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
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ABSTRACT |
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acid; brain stem; intracellular pH; pyranine; respiratory control; whole cell
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).
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MATERIALS AND METHODS |
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Preparation of pontine brain slices.
Transverse pontine slices (200300 µm) were prepared from preweanling Sprague-Dawley rats (postnatal days P0P12) beginning at the level of cranial nerve VII and extending rostrally for 11.5 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:
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Sharp electrode recordings.
Sharp electrodes (125150 M) 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) 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 0.20.4 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
) 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 510 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 (025 µm, 2550 µm, and 50100 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 515 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 Students 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.
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RESULTS |
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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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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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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 50100 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 50100 µm from the edge of the soma than at regions of the dendrite closer to the soma (Fig. 6D).
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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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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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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.
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DISCUSSION |
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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 0.20.3 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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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2. Ballantyne D and Scheid P. Central chemosensitivity of respiration: a brief overview. Respir Physiol 129: 512, 2001.[CrossRef][ISI][Medline]
3. Ballanyi K, Mückenhoff K, Bellingham MC, Okada Y, Scheid P, and Richter DW. Activity-related pH changes in respiratory neurones and glial cells of cats. Neuroreport 6: 3336, 1994.[ISI][Medline]
4. Boron WF. Intracellular pH transients in giant barnacle muscle fibers. Am J Physiol Cell Physiol 233: C61C73, 1977.
5. Boron WF and De Weer P. Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J Gen Physiol 67: 91112, 1976.[Abstract]
6. Bradley SR, Pieribone VA, Wang W, Severson CA, Jacobs RA, and Richerson GB. Chemosensitive serotonergic neurons are closely associated with large medullary arteries. Nat Neurosci 5: 401402, 2002.[CrossRef][ISI][Medline]
7. Chambers-Kersh L, Ritucci NA, Dean JB, and Putnam RW. Response of intracellular pH to acute anoxia in individual neurons from chemosensitive and nonchemosensitive regions of the medulla. Adv Exp Med Biol 475: 453464, 2000.[ISI][Medline]
8. Dean JB and Reddy RB. Effects of intracellular dialysis on CO2/H+ chemosensitivity in brain stem neurons. In: Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure, edited by Trouth CO and Millis RM. New York: Dekker, 1995, p. 453461.
9. Diaz-Cintra S, Cintra L, Kemper T, Resnick O, and Morgane PJ. The effects of protein deprivation on the nucleus locus coeruleus: a morphometric Golgi study in rats of three age groups. Brain Res 304: 243253, 1984.[CrossRef][ISI][Medline]
10. Dickens CJ, Gillespie JI, and Greenwell JR. Interactions between intracellular pH and calcium in single mouse neuroblastoma (N2A) and rat pheochromocytoma cells (PC12). Q J Exp Physiol 74: 671679, 1989.[ISI][Medline]
11. Erlichman S, Budd T, Graham M, Lawless R, Murray S, Zugermayr M, and Leiter JC. Role of the locus coeruleus in ventilatory control in the awake rat (Online). Abstr Soc Neurosci Program no. 609.5, 2003.
12. Filosa JA, Dean JB, and Putnam RW. Role of intracellular and extracellular pH in the chemosensitive response of rat locus coeruleus neurones. J Physiol 541: 493509, 2002.
13. Filosa JA and Putnam RW. Multiple targets of chemosensitive signaling in locus coeruleus neurons: role of K+ and Ca2+ channels. Am J Physiol Cell Physiol 284: C145C155, 2003.
14. Huang RQ, Erlichman JS, and Dean JB. Cell-cell coupling between CO2-excited neurons in the dorsal medulla oblongata. Neuroscience 80: 4157, 1997.[CrossRef][ISI][Medline]
15. Kawai A, Ballantyne D, Mückenhoff K, and Scheid P. Chemosensitive medullary neurones in the brainstem-spinal cord preparation of the neonatal rat. J Physiol 492: 277292, 1996.[Abstract]
16. Korngreen A and Sakmann B. Voltage-gated K+ channels in layer 5 neocortical pyramidal neurones from young rats: subtypes and gradients. J Physiol 525: 621639, 2000.
17. Mulkey DK, Henderson RAI, Ritucci NA, Putnam RW, and Dean JB. Oxidative stress decreases pHi and Na+/H+ exchange and increases excitability of solitary complex neurons from rat brain slices. Am J Physiol Cell Physiol 286: C940C951, 2004.
18. Nattie EE. Central chemosensitivity, sleep, and wakefulness. Respir Physiol 129: 257268, 2001.[CrossRef][ISI][Medline]
19. Nattie EE and Li A. Brainstem catecholaminergic neurons participate in central chemoreception in NREM sleep and wakefulness (Online). Abstr Soc Neurosci Program no. 145.9, 2004.
20. Nottingham S, Leiter JC, Wages P, Buhay S, and Erlichman JS. Developmental changes in intracellular pH regulation in medullary neurons of the rat. Am J Physiol Regul Integr Comp Physiol 281: R1940R1951, 2001.
21. Oyamada Y, Ballantyne D, Mückenhoff K, and Scheid P. Respiration-modulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem-spinal cord of the neonatal rat. J Physiol 513: 381398, 1998.
