Department of Cell and Molecular Physiology and Curriculum in Neurobiology, University of North Carolina, Chapel Hill, North Carolina 27599-7545
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ABSTRACT |
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Light, Alan R. and
Helen H. Willcockson.
Spinal Laminae I-II Neurons in Rat Recorded In Vivo in Whole
Cell, Tight Seal Configuration: Properties and Opioid Responses.
J. Neurophysiol. 82: 3316-3326, 1999.
Using the in vivo whole cell recording procedure described previously,
we recorded 73 neurons in laminae I and II in the lumbar spinal cord of
the rat. Input impedances averaged 332 M, which indicated that prior
sharp electrode recordings contained a significant current shunt.
Characterization of the adequate stimuli from the excitatory hindlimb
receptive field indicated that 39 of 73 neurons were nociceptive, 6 were innocuous cooling cells, 20 responded maximally to brush, and 8 cells were not excited by stimulation of the skin of the hindlimb. The
locations of 15 neurons were marked with biocytin. Nociceptive neurons
were mostly found in lamina I and outer II, cooling cells in lamina I,
and innocuous mechanoreceptive cells were mostly found in inner II or
in the overlying white matter. The µ-opioid agonist
[D-Ala2, N-Me-Phe4,
Gly5-ol]-Enkephalin (DAMGO) hyperpolarized 7 of 19 tested
neurons with a conductance increase. This hyperpolarization was
reversed by naloxone in the neurons in which it was applied. DAMGO also decreased the frequency of spontaneous PSPs in 13 neurons, 7 of which
were also hyperpolarized by DAMGO. Five of the seven hyperpolarized neurons were nociceptive, responding to both heat and mechanically noxious stimuli, whereas two responded to slow, innocuous brush. These
results indicate that whole cell, tight seal recordings sample a
similar population of lamina I and II neurons in the rat as those found
with sharp electrode recordings in cat and monkey. They further
indicate that DAMGO hyperpolarizes a subset of the nociceptive neurons
that have input from both heat and mechanical nociceptors and that
presynaptic DAMGO effects can be observed in nociceptive neurons that
are not hyperpolarized by DAMGO.
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INTRODUCTION |
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Laminae I and II (the marginal zone and substantia
gelatinosa, respectively) of the spinal cord have been implicated as a primary integration center for pain processing and potent sites at
which opioid analgesics act. These laminae receive input from both A
and C primary afferent nociceptors and from A
and C innocuous thermal receptors and innocuous mechanoreceptors (e.g., reviewed in
Light 1992
; Light and Perl 1979
;
Réthelyi et al. 1982
; Sugiura et al. 1986
,
1993
).
The inputs that activate laminae I and II neurons and the anatomy of
the dendrites and axons of these neurons have been well defined in the
cat and the monkey (Bennett et al. 1980, 1981
; Christensen and Perl 1970
; Han et al.
1998
; Jones et al. 1990
; Kumazawa
et al. 1975
; Light and Kavookjian 1988
;
Light et al. 1979
, 1993
; Randic and Miletic
1978
; Réthelyi et al. 1989
). Thus neurons
in lamina I and outer lamina II (IIo) are usually excited by noxious stimuli applied to the appropriate receptive field, by
innocuous thermal stimuli, or by both types of inputs. Neurons in inner
lamina II (IIi) are usually excited by innocuous mechanical stimuli. However, this differentiation has not been documented in the rat.
Only a few intracellular and extracellular studies have documented the
input properties of neurons in lamina I and II in vivo in the rat with
most recording only from lamina I. Furthermore, the results in the rat
are inconsistent. Some investigators report neurons located above
lamina I that respond only to innocuous mechanoreceptive stimulation.
Some report neurons in and above lamina I that respond only to
innocuous cooling. Others report mostly multireceptive neurons in
lamina I and II and others describe mostly nociceptive specific neurons
in lamina I and II (Hope et al. 1990; Hylden et
al. 1989
; McMahon and Wall 1985
, 1988
;
McMahon et al. 1984
; Mokha et al. 1987
;
Woolf and Fitzgerald 1983
). In most cases neurons were
not intracellularly labeled, complicating localization of the neurons.
This is largely because of the difficulty of recording and labeling a
population of the very small neurons in laminae I and II of the rat.
In the rat the biophysical properties of neurons in the superficial
laminae and their response to opioids have been studied intensively in
vitro (Baba et al. 1994; Glaum et al.
