Department of Physiology, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
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Leonard, Christopher S., Sanjai R. Rao, and Takafumi Inoue. Serotonergic Inhibition of Action Potential Evoked Calcium Transients in NOS-Containing Mesopontine Cholinergic Neurons. J. Neurophysiol. 84: 1558-1572, 2000. Nitric oxide synthase (NOS)-containing mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT) are hypothesized to drive the behavioral states of waking and REM sleep through a tonic increase in firing rate which begins before and is maintained throughout these states. In principle, increased firing could elevate intracellular calcium levels and regulate numerous cellular processes including excitability, gene expression, and the activity of neuronal NOS in a state-dependent manner. We investigated whether repetitive firing, evoked by current injection and N-methyl-D-aspartate (NMDA) receptor activation, produces somatic and proximal dendritic [Ca2+]i transients and whether these transients are modulated by serotonin, a transmitter thought to play a critical role in regulating the state-dependent firing of MPCh neurons. [Ca2+]i was monitored optically from neurons filled with Ca2+ indicators in guinea pig brain slices while measuring membrane potential with sharp microelectrodes or patch pipettes. Neither hyperpolarizing current steps nor subthreshold depolarizing steps altered [Ca2+]i. In contrast, suprathreshold currents caused large and rapid increases in [Ca2+]i that were related to firing rate. TTX (1 µM) strongly attenuated this relation. Addition of tetraethylammonium (TEA, 20 mM), which resulted in Ca2+ spiking on depolarization, restored the change in [Ca2+]i to pre-TTX levels. Suprathreshold doses of NMDA also produced increases in [Ca2+]i that were reduced by up to 60% by TTX. Application of 5-HT, which hyperpolarized LDT neurons without detectable changes in [Ca2+]i, suppressed both current- and NMDA-evoked increases in [Ca2+]i by reducing the number of evoked spikes and by inhibiting spike-evoked Ca2+ transients by ~40% in the soma and proximal dendrites. This inhibition was accompanied by a subtle increase in the spike repolarization rate and a decrease in spike width, as expected for inhibition of high-threshold Ca2+ currents in these neurons. NADPH-diaphorase histochemistry confirmed that recorded cells were NOS-containing. These findings indicate the prime role of action potentials in elevating [Ca2+]i in NOS-containing MPCh neurons. Moreover, they demonstrate that serotonin can inhibit somatic and proximal dendritic [Ca2+]i increases both indirectly by reducing firing rate and directly by decreasing the spike-evoked transients. Functionally, these data suggest that spike-evoked Ca2+ signals in MPCh neurons should be largest during REM sleep when serotonin inputs are expected to be lowest even if equivalent firing rates are reached during waking. Such Ca2+ signals may function to trigger Ca2+-dependent processes including cfos expression and nitric oxide production in a REM-specific manner.
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INTRODUCTION |
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The change in concentration of cytoplasmic free
calcium regulates a remarkable array of cellular processes, including
ion channel activity, receptor-effector coupling, enzyme activity, secretion, neurite elongation, differentiation, and gene expression (for review see Ghosh and Greenberg 1995). In neurons,
voltage-gated and ligand-gated ion channels can effect large, rapid,
and spatially discrete increases in cytoplasmic
Ca2+ levels which can be evoked by both
subthreshold (Eilers et al. 1995
; Markram and
Sakmann 1994
) and suprathreshold (Jaffe et al. 1992
; Tank et al. 1988
) membrane depolarization.
Mammalian central neurons differ substantially in their morphology and
electrophysiological properties (Llinas 1988
), and
therefore patterns of intracellular Ca2+ signaling are likely to vary
across neuronal classes. Little is known about the regulation of
[Ca2+]i by mesopontine
cholinergic (MPCh) neurons of the laterodorsal tegmental (LDT) nucleus,
although it is expected that the dynamics of this process is of
particular importance since virtually all of these cells express high
levels of the Ca2+/calmodulin-dependent enzyme
neuronal nitric oxide synthase (NOS) (Bredt et al. 1991
;
Hope et al. 1991
) throughout their cytoplasm. NOS
catalyzes the synthesis of nitric oxide (NO; for review see Mayer 1995
), which is a mobile, membrane-permeable
molecule that has been implicated as an intercellular signal in
numerous physiological processes including vasodilation, synaptic
plasticity, modulation of cell excitability, and neurotoxicity (for
review see Garthwaite and Boulton 1995
). Since the
synthesis of NO by NOS has an absolute requirement for elevated
[Ca2+], the regulation of
[Ca2+ ]i in MPCh neurons
is expected to play a critical role in regulating the spatial and
temporal patterns of NO signaling by these neurons. Indeed, recent
findings indicate that the Ca2+-dependent
production of NO can be stimulated both within the LDT (Mitchell
et al. 1995
) and at the synaptic targets of MPCh neurons in the
thalamus (Williams et al. 1997
) and the pontine tegmentum (Leonard and Lydic 1997
).
Functionally, MPCh neurons are hypothesized to play a pivotal
role in generating the electroencephalographic (EEG)-desynchronized states of waking and REM sleep via their
projections to the thalamus and medial pontine reticular formation (for
review see Steriade and McCarley 1990). They begin to
increase their firing rate seconds prior to the expression of these
states and then maintain an elevated level of tonic firing for the
duration of the states (El Mansari et al. 1989
;
Kayama et al. 1992
; Steriade et al.
1990
). We have investigated whether physiological rates of
repetitive firing engender Ca2+ transients in the
somata and proximal dendrites of MPCh neurons.
It has recently been established that excitatory synaptic input to MPCh
neurons utilizes a mixture of
N-methyl-D-aspartate (NMDA) and non-NMDA
subtypes of excitatory amino acid receptors (Sanchez and Leonard
1994, 1996
). NMDA receptor activation can lead to increases in
intracellular [Ca2+] (MacDermott et al.
