Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Jiang, M. C., L. Liu, and G. F. Gebhart. Cellular properties of lateral spinal nucleus neurons in the rat L6-S1 spinal cord. Conventional intracellular recordings were made from 26 lateral spinal nucleus (LSN) neurons in slices of L6-S1 spinal cord from 10- to 15-day-old rats. At rest, LSN neurons did not fire spontaneous action potentials. With injection of a positive current pulse, action potentials had an amplitude of 72 ± 7 (SD) mV and duration at half-peak height of 0.75 ± 0.22 ms. Action potentials were followed by an afterpotential. Most LSN neurons (13/17) exhibited only an afterhyperpolarization (AHP); four neurons exhibited both a fast and a slow AHP separated by an afterdepolarization (ADP). For LSN neurons that exhibited only an AHP, a slow ADP could be identified during bath application of apamin (100 nM). Four of 11 LSN neurons showed a postinhibitory rebound (PIR). Two types of PIR were noted, one with high threshold and low amplitude and the other with low threshold and high amplitude. The PIR with high amplitude was partially blocked in 0 mM Ca2+/high Mg2+ (10 mM) recording solution. Repetitive firing properties were examined in 17 LSN neurons. On the basis of the ratio of the slopes between initial instantaneous firing and steady-state firing frequencies, neurons with low spike frequency adaptation (SFA, 8/17) and high SFA (4/17) were identified. In addition, 2/17 LSN neurons exhibited biphasic repetitive firing patterns, which were composed of a fast SFA, delayed excitation, and low SFA; another two neurons showed only delayed excitation. Plateau potentials also were found in two LSN neurons. Dorsal root stimulation revealed that most LSN neurons (12/13) had polysynaptic postsynaptic potentials (PSP); only one neuron exhibited a monosynaptic PSP. Electrical stimulation of the dorsal root evoked prolonged discharges in low SFA neurons and a short discharge in high SFA neurons. Intrinsic properties were modulated by bath application of substance P (SP). Membrane potentials were depolarized in all eight LSN neurons tested, and membrane resistance was either increased (n = 3) or decreased (n = 2). Both instantaneous firing and steady-state firing were facilitated by SP. In addition, oscillation of membrane potentials were induced in three LSN neurons. These results demonstrate that LSN neurons exhibit a variety of intrinsic properties, which may significantly contribute to sensory processing, including nociceptive processing.
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INTRODUCTION |
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Located ventrolateral to the superficial spinal
dorsal horn, the lateral spinal nucleus (LSN) first was described by
Gwyn and Waldron (1968, 1969
). Morphological studies
found ascending projections of LSN neurons to a wide variety of
supraspinal sites, including the thalamus and lateral periaqueductal
gray (PAG) (Battaglia and Rustioni 1992
; Harmann
et al. 1988
), the hypothalamus and telencephalon
(Burstein et al. 1987
), the amygdala and orbital cortex
(Burstein and Potrebic 1993
), the tractus solitarius
nucleus (Esteves et al. 1993
), and the parabrachial
nucleus (Ding et al. 1995
; Feil and Herbert
1995
). LSN neurons also receive descending projections from the
raphe nuclei, brain stem reticular formation nuclei, dorsal column
nuclei, and PAG (Carlton et al. 1985
; Masson et
al. 1991
). In addition, LSN neurons can be activated by
peripheral mechanical stimulation (Menetrey et al.
1980
).
LSN neurons project to neurons in spinal lamina I, II, V, and VII
(Jansen and Loewy 1997) and receive peptidergic input
from local spinal cord neurons (Cliffer et al. 1988
).
Using immunochemical techniques, LSN neurons were found to contain a
variety of peptide receptors, including substance P (Battaglia
and Rustioni 1992
; Ding et al. 1995
; Li
et al. 1997
; Marshall et al. 1996
), neuropeptide Y (Zhang et al. 1995
), and kappa-opioid receptors
(Schafer et al. 1994
). LSN neurons also were found to
contain peptides, such as calcitonin gene-related peptide
(Conrath et al. 1989
) and vasoactive intestinal
polypeptide (Fuji et al. 1983
; Leah et al.
