Department of Molecular and Integrative Physiology, Neuroscience Program, and College of Medicine, University of Illinois, Urbana, Illinois 61801
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ABSTRACT |
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Horn, Eric M., Glenn H. Dillon, Yi-Ping Fan, and Tony G. Waldrop. Developmental aspects and mechanisms of rat caudal hypothalamic neuronal responses to hypoxia. Previous reports from this laboratory have shown that a high percentage of neurons in the caudal hypothalamus are stimulated by hypoxia both in vivo and in vitro. This stimulation is in the form of an increase in firing frequency and significant membrane depolarization. The goal of the present study was to determine if this hypoxia-induced excitation is influenced by development. In addition, we sought to determine the mechanism by which hypoxia stimulates caudal hypothalamic neurons. Caudal hypothalamic neurons from neonatal (4-16 days) or juvenile (20-40 days) rats were patch-clamped, and the whole cell voltage and current responses to moderate (10% O2) or severe (0% O2) hypoxia were recorded in the brain slice preparation. Analysis of tissue oxygen levels demonstrated no significant difference in the levels of tissue oxygen in brain slices between the different age groups. A significantly larger input resistance, time constant and half-time to spike height was observed for neonatal neurons compared with juvenile neurons. Both moderate and severe hypoxia elicited a net inward current in a significantly larger percentage of caudal hypothalamic neurons from rats aged 20-40 days (juvenile) as compared with rats aged 4-16 days (neonatal). In contrast, there was no difference in the magnitude of the inward current response to moderate or severe hypoxia between the two age groups. Those cells that were stimulated by hypoxia demonstrated a significant decrease in input resistance during hypoxic stimulation that was not observed in those cells unaffected by hypoxia. A subset of neurons were tested independent of age for the ability to maintain the inward current response to hypoxia during synaptic blockade (11.4 mM Mg2+/0.2 mM Ca2+). Most of the neurons tested (88.9%) maintained a hypoxic excitation during synaptic blockade, and this inward current response was unaffected by addition of 2 mM cobalt chloride to the bathing medium. In contrast, perfusion with the Na+ channel blocker, tetrodotoxin (1-2 µM) or Na+ replacement with N-methyl-D-glucamine (NMDG) significantly reduced the inward current response to hypoxia. Furthermore, the input resistance decrease observed during hypoxia was attenuated significantly during perfusion with NMDG. These results indicate the excitation elicited by hypoxia in hypothalamic neurons is age dependent. In addition, the inward current response of caudal hypothalamic neurons is not dependent on synaptic input but results from a sodium-dependent conductance.
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INTRODUCTION |
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The caudal portion of the mammalian hypothalamus
has long been known to play a modulatory role in cardiovascular and
respiratory regulation (Kabat 1936; Kabat et al.
1935
; Spencer 1894
). Several decerebration studies have shown
the caudal hypothalamus to be facilitatory to the cardiorespiratory
responses to hypoxia and hypercapnia (Hayashi and Sinclair
1991
; Nielsen et al. 1986
; Tenney and Ou
1977
). Neurons recorded in the caudal hypothalamus of rats, cats, and rabbits are stimulated by hypoxia and/or hypercapnia, and a
significant portion of these neurons have cardiovascular or respiratory
related rhythms (Cross and Silver 1963
; Dillon and Waldrop 1993
; Ryan and Waldrop 1995
). When
recorded in vitro, these neurons are stimulated by hypoxia and
hypercapnia, extending the possibility that these neurons may be
central chemoreceptors (Dillon and Waldrop 1992
).
Moreover, a significant number of neurons stimulated by hypoxia in the
rat caudal hypothalamus that are responsive to hypoxia project to the
periaqueductal gray in the midbrain; an area also known to modulate
cardiorespiratory function (Ryan and Waldrop 1995
).
During early mammalian development, hypoxia elicits a transient
increase in ventilation followed by an overall depression. This
response contrasts with the sustained ventilatory increase observed in
adults (Rigatto 1984). Although the reasons for this depression observed in early development are unknown, many
developmental studies examining the role of various brain regions
during hypoxia have been performed to address this question.
