Department of Molecular and Integrative Physiology and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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ABSTRACT |
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Fan, Yi-Ping,
Eric M. Horn, and
Tony G. Waldrop.
Biophysical Characterization of Rat Caudal Hypothalamic Neurons:
Calcium Channel Contribution to Excitability.
J. Neurophysiol. 84: 2896-2903, 2000.
Neurons in the
caudal hypothalamus (CH) are responsible for the modulation of various
processes including respiratory and cardiovascular output. Previous
results from this and other laboratories have demonstrated in vivo that
these neurons have firing rhythms matched to the respiratory and
cardiovascular cycles. The goal of the present study was to
characterize the biophysical properties of neurons in the CH with
particular emphasis in those properties responsible for rhythmic firing
behavior. Whole cell, patch-clamped CH neurons displayed a resting
membrane potential of 58.0 ± 1.1 mV and an input resistance of
319.3 ± 16.6 M
when recorded in current-clamp mode in an in
vitro brain slice preparation. A large proportion of these neurons
displayed postinhibitory rebound (PIR) that was dependent on the
duration and magnitude of hyperpolarizing current as well as the
resting membrane potential of the cell. Furthermore these neurons
discharged tonically in response to a depolarizing current pulse at a
depolarized resting membrane potential (more positive than
65 mV) but
switched to a rapid burst of firing to the same stimulus when the
resting membrane potential was lowered. The PIR observed in these
neurons was calcium dependent as demonstrated by the ability to block
its amplitude by perfusion of Ca2+-free bath
solution or by application of Ni2+ (0.3-0.5 mM)
or nifedipine (10 µM). These properties suggest that
low-voltage-activated (LVA) calcium current is involved in the PIR and
bursting firing of these CH neurons. In addition, high-voltage-activated calcium responses were detected after blockade of outward potassium current or in
Ba2+-replacement solution. In addition, almost
all of the CH neurons studied showed spike frequency adaptation that
was decreased following Ca2+ removal, indicating
the involvement of Ca2+-dependent
K+ current
(IK,Ca) in these cells. In conclusion,
CH neurons have at least two different types of calcium currents that
contribute to their excitability; the dominant current is the LVA or
T-type. This LVA current appears to play a significant role in the
bursting characteristics that may underlie the rhythmic firing of CH neurons.
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INTRODUCTION |
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The caudal
hypothalamus (CH) in the rat is bordered medially by the third
ventricle and laterally by the lateral hypothalamus (Paxinos and
Watson 1986; Veazey et al. 1982
). The function
of neurons in this area has mainly been determined by electrical or
chemical stimulation, which results in increases of respiratory output,
sympathetic nerve discharge, arterial pressure, and heart rate
(DiMicco and Abshire 1987
; Kabat 1936
;
Spencer et al. 1990
; Waldrop et al.
1988
). In addition, results from this laboratory have shown
that neurons in the CH have firing rhythms temporally related to either
the cardiac or respiratory cycles in rats and cats (Barman
1990
; Dillon and Waldrop 1993
; Ryan and
Waldrop 1995
).
Of particular interest is the role that neurons in this region play in
pathophysiological processes such as hypertension. Studies in a rodent
hypertension model, the spontaneously hypertensive rat (SHR), have
determined that sympathetic-related neurons in this region have a
higher discharge rate than normotensive rats (Shonis and Waldrop
1993). In addition, a reduction in the GABAergic function of CH
neurons in hypertensive rats compared with normotensive rats is
strongly associated with an increased cardiovascular tone initiated by
neurons in this region (Shonis et al. 1993
). Thus the
increased sympathetic tone in the SHR, which may play a role in the
hypertensive state in these animals, is possibly due to an increased
activity of CH neurons (Judy et al. 1976
).