1988
; Sasek et al. 1991
). A significant property
of LSN neurons (Leah et al. 1988
) is that their axons
that project to supraspinal sites contain the highest percentage of
neuropeptides. The foregoing suggests that the LSN may be involved in
the transmission and modulation of afferent input, including
nociception. In support of this, Herdegen et al. (1994)
reported expression of nitric oxide synthase in LSN neurons after
noxious stimulation of the rat hindpaw with formalin.
The behavior and properties of LSN neurons, however, have received
little attention. For example, limited information is available about
their receptive fields (Menetrey et al. 1980) and
synaptic input and intrinsic properties of LSN neurons, which are
important factors in determining neuron firing behavior
(Lopez-Garcia and King 1994
; McCormick
1990
; McCormick et al. 1992
; Thomson et
al. 1989
), have not been described. In spinal dorsal horn
neurons, synaptic responses can be divided into monosynaptic and
polysynaptic excitatory and inhibitory postsynaptic potentials (EPSPs
and IPSPs) (Inokuchi et al. 1992
; Jiang et al.
1995
; King et al. 1988
; Thomson et al.
1989
; Yajiri et al. 1997
; Yoshimura and
Jessell 1989b
). Dorsal horn neurons also express a variety of
intrinsic properties, such as repetitive firing patterns, delayed
excitation, plateau potentials, and oscillation (Hochman et al.
1994
; Jiang et al. 1995
; Lopez-Garcia and
King 1994
; Murase and Randic 1983
;
Yoshimura and Jessell 1989a
). A good correlation between
firing patterns evoked by synaptic input and intrinsic properties was
found in superficial and deep spinal dorsal horn neurons
(Lopez-Garcia and King 1994
; Thomson et al.
1989
). In addition, intrinsic properties can be modulated by
activation of excitatory amino acid or peptide receptors
(Hochman et al. 1994
; Morisset and Nagy
1996
; Russo et al. 1997
), which then change
neuron excitability. Accordingly, the goals of these experiments were
to examine the intrinsic properties of LSN neurons and their synaptic
response to afferent input and to study modulation of these properties
by substance P (SP). Parts of this work have been presented in abstract
form (Jiang et al. 1997
).
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METHODS |
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Slice preparation
Rat pups of both sexes aged postnatal days 10-15 were used. Under ether anesthesia, body temperature was reduced by immersing the pup below the cervical level into an ice-water pool. The dissection began when skin temperature fell to 20-22°C (~5-10 min) and respiration became shallow. A laminectomy was performed to expose the lower-thoracic and lumbosacral spinal cord. A block of lumbosacral spinal cord (~8-10 mm) with attached dorsal roots was excised quickly and placed in oxygenated (95% O2-5% CO2) Ringer solution, which contained (in mM): 124 NaCl, 5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSo4, 26 NaHCO3, and 10 glucose, pH 7.4. The pia matter on the spinal cord surface was removed using surgical forceps and the spinal cord was trimmed manually to a 4-to 5-mm lumbosacral block. The block was placed in a Plexiglass cutting chamber in an Oxford vibratome and fixed to an agar block with cyanoacrylic glue. The chamber was filled with aerated Ringer solution and maintained at 24°C. The spinal cord block was sectioned to yield several transverse slices of 300-400 µm with short (3-4 mm) dorsal rootlets; two to four slices from the L6-S1 region (middle of the block) were collected and removed to an incubation chamber. The procedure from removal of the spinal cord block to sectioning of slices for recording was performed in 4-5 min. The slices were incubated in Ringer solution at 30-31°C for ~1 h before recording.
Intracellular recording
Slices were transferred to a recording chamber, placed on a
nylon mesh, and perfused with oxygenated Ringer solution (95% O2-5% CO2) at rate of 2.5-3 ml/min. Using an
inverted microscope, the LSN was identified as a semitransparent
triangle lateral and ventral to the substantia gelatinosa.
Intracellular recordings were performed using conventional glass
electrodes with impedances of 100-120 M when filled with 2 M
potassium acetate and 2% neurobiotin. The electrode was driven by a
micromanipulator (Newport, Irvine, CA), which was controlled by a pulse
generator, in steps of 2-4 µm. To assist penetration into a neuron,
a positive current pulse (20 nA, 100 ms in duration) was passed through
the electrode or excessive capacitance compensation was applied for 5 ms. Immediately after a neuron was impaled, hyperpolarizing current was
applied to eliminate spontaneous firing. The hyperpolarizing current
was gradually withdrawn during 1-5 min, and stable intracellular
recordings could be maintained for 0.5-4 h.