Decerebration at the midbrain/pontine junction in rabbit pups abolished
the ventilatory depression during hypoxia, implicating more rostral
brain areas in this response (Martin-Body and Johnston
1988
). In contrast, studies in the lamb, rabbit, and rat
provided evidence that areas in the pons and medulla were all that were
needed for the ventilatory depression during hypoxia in neonates
(Fung et al. 1996
; Gluckman and Johnston
1987
; Waites et al. 1996
). Given these
differences, the exact site(s) in the brain causing the ventilatory
depression in the newborn is still not completely known, nor is the
role of caudal hypothalamic neurons in this response.
The aforementioned studies implicate the caudal hypothalamus as a potential region that may influence the developmental changes in the response to hypoxia. Thus the purpose of the present study was to elucidate any differences in oxygen sensitivity of caudal hypothalamic neurons during development. In addition, the involvement of sodium and calcium channels in the hypoxia response of these neurons was studied. The results of this work have determined that there are significant differences in the hypoxic responses of neonatal and juvenile caudal hypothalamic neurons and these responses are due to a sodium-dependent conductance mediated through tetrodotoxin-sensitive sodium channels.
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METHODS |
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Oxygen tension in caudal hypothalamic tissue during development
All animal protocols and procedures described were performed in
compliance with the National Institutes of Health Guide for the Care
and Use of Laboratory Animals and the Guidelines of the Laboratory
Animal Care Advisory Committee at the University of Illinois. All
chemicals listed were obtained from Sigma, (St. Louis, MO).
Sprague-Dawley rats obtained from a regulated colony were classified
into two age groups: neonatal (4-16 days old) and juvenile (20-40
days old). The animals were decapitated and the brains carefully
removed and placed in ice-cold artificial cerebrospinal fluid bubbled
with 95% O2-5% CO2 [which contained (in mM)
116 NaCl, 10 glucose, 5.4 KCl, 1.8 CaCl2, 0.9 NaH2PO4, 26.2 NaHCO3, and 15 HEPES,
pH 7.35, 295 mOsm]. The caudal hypothalamus then was blocked off
according to visual cues on the ventral portion of the brain and
sectioned into 400- to 600-µm coronal slices on a tissue chopper. The
slices then were rapidly transferred into a brain slice chamber and
perfused with 5% CO2-95% O2 equilibrated artificial cerebrospinal fluid at a flow rate of ~1 ml/min. The slices had a humidified 5% CO2-95% O2 gas
mixture flowing over their surface, and the temperature of the chamber
was stabilized at 35°C using a servo-controlled water circulator.
After the tissue had stabilized for 1 h, a fine-tipped (5- to 10-µm
diam, 0.1-torr resolution) Clark style polarographic oxygen electrode
(Diamond General, Ann Arbor, MI) was passed at increasing depths
through the posterior hypothalamic area, as identified from stereotaxic atlases (Paxinos 1991
; Paxinos and Watson
1986
). At each depth (0-150 µm, 50-µm increments) the
oxygen tension was measured during control (95% O2),
moderate hypoxic (10% O2-5% CO2-85%
N2) and severe hypoxic (0% O2-5%
CO2-95% N2) conditions for both neonatal and
juvenile tissue.