1986
), can trigger Ca2+-dependent changes
in gene expression (Bading et al. 1995
; Xia et
al. 1996
), and can stimulate NO production in cerebellar
granule cells (Garthwaite et al. 1988
). We, therefore,
investigated the possibility that activation of NMDA receptors could
elevate somatic [Ca2+]i
in MPCh neurons.
Finally, serotonin (5-HT) is an important transmitter in the control of
behavioral state (for review see Steriade and McCarley 1990). Its actions in the CNS are mediated by a large number of receptors, some of which (5-HT1c and
5-HT2) can stimulate phosphoinositide hydrolysis
and the mobilization of intracellular Ca2+ (for
review see Julius 1991
). MPCh neurons are strongly
inhibited by exogenous 5-HT (Leonard and Llinás
1994
; Luebke et al. 1992
), apparently through
activation of 5-HT1a receptors which increases an
inwardly rectifying K+ current. These neurons
have also been reported to colocalize 5-HT2
receptor immunoreactivity (Morilak and Ciaranello 1993
) We, therefore, also investigated whether 5-HT application triggers or
modulates activity-dependent Ca2+ transients in
MPCh neurons.
Our findings indicate that physiological rates of repetitive firing
effectively generate somatic and proximal dendritic
Ca2+ transients while subthreshold depolarization
produced by DC injections or NMDA application did not. Moreover, we
found that while 5-HT did not detectably alter resting
[Ca2+ ]i, it did suppress
activity-dependent changes in
[Ca2+]i by inhibiting
action potential occurrence and by reducing the Ca2+ transients evoked by individual action
potentials. Some of these data have been presented in preliminary form
(Leonard and Rao 1995; Leonard et al.
1995
).
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METHODS |
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Slice preparation, solutions, and drugs
Brain slices used for sharp electrode recordings (350-400 µm)
were prepared according to previously published methods (Leonard and Llinás 1994) in accordance with the National
Institutes of Health policy on humane care and use of
laboratory animals. Female guinea pigs (175-300 g) were deeply
anesthetized with pentobarbital sodium (50-75 mg/kg, ip) and
decapitated. Coronal slices were cut in an ice-cold artificial
cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5 KCl, 1.2 NaH2PO4, 2.7 CaCl2, 3 MgSO4, 26 NaHCO3, 10 glucose and saturated with 95%
O2-5% CO2 using a
vibrating microtome (Ted Pella Instruments). After at least 1 h of incubation in ACSF at room temperature, slices were submerged in a
recording chamber and superfused with ACSF at room temperature for recording.
Brain slices used for whole-cell patch recordings were prepared from female guinea pigs as indicated above except that following induction of deep anesthesia, animals were perfused through the heart with the ice-cold ACSF (10-20 mL) prior to decapitation. Slices (300 µm) were cut using a vibrating blade microtome (VT1000S, Leica) and were then incubated at 35°C for 15-30 min prior to returning the solution to room temperature. After at least 1 h, slices were submerged in a recording chamber that was perfused at 3-5 mL/min with ACSF maintained at 22 ± 1°C.
5-HT, TTX (Sigma), and NMDA (Research Biochemicals) were dissolved in ACSF for superfusion or in ultrapure water for focal pressure application. Focal application was accomplished with controlled pressure pulses (Picospritzer; General Valve) applied to a patch pipette (1-2 µm opening) positioned just above the slice.
Sharp microelectrode recording and imaging
Sharp borosilicate (cat. No. 6030, AM-systems) micropipettes
were pulled horizontally (Sutter Instruments, P87) and filled with the
potassium salt of either Calcium Green-1 (2-3 mM), Fura-2 (10-14 mM),
or Fluo-3 (2-3 mM; Molecular Probes) dissolved in 2 M KCl. In some
cases, 1-2% biocytin (Sigma) was included for cell visualization
following histochemistry (Leonard and Llinás 1994;
Sanchez and Leonard 1994
). Micropipettes had DC
resistances of 80-120 M
. Cells were injected for 10-30 min with
0.2 to
0.4 nA current pulses (2 Hz, 60% duty cycle). Membrane
potential was recorded using an Axoclamp-2A (Axon Instruments) operated
in bridge mode. Pulses were delivered and voltages were recorded using
PCLAMP software (Axon Instruments). Current, voltage, and shutter
timing pulses were recorded on a Neurocorder (Neurodata) having a
bandwidth of 0-11 KHz/channel.
Fura-2 was excited by a 100-W Hg source that was reduced two to
fourfold by neutral density filters and filtered at 380 ± 5 nm.
Fluo-3 and Calcium Green-1 were excited at 480 ± 20 nm by the Hg
or a 75-W Xe source (reduced 2-4 fold). Illumination was limited to
acquisition time by shuttering. Neurons were imaged through a 10×
objective (Nikon; 0.3 NA) with an intensified (Gensys 2) charge coupled
device camera (Dage 72) and acquired at 1-4 Hz with a frame
grabber (Data Cube) controlled by software (Ratiotool; Innovision)
running on a workstation (Sun). Each image acquired was an average of
four to eight video frames. No significant photobleaching or
phototoxicity was detected during recordings lasting more than 1 h
under these conditions. The slice chamber and micromanipulators were
mounted on a three-plate microscope stage so that the image field could
be shifted while maintaining cell impalement. Background images were
acquired just prior to the experiment from an adjacent image field
lacking filled cell processes for subtraction from data images to
compensate for tissue autofluorescence and shading. Fluorescence was
measured as the average pixel value over selected somatic and proximal
dendritic regions from background-subtracted images. Fluorescence
versus time data and electrophysiology data were imported into Igor Pro
software (Wavemetrics, Lake Oswego, OR) running on a Macintosh computer
for off-line analysis. Data are reported as mean ± SE.