Electrical activity of LSN neurons was amplified using an Axoclamp-2A (Axon Instruments, Burlingame, CA) in bridge mode and recorded on video tape. Data were collected both on-line and off-line; off-line analysis employed VTX (Keithley, Taunton, MA) and Visual Basic programming software. To generate synaptic input, a co-axial stainless steel stimulating electrode was positioned on the dorsal root. Intrinsic properties of LSN neurons were studied by intracellular injection of either positive or negative current pulses with various durations and intensities.
Intracellular staining
Recording electrodes contained 2% neurobiotin (Vector, Burlingame, CA). At the end of experiments, a 1-nA positive current was injected through the electrode (200-ms pulse duration at 3 Hz for 10 min). Slices were fixed in 4% paraformaldehyde and 0.2% picric acid in a 0.1 M sodium phosphate buffer solution (PBS, pH 7.4) overnight at 4°C. The tissue was sectioned into 40 µm slices and transferred to PBS containing 0.5% Triton-X on a shaker for 2 h. After two PBS washes, the slices were treated with avidin conjugate (avidin-biotin-horseradish peroxidase complex, diluted 1,000 times in PBS) for 2 h on a shaker. Afterward, the slices were washed and treated with 0.05% diaminobenzidine and 0.003% H2O2 in PBS. The sections were mounted on gelatin-coated slides, dried, defatted, and coverslipped.
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RESULTS |
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Under an inverted microscope, the LSN appears as a semitransparent
region ventrolateral to the substantia gelatinosa. Figure 1 shows the location of the LSN and a
neuron labeled with neurobiotin in the upper part of the LSN. LSN
neurons having a resting membrane potential more negative than 55 mV,
action potentials of more than 60 mV amplitude, and overshoot were
considered healthy and subjected to the experimental protocols. All
reported values are mean ± 1 SD.
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Action potentials and afterpotentials
The average resting membrane potential of the 26 LSN neurons
studied was 71 ± 6.2 mV. At rest, LSN neurons did not fire action potentials. Thus a positive pulse (10 ms) was injected into LSN neurons
to generate action potentials. Characteristics of these LSN neurons are
summarized in Table 1. Most LSN neurons
(13/17) exhibited a monophasic afterhyperpolarization (AHP; Fig.
2A). The remaining four LSN
neurons exhibited compound afterpotentials: a fast and a slow AHP and a
afterdepolarization (ADP) (Fig. 2A). To examine the duration
and amplitude of afterpotentials, these 17 LSN neurons were injected
with 500-ms positive pulses or a positive DC current adjusted manually
to produce action potentials; we did not attempt to separate
afterpotentials in the four neurons with compound afterpotentials. The
amplitude and duration of the AHP are summarized in Table 1. It is
known that fast and slow AHPs are mediated through different channels
(Sah 1996). The absence of an ADP in most LSN neurons
made it impossible to distinguish AHP components. Thus we used apamin
(100 nM), a calcium-dependent potassium channel blocker that is
reported to block the slow AHP (Sah 1996
), to examine
the composition of AHPs. The results (Fig. 2B) showed that
the duration of AHP in four LSN neurons studied was reduced
significantly (control: 93.4 ± 26.3 ms; apamin: 30 ± 10 ms)
after bath application of apamin, during which an ADP became apparent
(peak at 42.5 ± 6.3 ms) (Fig. 2B), suggesting that the
apparent monophasic AHP in these neurons is conducted by multiple
channels.
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Current-voltage relationship
Current-voltage (I-V) relationships were examined
using hyperpolarizing pulses (300 ms) in 11 LSN neurons. The majority
of LSN neurons (7/11) exhibited a predominately linear I-V
relation, in which the average of four steady-state membrane potentials in response to the same intensity of intracellular stimulation was
taken and plotted against each current step (Fig.
3A). In four LSN neurons, a
time-dependent inward rectification was observed (Fig. 3B),
in which the initial hyperpolarization of membrane potentials in
response to negative pulses decayed to steady-state levels 200 ms after
onset of the current pulses. The I-V relationships (average
of 4 trials) were plotted as peak and steady-state membrane potentials
against each current step. Whereas the steady-state I-V plot
is characterized by an upward bend (Fig. 3B, ), the peak
I-V relationship is linear.