The CH has also been linked to the control of other functions including
locomotion, antinociception, sleep/wakefulness, and the modulation of
the hippocampal electroencephalogram (Bland et al. 1995;
Carstens 1982
; Eldridge et al. 1981
;
Nitz and Siegel 1996
). For example, lesions in the
caudal hypothalamus abolish or attenuate hippocampal theta activity
(Anchel and Lindsley 1972
; Robinson and Whishaw
1974
), and high-frequency (100 Hz) stimulation in the CH has
been shown to be particularly effective in eliciting hippocampal
theta-activity (Bland and Vanderwolf 1972
). Recent evidence indicates that theta-related tonic discharge in the CH is
important in controlling hippocampal theta activity, which may be
particularly important in hippocampal processing of spatial information
(Kirk et al. 1996
; Skaggs and McNaughton
1996
).
Despite the extensive morphological and functional studies of the CH, little is known about the basic biophysical properties of neurons in this area. Of particular importance is the type of currents inherent to these neurons that underlie their rhythmic firing behavior. In view of a key role for the CH in various physiological and pathological states, we have analyzed the basic membrane and activity-related properties of neurons in the CH by using whole cell recordings in rat brain slices.
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METHODS |
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Preparation of brain slices
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. Male Sprague-Dawley rats (3-4 wk old, Harlan, Indianapolis, IN) were rapidly decapitated, and the brain was quickly removed and submerged in ice-cold artificial cerebrospinal fluid (ACSF) solution (see following text for composition). The section of the brain containing the caudal hypothalamus was cut into 400-µm-thick coronal slices with a tissue chopper. The slices were then transferred to an interface style recording chamber in which they were continuously perfused with a warmed (35°C), oxygenated ASCF solution. A 100% humidified gas (95% O2-5% CO2) flowed continuously over the slices, and the temperature was set at 35°C using a servo-controlled heating bath (Haake). The slices were allowed to stabilize for at least 1 h before electrical recording commenced.
Solutions and drugs
The composition of the ASCF solution was (in mM) 116 NaCl, 5.4 KCl, 0.81 MgSO4, 1.8 CaCl2,
1 NaH2PO4, 26.2 NaHCO3, 15 N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES), and 10 glucose. All solutions were gassed with a mixture of
95% O2-5% CO2 and
maintained at pH of 7.35-7.37. The osmolality was adjusted to 295-307
mOsm using a vapor pressure osmometer (Wescor). Due to the possible
modulatory effects on several kinds of voltage-gated
K+ channels, the common calcium channel blockers,
such as Co2+, Cd2+, or
Mn2+, were not used (Mayer and Sugiyama
1988). For Ca2+-free solution,
Ca2+ was omitted and Mg2+
was raised to 5 mM. In a few experiments, 0.5 mM ethylene
glycol-bis(
-aminoethylether)-N, N,N',N'-tetra-acetic acid
(EGTA) was added to chelate the residual calcium. In
Ca2+-replacement solution,
CaCl2 was replaced with 1.8 mM
BaCl2 to enhance the current through specific
calcium channels. In addition, some experiments utilized the specific
low-voltage-activated (LVA) calcium channel blocker
NiCl2 (0.3-0.5 mM). When
Ba2+ or Ni2+ was added to
the solution, NaH2PO4 and
MgSO4 were omitted to prevent precipitation.
Other drugs used included 1-2 µM tetrodotoxin (TTX, a sodium channel
blocker), 10 mM tetraethylammonium chloride (TEA, a potassium channel
blocker), 10 µM nifedipine (a specific L-type calcium channel
blocker), 1 mM
-amino-n-butyric acid (GABA), and 0.1 mM
bicuculline (a specific GABAA receptor
antagonist). Concentrated GABA, TEA, and NiCl2
were first dissolved in perfusion solution, while concentrated TTX was
first dissolved in citrate buffer. They were then added to the
perfusion solution to reach the final concentration. When bicuculline
was used, it was added to the GABA-containing solution. Nifedipine was
dissolved in absolute ethanol and added to the perfusion solution to
give the final concentration with a final alcohol content of 0.1%; a
control solution contained only 0.1% alcohol. Drug and solution
changes were performed by switching the inflow to reservoirs containing the desired test solution, and at least 15 min were allowed for equilibration before the data were collected or the effect of drug
reached its plateau. All drugs were obtained from Sigma Chemical (St.
Louis, MO).
