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Introduction
Orexin-A and orexin-B, also known as hypocretin-1 and
hypocretin-2, are members of a new family of neuropeptides
synthesized in the lateral hypothalamus and perifornical area
neurons[1,2]. Orexins have been shown to participate in many
functions, including sleep-wakefulness, feeding, and
neuroendocrine function[3-5]. In addition, many studies have
found that orexins also participate in the neural control of
cardiovascular functions. And, although most studies have
focused on the excitatory effect of orexins on cardiovascular
sympathetic activity[6-11], some evidence suggests that
orexins might also have a role in the parasympathetic control
of heart rate and cardiac functions.
Cardiac vagal nerves predominate over cardiac
sympathetic nerves in the control of heart rate and cardiac functions,
and their preganglionic fibers primarily originate from the
cardiac vagal neurons (CVN) in the nucleus ambiguus (NA)
and the dorsal motor nucleus of the vagus
(DMNX)[12-15]. CVN are intrinsically silent and their activity relies completely
on their synaptic inputs[15]. CVN receive excitatory
gluta-matergic and cholinergic
inputs[16,17], and inhibitory GABAergic
and glycinergic inputs[14,17,18]. Although previous studies
have suggested that orexins are involved in the vagal
control of heart rate and cardiac functions, the reported effects
are largely contradictory. Microinjection of orexin-A into
the NA of adult male rats elicited a dose-related decrease in
heart rate[19]. Microinjection of orexin-A into the
nucleus tractus solitarius (NTS), where GABAergic neurons
and glutamatergic neurons monosynaptically project to
CVN[14,16], elicited a decrease[20]
or increase[21] in heart rate. In a very recent
in vitro study using brainstem slices of
new-born rats, orexin-A was found to inhibit CVN indirectly, via
presynaptic enhancement of the actionpotential-dependent
GABAergic and glycinergic inputs, and via presynaptic
attenuation of the actionpotential-dependent glutamatergic
inputs[22].
The actual concentration of orexin-A in rat brainstem that
acts to modulate the physiological synaptic control of CVN
is not clear; however, the experimentally measured
concentration of orexin-A in the cerebrospinal fluid is less than
1 nmol/L in rats[23], and is even less in dogs and human
beings [24,25]. Surprisingly, the concentration of orexin-A used
in the in vitro study of Dergacheva et
al[22] was 1 μmol/L, and was as high as 1 μmol/L in microinjection
studies[19-21]. It is possible that these previous studies have used
overdoses of orexin-A, which might have resulted in a
non-specific effect.
The response of each kind of synaptic input of CVN to
orexin-A might be different at different concentrations. At the same concentration, the sensitivity of each kind of
synaptic input to orexin-A might also be different. These
possibilities make it necessary to reevaluate the effect of orexin-A
on CVN at lower and more divided concentrations. The
purpose of the present study is to reevaluate the effect of
orexin-A on the glycinergic and GABAergic spontaneous
inhibitory postsynaptic currents (sIPSC) at lower concentrations
(20 nmol/L, 100 nmol/L, and 500 nmol/L). Using retrograde
fluorescent labeling of CVN and the voltage patch-clamp
technique, we have demonstrated that orexin-A
dose-dependently increases the frequency of both the glycinergic and
the GABAergic sIPSC. However, at a lower concentration
(20 nmol/L) of orexin-A, although the frequency of the
glycinergic sIPSC was significantly increased, the frequency
of the GABAergic sIPSC was not significantly changed.
These results suggest that the glycinergic inputs and the
GABAergic inputs have different sensitivities to orexin-A,
and suggest that the two kinds of inhibitory inputs might
play different roles in the synaptic control of cardiac vagal
functions.
Materials and methods
Retrograde fluorescent labeling of CVN The inhalation
agent halothane (0.5 mL) was dripped into a glass box
(5 cm×5 cm×5 cm) with a lid and a bottom cotton pad. Three
to four-day-old Sprague-Dawley rats (Shanghai Institute for
Family Planning) were put in the box for 30 s with the lid
covered. This procedure anesthetized the rats but
maintained their breathing in a relatively normal state. The rats
were then buried in ice-filled bags to decrease the rats¡¯ body
temperatures and slow their hearts. After autonomic
breathing stopped (usually within 2 min) a right thoracotomy was
carried out to expose the heart, and rhodamine (2% solution,
20-50 μL; XRITC, Molecular Probes, Carlsbad, California,
USA) was injected into the pericardial sac with a glass
pipette (tip diameter 50 μm). The incision was sutured and the
animals were heated with a thermo-pad to help recovery.