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Postinhibitory rebound
Postinhibitory rebound (PIR) is characterized by a transient
depolarization of the membrane potential at the end of current injection (Fig. 3B). Although PIR is relatively rare in LSN
neurons (4/11), two types of PIR were noted as shown in Fig.
4. One type of PIR (2/4 LSN neurons)
required a higher stimulating intensity to be activated (Fig.
4A). In contrast, a lower stimulating intensity was required
to activate PIR in two other LSN neurons (Fig. 4B). In
addition, burst-like firing could be produced in these two LSN neurons
by higher intensity current. Because Ca2+ current has been
reported to underlie burst firing (Huguenard 1996), we
applied a low Ca2+ (0 Ca2+ and 10 mM
Mg2+) bath solution to test whether Ca2+
current participates in PIR; PIR was blocked partially
by the low Ca2+ bath solution (Fig. 4C).
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Repetitive firing
Repetitive firing properties of LSN neurons were studied by injection of positive current pulses (3-s duration). The majority of LSN neurons studied (12/16) showed a quick onset of action potentials in response to current steps. Although firing frequency of these neurons increased with increase in current intensity, it adapted (spike frequency adaptation, SFA) during each current step. The LSN neurons studied can be divided into low SFA (n = 8) and high SFA (n = 4) neurons (Fig. 5Ab) according to the ratio of frequency slopes (Fig. 5Aa). High SFA neurons fired action potentials that adapted quickly (Fig. 5Ab), whereas low SFA neurons continued firing action potentials during current pulses with little adaptation (Fig. 5Aa).
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Other types of repetitive firing also were observed in LSN neurons. Two LSN neurons apparently showed a delayed onset of action potentials (delayed excitation, DE) at the beginning of current injection (Fig. 6A). Unlike LSN neurons that adapted during stimulation, the firing rate of these neurons increased during current injection. In addition, another two LSN neurons exhibited a mix of SFA and DE (Fig. 6B). In these two neurons, current pulses with low intensities produced a gap in action potentials between the initial and steady-state firing. As the current intensity increased, the gap was filled with firing that had the lowest rate during the current pulses. Prolonged repetitive firing was observed after termination of the current pulse in another two LSN neurons (Fig. 6C), which was apparently due to a prolonged depolarization of membrane potential after the positive pulses (plateau potential). One of these neurons also showed a DE (Fig. 6Cb).
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Synaptic responses
Synaptic responses were examined in 13 LSN neurons by electrical stimulation of an attached dorsal root. For subthreshold postsynaptic responses, most LSN neurons (12/13) gave polysynaptic potentials; one neuron exhibited an apparent monosynaptic postsynaptic potential (Fig. 7A). For high-intensity stimulation, either short (n = 5) or prolonged firing (n = 7) was produced in these LSN neurons. A correlation between intrinsic firing property and synaptic input-induced firing was examined in eight LSN neurons. It was found that LSN neurons with low SFA (n = 3) had prolonged firing (Fig. 7B), whereas LSN neurons with high SFA (n = 3) had short firing (Fig. 7C) in response to electrical stimulation. However, the two LSN neurons with mixed repetitive firing properties (initial SFA and DE) responded to electrical stimulation with short firing (data not shown).
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Effects of SP on cellular properties
The effect of SP was examined in eight LSN neurons. Similar to
neurons in the spinal dorsal horn (Murase et al. 1989),
SP dose-dependently depolarized all eight LSN neurons (for
10
7 M: 3.61 ± 1.12 mV, 4.2 ± 1.7 min; for
10
6 M: 5.79 ± 1.42 mV, 5.3 ± 2.1 min; for
10
5 M: 8.64 ± 2.23 mV, 6.5 ± 1.8 min).
Membrane resistance showed an increase (n = 3) from 20 to 33% and decrease (n = 2) from 16 to 24% in
response to SP bath application at different concentrations. Figure
8A shows that SP
dose-dependently depolarized membrane potential and increased membrane
impedance in a LSN neuron. To examine the effect of SP on repetitive
firing properties, the membrane potential of LSN neurons was held
manually at control levels by injecting a hyperpolarizing current. The
results from five LSN neurons studied showed that both the
instantaneous and steady-state firing were all facilitated by SP and
the effect lasted ~4 min (Fig. 8B). In three of eight LSN
neurons, rhythmic changes in membrane potentials were produced by SP
application (frequency range: 0.08-0.16 Hz, Fig. 8C).