Comparisons of means were accomplished with paired t-tests
corrected for multiple comparisons, where appropriate, using DataDesk
Software (Data Description, Ithaca, NY). Intracellular
[Ca2+] was monitored by computing
F/F from the measured fluorescence according
to
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Whole-cell recording and imaging
Patch pipettes (4-8 M) were fabricated from Corning
7052 glass and filled with a solution containing (in mM) 144 K-gluconate, 0.1 Calcium Green-1, 3 MgCl2, 10 HEPES, 0.3 NaGTP, 4 Na2ATP. Neurons were visualized for whole-cell
recording at 100× magnification by visible-light differential
interference contrast optics, using a nuvicon tube camera (Dage
VE-1000) mounted on a fixed-stage microscope (Olympus BX50WI). Cells
for recording were chosen from within the bounds of the LDT nucleus
which was identified using a 4× objective. Gigaseals were obtained
under visual control using an Axoclamp 2A amplifier (Axon Instruments)
operated in continuous voltage-clamp mode to monitor seal resistance
with the output filter set at 3 KHz. After establishing the whole-cell
recording configuration, cells were filled with Calcium Green-1 by
diffusion from the pipette. Previous experiments indicated that
solution exchange occurs with a time constant of about 6 min
(Sanchez et al. 1998
) so imaging experiments were not
begun until at least 20 min after the time of breakthrough. The
neuron's electrophysiological properties were characterized in this
initial period using the current-clamp mode of the amplifier with the
output filter set to 10 KHz. Current and voltage traces were digitized
(20-100 KHz) and command pulses were generated with custom software
(TI Workbench; Inoue et al. 1998
) run on a
Macintosh OS computer which controlled an ITC-18 interface
(Instrutech). Neurons were imaged through a 40× objective (Olympus
LumaPlan Fluor, 0.8 NA) using a cooled, CCD camera equipped with a
back-illuminated, EEV57 frame-transfer chip having an imaging area of
512 × 512 pixels (MicroMax, Roper Scientific). Calcium Green-1
was excited at 480 ± 20 nm with light from a 75-W Xe lamp that
was reduced in intensity with a neutral density filter (12%
transmittance) and shuttered to restrict tissue illumination to the
frame acquisition epochs. TI Workbench software also controlled the
camera and shutter, allowing precise synchronization of the image
sequence with the electrophysiological data. The camera was read-out
with a 1 MHz., 14-bit A/D converter. Images were binned on the
chip at either 4 × 4 or 5 × 5 pixels and acquired every
35-50 ms. Changes in fluorescence were quantified by the average pixel
values within regions of interest (ROIs) that were positioned on
background-subtracted fluorescence images as described above. For
whole-cell experiments, the background was determined from an ROI which
was positioned at a location remote from any fluorescent processes.
Spike shape analysis
Action potential shape was measured using Igor Pro Software. Spike amplitude was determined as the difference between the peak and the baseline potentials. Because spikes were evoked by 2-ms-duration current pulses, current artifacts sometimes contaminated the rising phase of the action potentials making it impossible to measure spike width. We, therefore, used the time from the spike peak to the time of the maximum repolarization rate as an estimate of spike width. The maximum repolarization rate was used to characterize the speed of spike repolarization.
Histochemistry
Slices containing biocytin-filled cells were processed for
NADPH-diaphorase as previously reported (Leonard and
Llinás 1994; Sanchez and Leonard 1994
).
Injected cells were labeled with avidin-Texas Red and NOS-containing
cells were labeled by NADPH-diaphorase histochemistry. Injected neurons
were imaged with a confocal microscope (Zeiss LSM 310) using a HeNe
laser (543 nm) to visualize Texas-Red fluorescence. The transmitted
light from the same laser was used to visualize the NADPH-diaphorase
reaction product.
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RESULTS |
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Repetitive firing evoked by current injection produced somadendritic Ca2+ transients
All neurons studied with sharp electrodes (n = 21) had stable resting potentials (55.1 ± 2.6 mV),
overshooting action potentials, and displayed the characteristic
electrophysiological properties of the principal NOS-containing cells
found in guinea pig LDT (Fig. 1,
A1-A3)
which have been termed type II neurons (Leonard and Llinás
1990
, 1994
). These cells express a prominent sub-threshold A
current (Sanchez et al. 1998
), which characteristically
produces a delay in the return of the membrane potential to baseline
following a hyperpolarizing current pulse (Fig.
1A2). These cells also lacked low-threshold Ca2+ spikes. Following injection of
the indicator dye, somata were readily visible, and in some cases
(n = 4), proximal dendrites were also observed with the
intensifier and camera gains used to measure somatic fluorescence (Fig.
1B1, arrows). Direct current injection
sufficient to evoke action potentials also produced changes in cell
fluorescence in both the soma and proximal dendrites (middle
panel Fig. 1, B1 and
B2).
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To estimate the time course of the fluorescence changes and the
relation between steady-state firing and steady-state
Ca2+ fluorescence, we utilized a paradigm
consisting of 10-s-duration current pulses separated by 50-70 s. This
allowed the firing rate to reach a steady value after spike frequency
accommodation was complete and also allowed for a sufficient number of
fluorescence samples during cell firing since our imaging rate was
limited to between 1 and 4 per second. This is illustrated for another type II LDT neuron in Fig. 2. The change
in fluorescence (F/F) was computed from a
small region selected over the soma. A stable baseline measure was
established after first having electrophysiologically characterized the
cell and injecting it with indicator dye. Membrane hyperpolarization
produced no measurable change in fluorescence (Fig. 2A,
0.05 nA; 2B, left panel) suggesting that
voltage-dependent Ca2+ currents contribute little
to the resting Ca2+ levels in these cells, unlike
hippocampal pyramidal neurons (Magee et al. 1996
). In
contrast, a just-suprathreshold depolarization, which produced a firing
rate of 0.5 spikes/s (averaged over 10 s), evoked an increase in
fluorescence that appeared correlated with individual action potentials
(Fig. 2A, 0.02 nA; 2B, middle panel).