Recording and data analysis
Blind, whole cell patch-clamp recordings were made using the
technique described by Blanton et al. (1989). When
filled with pipette solution [(in mM) 135 potassium gluconate, 5 KCl,
2 MgCl2, and 10 HEPES, pH 7.2 buffered with KOH,
osmolality 275 mOsm], the DC resistance of the pipette was 4-7 M
.
Recordings were obtained with an Axoclamp 2A (Axon Instruments)
patch-clamp amplifier operating in bridge mode with the low-pass Bessel
filter set at 3 kHz. Data were acquired on-line at 10 kHz for off-line
analysis using analysis software (PClamp 5.5 and 6.0, Axon
Instruments). Whole cell patch-clamp configuration was obtained
following the formation of at least a 2-G
seal. Data were not
collected for at least 3 min after obtaining the whole cell
configuration to permit the pipette and cell interior to equilibrate.
In most neurons, several different protocols were utilized to obtain
biophysical properties. To overcome any effects of the patch pipette on
the membrane potential and thus the basal membrane properties, the
membrane potential for each cell was set at 60 mV. Thus the resting
membrane potentials of quiescent cells were recorded and then set at
60 mV, while the membrane potentials of spontaneously firing neurons
were simply adjusted to
60 mV to inhibit spike activity. The input
resistance (Rin) was measured by
injecting a hyperpolarizing current pulse (
100 pA, 150 ms) and
measuring the voltage response. The membrane time constants were
calculated by injecting a hyperpolarizing current pulse (
100 pA, 150 ms) and fitting the induced membrane voltage transient to the
exponential charging function [V = A1 × exp(
t/
0) + A2 × exp(
t/
1) + C] using
the Chebyshev fitting method in the software package, where
0 represents the longest or somatic membrane
time constant,
1 represents a shorter
equalizing time constant, and C is a constant (Rall
1969
). According to the relationship
0 = RmCm, where
Rm and
Cm are the specific membrane
resistance and capacitance respectively, we obtained
Rm from
0
(Cm was assumed to be 1 µF/cm2). The rheobase, which was obtained with
a 50-ms current pulse, was defined as the minimum current that produced
an action potential (AP) in 100% of five trials. Depolarizing pulses
of variable amplitude and duration were used to determine the following
AP properties: AP amplitude (APamp), duration at
half the AP peak (APW1/2) and afterhyperpolarization potential (AHP, postspike nadir subtracted from
the prespike membrane potential).
Analysis of firing adaptation was determined by a procedure modified
from a prior study in a separate laboratory (Viana et al.
1995). The procedure was modified because the strong adaptation of caudal hypothalamic neurons created difficulties in determining adaptation when a long (2 s) current pulse was employed. Thus repetitive firing was evoked by a series of 400-ms depolarizing current
injections of increasing amplitude. The instantaneous firing frequency
(fonset) was obtained from the first
interspike interval. The maintained firing frequency
(fmain) was the frequency of APs during
the last 300 ms of the 400-ms depolarization. Frequency adaptation or
acceleration was quantified by a spike frequency index (SFI), where
SFI = fonset/fmain.
For the analysis, if the SFI > 1, the firing pattern was
considered to show adaptation; if the SFI < 0.1, the firing
pattern was considered to show acceleration; and if the SRF = 1, there was no modulation of firing pattern.
A subset of neurons was tested for their pharmacological properties by
first recording in ACSF, then adding a particular agonist or antagonist
to the ACSF (see preceding text), and finally washing with ACSF. When
the membrane potential was changed due to a drug or ion replacement, it
was kept at the original resting potential using injection of bias
current. In some neurons, a hypoxic stimulus (10%
O2-5% CO2-85%
N2) was presented by altering the gas flowing in
the chamber for 90 s, as previously described (Dillon and
Waldrop 1992).
The liquid junction potential (12 mV) was measured using a separate
set of experiments as previously described and subtracted from the
recorded membrane potential (Horn et al. 1999
). After a
successful recording (membrane potential at least
40 mV and AP
passing through 0), the position of the pipette was noted on a standard
series of diagrams of the brain slices (Paxinos and Watson
1986
). The recording depth varied between 5 and 230 µm, and
all the data are presented as means ± SE. Statistical
significance was determined by Student's paired and unpaired
t-tests with P < 0.05 deemed significant.
