1Department of Physiology, New York Medical College, Valhalla, New York 10595; and 2Department of Pharmacology and Toxicology and Center for Neurobiology and Immunology Research, University of Kansas, Lawrence, Kansas 66047
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ABSTRACT |
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Leonard, C. S., E. K. Michaelis, and K. M. Mitchell. Activity-Dependent Nitric Oxide Concentration Dynamics in the Laterodorsal Tegmental Nucleus In Vitro. J. Neurophysiol. 86: 2159-2172, 2001. The behavioral-state related firing of mesopontine cholinergic neurons of the laterodorsal tegmental nucleus appears pivotal for generating both arousal and rapid-eye-movement sleep. Since these neurons express high levels of nitric oxide synthase, we investigated whether their firing increases local extracellular nitric oxide levels. We measured nitric oxide in the laterodorsal tegmental nucleus with a selective electrochemical microprobe (35 µm diam) in brain slices. Local electrical stimulation at 10 or 100 Hz produced electrochemical responses that were attributable to nitric oxide. Stimulus trains (100 Hz; 1 s) produced biphasic increases in nitric oxide that reached a mean peak concentration of 33 ± 2 (SE) nM at 4.8 ± 0.4 s after train onset and decayed to a plateau concentration of 8 ± 1 nM that lasted an average of 157 ± 23.4 s (n = 14). These responses were inhibited by NG-nitro-L-arginine-methyl-ester (1 mM; 92% reduction of peak; n = 3) and depended on extracellular Ca2+. Chemically reduced hemoglobin attenuated both the electrically evoked responses and those produced by authentic nitric oxide. Application of the precursor, L-arginine (5 mM) augmented the duration of the electrically evoked response, while tetrodotoxin (1 µM) abolished it. Analysis of the stimulus-evoked field potentials indicated that electrically evoked nitric oxide production resulted from a direct, rather than synaptic, activation of laterodorsal tegmental neurons because neither nitric oxide production nor the field potentials were blocked by ionotropic glutamate receptor inhibitors. Nevertheless, application of N-methyl-D-aspartate also increased local nitric oxide concentration by 39 ± 14 nM (n = 8). Collectively, these data demonstrate that laterodorsal tegmental neuron activity elevates extracellular nitric oxide concentration probably via somatodendritic nitric oxide production. These data support the hypothesis that nitric oxide can function as a local paracrine signal during the states of arousal and rapid-eye-movement sleep when the firing of mesopontine cholinergic neurons are highest.
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INTRODUCTION |
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Mesopontine cholinergic cells of
the laterodorsal tegmental (LDT) and the adjacent pedunculopontine
tegmental (PPT) nuclei provide the major cholinergic input to the
thalamus (Hallanger et al. 1987; Paré et
al. 1988
; Satoh and Fibiger 1986
; Semba and Fibiger 1992
; Sofroniew et al. 1985
;
Steriade et al. 1988
; Woolf and Butcher
1986
) and can profoundly influence thalamocortical processing
(Munk et al. 1996
; Steriade et al.
1991
). Mounting evidence indicates these cells play an
instrumental role in the induction of rapid-eye-movement (REM) sleep
and arousal by releasing acetylcholine (Ach) in the thalamus and medial
pontine reticular formation (for review, see Steriade and
McCarley 1990
). While the function of these neurons has been
considered mainly in terms of acetylcholine release, they probably also
release other chemical messengers including peptides (Standaert
et al. 1986
; Sutin and Jacobowitz 1988
;
Vincent et al. 1983b
, 1986
) and nitric oxide (NO)
(Leonard and Lydic 1997
; Williams et al.
1997
). Indeed, virtually all mesopontine cholinergic neurons
are intensely labeled by reduce
-nicotinamide adenine dinucleotide
phosphate (NADPH)-diaphorase histochemistry (Vincent et
al. 1983a
), which vividly reveals their somatic, dendritic, and
axonal morphology (Leonard et al. 1995a
; Vincent
et al. 1983a
). This staining results from the cytoplasmic distribution of high levels of the enzyme neuronal NO synthase (nNOS)
(Dawson et al. 1991a
; Hope et al. 1991
),
which catalyzes the formation of NO from L-arginine
(L-Arg) in a Ca/calmodulin-dependent manner (Bredt
and Snyder 1990
; Mayer et al. 1991
).
NO is an ubiquitous, membrane-permeant, intercellular signaling
molecule that functions in several diverse physiological processes (for
review, see Moncada et al. 1991), including the control
of vascular tone, where it was first identified. Evidence suggests that
NO is also an important molecule in the CNS (Garthwaite et al.
1988
; Knowles et al. 1989
), where it may
modulate synaptic transmission and cellular excitability (for review,
see Garthwaite and Boulton 1995
) and may have both
excitotoxic (Dawson et al. 1991b
) and neuroprotective
actions (Lipton et al. 1993
). The control of neuronal NO
production has been associated with Ca2+ entry
following activation of the N-methyl-D-aspartate
(NMDA) subtype of glutamate receptors (Garthwaite 1991
;
Garthwaite et al. 1988
; Kiedrowski et al.
1992
). Because mesopontine cholinergic neurons both express
voltage-dependent Ca2+ channels (Kamondi et al.