Depending on the resting membrane potential, the neurons either only
expressed oscillation of membrane potential (Fig. 8Ca) or
fired action potentials at the peak of depolarization (Fig.
8Cb).
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DISCUSSION |
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LSN neurons have been found to receive peripheral input and to
have reciprocal connections with supraspinal sites (Burstein et
al. 1987; Feil and Herbert 1995
; Harmann
et al. 1988
; Masson et al. 1991
; Menetrey
et al. 1980
), suggesting that the LSN may participate in
sensory processing, including nociception. To better understand the
role of the LSN in sensory processing, the intrinsic cellular
properties and the synaptic responses of LSN neurons were examined
here. LSN neurons in segments L6-S1 in the rat
spinal cord were found to exhibit a variety of cellular properties:
inward rectification, PIRs, low and high SFAs, DE, biphasic firing, and SP-induced oscillation. It also was demonstrated that the majority of
LSN neurons receive polysynaptic input, consistent with the absence of
evidence for direct input via the dorsal roots, and exhibit a strong
correlation between SFA properties and responses produced by synaptic
input. Thus the cellular properties likely contribute to the mechanisms
by which LSN neurons process synaptic input. In addition, we found in
the small sample studied that cellular properties of LSN neurons could
be modified by SP.
Cellular properties
The passive membrane properties, membrane resistances, and time
constants reflect whole membrane channels and cell size. Depending on
animal age, location, and the experimental preparation, passive membrane properties can vary significantly. The membrane resistance (mean 78.5 M, range 37-167 M
) of LSN neurons is similar to
membrane resistances of deep dorsal neurons, both in in vitro slice
(54.5 M
, range 11-138 M
) (King et al. 1988
) and
in vivo adult rat preparations (mean 38 M
, range 14-141 M
)
(Jiang et al. 1995
). However, the membrane resistance of
LSN neurons differed from those of superficial dorsal horn neurons,
both in adult rat slice [257 ± 17.7 M
(Yoshimura and
Jessell 1989a
) and 241 ± 12 M
(Yoshimura and
Jessell 1989b
)] and immature rat slice preparations (48-267 M
) (Murase and Randic 1983
). The membrane time
constant (mean 10.6 ms, range 5.6-21.1 ms) in LSN neurons was also
similar to that of deep dorsal horn neurons (mean 9.1 ms, range
1.8-19.7 ms) (Jiang et al. 1995
). The time constants
reported in superficial dorsal horn neurons differ depending on the
animal and the preparation. Whereas the membrane time constants of
superficial dorsal horn neurons in an in vivo cat preparation ranged
between 0.8 and 2.0 ms (Iggo et al. 1988
), they were
21.3 ± 1.9 ms in an in vitro rat slice preparation
(Yoshimura and Jessell 1989b
). This difference may be
due to the different animal species used in these experiments or the
different experimental preparation. Nevertheless the similarities in
passive membrane properties between LSN neurons and deep dorsal horn
neurons suggest that these two groups of neurons may have similar size.
The mean amplitude of action potentials (72.3 ± 6.6 mV) and
resting membrane potentials (71.0 ± 6.2 mV) of LSN neurons were also similar to those of deep dorsal horn neurons (amplitude of action
potentials, 77 ± 11.8; resting membrane potentials,
67 ± 8 mV) (King et al. 1988
). However, the resting membrane
potentials of LSN neurons differ from those found in an in vivo adult
rat preparation (
60.9 ± 3.9 mV) (Jiang et al.
1995
), which may be due to the different experimental
preparations. The mean width of the action potential at half-amplitude
of LSN neurons (0.75 ± 0.22 ms) was also different from that
determined in the in vivo adult preparation (0.33 ± 0.15), which
may be due to age because the animals used in this study were immature.
The conflicting results regarding the width of action potentials in the
young rat slice preparation [1.4 ± 0.5 ms (King et al.