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RESULTS |
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Basic membrane properties of CH neurons
All cells were recorded within the caudal hypothalamus as
identified using highly recognizable visual landmarks (mamillothalamic tracts, fornix, 3rd ventricle, etc.) according to a rat stereotaxic atlas (Paxinos and Watson 1986). The basal
electrophysiological properties of the 50 recorded CH neurons are
summarized in Table 1.
Of the 50 cells tested, 33 were quiescent at their resting membrane
potential and the other 17 spontaneously fired APs, thus only the
quiescent cells were used for the determination of the average resting
membrane potential in Table 1. For the spontaneously active cells, the
resting membrane potential was adjusted to approximately
60 mV to
calculate the input resistance (Rin)
and time constants (
0 and
1). No differences were observed in any of the
basal or active membrane properties between the quiescent and
spontaneously active cells.
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Postinhibitory rebound and burst firing of CH neurons
Postinhibitory rebound (PIR) refers to transient
depolarization immediately after release from hyperpolarization that is
manifested as a subthreshold depolarization, an AP or a burst of APs
(Huguenard 1996; Jiang et al. 1999
). In
CH neurons, two different kinds of PIR were observed in 28/50 cells
studied (Fig. 1), and it is important to
note that there were no observed differences in basal membrane properties between cells that displayed PIR and those that did not
display PIR. The first type of PIR (type I) was the most prevalent type
(23 of the 28 cells exhibiting PIR were type I) and consisted of a
burst of APs following the end of the hyperpolarizing current pulse
(Fig. 1A, left). These APs were eliminated after
perfusion with TTX, but a delayed transient depolarization (DTD)
remained that increased in amplitude during perfusion with
Ba2+ replacement solution (Fig. 1A,
right). The PIR in type I cells was strongly dependent on
the hyperpolarizing potential amplitude and duration as well as the
level of prepulse membrane potential (see following text). The second
type of PIR (type II, 5/28) showed a single AP following the end of the
hyperpolarization pulse (Fig. 1B, left) that was
eliminated when the cell was perfused with TTX. In contrast to the type
I response, there was no DTD observed in these neurons following the
hyperpolarizing pulse during TTX perfusion (Fig. 1B,
right). Since the type I PIR was the most prevalent, the
rest of the results refer to this type unless otherwise noted.
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We found that type I PIR was affected by three factors, amplitude of
the hyperpolarizing pulse, duration of the hyperpolarizing pulse, and
prepulse membrane potential. As shown in Fig.
2A, a minimum amount of
hyperpolarizing current at a constant duration was required to trigger
the PIR. When the amplitude was kept constant in another cell, the
duration of the hyperpolarizing pulse was directly related to its
ability to elicit PIR (Fig. 2B). While increases in the
amplitude of the hyperpolarizing pulse caused a graded enhancement of
the PIR response, additional increases in the duration of the
hyperpolarizing pulse had no further effect on the PIR once elicited.
In general, the PIR was observed at the normal membrane potential for
each cell but would completely disappear when the resting membrane
potential was lowered (usually below 70 mV) prior to the
hyperpolarizing pulse (Fig. 2C).
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In all of those neurons tested that showed Type I PIR, the pattern of
AP firing in response to a depolarizing pulse was strongly influenced
by the resting membrane potential, as illustrated in Fig.
3. When the neuron was at its normal
resting membrane potential (55 mV), the response to a depolarizing
current pulse was characterized by regular repetitive firing throughout
the entire pulse duration (Fig. 3A). Depolarization from a
more hyperpolarized membrane potential (usually more negative than
65
mV) evoked a qualitatively different response, i.e., a burst discharge
was induced (Fig. 3B). Five of these neurons were given the
same depolarizing current step from the more hyperpolarized membrane
potential in the presence of TTX, which abolished the fast spikes but
not the DTD (Fig. 3C). In four separate neurons,
hyperpolarizing pulses initiated from their normal resting membrane
potentials elicited oscillatory activity that was dependent on the
duration of the test pulse (Fig.