Increasing the strength of the current pulses evoked more spikes and
larger increases in
F/F (Fig. 2A
and 2B, right). At spike frequencies higher than
the imaging rate,
F/F appeared to increase and
decrease with a smooth time course that was well fit by an exponential
function. The decay time constant was measured for different amplitude
transients from six cells. Since the time constant did not strongly
depend on the amplitude of the transient, the data were pooled. Decay
time constants ranged from 2.5 to 9.2 s with an average value of
5.0 ± 0.24 s (n = 77 from 6 cells). It was
also apparent from these data that in addition to the rapid rise and
fall of
F/F which accompanied current
injection, there was a slow increase in
F/F
that built up over the entire sequence of current injections (Fig.
2A). Following termination of the last current pulse, this
accumulated signal decayed very slowly. To estimate the time course,
the
F/F signal following the final current
pulse was fit with a double exponential function. The average slow time
constant was 134.4 ± 19.5 s (n = 5 from 5 cells), which was significantly longer than the average fast time
constant (P < 0.0001).
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Current-evoked Ca2+ transients were TTX sensitive
To determine if the Ca2+ influx observed
during DC injection was related to the genesis of action potentials, we
examined the effect of TTX on the evoked changes in fluorescence.
Superfusion of the slice with Ringer containing TTX (1 µM) completely
abolished action potentials (n = 3) and strongly
reduced the current-induced increase in fluorescence (Fig.
3, A and B).
Moreover, the relation between the magnitude of the evoked
F/F and the strength of the injected current
was attenuated by spike blockade over the entire range of currents
tested (Fig. 3D). Application of the potassium channel
blocker tetraethylammonium (TEA, 20 mM) resulted in the production of
Ca2+ spikes following DC injection (Fig.
3C). The occurrence of Ca2+ spikes
restored the magnitude of the current-induced
F/F to the levels achieved prior to
Na+ channel blockade. Indeed, as few as two
Ca2+ spikes evoked nearly maximal increases in
fluorescence (Fig. 3D). These data indicate that the
majority of Ca2+ signal resulting from the
current-evoked increases in
F/F resulted from
activation of high-voltage activated Ca2+
channels.
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F/F is related to firing rate
The steady-state relation between the F/F
and the firing rate was estimated by comparing the change in
F/F to the total number of spikes elicited
during each current pulse. The number of spikes evoked during the 10-s
current injection was linearly related to current strength over the
range of currents studied and is summarized for six type II cells in
Fig. 4A. Comparing the peak
F/F to the total number of spikes revealed
that this relation was well approximated by a straight line for average firing rates below about 2 spikes/s and then began to saturate at
higher firing rates (Fig. 4B). A linear fit of the data
below 2 spikes/s indicated that cells ranged in sensitivity between 7.5 and 19.2%
F/F/spike/s with a mean of
15.6 ± 1.8%
F/F/spike/s (n = 6). These data indicate that action potentials
were very effective at triggering somatic
[Ca2+] transients. Indeed, even low firing
rates averaging 1.3 ± 0.27 spikes/s resulted in average increases
in
F/F of 25.4 ± 0.46% (n = 6).
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NMDA receptor activation produced somatic Ca2+ transients that were TTX sensitive
Application of NMDA also produced membrane depolarization and
triggered increases in somatic [Ca2+]. Both
superfusion and local pressure-ejection caused robust increases in
somatic F/F (n = 10) which
appeared to depend on the generation of action potentials. For example,
NMDA produced dose-dependent increases in
F/F
(Fig. 5, A1-6);
however, subthreshold doses produced only marginal increases (Fig. 5,
A1 and A2) and suprathreshold doses produced
increases that lagged the depolarization onset, but closely followed
spiking onset (See Fig. 5A3). The NMDA-evoked
Ca2+ transients were mimicked by DC injection
(Fig. 3, B6 and B7), suggesting that action
potentials were primarily responsible for triggering these
Ca2+ transients. This was confirmed by comparing
the effects of identical NMDA applications before and after TTX
blockade of action potentials which resulted in a 65.4 ± 8.6%
reduction in the peak
F/F (n = 4; compare Fig. 5A6 and B1). Nevertheless, the
residual increase in
F/F evoked by NMDA
following TTX was not completely mimicked by direct depolarization
(Fig. 5, B1 and B2), suggesting that a small
portion of the increase might be mediated by Ca2+
influx through NMDA receptor channels.
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5-HT inhibited current- and NMDA-evoked calcium transients
Neither superfusion (100 µM; n = 2) nor local
pressure-ejection of 5-HT (n = 5) had any detectable
effects on baseline F/F, although both
produced strong membrane hyperpolarization as previously reported
(Leonard and Llinás 1994
; Luebke et al.
1992
). In contrast, even brief 5-HT application effectively and
reversibly reduced Ca2+ transients evoked by
suprathreshold constant-current stimulation (Fig.
6A), apparently by suppressing
the number of evoked action potentials. This conclusion was supported
by experiments in which the effect of halting steady firing with either
5-HT or DC current was compared. Under these conditions, 5-HT was no
more effective at decreasing
F/F than was DC
(Fig. 6B).