1988
); 0.82 ms (Thomson et al. 1989
)] also may
arise from different locations of the neurons studied or different
experimental conditions.
Afterpotentials are important in shaping neuron firing patterns
(Fulton and Walton 1986; Gorelova and Reiner
1996
; Llinas and Yarom 1981
). A variety of
afterpotentials in spinal cord neurons have been reported in different
animal preparations (Jiang et al. 1995
; King et
al. 1988
; Thomson et al. 1989
; Yoshimura
and Jessell 1989b
), including fast AHP, slow AHP, fast ADP, and
slow ADP. The mean duration and amplitude of afterpotentials in this study (duration: 93.4 ± 26.3 ms; amplitude: 8.1 ± 2.4 mV)
were similar to those found in deep dorsal horn neurons (duration
range, 14-268 ms; amplitude range, 2-12.6 mV) (Jiang et al.
1995
). It has been demonstrated that the slow AHP is mediated
by an apamin-sensitive Ca2+-dependent K+
channel (for review, see Sah 1996
). For neurons
displaying a mono-phasic AHP in this study, the duration of the AHP was
reduced and an ADP was unmasked after bath application of apamin. This indicates that multiple ion channels underlie the monophasic AHP in LSN
neurons. It has been shown that inhibition of the slow AHP can either
increase or decrease SFA based on the locations of neurons
(Gorelova and Reiner 1996
; Spanswick et al.
1995
). It would be interesting to characterize the contribution
of the slow AHP to cellular excitability in LSN neurons.
Membrane rectification has been shown to participate in shaping
discharge patterns in spinal dorsal horn neurons (Yoshimura and
Jessell 1989a). Fast and time-dependent inward rectification, outward rectification, and linear I-V relations all were
observed in both superficial and deep dorsal horn neurons of the spinal cord (Jiang et al. 1995
; Yoshimura and Jessell
1989a
). It appears that the I-V relationship in LSN
neurons is relatively simple. Most LSN neurons (7/11) exhibited a
linear I-V relation; some (4/11) showed a time-dependent
inward rectification.
PIR transiently increases neuron excitability by quickly depolarizing
membrane potential after hyperpolarization, by which action potentials
can be triggered depending on the magnitude of PIR. PIR has been
observed throughout the CNS (Dekin 1993; Johnson
and Getting 1991
; Stewart and Wong 1993
),
including both the superficial and deep dorsal horn in spinal cord
(Jiang et al. 1995
; Lopez-Garcia and King
1994
; Yoshimura and Jessell 1989a
). It has been
shown that PIR can be blocked partially by Cs+
(Yoshimura and Jessell 1989a
). In the present study, two
types of PIR, low and high threshold, were observed. In low-threshold PIR neurons, burst-like firing also was observed at higher intensity stimulation. It was noted that bath application of low Ca2+
solution switched the low-threshold PIR to high-threshold PIR, in which
burst-like firing also disappeared. High-threshold PIR in the present
study, on the other hand, is similar to that reported by
Yoshimura and Jessell (1989a)
. These results suggest
that there are at least two membrane currents, K+ and
Ca2+, participating in PIR.
The plateau potential is a prolonged membrane depolarization after the
release from a depolarizing pulse, which can trigger action potentials.
It has been demonstrated that the plateau potential participates in
synaptic integration (Russo and Hounsgaard 1996) and
contributes to the mechanism of wind-up (Russo and Hounsgaard 1994
; Russo et al. 1997
). In the current study,
a plateau potential was observed in only 2/16 LSN neurons, which is
less than reported for deep dorsal horn neurons (Morisset and
Nagy 1996
). However, because the plateau potential can be
induced and enhanced by SP and/or
cis-(±)-1-aminocyclopentane-1,3-dicarboxylic acid
(Russo et al. 1997
), and LSN neurons contain receptors
for SP (Battaglia and Rustioni 1992
; Ding et al.
1995
; Li et al. 1997
; Marshall et al.
1996
), whether plateau potentials can be induced in LSN neurons
awaits further investigation.