4A). Interestingly, bursting
type firing responses and oscillatory membrane potentials were also
elicited with a depolarizing current step in a separate group of cells
with intrinsically lower resting membrane potentials such as the one
shown in Fig. 4B. Furthermore, spontaneously occurring
rhythmic burst firing in CH neurons of varying resting membrane
potentials was encountered in three cells (Fig. 4C). This is
a lower frequency than reported previously (~15%) in neurons
recorded extracellularly from Wistar-Kyoto rats (Shonis and
Waldrop 1993
). Since the use of whole cell patch recording techniques can cause significant perturbations such as whole cell washout in neurons, the differences in discharge frequency between this
study and the prior one may be due to different recording techniques.
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Mechanism and modulation of PIR in CH neurons
The presence of a DTD following the hyperpolarizing pulse during perfusion with TTX suggests the entry of Ca2+ might be a key factor for the PIR. As shown in Fig. 5A, the DTD was eliminated when Ca2+ was removed from the bathing medium in all six neurons tested. Addition of 300-500 µM Ni2+ (specific LVA Ca2+ channel blocker) to the normal solution completely blocked the DTD in three neurons tested as shown in Fig. 5B. We also tested the effects of the L-type Ca2+ channel blocker nifedipine on PIR. As shown in Fig. 5C, 10 µM nifedipine caused a complete block of the DTD in the two neurons tested, while the solution containing 0.1% alcohol in which nifedipine was dissolved had little influence on the DTD. Thus LVA, or T-type Ca2+ channels, appear to be a major mechanism of PIR in these cells, although there may be a significant influence from L-type Ca2+ channels.
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To determine how this PIR may be modulated, we explored the effects of GABA and hypoxia on the PIR in the presence of 2 µM TTX. In all three neurons tested, the DTDs were completely blocked by the application of 1 mM GABA in the normal bath solution, as shown in Fig. 6A. The effect of GABA on the PIR was almost completely reversed by application of 0.1 mM bicuculline in one cell tested. Furthermore exposure to 90 s of hypoxia (10% O2) in three cells tested caused an increase in the duration (30.2 ± 3.1 vs. 35.0 ± 3.3 ms, P < 0.05) and amplitude (15.7 ± 1.5 vs. 21.4 ± 1.8 mV, P < 0.05) of the PIR even though the input resistance decreased during this exposure, suggesting a possible direct effect of hypoxia on the channel (Fig. 6B).
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Influence of Ca2+ on membrane and firing properties in CH neurons
In the presence of the potassium channel blocker TEA (10 mM) and
TTX, depolarizing current steps in two neurons tested elicited oscillatory potential responses and an afterdepolarizing potential (ADP) that were inhibited following perfusion in
Ca2+-free solution (Fig.
7A). These depolarization
responses needed a high-intensity stimulation to evoke in normal
Ca2+ solution (with TEA); however, in four
separate neurons perfused with Ba2+ replacement
solution (without TEA), the Ca2+ oscillatory
responses and larger ADP could be evoked with a smaller depolarizing
current (Fig. 8B). Since
Ba2+ is a better charge carrier than
Ca2+ through L-type calcium channels (Bean
1989), the oscillations and ADP observed were most likely due
to this channel type. Barium also decreased the voltage threshold and
increased both the amplitude and duration of the DTD in these four
neurons (all type I as shown in Fig. 1A, right),
suggesting that Ba2+ is also a better charge
carrier through T-type calcium channels in these neurons.
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Considering the abundance of calcium channels in the CH neurons, we also evaluated the possible role of Ca2+-dependent K+ channels in repetitive firing and AP properties. In a total of 31 neurons tested, all but 2 showed a decrementing firing pattern, signifying adaptation in these cells. In addition, one cell had such a strong adaptation that the value for SFI could not be calculated (denominator = 0), thus the average SFI for the remaining 30 cells was 3.52 ± 0.71 (range = 0.92-18.23, Table 1) with the two cells not showing adaptation having SFI values of 0.92 and 0.96. The removal of Ca2+ in 11 of the neurons showing adaptation caused a significant amplitude decrease in the AP and AHP with no significant change in AP duration (Table 2, Fig. 8). The decrease in AP amplitude, however, may possibly be due to the more depolarized baseline and decreased sodium electrical gradient. Furthermore, in all eight of the neurons tested, Ca2+ removal significantly reduced the frequency adaptation (Table 2, Fig. 8).