1992
; Leonard and Llinás 1990
;
Takakusaki and Kitai 1997
) and display elevated
somatodendritic [Ca2+]i
following action potentials (Leonard et al. 1995b
, 2000
)
and because excitatory synaptic input is mediated partly by NMDA
receptors (Sanchez and Leonard 1994
, 1996
), NO may, in
principle, be generated by these cells at both somatodendritic regions
as well as axonal terminals during periods of activity.
Evidence indicates that NO is released from the terminals of these
neurons in the thalamus (Williams et al. 1997
) and
medial pontine reticular formation (Leonard and Lydic
1997
) in relation to behavioral state. We have sought to
directly determine whether extracellular NO is generated within the LDT
in response to activity of LDT neurons.
Measurement of neuronal NO production has been limited to a few brain
regions and has relied mainly on indirect methods, including bioassay
(Garthwaite et al. 1988), cGMP assays (DeVente et
al. 1990
; East and Garthwaite 1991
;
Garthwaite et al. 1988
; Morris et al.
1994
), [3H] citrulline production
(Kiedrowski et al. 1992
; Toms and Roberts 1994
), and the measurement of nitrates from brain microdialyses (Luo et al. 1993
; Shintani et al. 1994
),
although direct measurement of NO by microdialysis has also been made
(Williams et al. 1997
). While these methods demonstrate
NOS activity, they provide little information about the bioavailability
or spatiotemporal patterns of NO in tissue. Recent electrochemical
techniques have been developed (Bedioui et al. 1997
;
Christodoulou et al. 1996
; Fabre et al. 1997
; Friedemann et al. 1996
; Iravani et
al. 1998
; Malinski and Taha 1992
;
Meulemans 1993
; Mitchell and Michaelis
1998
; Park et al. 1998
; Shibuki
1990
) that directly measure NO at precise locations, and a
growing number of studies have applied these methods to neural tissue
(Burlet and Cespuglio 1997
; Desvignes et al.
1997
; Iravani et al. 1998
; Kimura et al.
1998
; Malinski et al. 1993a
; Meulemans
1994
; Meulemans et al. 1995
; Rivot et al.
1997
, 1999
; Shibuki and Kimura 1997
;
Shibuki and Okada 1991
; Zhang et al. 1995
).
We have utilized a recently developed porphyrinic microprobe, modified
for enhanced NO selectivity (Mitchell and Michaelis 1998), to investigate the activity-dependent production of NO within the LDT. We provide, for the first time, direct evidence that NO
is produced in the LDT following electrical and chemical stimulation.
This finding implies that the local [NO] is modulated by the
behavioral state-dependent firing of mesopontine cholinergic neurons
and therefore that NO may function as a paracrine signal within the
mesopontine tegmentum.
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METHODS |
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Brain slice preparation
Brain slices of the mesopontine tegmentum were prepared
according to standard methods (Leonard and Llinás
1994) in accordance with the National Institutes of Health
policy on humane care and use of laboratory animals (NIH Publication
80-23). The minimum number of animals were used to reach statistically
meaningful results. Briefly, female guinea pigs (175-300 g) were
anesthetized with pentobarbital sodium (75-100 mg/kg ip) and
decapitated. The section of the brain stem containing the LDT was
rapidly removed and placed into ice-cold standard Ringer solution
containing (in mM) 124 NaCl, 5 KCl, 1.2 NaH2PO4, 2.7 CaCl2, 3 MgSO4, 26 NaHCO3, and 10 glucose. The tissue block was then
affixed to a vibratome stage, and slices were cut at a nominal
thickness of 400 µm. Slices were then incubated at room temperature
in standard Ringer solution and continuously bubbled with 95%
O2-5% CO2. After an
incubation period of
1 h, slices were submerged in a recording
chamber and continuously superfused with standard solution at room
temperature at ~1 ml/min.
Electrochemical NO probe
The NO-selective microprobe has been described in detail
previously (Mitchell and Michaelis 1998). Briefly, it
was constructed from a carbon fiber encased in a tapered glass
capillary. The electroactive surface was 35 µm in diam and 300 µm
long and was coated with several thin-layer membranes to enhance the
sensitivity and selectivity. Optimal sensitivity was achieved by
catalysis of the electron transfer from NO oxidation by a conductive
polymeric membrane of metal porphyrin,
tetrakis(3-methoxy-4-hydroxyphenyl)-nickel (II) porphyrin, which was
electropolymerized onto the carbon surface as has been described
(Malinski and Taha 1992
; Malinski et al. 1993b
). The probe was then coated with Nafion, a perfluorinated negatively charged polymer, to exclude negatively charged electroactive species from the sensor surface (e.g., NO
Electrochemical measurements
Electrochemical measurements in the slice and in vitro
calibrations of the probe sensitivity and selectivity were made with a
standard three-electrode cell configuration with Ag/AgCl reference and
auxiliary electrodes. The probes were mounted on a micromanipulator and
inserted into the LDT from either the ventral or dorsal aspect. The
angle of the probe with respect to the slice surface was adjusted so
the entire sensing surface of the probe was buried within the tissue.