22. Pilowsky P, Llewellyn-Smith IJ, Arnolda L, Lipski J, Minson J, and Chalmers J. Are the ventrally projecting dendrites of respiratory neurons a neuroanatomical basis for the chemosensitivity of the ventral medulla oblongata? Sleep 16, Suppl 8: S53S55, 1993.
23. Pineda J and Aghajanian GK. Carbon dioxide regulates the tonic activity of locus coeruleus neurons by modulating a proton- and polyamine-sensitive inward rectifier potassium current. Neuroscience 77: 723743, 1997.[CrossRef][ISI][Medline]
24. Putnam RW. Intracellular pH regulation of neurons in chemosensitive and nonchemosensitive areas of brain slices. Respir Physiol 129: 3756, 2001.[CrossRef][ISI][Medline]
25. Putnam RW, Conrad SC, Gdovin MJ, Erlichman JS, and Leiter JC. Neonatal maturation of the hypercapnic ventilatory response and central neural CO2 chemosensitivity. Resp Physiol Neurobiol (In Press). First published May 2, 2005; doi:10.1016/j.resp.2005.03.004.
26. Putnam RW, Filosa JA, and Ritucci NA. Cellular mechanisms involved in CO2 and acid signaling in chemosensitive neurons. Am J Physiol Cell Physiol 287: C1493C1526, 2004.
27. Putnam RW, Ritucci NA, and Dean JB. Dendritic vs. somatic changes of intracellular pH (pHi) in response to hypercapnia in chemosensitive neurons from the locus coeruleus (LC) (Online). Abstr Soc Neurosci Program no. 889.17, 2004.
28. Putnam RW, Ritucci NA, and Dean JB. Somatic vs. dendritic responses to hypercapnia in chemosensitive locus coeruleus neurons from neonatal rats. Abstr Soc Neurosci Program No. 352.6, 2005.
29. Putnam RW and Roos A. Intracellular pH. In: Handbook of Physiology: Cell Physiology, edited by Hoffman JF and Jamieson JD. Bethesda, MD: Am. Physiol. Soc., 1997, sect. 14, p. 389440.
30. Riazanski V, Becker A, Chen J, Sochivko D, Lie A, Wiestler OD, Elger CE, and Beck H. Functional and molecular analysis of transient voltage-dependent K+ currents in rat hippocampal granule cells. J Physiol 537: 391406, 2001.
31. Richerson GB. Response to CO2 of neurons in the rostral ventral medulla in vitro. J Neurophysiol 73: 933944, 1995.
32. Ritucci NA, Chambers-Kersh L, Dean JB, and Putnam RW. Intracellular pH regulation in neurons from chemosensitive and nonchemosensitive areas of the medulla. Am J Physiol Regul Integr Comp Physiol 275: R1152R1163, 1998.
33. Ritucci NA, Dean JB, and Putnam RW. Intracellular pH response to hypercapnia in neurons from chemosensitive areas of the medulla. Am J Physiol Regul Integr Comp Physiol 273: R433R441, 1997.
34. Scheid P, Putnam RW, Dean JB, and Ballantyne D. Special issue: Central chemosensitivity (Entire Issue). Respir Physiol 129: 1278, 2001.[CrossRef][ISI]
35. Schwiening CJ and Willoughby D. Depolarization-induced pH microdomains and their relationship to calcium transients in isolated snail neurones. J Physiol 538: 371382, 2002.[Medline]
36. Stunden CE, Filosa JA, Garcia AJ, Dean JB, and Putnam RW. Development of in vivo ventilatory and single chemosensitive neuron responses to hypercapnia in rats. Respir Physiol 127: 135155, 2001.[CrossRef][ISI][Medline]
37. Thomas JA, Buchsbaum RN, Zimniak A, and Racker E. Intracellular pH measurements in Erhlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 22102218, 1979.[CrossRef][ISI][Medline]
38. Tombaugh GC. Intracellular pH buffering shapes activity-dependent Ca2+ dynamics in dendrites of CA1 interneurons. J Neurophysiol 80: 17021712, 1998.
39. Trapp S, Lückermann M, Brooks PA, and Ballanyi K. Acidosis of rat dorsal vagal neurons in situ during spontaneous and evoked activity. J Physiol 496: 695710, 1996.[Abstract]
40. Vincent A and Tell F. Postnatal changes in electrophysiological properties of rat nucleus tractus solitarii neurons. Eur J Neurosci 9: 16121624, 1997.[ISI][Medline]
41. Wang W, Bradley SR, and Richerson GB. Quantification of the response of rat medullary raphe neurones to independent changes in pHo and PCO2. J Physiol 540: 951970, 2002.
42. Wiemann M and Bingmann D. Ventrolateral neurons of medullary organotypic cultures: intracellular pH regulation and bioelectric activity. Respir Physiol 129: 5770, 2001.[CrossRef][ISI][Medline]
43. Willoughby D and Schwiening CJ. Electrically evoked dendritic pH transients in rat cerebellar Purkinje cells. J Physiol 544: 487499, 2002.
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