Whole cell current responses to hypoxia during development
Caudal hypothalamic slices of both neonatal and juvenile rats
were prepared as in the preceding section. Electrodes with a tip
impedance of 5-7 M were pulled on a vertical, two-stage pipette puller (Narishige, PP-83, Tokyo, Japan), fire-polished and filled with
an intracellular solution [which contained (in mM) 130 K+
gluconate, 10 EGTA, 10 HEPES, 1 CaCl2, 2 MgCl2,
and 1 ATP, pH 7.25, 275 mOsm]. Whole cell recordings of caudal
hypothalamic neurons were obtained using the "blind-patch"
technique (Blanton et al. 1989
). Membrane currents were
recorded in voltage-clamp mode using an AxoClamp 1D patch-clamp
amplifier (Axon Instruments, Foster City, CA) with the signal filtered
at 5 kHz using a four-pole low-pass Bessel filter. While in the
voltage-clamp mode, the series resistance and whole cell capacitance
were determined using the circuitry of the amplifier. Because the
average whole cell current recorded was <200 pA, the series resistance
was not compensated. All of the cells also were recorded briefly in
current-clamp mode to assess basal and active membrane potential
characteristics. Basal electrophysiological and hypoxic response data
were obtained from only those cells that had a resting membrane
potential of at most
40 mV and could be held at
60 mV with no more
that 150 pA of current. Once the basal electrophysiological
characteristics of each cell had been analyzed, the membrane current
responses to moderate and/or severe hypoxia (2-5 min) were determined.
Cells that displayed an inward current during hypoxia of >20 pA were considered stimulated, whereas those that had a response <20 pA were
considered unstimulated. This level was chosen based on minor baseline
shifts in the recordings and the resolution limits of the amplifier (10 pA). Additionally, some cells were also tested for a membrane response
to hypercapnia (7% CO2-93% O2).
Ionic properties of the cellular response to hypoxia
Some cells displaying inward current responses to hypoxia
subsequently were perfused with a low-calcium (0.2 mM), high-magnesium (11.4 mM) medium (synaptic blockade) to block all classical chemical synaptic transmission (Dean and Boulant 1989). This
medium has been shown in this laboratory to reversibly abolish evoked
synaptic potentials recorded in the CA1 region of the hippocampus on
stimulation of the Schaffer collaterals (Dillon and Waldrop
1992
; Nolan and Waldrop 1993
). A cell responsive
to hypoxia during synaptic blockade indicated the cell had an innate
ability to respond to hypoxia that was not due to input from other
cells. After testing for the response to hypoxia during synaptic
blockade, the current component of this response was analyzed further
using ion channel modifying agents including tetrodotoxin (TTX, 1-2
µM), cobalt chloride (2 mM), or after replacing the extracellular
sodium with N-methyl-D-glucamine (NMDG). To
assess for possible changes in input resistance during hypoxia, the
current responses to step voltage commands were digitized at 10-20 kHz
and analyzed using PClamp 5.5 software in a subset of cells during
control and hypoxic conditions.
Current-clamp experiments also were performed on a separate set of neurons. For these experiments, the membrane voltage responses to hypoxia (10% O2 and 0% O2) were recorded with an Axoclamp 2A amplifier (Axon Instruments). Slice preparation, seal formation, and hypoxic stimulation were performed identically as in the voltage-clamp recordings. In a subset of these neurons, the fluorescent dye Lucifer yellow (0.1-0.2%) was included in the pipette for later morphological characterization of the cells studied.
Correction of junction potential
Separate experiments were performed to correct for the slight
junction potential that develops at the tip of the electrode caused by
the different ion components between the bathing media and the
intracellular pipette solution (Fenwick et al. 1982). The junction potential experiments consisted of placing the
microelectrode containing the intracellular solution into the normal
bathing media, adjusting the DC offset to zero and then placing the tip into intracellular media. The potential that developed on switching between the two media was recorded using the patch-clamp amplifier and
used to correct the resting membrane potentials recorded from the cells
studied. In all of the cells tested, the membrane potential was
adjusted by
11.0 mV to correct for the observed junction potential
(actual value:
11.3 ± 0.3 mV, n = 27).
Data and statistical analysis
Tissue oxygen recordings were reported as means ± SE and
compared between neonatal and juvenile tissue using Student's
t-test (P < 0.05). All electrophysiological
data were recorded on a Gould chart recorder and digitized and stored
on video cassette tape using data-acquisition software (P-Clamp, Axon
Instruments), and a modified four-channel video cassette recorder
(A. R. Vetter, Rebersburg, PA). Data were reported as means ± SE (except percentages). Comparisons were made between: neonatal and
juvenile cell characteristics and responses to hypoxia, stimulated and
unstimulated neuronal characteristics, and the cellular response to
hypoxia during perfusion with various pharmacological agents.