Measurements were performed using a PC-controlled amplifier and
interface (Cypress Systems, Lawrence, KS) operated in the coulometric
mode. The current resulting from NO oxidation at the electrode
interface was integrated for either a 500-ms or 5-s period yielding a
continuous on-line recording of the electrode response in picocoulombs
versus time. These measurements were then converted via calibration of
each individual probe to apparent NO concentration (typically
nanomolar) versus time. Some additional amperometry experiments were
also conducted using an Axopatch 200A amplifier (Axon Instruments)
modified for an extended range of operation. Measurements with both
systems were performed using an applied potential at the NO probe of
+700 mV versus Ag/AgCl. Following placement of the NO probes, the
potential was applied and the probes were allowed to equilibrate for
1 h before further experimentation. Acquired waveforms were imported
into the computer program Igor Pro (Wavemetrics), for calibration,
filtering and graphing. Amperometry records were integrated over a
period of 480 ms off-line using Igor Pro.
Calibration of the NO probe was performed in an oxygenated and stirred
standard slice solution before and then again after tissue measurements
to account for any changes that may have resulted from exposure of the
probe to tissue, e.g., adsorption of protein. Identical coulometry was
employed to measure the electrode response to small volume aliquots of
standard solutions of diethylamine NO complex (DEA-NO). The
concentration of NO in the DEA-NO solution was established using a
spectrophotometric method based on the conversion of oxyhemoglobin to
met-hemoglobin by NO (Doyle and Hoekstra 1981). This
assay was performed as described previously (Mitchell and
Michaelis 1998
). In some experiments, a second calibration was
not performed and the data are expressed in picocoulombs.
Two additional tests were performed on these probes. First, because
hemoglobin was used to scavenge tissue NO in some experiments (see
following text), the sensitivity of the probe in the presence of
hemoglobin was determined. A reduced hemoglobin (Hb) preparation (see
following text) was quantitatively oxidized to methemoglobin (Di
Iorio 1981) by preexposure to NO prior to testing the NO
probe response. This oxidized Hb sample had no discernable
effect on the NO or background response of the NO probes. Second,
because carbon fibers electrodes can exhibit pH sensitivity under some conditions, e.g., with certain surface treatments and for measurements made in particular applied potential ranges (Rice and Nicholson 1989
; Runnels et al. 1999
), the pH sensitivity
of the NO probe was also determined. Neuronal activity evoked by
electrical stimulation is associated with extracellular pH changes of a
few tenths of a pH unit (Chesler and Chan 1988
;
Kraig et al. 1983
) and typically consist of a transient
alkaline shift followed by an acidic shift of longer duration (~110 s
to return to the basal pH). We found that the NO probe was insensitive
to pH changes over the range pH 6.0-7.8 when operated at the potential
used for detection in these experiments.
Extracellular and intracellular recordings
Electrophysiological recordings were made in experiments
separate from the NO measurements. Extracellular recordings of
electrically evoked field potentials were made with borosilicate patch
electrodes (6 M) filled with Normal Ringer solution. Intracellular
recordings were performed with sharp microelectrodes (80-100 M
)
filled with 3 M KCl. Electrical measurements were obtained using an
Axoclamp 2A amplifier (Axon Instruments) operated in bridge mode.
Current and voltage traces were digitized using pClamp software (Axon Instruments) running on a personal computer. pClamp data were imported
into Igor Pro (Wavemetrics) for graphing and measurement. For both of
these sets of experiments, a stimulating electrode was positioned at
the location used in the NO measurements.
Electrical and chemical stimulation
Electrical stimulation of the slice was accomplished with
isolated constant-current pulses (Neurodata Instruments) applied to a
Teflon-coated stainless steel bipolar stimulating electrode (114 µm
OD) bared only at the tips. The electrodes were positioned at the
ventral edge of the LDT as determined by inspection of the slice
surface (see Fig. 1B).
Chemical stimulation was accomplished by either superfusion of the
compound dissolved at final concentration in normal Ringer solution or
by pressure pulses (Picospritzer II; General Valve) applied to a patch
pipette positioned near the surface of the slice at various
distances from the electrochemical probe. In some experiments, which
are indicated in the text, the pipette was positioned in the tissue for
drug delivery. The drugs in the pipette were dissolved in either
deionized water or in normal Ringer solution. All drugs were
obtained from Research Biochemicals International unless
otherwise indicated. The applied drugs were NMDA,
NG-nitro-L-arginine-methyl-ester
(L-NAME), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (APV) and
L-Arg (tissue culture grade; Sigma). Chemically reduced Hb
was prepared from a Ringer solution containing 1 mM hemoglobin (Sigma)
in 10 mM sodium dithionite (Sigma). This solution was dialyzed in the
dark against a total of 2,500 volumes of oxygenated Ringer at 4°C. The resulting solution was then diluted to its final concentration in
Ringer. In some experiments a solution of authentic NO was pressure-ejected into the tissue. This NO solution was produced by
bubbling double-distilled deoxygenated water with NO gas (99%, Matheson, Joliet, IL), which was purified of higher oxides of nitrogen
prior to use as described previously (Mitchell and Michaelis 1998). The transients evoked by this solution were reproducible and were specifically related to the NO and not the hypoosmotic vehicle
(see Fig. 6) (Mitchell and Michaelis 1998
). Under
similar recording and ejection conditions, comparable NO transients
were also obtained by pressure ejection of DEA-NO in normal Ringer (data not shown). Tetrodotoxin (TTX) was purchased from Sigma and was
dissolved in Ringer solution to a concentration of 1 µM.