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5-HT also effectively reduced somatic Ca2+
transients evoked by NMDA (n = 3). By preceding NMDA
application with a pulse of 5-HT, the magnitude and time course of the
NMDA-evoked F/F was dramatically reduced (Fig.
7A). As with DC, however, the
suppressive effect of 5-HT appeared related to membrane
hyperpolarization and the reduction in the number of action potentials.
Results from experiments in which DC was used to mimic the effect of
5-HT supported this conclusion (Fig. 7B). In this case, two
different doses of NMDA were applied at two membrane potentials. At a
membrane potential of
70 mV, the NMDA-evoked depolarizations were
ineffective at generating somatic Ca2+ transients
since the smaller dose was subthreshold and the larger dose produced
only a single spike (Fig. 7, B1 and B2). However, the same pair of doses resulted in dramatically larger
Ca2+ transients when delivered from resting
potential (
60 mV) since both responses were suprathreshold (Fig. 7,
B3 and B4). This effect of steady
hyperpolarization was mimicked by 5-HT. When the same pair of NMDA
doses was preceded by 5-HT application, which hyperpolarized the
membrane by about 5 mV, the Ca2+ transients were
suppressed. The smaller NMDA dose evoked only a subthreshold
depolarization and no Ca2+ signal, while the
larger dose evoked an intermediate number of spikes and an intermediate
Ca2+ signal (Fig. 7B5).
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5-HT inhibited action potential-evoked calcium transients
The previous data demonstrate that 5-HT reduces activity-dependent
calcium signals in LDT neurons by reducing the number of action
potentials fired in response to a given stimulus. To determine if 5-HT
also reduced the Ca2+ transients produced by
individual action potentials, we conducted whole-cell recording
experiments with simultaneous high-speed imaging in which action
potentials were evoked by a train of five brief current pulses (2 ms).
Pulses were delivered at 1-10 Hz following a 100-ms hyperpolarizing
current pulse which was used to estimate input resistance.
Sixteen type II neurons were patched under DIC optics and
filled with 100 µM Ca-Green-1 dissolved in patch solution (Fig.
8A).
Sufficient data concerning spike-evoked changes in
F/F and spike shape were obtained for six
cells from six slices each of which underwent one to three 5-HT
applications (total of 13). Since the results from each application
were qualitatively similar, the data were pooled. These neurons all had
resting potentials more negative than
50 mV and large overshooting
action potentials (mean amplitude: 91.11 ± 2.0 mV). As suggested
from the results obtained with sharp electrodes, individual action
potentials produced significant increases in
F/F measured from the soma and proximal dendrites (Fig. 8B1). Each spike
produced a transient increase in
F/F which
rose more rapidly than it decayed, such that
F/F summated over the time course of the five
stimuli. Spike-evoked fluorescence transients were routinely observed
in the proximal dendrites and were completely absent when spike-failure
occurred (data not shown). These dendritic transients were typically
larger and faster decaying than those measured at the soma. Since the data were qualitatively similar for each of the one or two dendritic branches recorded per cell and the recording distances from the soma
were similar (24.8 ± 2.1 µm; n = 11), dendritic
data were averaged for each cell.
|
As expected, application of 5-HT produced a reversible membrane
hyperpolarization (7.17 ± 1.00 mV) and large decrease in apparent input resistance (51.98 ± 7.12%; n = 13). These
membrane changes typically resulted in spike failure and required
increased current strength to overcome. Suprathreshold current pulses
delivered during 5-HT application revealed that the spike-evoked
increases in F/F were smaller than in control
conditions (compare Fig. 8B1 to
8C1). Moreover, this reduction
occurred throughout the soma and proximal dendrites as can be seen by
comparing images obtained at the peak of the spike-evoked fluorescent
transients (dotted line in Fig. 8B1 to
8C1) before and during 5-HT
application (compare Fig. 8B2 to
8C2). To measure the time course of
these effects, we tracked the peak
F/F evoked
by five spikes along with the membrane potential and input resistance
before, during, and after 5-HT application. We also measured the
maximum spike-evoked
F/F elicited from a
hyperpolarized membrane potential (
dc;
10.91 ± 1.14 mV) in
the absence of 5-HT and from the control potential during 5-HT
application (+dc) to examine the effect of membrane potential on the
spike-evoked Ca2+ transients. Results indicated that the
5-HT effects were reversible and that the inhibition of spike-evoked
F/F evolved with a time course similar to that
for Vm and input resistance following 5-HT application (Fig. 8D). The spike-evoked fluorescence
transients for each condition are superimposed in Fig. 8E
for a somatic and dendritic region (lowercase letters indicate the
corresponding condition in Fig. 8D). These data illustrate
the reduction in the spike-evoked Ca2+ transients
produced by 5-HT and indicate that this reduction was greater than any
change produced by altering baseline potential. Moreover, 5-HT
inhibition of the spike-evoked
F/F was not
associated with gross changes in spike amplitude or shape (Fig.
8E, bottom). Similar changes were observed for
each of the other cells studied. The group data show that while there
was no statistical difference between spike-evoked changes in
F/F measured at baseline and hyperpolarized
membrane potentials, 5-HT reduced the spike-evoked
F/F to 57 ± 1.4 and 68.7 ± 5.3%
of the control values measured at the soma and dendrites, respectively
(Fig. 9A). Moreover, even when
the membrane was depolarized back to baseline during the 5-HT
application, the spike-evoked changes in
F/F
were significantly smaller than control. The
F/F data and statistical differences for these
groups are summarized in Table 1.