2, Student's paired and unpaired t-tests
(with Bonferroni correction when necessary) and repeated measures ANOVA
(with Tukey's post hoc analysis) tests were used with the significance
level set at P < 0.05.
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RESULTS |
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Oxygen tension in caudal hypothalamic tissue during development
The oxygen tension measured during control conditions (95% O2) was significantly higher in neonatal (10.0 ± 0.4 days old, n = 6) when compared with juvenile (23.8 ± 0.7 days old, n = 6) tissue at depths equal to and >100 µm (Fig. 1A). During the moderate or severe hypoxic stimulus, there was no significant difference in oxygen tension between the two ages when tested at 50, 100, and 150 µm (Fig. 1B). Depths >150 µm were not tested for differences in oxygen tension because the average depth of all the cellular recordings was less than this level for both the neonates (115.9 ± 16.0 µm, n = 26) and juveniles (128.8 ± 8.9 µm, n = 76). On the basis of these findings, we are confident that the stimuli received by neurons at the different ages were identical and cannot account for the differences in the observed responses.
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Basal neuronal characteristics during development
Basal electrophysiological characteristics were examined in 49 caudal hypothalamic neurons from 24 neonatal rats and 96 neurons from 49 juvenile rats (Table 1). There were no significant differences in the average number of cells recorded per animal between neonates and juveniles (2.04 ± 0.26 vs. 1.96 ± 0.17; P > 0.05). No significant differences were observed in resting membrane potential or whole cell capacitance between neonatal and juvenile caudal hypothalamic neurons (Table 1). In contrast, the average resting input resistance, time constant and half-time to spike height was significantly larger in the neonatal neurons as compared with the juvenile neurons, signifying a greater amount of resting membrane current and faster action potential generation in the juvenile neurons (Table 1).
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Whole cell current responses to hypoxia during development
The most striking difference found between the two age groups was in the ability to respond to both a moderate or severe hypoxic stimulus. In the juvenile caudal hypothalamus, a significantly larger percentage (55.6%; 20/36) of the tested neurons were stimulated by moderate hypoxia as compared with neonatal (27.3%; 9/33) neurons (Fig. 2A). In addition, caudal hypothalamic neurons of juvenile rats (65.1%; 56/86) were significantly more likely than neonates (42.9%; 18/42) to be stimulated by the severe hypoxic stimulus (Fig. 2A). An example of the current response to severe hypoxia for both a neonatal and juvenile caudal hypothalamic neuron is shown in Fig. 3. In contrast to the differences observed in the proportions of caudal hypothalamic neurons stimulated by both moderate and severe hypoxia, there were no differences between neonates and juveniles in the magnitude of the inward current response of stimulated cells (Fig. 2B).
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As compared with the responses to hypoxia, the caudal hypothalamic responses to hypercapnia between both the neonates and juveniles were less robust. Only 22.2% (4/18) of neonatal and 10.5% (2/19) of juvenile caudal hypothalamic neurons tested displayed an inward current response to hypercapnia, and these neurons generally did not respond to hypoxia (only 1 cell from each group responded to hypoxia). Even though only a small number of neurons responded to hypercapnia, the average inward current response of juvenile neurons was significantly larger than neonatal neurons (42.5 ± 22.8 vs. 150.0 ± 14.1 pA; P < 0.05, Student's unpaired t-test). This suggests a difference in the development of the response to hypercapnia as compared with hypoxia in caudal hypothalamic neurons.