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Histochemistry
To mark the recording sites, the probes were gently agitated to
produce a small mechanical lesion after recordings were completed. The
slice was then removed and immersion fixed in 4% paraformaldehyde for
12-36 h. After fixation, the slice was equilibrated with 20% sucrose
in 0.1 M phosphate buffer (pH 7.4) for 24 h. The slice was then
resectioned on a freezing microtome, and the tissue was processed for
NADPH-diaphorase (NADPH-d) by a method modified from (Hope and
Vincent 1989
) as previously described (Leonard et al.
1995a
). The location of the NO probes were then determined with
respect to the pattern of NADPH-d staining centered on the LDT and PPT.
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RESULTS |
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Probe locations and the distribution of NOS in the LDT and DR of the guinea pig
Histological examination of sections prepared from brain slices
used for electrochemical measurement revealed that all LDT recording
sites contained numerous NADPH-d cells (n = 11). To visualize the precise probe locations with respect to the
NADPH-d-containing cells, it was necessary to make small mechanical
lesions after recording (Fig. 1A) because positioning of the
probes in the tissue did not result in observable damage (see LDT
region, Fig. 1B). The example in Fig. 1A was
taken from an experiment where an electrochemical probes was placed in
the LDT (Fig. 1A, ). Labeled cells were observed within
the LDT and ventral LDT (LDTv) as previously described in guinea pig
(Leonard et al. 1995a
). Numerous NOS-containing somata
were within a few soma diameters of the probes located within the LDT.
Moreover, these findings verified the absence of NADPH-d-containing
somata from the DR of the guinea pig as was previously reported
(Leonard et al. 1995a
) and is different from the rat
(Leger et al. 1998
).
Electrical stimulation experiments were conducted with the NO probe
located in the LDT and the stimulating electrodes placed on the surface
of the slice ventral to the LDT in the underlying tegmentum. An example
of the relation between the stimulating electrodes and the NADPH-d
cells of the LDT region is shown in Fig. 1B (, lesions
from stimulating electrodes). This location is effective in evoking
local EPSPs in NADPH-d-labeled LDT cells (Sanchez and Leonard
1994
).
Local electrical stimulation evoked a NOS-dependent electrochemical signal in the LDT
Electrical stimulation of the LDT resulted in electrochemical
signals that can be attributed to increases in extracellular [NO].
Single pulses typically used to evoke synaptic input to LDT neurons
(Sanchez and Leonard 1994, 1996
) did not evoke
detectable signals. However, trains of pulses delivered at either 10 or
100 Hz did evoke detectable signals. Pulses delivered at 10 Hz for 10 s evoked clear signals, but pulses delivered at 100 Hz for 0.1-1 s were preferred and were used in all experiments, unless otherwise indicated, because the shock artifacts subsided more rapidly.
Electrochemical signals resulting from pulse trains of increasing
current strengths (0.1 s at 100 Hz) are superimposed in Fig.
2A. With each successive
train, the electrochemical signal became larger until it reached a
maximal value for the highest stimulus strengths tested. Lengthening
the train duration to 1 s at the highest current strength produced
a striking increase in the amplitude and duration of the
electrochemical response (Fig. 2B) revealing a biphasic time
course (Fig. 2C). Responses to this stimulation paradigm
often consisted of a rapid increase that peaked (33 ± 2 nM;
n = 14) at 4.8 ± 0.4 s following stimulus onset and a variable duration plateau phase (8 ± 1 nM; measured at 90 s; n = 14), which lasted until
157 ± 23.4 s from stimulus onset (n = 14).
While such biphasic responses were common, other patterns were also
observed. In some cases, responses decayed monophasically (cf. Fig.
4B), and in other cases, the late-phase was comparable to,
or even larger than, the early peak (cf. Figs. 5B and
8B). Nevertheless these electrochemical signals were not artifacts associated with polarization of the stimulating electrode or
probe surface because identical shocks delivered in the absence of
tissue, and with less distance separating the probe and stimulating electrodes, produced only a brief artifact (Fig. 2C,
inset).
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The electrochemical response to local electrical stimulation was also sensitive to NOS inhibitors as indicated in Fig. 3. Following the superfusion of solution containing 1 mM L-NAME, the electrically evoked signal progressively declined so that by 48 min the signal was virtually abolished (Fig. 3, A-C). The time course of L-NAME inhibition was measured in three slices (Fig. 3D). For two of the slices, the electrochemical signal was completely abolished with a time constant of ~20 min, while in the third case, the signal was reduced by 77% with a similar time constant. The average steady-state inhibition was 92.3% (P < 0.01; n = 3). This indicates that the electrochemical signals evoked by local electrical stimulation required activation of NOS and further supported the idea that these signals arose from endogenous NO production.
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Electrochemical signal required extracellular Ca2+ and was diminished by hemoglobin
nNOS is a Ca/calmodulin-dependent enzyme so we investigated the
possibility that the electrically evoked electrochemical signals were
Ca2+ dependent. Superfusion of the slices with a
Ringer solution containing no added calcium and 2.7 mM EGTA reversibly
blocked these electrochemical signals (Fig.
4A). This was observed in two
of three slices where the response was reduced by 89.5%. In the third
slice, the response was reduced by 55%. These results indicate that
most, if not all, of the electrically evoked signal arose from
Ca-dependent processes. Because NO binds to the heme center of reduced
hemoglobin (Hb) with high affinity and rapid kinetics (Doyle and
Hoekstra 1981), we investigated whether the electrically evoked
electrochemical signals were also sensitive to Hb. Bath superfusion of
1 µm Hb produced a reversible inhibition of the electrochemical
signal (54.0 ± 1.8%, n = 3), suggesting again
that the electrochemical signal had properties attributable to NO (Fig.