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DISCUSSION |
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Using whole cell recording methods and pharmacological manipulation, we have described the biophysical properties of CH neurons that have at least two different endogenous calcium channels, LVA and HVA. The neurons in CH are not electrophysiologically homogeneous with many neurons (23/50) showing PIR that is mediated through the activation of LVA calcium channels. Most of the neurons also showed adaptation and had firing patterns partly mediated by the interaction of calcium current and calcium-dependent potassium current. The significance of these properties is discussed in relation to the known function of neurons in this brain region.
LVA Ca2+ current involvement in PIR
A major finding from this work was that most of the CH neurons
showed PIR. The major type of PIR encountered was characterized by the
ability of a previously quiescent neuron to fire in a transient burst
mode following a hyperpolarizing current injection and is referred to
as type I PIR. The bursts of firing and rebound depolarizations were
time and voltage dependent, requiring a sufficiently hyperpolarized membrane potential for several hundred milliseconds to obtain the full
effect. After exposure to TTX, the cells still showed a DTD after being
released from hyperpolarization, suggesting a mechanism different from
voltage-dependent sodium conductance. In fact, the DTD was strictly
dependent on the presence of extracellular calcium and was fully
blocked by low concentrations of Ni2+. At a more
depolarized membrane potential (more positive than 65 mV), direct
stimulation did not trigger a burst discharge, suggesting that the
ionic conductance underlying its generation was inactivated at these
potentials (Llinas and Yarom 1981
). De-inactivation of
the conductance occurred as the membrane potential hyperpolarized as
evidenced by the generation of the DTD. The properties of this DTD are
similar to those found in neurons of the adult inferior olivary complex
(Llinas and Yarom 1981
), thalamus (Jahnsen and Llinas 1984a
,b
), dorsal raphe nucleus (Burlhis and
Aghajanian 1987
), neonatal hypoglossal nucleus (Viana et
al. 1993
), and many other central and peripheral neuronal types
(Huguenard 1996
). Taken together, the LVA calcium
channel, or T-type channel, significantly affects the DTD and may
underlie the PIR observed in these cells, although definitive proof
needs assessment via voltage-clamp.
Interestingly, the LVA Ca2+ channels in the CH
neurons were extremely sensitive to the organic channel blocker,
nifedipine, which has also been shown to block these channels in mouse
embryonic dorsal root ganglion neurons and isolated neurons from the
ventromedial hypothalamus (Akaike et al. 1989;
Richard et al. 1991
). Recent evidence has indicated that
dihydropyridine-sensitive L-type channels on CA3 neurons can be
activated at potentials much more negative than other HVA calcium
channels and thus contribute to the LVA currents (Avery and
Johnston 1996
). This challenges the traditional designation of
L-type channel as exclusively HVA calcium channels and the T-type
channel as the only LVA calcium channels. Thus it is unknown whether
the nifedipine was affecting the PIR via LVA or HVA current in the CH
neurons in the present study.
Barium is known to be a better charge carrier than calcium in certain
types of HVA calcium channels, such as the L-type calcium channel
(Bean 1989). This increased conductance by barium
through putative HVA channels was observed in CH neurons. In contrast, LVA calcium current amplitude is normally unaffected or reduced by
Ba2+ except in the rat thalamic reticular
nucleus, where Ba2+ was shown to increase the LVA
current (Akaike et al. 1989
; Fox et al.
1987
; Huguenard and Prince 1992
). In type I
neurons in the CH, Ba2+ replacement decreased the
voltage threshold for the PIR-induced DTD, while increasing both its
amplitude and duration. Although this suggests CH neurons have LVA
current more sensitive to Ba2+ than
Ca2+, voltage-clamp studies are needed to
definitively attribute this response to the LVA and not HVA current.