Electrophysiological and morphological properties of neurons sensitive to hypoxia
Cells recorded in current-clamp conditions were classified into
those cells showing a membrane depolarization (stimulated) or no
membrane depolarization (unstimulated). In stimulated cells (average
membrane depolarization of 5.7 ± 1.1 mV), the basal
characteristics were: resting membrane potential = 52.4 ± 1.6 mV (n = 17), input resistance = 423.0 ± 56.6 M
(n = 17), spike amplitude = 58.3 ± 3.1 mV (n = 15), and half spike height duration = 0.26 ± 0.02 ms (n = 15). For those cells not
stimulated by hypoxia, the basal characteristics were: resting membrane
potential =
50.7 ± 1.8 mV (n = 13), input
resistance = 420.1 ± 36.8 M
(n = 13),
spike amplitude = 65.2 ± 3.6 mV (n = 11),
and half spike height duration = 0.30 ± 0.02 ms
(n = 11). None of these differences were statistically significant. In addition, the presence of postinhibitory rebound (PIR)
was ubiquitous throughout the entire population with 80.0% (12/15) of
stimulated and 85.7% (6/7) of unstimulated cells displaying this
property. An example of a stimulated cell that displayed PIR is shown
in Fig. 4, A and B.
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Voltage-clamped neurons stimulated by moderate hypoxia had a
significantly lower resting input resistance compared with unstimulated neurons (306.2 ± 15.3 vs. 354.6 ± 19.4 M). In addition,
neurons stimulated by severe hypoxia had a significantly lower resting input resistance (292.1 ± 9.4 vs. 318.5 ± 12.2 M
),
higher resting cell capacitance (41.6 ± 2.1 vs. 36.3 ± 1.9 pF) and shorter half-time to spike height (400 ± 20 vs. 450 ± 20 µs) compared with unstimulated cells. However, when these
analyses were corrected for age, all significant differences were
eliminated. Therefore the main differences between stimulated and
unstimulated cells appear to be related to the developmental stage of
the neuron.
Morphological characterization of 23 caudal hypothalamic neurons was performed. Neurons filled with Lucifer yellow showed no differences in basal characteristics or hypoxic depolarization compared with cells not filled (average hypoxic membrane response for filled = 4.8 ± 0.5 mV and unfilled = 6.2 ± 1.0 mV, P > 0.05). Neurons stimulated by hypoxia (10 and 0% O2, n = 16) were mostly multipolar and had dendritic fields extending to or toward the third ventricle or ventral surface of the brain. The average size of the soma of these neurons was 18.3 ± 1.7 × 28.8 ± 3.2 µm. The morphological attributes of a hypoxia-stimulated neuron are shown in Fig. 4, C and D. Neurons unstimulated by hypoxia were either bipolar (n = 3) or tripolar (n = 2); none showed the numerous dendritic extensions from the cell soma that frequently were observed in hypoxia-stimulated neurons. The bipolar unstimulated neurons were quite long and relatively narrow (53.0 ± 5.8 × 15.7 ± 0.8 µm), whereas the tripolar neurons in this category were similar in size to hypoxia-stimulated neurons. An example of a bipolar neuron that was inhibited by hypoxia is shown in Fig. 5, A-C.
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Current and ionic properties of the cellular response to hypoxia
Not every cell was tested with both moderate and severe hypoxia, but in those cells (n = 32) that were tested for both, the magnitude of the inward current response to hypoxia increased with decreasing levels of oxygen (10% O2: 90.3 ± 25.1 pA vs. 0% O2: 183.9 ± 49.5 pA, P < 0.05, Student's unpaired t-test). Because the magnitude of the response was greater during severe hypoxia, the ionic properties of the inward current response to hypoxia were tested during the more severe stimulus only. Current-voltage relationships were compared during normoxia and hypoxia to determine input resistance changes. In those cells that displayed an inward current response to hypoxia of >20 pA (stimulated), a significant decrease in input resistance was observed compared with cells with a response between 0 and 20 pA (unstimulated), indicating an inward cationic current is active during hypoxia (Table 2). Furthermore, the current-to-voltage relationships during hypoxia of stimulated neurons displayed a significant increase in the reversal potential of whole cell current during hypoxia as compared with unstimulated neurons (Fig. 6).
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In 36 of the cells responding to hypoxia with an inward current, the response to hypoxia was tested in a low-Ca2+/high-Mg2+ medium to inhibit chemical synaptic transmission. In 32 of these cells tested (88.9%), the response to hypoxia was maintained. The hypoxic inward current response of these cells during perfusion with control media was 196.9 ± 34.4 and 128.4 ± 25.4 pA during synaptic blockade (P > 0.05, Student's unpaired t-test).