4B). However, inhibition was not complete and occurred
slowly following superfusion. Because our probe was positioned to sense
NO throughout the thickness of the slice, one possible explanation for
the slow and incomplete effect was that the high-molecular-weight Hb
only entered the slice slowly. We tested this idea by pressure ejecting
small volumes of authentic NO into the brain slice and measuring the
effectiveness of superfused Hb in attenuating that signal. This showed
that even when the Hb concentration was raised to 10 µM it took 30 min to attenuate the electrochemical signal by 65% (Fig.
4C). This experiment also demonstrated that superfusion of
Hb reduced the basal electrochemical signal with a slow time course. We
further examined this point by pressure ejecting Hb into the slice at a
location situated between the NO source pipette and the microprobe as
schematically illustrated in the inset of Fig.
5A. Under these conditions,
four brief pulses of Hb (100 µM) produced small but rapid reductions
in the baseline electrochemical signal and reduced the effect of a
60-ms pulse of NO by nearly 50% even when delivered ~3 min after the
last Hb ejection (Fig. 5A1). Moreover, ejecting Hb near the
peak of the response produced by a 60-ms pulse of NO rapidly reduced
(within 1 s) the response by 50% (Fig. 5A2). These
data illustrate that Hb can rapidly attenuate the NO signal if it is
delivered into the slice between the source and the probe. We then
determined if Hb ejection into the slice would also rapidly attenuate
the electrically evoked electrochemical signal (Fig. 5B1).
Two ejections of Hb delivered after the peak of the electrically evoked
response rapidly attenuated the response by ~20% each. Moreover,
responses to subsequent stimulation were attenuated by 50%.
Following the third Hb ejection, the responses began to slowly recover.
The rapidity of the Hb attenuation is illustrated on a faster time
scale in Fig. 5B2. These data indicate that Hb has a similar
effect on both the electrically evoked electrochemical signal and
authentic NO, which strongly supports the idea that the microprobes
were detecting tissue NO or a NO-related product.
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Electrochemical signal results from direct activation of NOS-containing processes
The NO detected following electrical stimulation of the LDT may have arisen from several sources. These include NOS-containing axons and terminals that may have been directly stimulated to liberate NO, and the dendrites and somata of NOS-containing neurons, that may have been synaptically or directly stimulated to produce NO. Results from two types of experiments pertain to this point. First, we examined the possibility that activation of LDT neurons by glutamatergic EPSPs was necessary for the NO responses. To do this, we compared the electrically evoked NO signals from before and after inhibition of EPSPs with CNQX and APV. Because we previously had noticed that EPSPs recorded from LDT neurons undergo strong synaptic depression during high-frequency stimulation (Leonard, unpublished observations), we chose a stimulation paradigm of 10 Hz for 10 s to maximize the likelihood of observing excitatory postsynaptic potential (EPSP)-dependent signals for these experiments. However, as mentioned in the preceding text, this stimulation protocol produced a long-duration artifact which interfered with the early part of the electrochemical signal. We therefore measured the effect of ionotropic glutamate receptor antagonists in the late-phase of the evoked electrochemical signal. Bath superfusion of CNQX (10 µM) and APV (50 µM) produced no detectable suppression (107.6% of control; n = 5; P > 0.1) of these electrochemical responses (Fig. 6, A and B), indicating that fast EPSPs are not necessary for the stimulus-evoked increase in NO. Bath application of L-NAME verified these late signals were NOS dependent (Fig. 6C).
|
In another series of experiments, we examined the field potentials
produced in the LDT by high-frequency stimulation. Results from these
experiments indicated that substantial direct activation of LDT neurons
occurred for the stimulus strengths used to evoke detectable
electrochemical signals (Fig. 7). A
short-latency, graded field potential (1.6 mV, maximum amplitude; 1.15 ms, width at half-maximum amplitude; n = 2 slices) was
evoked with stimulus currents that ranged from 0.3 to 0.8 mA (Fig.
7A). The amplitude of this field potential decreased
monotonically as the interval between the stimuli was shortened without
evidence for paired-pulse facilitation (Fig. 7B) as
expected if this field potential resulted from synchronized firing of
LDT neurons rather than synaptic currents. Although the field potential
was reduced by ~50% at a pulse interval of 10 ms, the field
potential followed a stimulus frequency of 100 Hz (Fig. 7C).
The field potential was insensitive to bath superfusion of 10 µM CNQX
(Fig. 7, D and E), which always attenuated EPSPs
(Sanchez and Leonard 1994) and indicated that the field was not synaptic. Intracellular recordings verified that local electrical stimulation could directly fire LDT cells (Fig. 7, F and G). Collectively, these results indicate
that the electrically evoked electrochemical signal resulted from
direct activation of LDT neurons.
|
L-Arginine increased the duration of elevated [NO] in the LDT following electrical stimulation
The previous data indicate that electrical stimulation resulted in a prolonged activation of NOS in the LDT. We investigated the possibility that this long-duration response was limited by substrate availability. We compared the electrochemical signals evoked by electrical stimulation in normal Ringer solution to those evoked in a solution containing L-Arg. As illustrated in Fig. 8A, L-Arg (1 mM) did not effect the early peak but the plateau phase was increased in amplitude (181.1% of control; n = 4; P < 0.01) and the response duration (423.5 ± 39.8 s; n = 4) was longer than controls (157.1 ± 23.4 s; n = 14; P < 0.01). The prolonging of the plateau phase was reversed after washing out the L-Arg (Fig. 8B), suggesting that the plateau phase of the response was sensitive to the availability of L-Arg. Moreover, application of TTX (1 µM), which blocks voltage-gated Na+ channel-dependent action potentials, also abolished both the peak and the enhanced plateau responses (Fig. 8A, right), indicating that both phases of the response depends on the generation of TTX-sensitive currents.