After the surgery (about 5 min) the animals usually started
autonomic breathing within 3 min and started free moving
within another 5 min. The animals were allowed 1-2 d to
recover, and experiments were performed when animals were
4-6 d old. Rats at this age have been shown to have similar
respiratory-related and reflex-related parasympathetic heart
rate control as adult rats[17,26]. Similar surgical procedures
that selectively labeled CVN have been described previously
by Mendelowitz and Kunze[27], who proved that no brainstem
neurons were labeled if rhodamine was injected into the chest
cavity while the pericardial sac was kept intact, and if rhoda-
mine was injected into the pericardial sac while the cardiac
branch of the cardiac vagal nerve was sectioned. In addition,
it has also been proved that intravenous injection of up to 10
mg of rhodamine failed to label any
CVN[27].
Slice preparation The animals were anesthetized deeply
with halothane and decapitated at the supracollicular level.
The hindbrain was exposed, isolated, and submerged in cold
(4 ºC) artificial cerebral spinal fluid (ACSF) of the following
composition (in mmol/L): NaCl (124), KCl (3.0),
KH2PO4 (1.2),
CaCl2 (2.4), MgSO4 (1.3),
NaHCO3 (26), D-glucose (10), sucrose (10), and constantly bubbled with gas (95%
O2, 5% CO2; pH 7.4). The cerebellum was removed and the brainstem
was dissected using a dissection microscope. With the
rostral end facing upwards and the ventral surface facing the
razor, the brainstem was then secured in the slicing chamber
of a vibratome (Leica VT 1000 S, Heerbrugg, Switzerland)
filled with the same ACSF, and sequentially sectioned in
variable thickness in the transverse plane. Once the NA and
other landmarks[17] emerged under the microscope, a single
slice of 400 mm thickness was taken for experimentation. A
medulla slice of this thickness from a 4-6-d-old rat actually
includes the full range of the medulla, and such slices have
been used in many studies (eg, respiratory rhythm studies)
that require relatively intact in vitro neuronal networks. The
slice was transferred into the recording chamber and
submerged in the ACSF maintained at 22 ºC.
Electrophysiological recording Individual CVN in the
NA were identified by the presence of the fluorescent tracer
using an Olympus (Tokyo, Japan) upright microscope
through a 40×water immersion objective. These identified
CVN were then imaged with differential interference contrast
(DIC) optics, and infrared illumination and infrared-sensitive
video detection cameras to gain better spatial resolution and
to visually guide and position the patch pipette onto the
surface of the identified neuron. The pipette
(2.0-5.0 W) was advanced until a high resistance seal was obtained (>1
GW) between the pipette tip and the cell membrane of the
identified neuron. The membrane under the pipette tip was then
ruptured with a brief suction to obtain whole-cell patch-clamp
configuration, and the cell was voltage-clamped at a holding
potential of -80 mV. The pipette resistance and capacitance
was not compensated either before or after gaining
intracellular access. To record GABAergic and glycinergic synaptic
events, the patch pipettes were filled with a solution
consisting of (in mmol/L): KCl (150),
MgCl2 (2), ethyleneglycol-bis(b-aminoethyl
ether)-N,N,N¡¯,N¡¯-tetraacetic acid (EGTA) (2),
N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid
(HEPES) (10), Mg-ATP (2), at pH 7.35. With this pipette
solution the Cl- current induced by activation of the GABA (g-aminobutyric acid) receptors and/or glycinergic receptors
was recorded as an inward current. In some experiments a
2 ms, 5 mV, hyperpolarizing current was injected with a
frequency of 0.25 Hz to calculate and monitor the membrane
resistance change of CVN throughout the recording. All
animal procedures complied with the institutional guidelines
of Fudan University, and were in accordance with the
National Administration Guidelines for Experimental Animals.
