechanical disruption of the whole cell patch upon microinjection of solution and not due to the hypercapnic solution. We thus repeated the experiments shown in Fig. 8 but this time used microinjection pipettes filled with control solution equilibrated with 5% CO2. Microinjection of this control solution, whether on a dendrite or on the soma, did not result in any increase in the spontaneous firing rate (Fig. 9B). Thus the increased firing rate we observed with the microinjection of hypercapnic solution appears to be due to hypercapnia and not to any mechanical disturbance.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we have compared the somatic vs. the dendritic responses to hypercapnia in LC neurons from neonatal rats. The main findings are that, surprisingly, exposure of the soma but not a dendrite of chemosensitive LC neurons to hypercapnia resulted in an increased firing rate, while the response of pHi to hypercapnia in the dendrites and somata of LC neurons is the same. This study is the first to address the CO2 sensitivity of the dendrites vs. the somata in central chemosensitive neurons, and our findings have implications for models of cellular chemosensitive signaling.

Our model system. We studied chemosensitive neurons from the LC region of brain stem slices of neonatal rats. The LC is a dorsal pontine region that contains largely noradrenergic neurons, and its role in ventilatory control is uncertain. The LC contains the highest percentage (>80%) of CO2-activated neurons of any brain stem region (12, 21, 26). These neurons have been shown to be intrinsically sensitive to hypercapnia (21); focal acidification of the LC results in increased ventilation (18); and ablation of catecholaminergic neurons (including 80% of the LC neurons) blunts the response of ventilation to hypercapnia (19). On the other hand, LC neurons have the smallest firing rate response for a given acidic stimulus of any chemosensitive neuron (26), and the creation of lesions in LC neurons with kainic acid resulted in at most a modest effect on hypercapnia-induced ventilation (11). It has been proposed that the LC may also play a role in hypercapnia-induced anxiety (26).

Because the ventilatory response is known to change with development (25, 36), our findings with neonatal rats might not reflect the properties of adult LC neurons. We have previously shown that both the percentage of CO2-activated neurons and the magnitude of their response is the same in slices from neonatal rats aged P1P21 and that these values are similar to adult values (25, 36). Thus we think that the values measured in the present study are not affected by development and may well reflect the responses of adult LC neurons as well.

In the few previous studies in which neuronal pHi responses were compared between the somata and dendrites, researchers used isolated neurons and neurons stimulated electrically (35, 39, 43). Because we used slices superfused with hypercapnic solutions, our measured rates of hypercapnia-induced acidification represent a complex function of the rate of superfusion of the slice, the depth within the slice of the somata and dendrites measured, the surface area-to-volume ratio of the structure being acidified, and the cellular buffering power. We were able to control the depth of the structure being studied within the slice by measuring somata and dendrites at the same focal plane (and thus at the same depth within the slice) (e.g., Fig. 4). Furthermore, the intracellular buffering power was the same in the somata and the dendrites. Nevertheless, superfusion and diffusion are likely to contribute substantially to the rate of acidification of LC neurons within a slice.

The use of slices offers several advantages. Access to individual neurons (both soma and dendrite) can be obtained easily, facilitating the simultaneous measurement of Vm and pHi. With the exception of some dendritic pruning (see, e.g., Fig. 4), LC neurons maintain a near-normal morphology compared with LC neurons in vivo (9). Finally, the normal association of neurons with glia in the slice offered a more nearly physiological condition than that found in reduced preparations and allowed for the study of potential roles of glia in neuronal responses to hypercapnia.

We visualized and studied pHi simultaneously in the somata and dendrites to at least 100 µm from the edge of the soma (Fig. 5). We were able to visualize dye-loaded dendrites at distances >100 µm, but to do so, we had to increase the excitation light and increase system gain (see, e.g., Fig. 4). This situation resulted in saturation of the dye signal from the soma, which precluded accurate measurement of pHi from the soma. Other studies in which a similar technique was used to load either pyranine (43) or Oregon Green BAPTA (a Ca2+-sensitive dye) (38) into the somata and dendrites of neurons demonstrated a similar phenomenon of very high fluorescence in the soma, and some of the observed lack of response of pHi or Cai2+ in the somata vs. the dendrites may have been due to dye saturation. Thus, to measure fluorescence in both the soma and the dendrites, care must be taken not to saturate the fluorescence of the soma. More distal regions could be studied by using higher dye concentrations or greater system gain, by using confocal microscopy, or by using detached neurons whereby the neuron is loaded and the patch pipette is removed (Fig. 2). We used the latter approach to study dendrites as far as ~160 µm from the edge of the soma (Fig. 6).

Morphology of LC neurons. The morphology of our cells was most similar to multipolar neurons previously described in LC neurons (9). The neurons have large, pyramidal somata with an average of four primary and four secondary dendrites. These dendrites have a fairly uniform diameter of between 2 and 2.5 µm over most of their length (9). Primary dendrites in neurons from 30-day-old rats extend ~350 µm from the soma, although dendrites have been shown to increase in length after day P15 in other brain stem neurons (40). We were able to visualize much of the structure of LC neurons within ~160 µm from the edge of the soma (Figs. 4 and 6). Thus we probably measured pHi in at least the first half, and perhaps much more, of the primary dendrites in this study. However, we may not have studied pHi in dendrites to their farthest extension. Further study is necessary to determine whether the pHi response to hypercapnia in the farthest reaches of the dendrites may differ from the responses observed in the present study.