|
NMDA evoked NO production in the LDT
Since NMDA receptor activation has been specifically linked to NO
production, we also examined the possibility that NMDA elicits NO
production in the LDT, which is known to contain synaptic NMDA receptors (Sanchez and Leonard 1994, 1996
). A 3-min
superfusion of Ringer containing 100 µM NMDA produced a reversible
increase in the electrochemical signal measured by a probe located
within the LDT (Fig. 9A). Such
an increase was also observed following local ejection of NMDA with a
patch pipette placed above the slice near the probe. In this
configuration, NMDA produced a dose-dependent increase in the signal
(Fig. 9B) with an average time to peak of 42.1 ± 14.9 s. Such signals were observed following NMDA application either locally (n = 8) or by bath superfusion
(n = 2) in 5/6 slices. These signals were reversibly
inhibited (64.6 ± 4.1%; n = 2) by L-NAME (1 mM; Fig. 9C), indicating that a
majority of the signal could be attributed to activation of NOS. Such
partial inhibition of NMDA-evoked NO signals has been reported
previously under similar recording conditions using a different
NO-selective probe (Iravani et al. 1998
). The average
maximal increase in [NO] produced by local NMDA application in the
LDT was 39 ± 14 nM (n = 8).
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DISCUSSION |
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We have used an NO-selective electrochemical microprobe to measure changes in extracellular [NO] evoked by local electrical and chemical stimulation in a brain-slice preparation of the guinea pig LDT nuclei. Our central finding is that direct electrical stimulation of the LDT triggers a long-lasting electrochemical signal that is attributable to an increase in local [NO]. Moreover, this signal was increased in duration by exogenous L-Arg and was abolished by blocking voltage-gated Na+ channels with TTX. These findings, along with the observation that NMDA application increased tissue [NO] within the LDT, support the hypothesis that the activity-dependent production of NO by mesopontine cholinergic neurons functions as a local paracrine signal in the control of behavioral state.
Technical consideration
An important consideration relates to the specificity of the
probe. Several lines of evidence indicate that the electrochemical signals recorded in these experiments arose from tissue concentration changes of authentic NO. First, the basis of these measurements was the
use of a porphyrinic sensor that has been previously shown to be highly
sensitive to NO (Malinski and Taha 1992). The probes used in our experiments were modified to reduce interference from molecules such as monoamines and their metabolites, which are expected
to be found in brain tissue. The degree of interference from these and
other possible interferants were previously quantified and these probes
were shown to be highly selective for NO (Mitchell and Michaelis
1998
). Second, the individual probes used in these experiments
were tested for interference from monoamines and experimental reagents,
including Hb and changes in pH, and calibrated for NO as described in
METHODS to verify their selectivity under the current
experimental conditions. Third, the electrochemical signals were
sensitive to L-NAME indicating that NOS activity was
necessary for the production of the signals. Fourth, the electrically
evoked signals were Ca2+ dependent and were
attenuated by reduced hemoglobin in a manner similar to signals
produced by exogenous NO. Finally, an electrochemical signal was
also evoked by NMDA application, indicating that it was not an artifact
of electrical stimulation. Collectively, these data strongly support
the view that the measured electrochemical signals arose from
endogenous NO.
Possible sources of NO
Our data indicate that the measured NO signals most likely arose
from the NOS-containing cells of the LDT. First, the probes were shown
histochemically to have been positioned among the NADPH-d-containing cells of the LDT, and local electrical stimulation evoked NO production at these measurement sites. Second, tissue superfusion and local pressure application of NMDA increased the [NO] at these LDT sites. Third, field potential measurements indicated that the electrical stimuli that evoked NO production also evoked firing of LDT neurons. Finally, electrical-stimulation-evoked NO signals were blocked by TTX
but not by CNQX and APV, which abolish fast excitatory synaptic input
to LDT neurons (Sanchez and Leonard 1994, 1996
). This
indicates that NO production was evoked by direct rather than by
synaptic activation of action potentials in NOS-containing neurons and
processes within the LDT.
Because nNOS is distributed throughout the cytoplasm of neurons, NO
might be produced in axons, terminals, dendrites, and somata.
Electrochemical measurements of NO in the molecular layer of the
cerebellum (Shibuki and Kimura 1997), the substance
gelatinosa of the spinal cord (Kimura et al. 1999
), and
dialysis measurements in the thalamus (Williams et al.
1997
), which is innervated by axons of NOS-containing
mesopontine cholinergic neurons, suggest that activity-dependent NO
production in these structures arises from axon terminals. However,
given the high density of NOS-containing somata and dendrites within
the LDT (Leonard et al. 1995a
), it is more likely that
the electrically evoked changes in [NO] measured here were generated
at these somatic and dendritic sites. The measurement of spike-evoked
changes of [Ca2+]i in
NOS-containing LDT cells supports this view because during repetitive
firing, somatodendritic
[Ca2+]i can readily
achieve the levels reported to activate NOS (Leonard et al.