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While the action potentials in the control and 5-HT conditions appeared
grossly similar, we examined them in more detail to determine if there
were any subtle changes in spike shape that might correspond to the
changes in F/F produced by 5-HT. We first examined the possibility that 5-HT produced smaller amplitude action
potentials. Membrane hyperpolarization alone produced a small but
significant reduction (3.4 mV) in the peak value of the action
potential (33.8 ± 1.7 vs. 30.5 ± 2.0 mV; n = 20; P < 0.01). However, comparing action potential
peaks evoked during 5-HT application to those evoked from a
hyperpolarized membrane potential in the absence of 5-HT revealed no
statistical difference (P = 0.48; n = 30). This finding indicates that serotonergic inhibition of the
spike-evoked Ca transients could not result from inadequate depolarization of the soma during the action potential.
Since action potentials of MPCh neurons become narrower following
Ca2+-channel blockade with extracellular
Co2+ or Cd2+
(Leonard and Llinás 1990), we examined two
parameters of spike repolarization: the maximum repolarization rate and
the time from the action potential peak to the time of the maximum
repolarization rate. Membrane hyperpolarization alone did not produce a
statistically meaningful increase in the maximum spike repolarization
rate (
49.2 ± 1.8 mV/ms; n = 40 vs.
52.0 ± 2.4 mV/ms; n = 35; P > 0.08); however, it did significantly shorten the time from the peak to maximum
repolarization rate from 728.6 ± 31.6 µs (n = 40) to 674.8 ± 42.6 µs (n = 35;
P < 0.004). Application of 5-HT increased the maximum
spike repolarization rate to
55.4 ± 1.8 mV/ms
(n = 35) which was significantly faster than observed
for spikes produced during membrane hyperpolarization alone
(P < 0.0001). Moreover, 5-HT reduced the time from the
peak to the maximum repolarization rate to 601.0 ± 22.0 µs
(n = 55), which was significantly shorter than that
observed for spikes during membrane hyperpolarization alone
(P < 0.0001). An example of these effects on spike
shape is illustrated in Fig. 9B and is summarized for the
entire sample in Fig. 9C. Thus 5-HT resulted in a speeding
of spike repolarization and a shortening of the action potential to a
degree larger than expected for membrane hyperpolarization alone. These
parameters all recovered upon washout of 5-HT.
Recorded cells were NADPH-diaphorase positive
Previous studies combining intracellular labeling and
histochemistry found that >90% of guinea pig LDT neurons displaying "type II" electrophysiological properties were NOS-containing since
they robustly stained for NADPH-diaphorase (Leonard and Llinás 1994; Sanchez and Leonard 1994
).
All of the cells investigated in this study had type II physiological
characteristics and therefore were likely to have been NOS-containing.
To verify that the studied cells were, in fact, NOS-containing, some
neurons were co-injected with biocytin (n = 5) and
processed by NADPH-diaphorase histochemistry. Cells were injected with
biocytin so that they could be visualized after processing using
avidin-Texas Red, which is resistant to quenching by the
histochemistry. As expected from their location and their physiological
properties, all three of the biocytin-injected cells that were
recovered were also NADPH-diaphorase containing (Fig.
10, A-C). These data
confirm that the described pattern of Ca2+
transient regulation was typical of NOS-containing MPCh neurons.
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DISCUSSION |
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A principal finding of this study is that TTX-sensitive action
potentials are of prime importance in mediating
[Ca2+] transients evoked by DC injection in
NOS-containing LDT neurons. Results from other central neurons indicate
that action potentials are variable in their ability to evoke somatic
[Ca2+] transients. High rates of "simple"
spiking by cerebellar Purkinje cells result in relatively small
increases in somatic and dendritic [Ca2+]
unless a Ca2+ spike is triggered
(Lev-Ram et al. 1992; Midtgaard et al.
1993
; Tank et al. 1988
). In hippocampal
pyramidal cells (Jaffe et al. 1992
), action potentials
are somewhat more effective at elevating somatic
[Ca2+]i. Our estimates
indicate that even moderate firing rates of ~4 spikes/s in MPCh
neurons result in increases of
F/F which averaged 48.4 ± 3.8% (n = 6). Based on the
assumptions described in the METHODS, this corresponds to a
concentration of ~200-250 nM and suggests that even moderate firing
rates produce substantial increases in somatic
[Ca2+]i in MPCh neurons.
Moreover, two factors suggest this is an underestimate of the peak
concentration changes achieved during repetitive firing. First, our
maximal imaging rate in the sharp electrode experiments was low, which
resulted in undersampling of the transients associated with each action
potential. Second, the cytoplasmic concentration of indicator dye was
probably high enough to significantly buffer entering calcium, thereby
diminishing the peaks and slowing the changes in
[Ca2+]i. Evidence for
buffering by the indicator was suggested by the transient decay time
constants, which were long compared with those observed with minimal
dye concentrations in other neurons (Helmchen et al.
1996
). This was true even in our whole-cell recording where the
indicator concentration was 100 µM. However, several other factors
including the rate of pumping from the cytoplasm and the capacity of
the endogenous calcium buffers may also contribute to this longer decay
and will need to be determined for these cells. Thus these data support
the idea that a relatively large somatic Ca2+
influx occurs with each action potential. To our knowledge, the only
other brain NOS-expressing central neurons for which spiking has been
related to [Ca2+]i are
immature cerebellar granule cells (Connor et al. 1987
), which differ from MPCh neurons morphologically and
electrophysiologically. Nevertheless, following strong depolarization
produced by raising [K+]o
to 25 mM, TTX blocked about 50% of the early
[Ca2+]i increase. Thus
substantial spike-mediated elevation of somatic [Ca2+] may be a general feature of
NOS-containing neurons.
In cases where we were able to image the dendrites along with the cell
body, we observed action potential-dependent changes in fluorescence.