1994
; Jeftinija 1988
; Jeftinija and Urban
1994
; Magnuson and Dickenson 1991
;
Miletic and Randic 1981
; North and Yoshimura
1984
; Rusin et al. 1993
; Yoshimura and
Jessell 1989
; Yoshimura and Nishi 1993
). These
studies have documented both pre- and postsynaptic mechanisms for the effects of opioids on laminae I and II neurons. The postsynaptic hyperpolarization in response to opioids is caused by the activation of
a G protein-coupled, inward-rectifying potassium channel. The observed
presynaptic effect is a decrease in spontaneous excitatory postsynaptic
potentials (EPSPs), presumably mediated by a decrease in free
intracellular calcium in presynaptic terminals.
The relevance of the in vitro effects to clinically effective doses of
opioids and the specificity of opioid inhibition for nociception are
still questioned. Spinal neurons studied in vitro have no peripheral
inputs and thus cannot be functionally identified as nociceptive using
adequate stimuli applied to their "receptive fields." Both
intracellular and extracellular recording experiments on functionally
defined neurons in cats and monkeys have suggested that opioids produce
both excitatory and inhibitory effects on nociceptive as well as
nonnociceptive neurons (Craig and Hunsley 1991;
Craig and Serrano 1994
; Jones et al.
1990
; Willcockson et al. 1986
). However, in
vitro experiments in rats indicate that opioids produce a predominantly
inhibitory effect both pre- and postsynaptically (e.g., Glaum et
al. 1994
; Schneider et al. 1998
).
To overcome some of these difficulties, we have developed a procedure
in rat that relies on whole cell, tight-seal recordings of spinal
neurons in laminae I and II. This technique allows recording in the
whole cell mode for extended periods of time and allows for the
recorded neuron to be labeled. Recently, similar recordings have been
made in the kitten cortex (Nelson et al. 1994), the bat
inferior colliculus (Covey et al. 1996
), and the rat
cortex (Moore and Nelson 1998
), demonstrating the
utility of this recording mode both for recording and for manipulating
the internal environment of recorded neurons.
Our results demonstrate that the same functional types of neurons
(defined by stimulation of the receptive field) are found in the rat in
approximately the same proportions as in monkey (Kumazawa and
Perl 1978; Light et al. 1979
). The membrane and synaptic properties of these types of neurons recorded in vivo are
similar to those found in vitro. Furthermore, the responses to opioids
are similar to those reported in vitro with the additional finding that
most of the affected neurons are nociceptive. Finally, the low noise of
the patch pipettes and the high-input impedances of the recorded
neurons allow for detailed analyses of both normal and opioid affected
subthreshold synaptic inputs, which have not been resolved in vivo with
sharp electrode recordings in the past. Preliminary reports of some of
these findings have appeared (Light et al. 1997
;
Light and Willcockson 1996
).
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METHODS |
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The guidelines on ethical standards for the investigation of
experimental pain in animals were strictly followed and the following experimental protocol was approved by the Institutional Animal Care and
Use Committee of the University of North Carolina. Fifty adult female
Long-Evans rats (Charles River, 225-350 g) were used in these
studies. Long-Evans rats were used because their survival is more
robust under our experimental conditions and they routinely maintained
adequate physiology following unilateral pneumothorax, which was
necessary to achieve adequate stability of the preparation. Bilateral
pneumothorax further decreased the survival of the animals. In
addition, we have been using this strain in behavioral experiments because they exhibit better performance on a variety of behavioral tasks (e.g., Vierck et al. 1995).
The rats initially were anesthetized deeply and maintained in this state with sodium pentobarbital and chloral hydrate (50 mg/kg and 125 mg/kg, respectively) delivered i.p. Catheters were inserted in the jugular vein and carotid artery for administering drugs and measurement of blood pressure, respectively. The rats were placed in a head holder and stabilized in a spinal frame with hip pins and vertebral clamps attached laterally. A laminectomy exposed ~7 mm of lumbar spinal cord. A small reservoir (~150 µL) was formed with dental impression material (CutterSil Light; Heraeus Kulzer, South Bend, IN) and filled with oxygenated, artificial cerebral spinal fluid (ACSF) containing (in mM) 120 NaCl, 2.5 KCl, 2.5 CaCl2, 1.5 MgSO4, 1.25 NaH2PO4 · H2O, 26 NaHCO3, and 10 glucose at 37°C (pH 7.35-7.45, 305-310 mOsm).
After a unilateral pneumothorax, the rat was paralyzed with pancuronium bromide (4-8 mg/kg) and respirated with 100% O2. To assess adequate depth of anesthesia the animal was monitored for respiratory CO2, rectal temperature, heart rate, and blood pressure and kept within normal physiological limits with supplemental doses of anesthetic (sodium pentobarbital, 4-8 mg/kg). In addition, paralysis was occasionally allowed to wear off to assess that the rats were areflexic. The dura over the lumbar spinal cord was carefully removed and the arachnoid between the dorsal roots was dissected away over a small section of the lateral spinal cord with care being taken not to compress the cord or damage small blood vessels. Initially in some experiments, needle electrodes were inserted into the receptive field locations to electrically activate primary afferents.