2000
). This idea was further supported by our finding that NMDA
application, which produces large increases in somatodendritic [Ca2+] in NOS-containing LDT neurons
(Leonard et al. 2000
), also generated NO. Nevertheless,
the available evidence does not exclude the possibility that at least
some of the measured NO arose from NOS-containing axon terminals within
the LDT, especially because some of these axons are local collaterals
of the NOS-containing LDT neurons (Surkis et al. 1996
).
Furthermore, the possibility that some measured NO arose from blood
vessel endothelial cells cannot be ruled out, although this seems
unlikely, at least for NMDA receptor mediated NO production because
cerebrovascular microvessel endothelial cells appear to lack functional
glutamate receptors (Morley et al. 1998
).
NMDA receptor control of NO production
The entry of Ca2+ through NMDA receptors has
been suggested to be a key control mechanism for neuronal NO synthesis
(Garthwaite 1991). Both indirect methods used to infer
the production of NO such as bioassays (Garthwaite et al.
1988
), cGMP assays (DeVente et al. 1990
;
East and Garthwaite 1991
; Garthwaite et al.
1988
; Morris et al. 1994
),
[3H] citrulline assays (Kiedrowski et
al. 1992
; Toms and Roberts 1994
), and brain
dialyses (Luo et al. 1993
; Shintani et al.
1994
), and direct measures of NO (Desvignes et al.
1997
; Iravani et al. 1998
) indicate that NMDA
receptor activation stimulates NO production in the brain. The recent
finding that NMDA receptor blockade inhibits ~50% of the
electrically evoked increase in extracellular NO in layer 5 of auditory
cortex (Wakatsuki et al. 1998
) further indicates that
activation of synaptic NMDA receptors can stimulate NO
production in the CNS. Our finding that NMDA application increased
extracellular NO in the LDT provides additional direct support for the
general idea that NMDA receptor activation can stimulate NO production in the CNS. Nevertheless, our finding that electrically evoked NO
production did not depend on NMDA receptor activation indicates that
Ca2+-dependent NO production can also result from other
mechanisms such as the firing of TTX-sensitive action potentials.
In some neurons, nNOS appears to be selectively regulated by NMDA
receptor-evoked Ca2+ entry (Kiedrowski et
al. 1992). This may be mediated by the association of nNOS with
NMDA receptors and by their mutual targeting to postsynaptic densities
(Brenman et al. 1996
; Christopherson et al.
1999
) and spines (Aoki et al. 1998
) where
Ca2+ influx through NMDA receptors mediates large
increases in local [Ca2+] (Yuste et al.
1999
). However, the degree of functional compartmentalization in neurons remains unclear. The most intensely NOS-immunoreactive neurons in neocortex and those in the LDT are aspiney or sparsely spiney and have nNOS label distributed throughout their cytoplasm (Leonard et al. 1995a
; Vincent and Kimura
1992
). Elevation of cytoplasmic Ca2+ by
several different pathways might activate nNOS. Our
Ca2+ imaging studies from NOS-containing LDT
neurons indicate that even weak activation of NMDA receptors stimulates
large somatodendritic [Ca2+] transients
mediated by action potential-activated voltage-gated Ca2+ channels (Leonard et al.
2000
) rather than by Ca2+ influx through
NMDA receptors. Thus it seems unlikely that the observed NMDA-evoked
changes in [NO] could be mediated by a restricted influx of
Ca2+ through NMDA receptors. It is more likely
that Ca2+ influx through voltage-gated
Ca2+ channels played a common role in both the
NMDA- and electrically evoked NO production we have measured. Perhaps,
subthreshold activation of NMDA receptors produces the highly localized
NO signals necessary for synapse-selective plasticity (Schuman
and Madison 1994
) while suprathreshold activation recruits NOS
activity throughout the cytoplasm for a more general paracrine
signaling function.
Kinetics of tissue [NO] changes produced by electrical stimulation
Electrical stimulation resulted in an average peak increase of
33 ± 2 nM, which is comparable to peak concentration changes reported in the molecular layer of the cerebellum following white matter stimulation (Shibuki and Okada 1991). Considering
that NO is produced at sites removed from the immediate surroundings of
the microprobe surface and that NO can react with tissue and molecular
oxygen (Taha et al. 1992
), the measured [NO] was
undoubtedly lower than the actual [NO] at the synaptic sites
surrounding NO-producing cells.
The onset of the NO signal following electrical stimulation was evident
before the complete decay of the stimulus artifact, and the average
time to peak was 4.8 ± 0.4 s (n = 14), which
was comparable to the times of under five seconds reported for NO signals evoked by electrical stimulation in the molecular layer of the
cerebellum (Shibuki and Kimura 1997) and cerebral cortex (Wakatsuki et al. 1998
). Because our NO probes have
response times of ~0.5 s (Mitchell and Michaelis 1998
)
and our integration time was 0.5 s, the time to peak accurately
reflects the time course of the concentration change of extracellular
NO. This indicates that the dynamics of signals carried by
extracellular NO would be quite slow compared with conventional
synaptic mechanisms.