Role of calcium on the AP and repetitive firing activity
In a majority of the cells tested, the blockade of
Ca2+ entry caused a decrease in the AP amplitude
without changing its duration. These properties have also been found in
rat superior cervical ganglion and reflect the involvement of
large conductance Ca2+-activated
K+ channels activated during the AP in these
cells (Davies et al. 1996). Although these cells were in
the peripheral nervous system, a similar mechanism may underlie the
response to decreased Ca2+ in the CH neurons.
The role of calcium in the repetitive firing qualities of CH neurons was also highlighted after perfusion with calcium-free solution. The loss of Ca2+ led to a significant decrease in the afterhyperpolarization, increase in firing frequency and reduction in adaptation (as determined from the SFI). These responses can be explained by the large presence of Ca2+-activated K+ channels that modulate the adaptive abilities of these CH neurons. However, the adaptation was never completely blocked by calcium-free solution, suggesting that other, unknown factors are involved in the adaptation of these neurons.
Functional role of LVA calcium channels in CH neurons
The physiological function of the LVA calcium channels in CH
neurons has yet to be determined. In many of the CH neurons studied, LVA was the dominant calcium channel, which is similar to many areas in
the hypothalamus (Akaike et al. 1989; Alonso and
Llinas 1992
). Our present data indicate a strong influence of
LVA calcium channels on the firing pattern of CH neurons and suggest
that this channel plays a significant role in burst discharge. The role
of CH neurons in neuronal rhythms significant to physiological function
may be twofold. First, there is compelling evidence that neurons in the
CH have theta rhythm-related discharge implicating them in memory
function (Bland et al. 1995
; Kirk et al.
1996
; Skaggs and McNaughton 1996
). Second,
neurons in this area have been shown to modulate cardiorespiratory
activity and display rhythms related to sympathetic discharge
(Barman 1990
; Dillon and Waldrop 1993
;
Waldrop and Porter 1995
).
The role of the caudal hypothalamus in memory function was first shown
after lesions of the CH abolished or attenuated hippocampal theta
activity (Anchel and Lindsley 1972; Robinson and
Whishaw 1974
). A recent anatomical study has further shown that
the CH has dense projections to the medial septum and vertical limb of the diagonal band of Broca that are important relays in the generation of hippocampal theta activity (Vertes et al. 1995
).
Together with the results from the above-mentioned neuronal recordings,
these findings implicate the CH in the formation of the theta rhythm found in hippocampal neurons. The LVA calcium channels in CH neurons may be one of the underlying ionic mechanisms for the theta-related tonic discharge in these cells.
Neurons in the CH discharge with rhythms related to the
cardiovascular and sympathetic cycles in both rats and cats
(Barman 1990; Dillon and Waldrop 1993
;
Ryan and Waldrop 1995
). Furthermore neurons in this
region send dense projections to other cardiovascular modulating areas
such as the periaqueductal gray, ventrolateral medulla and nucleus
tractus solitarius (Barman 1990
; Ryan and Waldrop
1995
; Vertes and Crane 1996
). Because a
hyperpolarization is required to activate the LVA
Ca2+ current, the pattern of inhibitory input may
be a critical factor in determining the role of LVA current in
sympathetic-related CH neuronal discharge. One of the primary functions
of GABAergic input to the caudal hypothalamus is to depress the
activity of sympathoexcitatory neurons involved in cardiovascular
control (DiMicco and Abshire 1987
; Shonis et al.
1993
; Waldrop and Bauer 1989
). GABA completely
abolished the LVA calcium current in these neurons, suggesting an
effect of GABA on this channel. This GABAergic effect has been
previously shown in hippocampal cells, where the GABAB agonist baclofen inhibited the LVA current
(Fraser and MacVicar 1991
). Since intracellular
messenger systems are known to regulate calcium channel function, the
effect of GABAB receptor activation on calcium
channel function is not surprising (Gray et al. 1998
). Our data suggest, however, that the affect of GABA on calcium current
is mediated through the GABAA channel because it
is inhibited by bicuculline. A similar mechanism of GABA action on ion
channel properties has been demonstrated in astrocytes, where
GABAA-induced chloride flux modulated potassium
channel function over long durations (Bekar and Walz
1999
). Thus the relative amount of GABA through the involvement
of GABAA receptors in the CH may greatly affect the function of this current.