Dendritic vs. somatic pHi responses to hypercapnia. The responses of dendrites and somata can differ under various conditions. The pHi of neurites was 0.2–0.3 more alkaline than that of the somata in PC12 cells (10). Repetitive firing resulted in acidification of the soma but no change in dendritic pHi in expiratory neurons from the ventral respiratory group (3), as well as a faster and larger acidification in the lamellipodia of snail neurons (35) and in the dendrites of cerebellar Purkinje cells (43) than in the somata of these cells. Faster and larger responses in the dendrites vs. the somata also have been observed for depolarization-induced Ca2+ transients in snail (35) and hippocampal neurons (38). Furthermore, the complement of channels on the surface membrane of the dendrite may differ from those on the membrane of the soma. For example, voltage-gated K+ channels reside on the somatic membrane and may be largely absent in dendrites (30) or may decrease along the dendrite away from the soma (16). Also, in chemosensitive olfactory neurons, voltage-gated K+ channels are fairly uniformly distributed across the neuron, while voltage-gated Cl channels are present at a much higher density in the dendrites. Thus substantial differences can exist in both the membrane properties and intracellular responses of dendrites vs. somata.

We have obtained the first measurements of the effects of hypercapnia on dendritic vs. somatic pHi changes in chemosensitive neurons. We found the rate of acidification to be somewhat greater (Figs. 5 and 6) in the dendrites than in the somata, but this effect was far smaller than expected on the basis of the larger dendritic surface area-to-volume ratio. That dendrites showed only a slightly faster rate of acidification suggests that perfusion and diffusion contribute substantially to the rate of acidification of LC neurons within the slice. It may be that in vivo LC neuron dendrites respond even faster than the soma to hypercapnia.

In all other respects, the pHi responses to hypercapnia of the dendrites from LC neurons were remarkably similar to the responses of the soma. Both had a similar magnitude of acidification in response to 15% CO2 (~0.25 pH unit) (Fig. 6A), neither exhibited any pHi recovery from hypercapnia-induced acidification during maintained hypercapnia (Fig. 5B), both had a similar total buffering power (~75 mM/pH unit) and similar intrinsic buffering power (~45 mM/pH unit), and both showed a similar rate of recovery from NH4Cl prepulse-induced acidification (Fig. 7). The latter observation is interesting. Given the similar rates of pHi recovery and total buffering power in somata and dendrites, the larger volume-to-surface area ratio of the soma implies that there must be a larger membrane transport-mediated H+ flux across the somatic membrane vs. the dendritic membrane (29). Acid flux in LC neurons appears to be mediated nearly completely by Na+/H+ exchange (NHE) (unpublished observations). Thus NHE activity must be substantially higher in LC somata vs. LC dendrites. This could be accomplished by either a higher expression of NHE on the somatic membrane or a higher activity, possibly due to a different isoform, of the NHE present on the somatic membrane vs. the dendritic membrane.

Differences in the firing rate response to local hypercapnic exposure of dendrite vs. soma. Exposure of a dendrite to hypercapnia did not stimulate the LC neuron firing rate, while stimulation of the soma produced a near-maximal firing rate response (Figs. 8 and 9). This finding was not due to mechanical effects on the patched soma, because exposure of the soma to normocapnic solution did not result in any change in firing rate (Fig. 9B). These data suggest the surprising conclusion that the soma is most involved in chemosensitive signaling in LC neurons. These findings further suggest that the pH-sensitive ion channels that serve as chemosensitive targets (26) must reside preferentially on the soma and perhaps on the proximal dendrites of LC neurons.

Chemosensitive signaling has previously been proposed to involve the dendrites (2, 14, 15, 22). Chemosensitive neurons of the ventrolateral medulla send dendritic projections to the ventral surface (15, 22), LC neurons have some dendrites that end on the ependyma (1), and medullary raphe neurons have projections that run along the basilar artery (6), all of which would be consistent with the dendrites serving to sense CO2. These previous findings seem to be at odds with our findings of a major role played by the soma in chemosensitive signaling. However, our studies do not rule out a potential role for the dendrites. We have excited only one portion of one dendrite (Fig. 8, AC), and it may require hypercapnic exposure of a larger portion of the dendritic field to increase the LC neuron firing rate. Alternatively, dendritic endings closely associated with the brain stem surface (15, 22) or with large arteries (6) may be highly adapted to respond to hypercapnia, although this response would have to be powerful for a few endings to activate an action potential in the distant soma. Finally, chemosensitive signaling may be different in LC neurons than in neurons from other chemosensitive areas. No direct evidence for differences in signaling exists, but there are differences in the percentage and the magnitude of the response of chemosensitive neurons from various brain stem areas (26).

Given that a maintained decrease in pHi in response to hypercapnia is an important aspect of signaling in central chemosensitive neurons (12, 27), our findings of a maintained intracellular acidification induced by hypercapnia in both the dendrites and the somata are consistent with either a dendritic or a somatic source of chemosensitive signaling, or both. In contrast, our direct measurements of hypercapnia-induced increased firing rate in LC neurons shows that exposure of the soma is most important. These data imply that LC neurons seem to sense CO2 over a broad expanse of tissue, including the soma and perhaps the proximal regions of the dendrites, and not just in a localized region. This hypothesis would be consistent with a sensor that responds to global, rather than local, changes of CO2.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-56683.


    ACKNOWLEDGMENTS
 
We thank Phyllis Douglas for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. W. Putnam, Dept. of Neuroscience, Cell Biology and Physiology, Wright State Univ. School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435 (e-mail: robert.putnam{at}wright.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
 GRANTS
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