The time course of the electrically evoked [NO] transients often had
a form consisting of a peak followed by a plateau lasting over a
minute. In contrast to our findings, the [NO] in the molecular layer
of the cerebellum is reported to declined to baseline within 10 s,
following either white matter or molecular layer stimulation (Shibuki and Kimura 1997; Shibuki and Okada
1991
). While this may reflect methodological differences,
another consideration is the source of NO in these two studies. In the
LDT, measured NO appears generated mainly by the somatodendritic pool
of NOS while in the molecular layer, NO appears generated by NOS in the parallel fibers (see Shibuki and Kimura 1997
). It is
possible the differences in decay time course reflect a differential
regulation of these two pools of NOS. One possibility is that the
somatodendritic pool of nNOS remains activated by the residual
elevation of [Ca2+]i
which declines only slowly (
= 134 s) after repetitive
firing (Leonard et al. 2000
). Of course, other factors
might also have contributed to the plateau response and include a
secondary activation of NOS by other cellular messengers released
during electrical stimulation and/or the direct alteration of NOS
activity through interactions of its numerous, but poorly understood,
intracellular modulators (for review, see Michel and Feron
1997
). Further work will be necessary to clarify the underlying
mechanisms of these plateau responses.
Functional implications of NO produced by mesopontine cholinergic neurons
A role for NO in the control of behavioral state was initially
suggested from the effects of central L-NAME administration (Kapas et al. 1994). Several lines of evidence now
indicate that NO production by NOS-containing cholinergic neurons of
the LDT and PPT plays a role in this control. Mesopontine cholinergic neurons provide extensive NOS-containing input to the thalamus (Bickford et al. 1993
) and thalamic NO levels vary
according to behavioral state with the highest levels during waking and
REM sleep (Williams et al. 1997
). The action of this NO
appears to enhance visual and somatosensory activity (Cudeiro et
al. 1994a
,b
; Do et al. 1994
), perhaps by
shifting the voltage dependence of the H-current in thalamic relay
neurons (Pape and Mager 1992
).
Evidence for local NO actions within the mesopontine tegmentum also
supports a role for NO in behavioral state control. Potential targets
of local NO action include the locus coeruleus (LC) where NOS
inhibitors enhance EPSPs (Xu et al. 1994) and where NO
donors produce a cGMP-dependent depolarization (Pineda et al.
1996
). These neurons are closely adjacent to LDT in rat and are
extensively interdigitated among LDT neurons in the guinea pig and
other species including human (see discussion in Leonard et al.
1995a
), suggesting that they might be influenced by the
somatodendritic production of NO by mesopontine cholinergic neurons.
Mesopontine cholinergic neurons, themselves, may also be targets of
their own NO production. NO donor compounds influence NOS-containing
LDT cells in two ways (Leonard et al. 1995b). First, they reduce NMDA receptor-mediated excitation, which on average comprises ~10% of the excitatory synaptic current at
60 mV
(Sanchez and Leonard 1996
). Second, exogenous NO
inhibits EPSPs in NOS-containing LDT cells. Thus NO generated by
mesopontine cholinergic neurons could function as an inhibitory
feedback pathway by reducing excitatory synaptic input to the LDT.
Strong feedback control of this system is also mediated by the
postsynaptic inhibitory action of Ach on autoreceptors (Leonard
and Llinás 1994
; Luebke et al. 1993
). Interestingly, NO may also enhance this cholinergic feedback inhibition by promoting Ach release as suggested by the finding that NOS inhibition in the cat PPT reduces the local release of Ach
(Leonard and Lydic 1997
).
Although the specific roles need to be further elucidated, NO actions
at LDT and nearby sites appear to be functionally important in REM
sleep. NOS inhibitors applied to the cat PPT (Datta et al.
1997) and medial pontine reticular formation (Leonard
and Lydic 1997
) reduced REM sleep amounts. Microinjection of
NOS inhibitors in the closely adjacent dorsal raphe nucleus reduced REM
sleep in rat (Burlet et al. 1999
) and a consolidation of
REM episodes has been reported following local injection of
L-NAME into the mPRF (Okabe et al. 1998
).
In conclusion, our data indicate that neural activity evoked by
electrical stimulation and NMDA receptor activation increases extracellular [NO] within the LDT. This implies that NO can act as a
paracrine signal whose production is linked to the suprathreshold activity of LDT neurons. Given the evidence from single-unit studies (El Mansari et al. 1989; Kayama et al.
1992
; Koyama et al. 1998
; Steriade et al.
1990
) indicating that putative NOS-containing cells of
the LDT fire at their highest rates during waking and REM sleep
(~10-20 spikes/s in rodent) and are either quiescent or fire at low
rates during slow-wave sleep, our results and the work discussed in the
preceding text, support the hypothesis that NO functions as a local
paracrine signal within the mesopontine tegmentum in the control of
behavioral state.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Sophie Burlet for critical comments on the manuscript, B. Taylor for excellent technical assistance, and Dr. S. Rao for assistance in some of the experiments.
This study was supported by National Institutes of Health Grants NS-27881 and HL-64150 to C. S. Leonard, AG-12993 and AA-04732 to E. K. Michaelis, and NS-37777 to K. M. Mitchell, who also received a grant from the Marion Merrell Dow Scientific Education Partnership.
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FOOTNOTES |
---|
Address for reprint requests: C. S. Leonard, Dept. of Physiology, New York Medical College, Basic Science Building, Valhalla, NY 10595 (E-mail: chris_leonard{at}nymc.edu).
Received 5 February 2001; accepted in final form 25 July 2001.
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REFERENCES |
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