These data indicate the presence of voltage-gated Ca2+ channels in the proximal dendrites. It is
likely that action potentials generated in the somata propagate into
the dendrites to open these channels as described in some other cell
types (for review see Johnston et al. 1996), but further
work will be necessary to verify where action potentials are initiated
in these neurons.
Another finding of this study was that NMDA-receptor activation
produced large and rapid somatic Ca2+ transients
which strongly depended on action potentials. In the presence of action
potential blockade by TTX, only small and slow somatic
[Ca2+] transients were observed even though
application of NMDA produced substantial somatic membrane
depolarizations. This observation suggests that the major route by
which the somatic [Ca2+]i
becomes elevated following NMDA receptor activation is through activation of high-threshold voltage-gated Ca2+
channels by action potentials. The Ca2+ influx
through the NMDA receptors expressed by these cells (Sanchez and
Leonard 1994, 1996
) was probably not detectable because it was
highly localized to the synapses on the soma (Honda and Semba 1995
) or in the dendrites, as suggested for pyramidal neurons (Miyakawa et al. 1992
), where Ca2+
influx through NMDA receptors appears localized to spines (Yuste et al. 1999
). Ca2+ influx through ion
channels can produce very localized concentration changes (Simon
and Llinas 1985
) and this influx could have important consequences for the spatial control of NO signaling. This topic warrants further investigation.
Another principal finding of this study was that 5-HT inhibited somatic
and dendritic [Ca2+]i
transients produced by depolarizing stimuli both by inhibiting action
potential production and by inhibiting the increase in [Ca2+]i produced by
individual action potentials. Serotonin has multiple actions on central
neurons including the mobilization of
[Ca2+]i which, among
other things, can activate Ca2+-dependent
K+ currents (Uneyama et al. 1993).
Our data, however, indicate that 5-HT produced a membrane
hyperpolarization without any measurable change in
F/F. This finding supports our earlier
conclusion that the 5-HT activated K+ current in
MPCh neurons is not Ca2+ dependent
(Leonard and Llinás 1994
). Thus if functional
5-HT2 receptors are present on the somata of MPCh
neurons (Morilak and Ciaranello 1993
), they do not
directly trigger changes in resting [Ca2+]i. Our findings
also indicate that 5-HT inhibits NMDA- and current-evoked increases in
somatic [Ca2+ ]i by
inhibiting action-potential generation. While this is an indirect
action on [Ca2+]i, it
demonstrates that 5-HT-mediated signals can be cross-coupled to
Ca2+-dependent processes via the modulation of firing.
Finally, our data demonstrate that 5-HT inhibits about 40% of the
action potential-triggered Ca2+ transient in the
soma and proximal dendrites of type II LDT neurons. This inhibition did
not result from membrane hyperpolarization or a reduction in amplitude
of the somatic action potential. Rather, the inhibition correlated with
a speeding of action potential repolarization and shortening of action
potential duration. These action potential changes produced by 5-HT
were small but qualitatively similar to the changes in spike shape
observed following Ca2+-channel blockade with
solutions containing low extracellular Ca2+ and
Cd2+ or Co2+
(Leonard and Llinás 1990). Considering that 5-HT
has been shown to inhibit high-voltage-activated
Ca2+ channels in several types of neurons
(Bayliss et al. 1995
, 1997
; Foehring
1996
; Penington and Kelly 1990
), it is likely
that the 5-HT effects on action potential shape and spike-evoked
Ca2+ transients result from inhibition of
voltage-gated Ca2+ channels. This interpretation
is bolstered by previous reports showing that high-threshold
Ca2+ spikes in type II MPCh neurons are
generated, in part, by N- rather than L-type Ca2+
channels (Takakusaki and Kitai 1997
) and that N- and
P/Q-type Ca2+ channels are inhibited by 5-HT
(Bayliss et al. 1997
; Penington et al.
1991
), while L- (Foehring 1996
) and T-type
(Bayliss et al. 1995
) Ca2+
channels are not. Nevertheless, other possibilities may also account
for or contribute to this inhibition and further experiments will be
necessary to fully resolve the underlying mechanisms.
An inhibition of spike-evoked Ca2+ transients by
5-HT has recently been reported for hippocampal CA1 pyramidal neurons
(Sandler and Ross 1999). In that study, 5-HT
hyperpolarized the soma and dendrites but did not reduce the peaks of
somatic action potentials (although the peaks of dendritic action
potentials were reduced). Since somatic spikes were unchanged, and
spike-evoked Ca2+-transients were inhibited by
~30% throughout the cell, the authors suggested that the inhibition
of spike-evoked Ca2+ transients resulted from
inhibition of voltage-gated Ca2+ channels. In
general, our findings agree well with that study. One difference was
that in MPCh neurons, 5-HT produced detectable changes in the shape of
somatic action potentials, while in CA1 neurons, the somatic action
potentials were reported to be unchanged by 5-HT. One possible
explanation for this difference is that different channel types or
different relative channel densities contribute to spike repolarization
in these two cell types. Indeed, such differences are evident from the
effect of blocking Ca2+ currents with
Cd2+ or Co2+ on the spike.
In type II MPCh neurons, the action potential becomes narrower
(Leonard and Llinás 1990
), while in CA1 pyramidal
neurons, the action potential broadens (Lancaster and Nicoll
1987
; Storm 1987
), apparently due to the
importance of BK-type Ca2+-activated
K+ channels in spike repolarization (Shao
et al. 1999
). Preliminary evidence from mouse MPCh neurons
suggests that BK channels contribute little to spike repolarization
since Iberiotoxin, which selectively blocks BK-type channels, has
negligible effect on action potential repolarization (Tyler et
al. 1999
). Thus it is possible that 5-HT produces a negligible
effect on spike shape in CA1 pyramidal neurons due to a reduction in
both the Ca2+ current and the
Ca2+-activated K+ current
which would cancel. Nevertheless, the collective evidence discussed
above indicates that a common action of 5-HT is to inhibit high-threshold Ca2+ currents which suppresses
spike-evoked increases in
[Ca2+]i.