Twenty-one neurons that maintained the response to hypoxia in the synaptic blockade media were tested for the inward current response to hypoxia during perfusion with either CoCl2, TTX, or both as shown in Fig. 7. In eight of these cells, addition of CoCl2 (2 mM) alone had no effect on the response to hypoxia (Fig. 8A). In contrast, addition of TTX (1-2 µM) after synaptic blockade in a group of eight separate cells virtually abolished the response to hypoxia (Fig. 8B). The differing effects of CoCl2 and TTX were tested together in five neurons with similar results as when tested separately, namely TTX abolishing the response and CoCl2 having no effect (Fig. 8C). To verify further the influence of sodium current on this response, extracellular sodium was replaced with NMDG in six separate neurons. To a similar magnitude as perfusion with TTX, NMDG significantly attenuated the inward current response to hypoxia (TTX attenuation: 78.7 ± 4.5% and NMDG attenuation: 63.5 ± 12.9%, P > 0.05). Furthermore, in three of the neurons perfused with NMDG, the large decrease in input resistance observed during hypoxia was abolished (Control Ri decrease: 56.1 ± 5.9% and NMDG Ri decrease: 3.1 ± 11.9%, P < 0.05).
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DISCUSSION |
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A major finding in this study was the disparity in the ability of
neonatal caudal hypothalamic neurons to respond to moderate or severe
hypoxia as compared with juveniles. Our results indicate that fewer
neonatal than juvenile neurons respond to hypoxia; however, the
magnitude of the response to hypoxia does not differ between stimulated
neonatal and juvenile neurons. Other investigators have demonstrated
differential responses to hypoxia between neonatal and juvenile/adult
neurons in the rat brain stem (Haddad et al. 1990). A
possible explanation for the differences observed is a disparity in the
actual oxygen tensions between the neonatal and juvenile tissue during
hypoxia. A difference in the tissue oxygen concentration between
neonatal and adult rats was demonstrated previously in the hypoglossal
nucleus at all levels >21%, but there was no significant difference
when the oxygen concentration delivered to the tissue was <21%
(Jiang et al. 1991
). The results of the present study
clearly show that the oxygen tension during both levels of hypoxia
between neonatal and juvenile caudal hypothalamic tissue is not
significantly different. There is, however, a significant difference in
the oxygen tension between neonatal and juvenile tissue at depths
100
µM when perfused with 95% O2. This most likely is the
result of the neonatal brain being less dependent on aerobic metabolism
for energy (Clark et al. 1993
; Xia et al. 1992
). Therefore because almost all of the oxygen available to the tissue comes from the top of the chamber, the neonatal tissue is
less of an oxygen sink compared with the juvenile tissue at greater
depths. Therefore we believe that the decrease in the proportion of
neonatal caudal hypothalamic neurons responding to hypoxia is due to a
lack in the oxygen sensing ability of these neurons early in development.
In this study, no significant differences were found in the magnitude
of the inward current responses to moderate or severe hypoxia between
neonatal and juvenile caudal hypothalamic neurons. There is, however, a
trend showing a greater inward current response to moderate and severe
hypoxia in the juveniles as compared with neonate. One cannot rule out
the possibility of a difference in the response between the ages
because the heterogeneity of cell types in this area adds to the high
variance observed in the responses that was not overcome even by the
large sample size in this study. This is in contrast to results in
ventrolateral medullary neurons from this laboratory and hypoglossal
neurons from another laboratory that have shown larger membrane
potential responses to hypoxia in adult neurons as compared with
neonates (Haddad and Donnelly 1990; Nolan and
Waldrop 1996
). This difference may be due to the relatively
smaller range of membrane voltage responses (~1 orders of magnitude)
compared with the inward current response (~3 orders of magnitude),
leading to a smaller variance in the membrane-voltage analyses.