Drug application GABAergic synaptic currents were
isolated by inclusion in a perfusate of strychnine (1 μmol/L), D-2-amino-5-phosphonovalerate (AP5, 50 μmol/L), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μmol/L) to
block glycine, NMDA (N-methyl-D-aspartate), and
non-NMDA receptors, respectively, and were confirmed by
blockade of the synaptic currents by 1 μmol/L picrotoxin at the
end of the experiment. Glycinergic synaptic currents were
isolated by inclusion in a perfusate of picrotoxin (1 μmol/L),
AP5 (50 μmol/L), and CNQX (50 μmol/L) to block GABA,
NMDA, and non-NMDA receptors, respectively, and were
confirmed by blockade of the synaptic currents by 1 μmol/L
strychnine at the end of the experiment. Orexin-A (20 nmol/L,
100 nmol/L, 500 nmol/L) was used to activate the orexin-A
receptors. The duration of orexin-A application was 2 min
and each slice was applied only once to avoid
desensitiza-tion. All drugs were purchased from Sigma Aldrich (St Louis,
MO, USA).
Data analysis Spontaneous GABAergic and glycinergic
sIPSC were analyzed with MiniAnalysis (Synaptosoft,
version 4.3.1, Decatur, GA, USA) with a minimal acceptable
amplitude of 10 pA. Results are presented as mean±SEM,
and are statistically compared by using the nonparametric
Kolmogorov-Smirnov (K-S) test. Significant difference was
set at P<0.05.
Results
Responses of the glycinergic sIPSC to different doses
of orexin-A Orexin-A significantly increased the frequency
of the glycinergic sIPSC at all three concentrations tested.
At a concentration of 20 nmol/L orexin-A increased the
average frequency of the glycinergic sIPSC by 63%±4%, from
3.04±1.01 Hz to 4.97±0.91 Hz (P<0.01,
n=6). This frequency increase was observed in all the 6 CVN tested. At a
concentration of 100 nmol/L, orexin-A increased the average
frequency of the glycinergic sIPSC by 167%±5%, from 3.60±
0.90 Hz to 9.64±0.86 Hz (P<0.01,
n=8). At a concentration of 500 nmol/L orexin-A caused a frequency increase by
149%±7%, from 3.55±1.21 Hz to 8.85±2.71 Hz
(P<0.05, n=8), which was similar in degree to that produced by 100 nmol/L
orexin-A. These results indicate that orexin-A
dose-dependently increases the frequency of the glycinergic sIPSC, and
a maximal effect can be achieved at 100 nmol/L. A typical
CVN with an increased frequency of glycinergic sIPSC
induced by orexin-A at 100 nmol/L is shown in Figure 1A, 1B,
and the changes in average frequency caused by the three
concentrations of orexin-A are shown in Figure 1C, 1D.
In most CVN and at all the three concentrations of
orexin-A used, an increase in the amplitude of the glycinergic sIPSC
was also observed. Because Dergacheva et
al proved that this amplitude increase was presynaptically caused by
summated action-potential-dependent release of
glycine[22], the amplitude data were not statistically analyzed and were not
presented in this article. In addition, at no concentration of
orexin-A used in this study was there any significant change
in the membrane resistance, or visible change in the baseline
currents of CVN. The average membrane resistance before
and after 500 nmol/L orexin-A was 370.24±16.17
MW and 382.66±20.87 MW, respectively
(P>0.05, n=8).
Responses of the GABAergic sIPSC to different doses
of orexin-A Orexin-A significantly increased the frequency
of the GABAergic sIPSC only at 100 nmol/L and 500 nmol/L.
At a concentration of 20 nmol/L orexin-A changed the
frequency of the GABAergic sIPSC from 3.40±0.75 Hz to 2.88±0.53
Hz (P>0.05, n=6). In none of these 6 CVN was obvious
frequency change observed. At a concentration of 100 nmol/L
orexin-A increased the frequency of the GABAergic
sIPSC by 97%±11%, from 2.58±0.63 Hz to 5.07±1.22 Hz
(P<0.05, n=6). At a concentration of 500 nmol/L orexin-A increased the
frequency of the GABAergic sIPSC by 173%±14%, from
2.59±0.82 Hz to 7.08±0.86 Hz (P<0.01,
n=5). These results indicate that although orexin-A also dose-dependently increased the
frequency of the GABAergic sIPSC, a relatively higher
concentration (>20 nmol/L) is needed to cause a significant
change, and the maximal frequency increase could not be
reached at a concentration of 100 nmol/L. A typical CVN
with an increased frequency of the GABAergic sIPSC
induced by orexin-A at 100 nmol/L is shown in Figure 2A, 2B,
and the changes in average frequency caused by the three
concentrations of orexin-A are illustrated in Figure 2C, 2D.