Since the LVA calcium channel is modulated by GABA, a
differential level of GABA in the CH of the spontaneously hypertensive rat (SHR) may influence this current and thus significantly alter the
firing properties of these neurons. Recent work from this lab has found
that there is an elevated discharge rate of neurons in the CH of the
SHR, and a higher percentage of these neurons have a bursting-type
firing pattern of when compared with normotensive rats (Shonis
and Waldrop 1993). It has been proposed that these bursting-type cardiovascular neurons may exert a more efficient influence than other types of cells in eliciting the vasoconstrictor response and thus contribute to the occurrence of hypertension (Chan et al. 1991
; Shonis and Waldrop
1993
). Indeed, it has been suggested that neuronal bursts are
the best stimuli for exciting a cell and have been shown to make
unreliable synapses reliable (for review, see Lisman
1997
). Interestingly, the most obvious function of LVA calcium
channels is to promote burst-type firing. Since the LVA calcium channel
is found in a large number of CH neurons, it raises the possibility
that the higher number of burst-type firing CH neurons in the SHR might
be due to either a greater expression or a disinhibition of this
channel in the CH of the SHR. The latter explanation might be more
possible because the LVA channel is directly inhibited by GABA and the
GABAergic input to CH neurons is reduced in the SHR (Horn et al.
1998
). However, more work is needed to determine whether there
is a greater function of the LVA channel in CH neurons of the SHR.
Several studies have suggested CH neurons function as putative
oxygen-sensing neurons that modulate the cardiorespiratory response to
hypoxia (Dillon and Waldrop 1992, 1993
; Horn and
Waldrop 1997
; Horn et al. 1999
). The response of
these neurons to a hypoxic stimulus is a membrane depolarization
accompanied by a significant increase in firing frequency. The specific
ionic mechanism by which hypoxia causes this response in CH neuron has
not been fully understood, but several channels are candidate oxygen
sensors as shown in other studies. Work in hippocampal neurons has
demonstrated that voltage-dependent sodium channels decrease their
conductance during hypoxia, and human cardiac L-type
Ca2+ channels also decrease their conductance
during hypoxia (Fearon et al. 1997
; O'Reilly et
al. 1997
). Furthermore both LVA and HVA calcium currents as
well as persistent sodium currents are increased during hypoxia in rat
rostral ventrolateral medullary neurons responsible for
sympatho-excitatory output (Kawai et al. 1999
; Sun and Reis 1994
). In the present study, the duration
and magnitude of the PIR-induced DTD in CH neurons increased during the
hypoxic stimulus. Thus the LVA current may be directly affected by
oxygen and help in the cardiorespiratory changes attributed to these and other neurons during hypoxia.
In conclusion, neurons in the caudal hypothalamus of rats contain LVA calcium channels that are essential to the postinhibitory rebound properties inherent in these cells. These channels, along with calcium-activated potassium channels, help establish the bursting firing properties of caudal hypothalamic neurons. Undoubtedly, the bursting properties of these neurons underlie the theta and sympathetic rhythms inherent in the caudal hypothalamus. Further work is needed to determine if the same cells are responsible for both of these rhythms or if there are subpopulations of caudal hypothalamic neurons separately supplying each rhythm and ultimately contributing to memory and/or sympatho-excitatory function.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-06296 and an American Heart Association Grant-in-Aid to T. G. Waldrop and an American Heart Association Predoctoral Fellowship to E. M. Horn.
Present addresses: Y.-P. Fan, Dept. of Bioengineering, University of California, San Diego, La Jolla, CA 92093; E. M. Horn, Division of Neurosurgery, Barrow Neurological Institute, 350 Thomas Rd., Phoenix, AZ 85013.
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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 (E-mail: twaldrop{at}uiuc.edu).
Received 29 November 1999; accepted in final form 16 August 2000.
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REFERENCES |
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