This study also demonstrated that the input resistance, time constant
and half-time to spike peak in neonatal caudal hypothalamic neurons
were all significantly larger as compared with the juvenile. These
findings suggest that neonatal cells have a lower channel density and
therefore are less excitable. Similar results previously have been
shown in neurons of the genioglossal nucleus of the rat brain stem
(Nunez-Abades et al. 1993). In contrast, there was no
significant difference found in the average resting membrane potential
between neonatal and juvenile caudal hypothalamic neurons. These
differences in channel density appear not to affect those channels
responsible for the resting membrane potential, such as potassium leak
channels, but may be involved in initiating spiking behavior. Thus
compared with neonates, the juvenile neurons may be more able to
demonstrate increases in firing during periods of hypoxia even if the
specific mechanism of oxygen sensitivity is fully developed in neonatal neurons.
Prior work from this laboratory has established the ability of the
caudal hypothalamus to modulate the respiratory responses to hypoxia
(Horn and Waldrop 1997). Microinjection of the
excitatory amino acid antagonist, kynurenic acid, into the caudal
hypothalamus significantly attenuates the respiratory response to
hypoxia in anesthetized rats (Horn and Waldrop 1994
). In
addition, caudal hypothalamic neurons recorded in vivo are excited by
hypoxia in the absence of peripheral chemoreceptors and a large
percentage of these neurons have firing rhythms related to respiration
(Dillon and Waldrop 1993
; Ryan and Waldrop
1995
). Furthermore, a large number of neurons in the rat caudal
hypothalamus that are excited by hypoxia project to the midbrain
periaqueductal gray, another area known to modulate respiratory output
(Ryan and Waldrop 1995
). These results suggest that most
caudal hypothalamic neurons are responsive to changes in arterial
O2 concentration and this responsiveness helps modulate the
respiratory responses to hypoxia. Unfortunately the present study was
unable to identify the precise characteristics of hypoxia-responsive
caudal hypothalamic neurons that make them different from nonresponsive
cells. When electrophysiological parameters were compared between
responsive and nonresponsive neurons, no differences were found that
could not be explained by the age of the neuron. There were, however,
slight morphological differences between the responsive and
nonresponsive neurons. The hypoxia-responsive neurons were mostly
multipolar, whereas the nonresponsive neurons were usually bipolar with
less extensive projections; but the significance of these morphological
differences needs further investigation.
This study demonstrates that neurons in the rat caudal hypothalamus
develop the ability to respond to hypoxia during the first 3 wk of
postnatal development. The biphasic respiratory response to hypoxia
(initial increase then sustained decrease in ventilation) in rats early
in development has been well characterized, although the exact
mechanism for this response is unknown (Eden and Hanson 1987). The favored hypothesis for the depression stage in the biphasic response to hypoxia is an underdeveloped facilitatory area in
the CNS of neonates that usually counteracts inhibitory respiratory
drive during hypoxia (Waites et al. 1996
). The caudal hypothalamus is one such facilitory area that counteracts inhibitory ventilatory drive from other areas of the brain during hypoxia (Tenney and Ou 1977
). Previous findings have shown that
decerebration at the midbrain/pontine junction abolishes the depression
of respiration during hypoxia in neonatal rabbits (Martin-Body
and Johnston 1988
). Furthermore, the biphasic response to
hypoxia is present when the brain is sectioned rostral to the caudal
hypothalamus but is absent after specific bilateral lesions in the
upper lateral pons or red nucleus of lambs and rabbits (Dawes et
al. 1983
; Gluckman and Johnston 1987
;
Waites et al. 1996
). Because the studies implicating discrete nuclei in the pons and midbrain in the biphasic response used
electrolytic instead of chemical lesions, the results observed could be
due to destruction of caudal hypothalamic projections through these
areas (Vertes and Crane 1996
). The present findings suggest that the lack of excitation from caudal hypothalamus neurons may be involved in the depression stage of the biphasic response to
hypoxia in neonates. The overall oxygen sensitivity of neurons in this
area may not be fully developed in neonates leading to an inability to
counteract cortical inhibitory drive during hypoxia. However, a recent
study counteracted this hypothesis by demonstrating the maintenance of
the depression stage of the biphasic response to hypoxia after a
midcollicular decerebration (Fung et al. 1996
). The
investigators in this study, however, still could not rule out the
involvement of diencephalic areas in the biphasic response to hypoxia
because such a large area was eliminated (Fung et al. 1996
).