Orexin-A at concentrations of 100 nmol/L and 500 nmol/L
also caused an amplitude increase of the GABAergic sIPSC
in most CVN tested. Because Dergacheva et
al have proved that this amplitude increase is also presynaptically caused
by summated action-potential-dependent release of
GABA [22] the amplitude data were not statistically
analyzed and are not presented in this article.
Discussion
There are two major findings in the present study. One is
that the frequency increase of both the glycinergic and the
GABAergic IPSC caused by orexin-A is dose-dependent. The
other is that orexin-A at a lower concentration (20 nmol/L)
significantly enhances the glycinergic inputs but does not
cause significant alteration in the GABAergic inputs.
Ever since the discovery of the GABAergic inputs and
the glycinergic inputs in CVN, these two inhibitory inputs
have shown identical roles in the regulation of CVN. The
synaptic activities of both kinds of inputs are
action-potential-dependent and are absent in the presence of
tetrodo-toxin (TTX)[14,18]; both kinds of inputs are enhanced by
endogenous acetylcholine and exogenous
nicotine[18], are inhibited by opioids, and are rhythmically augmented during
inspiration[17,28]. Little is known about whether and how these
two inhibitory inputs play different roles in the synaptic
control of CVN, except that dihydro-b-erythroidine, a nicotinic
antagonist, prevents the inspiratory-related augmentation
of the GABAergic, but not the glycinergic, inputs to CVN
when focally applied at 3 mmol/L, a concentration under
which dihydro-b-erythroidine specifically binds to the
a4b2 type of nicotinic receptors [17]. The present study
demonstrated that orexin-A at a lower concentration (20 nmol/L)
significantly increased the frequency of the glycinergic
sIPSC, but did not cause a significant frequency alteration in
the GABAergic sIPSC. These results indicate that the
glycinergic inputs and GABAergic inputs might have
different roles in the orexinergic control of CVN. At lower
concentrations (£ 20 nmol/L) of orexin-A, glycinergic inputs might
dominate, and at higher concentrations (³20 nmol/L) of
orexin-A, a synergistic action of both kinds of inputs might
occur.
Although in the present study lower concentrations of
orexin-A were used, the results were still quite consistent
with those from the study of Dergacheva et
al[22]. The results of the present study as well as the results of Dergacheva
et al indicate that the responses of the glycinergic inputs
and the GABAergic inputs to different concentrations of
orexin-A differ in degree, but do not differ in the direction of
responses. The present study still cannot reveal why CVN
are excited by microinjection of orexin-A into the NA
in vivo and are inhibited in vitro. Further study is necessary.
The physiological roles of the glycinergic and the
GABAergic inputs to CVN in cardiac vagal control are not
fully understood. Currently, the only confirmed function of
these two inputs to CVN is that they are involved in the
generation of respiratory-related heart rate rhythm. During
the inspiratory phase of the respiratory cycle, both the
GABAergic and the glycinergic inputs are facilitated by ace
tylcholine to inhibit CVN and speed up the
heart[18,27]. These two inhibitory inputs to CVN have also long been
postulated to be involved in the reflex parasympathetic control of
the heart. The serotonergic neurons in the brainstem or in
the nodose ganglion have been reported to be activated
during activation of the cardiovascular chemo- or
baro-reflex[29]. The activation of the serotonergic system, via
inhibition of both the GABAergic and the glycinergic inputs to
CVN, possibly excites CVN and mediates the reflex
parasympathetic inhibition of the heart. The physiological
significance of the modulation of the GABAergic and the glycinergic
inputs to CVN by orexin-A is also not clear, and nothing is
known about why the glycinergic neurons preceding CVN
were more sensitive to orexin-A than the GABAergic neurons.
Perhaps the receptors for orexin-A binding in these two kinds
of neurons are different in density. Orexin-A is known to
strengthen preying behavior and increases food uptake.
Because the action of orexin-A on CVN is assumed to
accelerate the heart, orexin-A might help mammals to adapt to
their preying and eating behavior via speeding up the heart.
In conclusion, the present study demonstrated that
orexin-A dose-dependently increased the frequency of both
the glycinergic and the GABAergic sIPSC of CVN; the
glycinergic neurons and the GABAergic neurons preceding
CVN had different sensitivities to orexin-A, which might
indicate that these two kinds of inhibitory neurons play
different roles in the synaptic control of cardiac vagal functions.
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