Functional implications
Extracellular recordings of neural activity in regions containing
mesopontine cholinergic neurons have identified neurons whose firing
rates vary with behavioral state in cat and rat (El Mansari et
al. 1989; Kayama et al. 1992
; Steriade et
al. 1990
). One population of these neurons has higher firing
rates during waking and REM sleep than during slow-wave sleep and
another population fires selectively during REM sleep. Evidence that
the neurons that display these two firing patterns are cholinergic is
mostly indirect; however, a recent study using juxtaneuronal
applications of biocytin has provided strong support for this idea in
rat (Koyama et al. 1998
). These "presumed"
cholinergic neurons fire slowly, have broad action potentials
(Kayama et al. 1992
), and are inhibited by cholinergic
agonists in rat (Koyama and Kayama 1993
) and cat (Sakai and Koyama 1996
). In these ways, they resemble
the properties of mature identified mesopontine cholinergic neurons in
vitro (Leonard and Llinás 1994
). Our finding that
somatic [Ca2+]i is
related to the steady firing rate indicates that state-dependent firing
of mesopontine cholinergic neurons should lead to state-dependent fluctuations of [Ca2+]i.
Moreover, our finding that serotonin inhibits spike-evoked somadendritic Ca2+ -transients in these neurons
suggests that [Ca2+]i
would become most elevated by firing during REM sleep, when the
serotonergic input to MPCh neurons is expected to be lowest (for review
see Portas et al. 2000
). Such state-dependent increases in [Ca2+]i could then
function to control Ca2+-dependent cellular
processes such as gene expression and NO synthesis in a state-dependent manner.
Evidence that gene expression varies with behavioral-state in brainstem
regions containing mesopontine cholinergic neurons has come from
studies examining immunostaining for the product of the immediate early
gene cfos, which has been used extensively as a marker of
activity in the CNS (Hunt et al. 1987; Morgan et al. 1987
; Sagar et al. 1988
). Increased numbers
of cFos-labeled cells in these regions have been
found following periods of carbachol-induced REM sleep in cats
(Shiromani et al. 1992
; Yamuy et al.
1993
) and periods of natural REM rebound in rats
(Maloney et al. 1999
). While only a fraction of these
cFos-labeled cells were cholinergic, the number of
cholinergic neurons stained for cFos was also increased, and
it was increased in proportion to the percentage of time spent in REM
sleep following periods of REM deprivation (Maloney et al.
1999
). Since cfos transcription can be stimulated
through Ca2+-dependent signals (Morgan and
Curran 1986
) arising from activation of voltage-dependent
Ca2+ channels in neurons (Bading et al.
1993
), our observation that somatic
[Ca2+]i varies as a
function of steady firing, supports the hypothesis that state-dependent
changes in action potential frequency regulates the expression of
cfos, and presumably other genes, in mesopontine cholinergic
neurons. This observation also supports the interpretation that
increased cFos immunoreactivity reflects changes in action potential frequency in this system. This point was further supported by
our observation that both NMDA and 5-HT receptor activation, which
might have directly altered intracellular
[Ca2+]i, had their
primary influence on somatic
[Ca2+]i by altering
firing rate and/or spike-evoked Ca2+ transients.
Functionally, our findings also support the hypothesis that NO
production can be triggered by a spike-related increase in somatic
[Ca2+]i in MPCh neurons.
Although the Ca2+ dependence of NOS has not been
measured in situ, the purified enzyme is inactive at
[Ca2+] <80 nM and fully active at
concentrations >400-500 nM (Schmidt et al. 1992). We
found that even moderate rates of firing (~4 spikes/s) are estimated
to produced increases in average
[Ca2+]i sufficient to
activate the enzyme. During waking or REM sleep, putative MPCh neurons
in rat are reported to fire at rates of ~10 spikes/s (Kayama
et al. 1992
), which we estimate could produce Ca2+ transients >1 µM. Thus the somatic
Ca2+ levels achieved by physiological rates of
firing appear sufficient to stimulate somatic NOS activity. This is
supported by results from our experiments in which NO was
electrochemically detected within the LDT in response to local
electrical stimulation in brain slices (Mitchell et al.
1995
).
Conclusion
Our findings support the hypothesis that the increased repetitive firing of MPCh neurons associated with the behavioral states of waking and REM sleep evoke significant rises in soma-dendritic [Ca2+]i. Moreover, our data suggest that synaptic activation of NMDA receptors would mainly influence somatic [Ca2+]i by generating action potentials. In addition, we found that 5-HT reduces activity-dependent increases in [Ca2+]i both indirectly, by inhibiting action potential generation, and directly, by inhibiting spike-evoked Ca2+ transients, most likely by inhibiting voltage-gated Ca2+ channels. We therefore predict that the greatest increases in [Ca2+]i occur during REM sleep, when MPCh neurons fire at high rates and when the serotonergic input to LDT neurons is expected to be lowest.
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ACKNOWLEDGMENTS |
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We thank Dr. D. Sanes for numerous useful discussions, Dr. William Ross for comments on the manuscript, Dr. G. E. Stutzmann for participation in some experiments, and B. Taylor for excellent technical assistance.
This study was supported by National Institutes of Health Grants NS-27881 and HL-64150 to C. S. Leonard.
Present address of T. Inoue: Dept. of Molecular Neurobiology, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
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FOOTNOTES |
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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).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 July 1999; accepted in final form 30 May 2000.
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REFERENCES |
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