Previous reports looking at the mechanism of hypoxic sensitivity in
central neurons demonstrated the importance of sodium currents in the
membrane response to hypoxia in the hypoglossal motor nucleus and
hippocampus (Cummins et al. 1991; Haddad and Jiang 1993
). The present results further support the
involvement of sodium channels in the cellular response of hypoxia in
central neurons. Because the characteristics of this response in caudal hypothalamic neurons is a slow, sustained inward sodium current, the
involvement of fast-inactivating sodium currents is unlikely even
though the present current was inhibited by TTX. One sodium current
that could mediate this type of response, however, is the persistent
sodium current, which is a noninactivating TTX-sensitive sodium current
dictated by differential gating of the same protein channel as the
fast-inactivating current (Alzheimer et al. 1993
). The
persistent sodium current is involved mainly in modulating neuronal and
muscle excitability and recently has been shown to display increased
activity during hypoxia in cardiac myocytes (Crill 1996
;
Ju et al. 1996
). Thus the persistent sodium current is a
likely candidate causing the observed response in these neurons, although direct evidence is needed.
In contrast to sodium current, the inward current response to hypoxia
of caudal hypothalamic neurons was shown to be unaffected by calcium
current, although there was a tendency for a decrease in the current
response after perfusion with low-calcium solution. However, this
tendency to decrease was most likely due to blockade of excitatory
synaptic input onto these cells that has been shown in prior reports to
facilitate the response to hypoxia in vivo (Horn and Waldrop
1994). Moreover, the lack of influence by calcium channels was
supported further by the observation that perfusion with
CoCl2 had no effect on the inward current response to
hypoxia. These findings are in contrast to results showing the
involvement of calcium channels in the cellular response to hypoxia in
ventrolateral medullary neurons and myocardial cells (Fearon et
al. 1997
; Sun and Reis 1994
). Furthermore,
several types of oxygen-sensitive potassium channels have been
described in peripheral (carotid body glomus, arterial smooth muscle
cells) and central (neocortical, substantia nigra neurons) cell types
that display both increases and decreases in conductance during anoxia
(Ganfornina and Lopez-Barneo 1991
; Jiang and
Haddad 1994
; Weir and Archer 1995
). Because a decrease in potassium conductance causes membrane depolarization, these
channels were not analyzed in the present study due to the inward
current response arising from an increase in membrane conductance. Thus
there appears to be a panoply of oxygen sensitive channels in a variety
of cell types involved with cardiorespiratory control. More work is
needed to determine the exact role of these channels in both the
cellular and respiratory responses to hypoxia.
In conclusion, a smaller proportion of neurons in the caudal hypothalamus of neonatal rats become stimulated during hypoxia as compared with neurons in more mature rats. In addition, the magnitude of the inward current response to hypoxia of caudal hypothalamic neurons is similar during rat development. Furthermore, the inward current response to hypoxia in these neurons appears to be due to TTX-sensitive sodium channels. The sum of these results implies that caudal hypothalamic neurons act as central chemoreceptors; this lack of oxygen sensitivity in many of the neurons during development may be a contributing factor to the respiratory depression observed in neonatal rats during hypoxia.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-32864 and HL-06296 to T. G. Waldrop and American Heart Association Grant 9704427A to E. M. Horn.
Present address of G. H. Dillon: Dept. of Pharmacology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107.
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FOOTNOTES |
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Address for reprint requests: T. G. Waldrop, Dept. of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, MC-114, 407 S. Goodwin Ave., Urbana, IL 61801.
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 soley to indicate this fact.
Received 9 September 1998; accepted in final form 16 November 1998.
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