SFA has been reported in both superficial and deep dorsal horn neurons
(Jiang et al. 1995; Thomson et al. 1989
;
Yoshimura and Jessell 1989a
). In LSN neurons, SFA could
be divided clearly into high- and low-adapting groups based on the
ratio of the slopes between initial instantaneous firing and
steady-state firing. A unique, biphasic SFA was observed in two LSN
neurons in the present study. These two neurons appeared to have a
combination of high SFA and delayed excitation (but adapted in firing
frequency). In the majority of LSN neurons tested, the firing behavior
evoked by synaptic input correlated well with SFA (low and high SFA). Similar to neurons in the dorsal horn (Lopez-Garcia and King
1994
; Thomson et al. 1989
), the low SFA neurons
in the present study had prolonged discharges, whereas the high SFA
neurons fired briefly to electrical stimulation. In the two biphasic
firing neurons, synaptic input only produced a few discharges.
Effect of SP on cellular properties
SP is well documented to participate in nociceptive mechanisms
(for reviews, see Henry 1982; Jessell
1981
; also Moochhala and Sawynok 1984
). It has
been shown that SP has multiple effects on membrane currents
(Adams et al. 1983
; Dun and Minota 1981
; Murase et al. 1989
; Stanfield et al.
1985
) and on intrinsic properties (Russo et al.
1997
) of spinal dorsal horn neurons. These include depolarizing
the membrane potential, changing membrane resistance, modulating
plateau potential, and increasing neuronal excitability. In the present
study, similar findings were made on eight LSN neurons after bath
application of SP (10
7 M to 10
5 M). The SP
effects lasted for 4.5-6.8 min depending on SP concentration. In all
eight LSN neurons studied, SP depolarized membrane potential and
increased neuron excitability by increasing initial instantaneous and
steady-state firing frequencies. Membrane resistance was increased in
three or decreased in two LSN neurons by SP, which also has been
observed by other investigators (Krnjevic 1977
;
Murase and Randic 1984
; Murase et al.
1982
; Russo et al. 1997
). The effect of SP was
suggested by Murase et al. (1989)
to be due to the
balance between the voltage-sensitive Ca2+ current and the
voltage-insensitive, Ca2+-sensitive cationic conductance.
In addition, we observed that SP could cause rhythmic changes in
membrane potential (oscillation) in three LSN neurons. Oscillation of
membrane potential has been reported in spinal cord neurons, the
occurrence of which can be a consequence of intrinsic mechanisms (Hochman et al. 1994; Jiang et al.
1995
), N-methyl-D-aspartate receptor
activation (Hochman et al. 1994
) or neural network
mechanisms (Sandkühler and Eblen-Zajjur 1994
).
Synaptic input
Extracellular recordings of LSN neurons by Menetrey et al.
(1980) documented that a small population of LSN neurons can be activated by peripheral mechanical stimulation and that the receptive fields for these neurons are large. We found in the present study that
all LSN neurons studied responded to electrical stimulation of dorsal
roots and that most of them (11/12) had polysynaptic input. Once
activated, the LSN could contribute to nociceptive modulation due to
its high concentration of nociception-related peptides and its synaptic
connection with other brain structures.
Conclusion
The current study of rat LSN neurons in the
L6-S1 spinal segments in an in vitro slice
preparation has revealed a diversity of intrinsic cellular properties
and polysynaptic communication in the LSN. The results also suggest
that while intrinsic properties could control firing behavior of LSN
neurons, which is supported by the correlation between synaptic evoked
firing and SFA, synaptically released neuromodulators such as SP are
able to modulate these intrinsic properties. This suggests that the LSN
is able to participate in sensory processing, including nociception.
However, as it has been shown that synaptic activities of LSN neurons
are less responsive to natural stimulation (Menetrey et al.
1980) than electrical stimulation of the dorsal root, the role
of LSN in sensory processing may be specifically important in certain
conditions, such as hyperalgesia.
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ACKNOWLEDGMENTS |
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The authors thank S. Birely for secretarial assistance and M. Burcham for preparation of the figures.
This work was supported by National Institute of Neurologcial Disorders and Stroke Grant NS-19912.
Present address of M. C. Jiang: Dept. of Physiology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611.
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FOOTNOTES |
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Address for reprint requests: G. F. Gebhart, Dept. of Pharmacology, Bowen Science Bldg., University of Iowa, Iowa City, IA 52242.
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 16 November 1998; accepted in final form 8 February 1999.
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