Microelectrodes
Whole cell, patch pipettes (N-51A custom borosilicate glass;
Drummond Scientific, Broomall, PA) with a DC resistance of 6-8 M
were fabricated on a Model P-87 Flaming-Brown puller (Sutter Instrument
Co., Novato, CA) and the tip was filled with an internal solution
containing (in mM) 130 K-gluconate, 5 NaCl, 1 CaCl2, 1 MgCl2, 11 EGTA, 10 HEPES, 0.1 GTP, and 2 Mg-ATP (pH 7.30, 280-290 mOsm) (Schneider
et al. 1998
). Some electrodes were then backfilled with 0.1 to
1% biocytin (free base, FW = 372.5; Sigma, St. Louis, MO)
dissolved in the internal solution (Horikawa and Armstrong 1988
). Because biocytin labeling of unrecorded neurons could
occur inadvertently if multiple attempts were made with a single
electrode, and because we had observed some possible effects of
biocytin on patch sealing and on opioid responsiveness in our in vitro studies, later experiments used a reduced concentration of biocytin (0.1%) in the recording pipette.
Recording procedure
Patch electrodes were lowered into the spinal cord via a hydraulic microdrive (Model 607-C; David Kopf Instruments, Tujunga, CA) attached to an electrode holder with a pressure port (Axon Instruments, Foster City, CA). Pulses of hyperpolarizing current (0.1 nA, 60 ms, 1 Hz) were used to continuously monitor series resistance. Recordings were made with an AxoClamp 2 electrometer (Axon Instruments) in bridge mode.
When approaching the surface of the cord, positive pressure was applied to the pipette to prevent plugging the tip of the electrode with pia or overlying white matter. This pressure was released following penetration of the cord surface. Seals were formed by initially reducing the pressure to atmospheric and applying gentle suction to the pipette. Neurons were recognized by negatively directed action potentials in response to electrical stimulation of the periphery, spontaneous action potentials, or simply by the increase in impedance observed when forming seals. Seals often formed on glial cells or on unidentified debris. Seals on nonneuronal elements were broken by applying positive pressure to the pipette. After an unsuccessful attempt to attain a neuron, electrodes were replaced to prevent leakage of biocytin into surrounding tissue and the subsequent undesired labeling of neuropil. Whereas neurons deeper in the dorsal horn could be "patched" in this same manner, we restricted our attempts to the first 500 µm to more thoroughly sample laminae I and II.
After acquiring a seal >1G (usually 2-10 G
), the membrane patch
was ruptured by further gentle suction to the electrode, establishing a
whole cell recording configuration and the pressure was immediately
returned to atmospheric. Electrode signals were displayed on a storage
oscilloscope and also digitized and saved on videotape for later
analysis using Axotape and pClamp software (Axon Instruments). Using
chart recordings at moderate speeds with high resolution, we measured
~5 consecutive conductance pulses (occasionally contamination by
action potentials made it possible to obtain only 4 consecutive pulses)
from before and during the ([D-Ala2,
N-Me-Phe4,
Gly5-ol]-Enkephalin (DAMGO) applications (see
Fig. 6C). Measurements were made from the mid-baseline to
the middle of the noise in the pulse. A t-test
(P < 0.05) was used to determine significant changes
in conductance during DAMGO application. Only units with significant
conductance changes were classified as affected postsynaptically by DAMGO.
A neuron was characterized according to the type of natural stimuli
that evoked action potentials (i.e., brush, pinch, cool, heat, or
combinations of each) to allow comparisons with previous samples from
our lab (Light 1992; Light et al. 1993
,
1997
) and others that have used extracellular recordings to
characterize neurons (see INTRODUCTION and
DISCUSSION for references). We often observed subliminal
PSPs [EPSPs, inhibitory postsynaptic potentials (IPSPs), and slow
potentials] from other inputs that did not activate action potentials.
These PSPs appeared to be variable in their appearance, and could be
affected by a number of factors. However a thorough characterization of
these inputs under various conditions is beyond the scope of this
paper. We used our standard tests to determine whether the cell had
specific innocuous mechanical input (responded only to very slowly
moving stimuli), innocuous thermal inputs (<43°C for heat and
>20°C for cooling, using a feedback controlled thermal stimulator),
and nociceptive input (responded only to intense mechanical and/or
intense thermal stimulation, >45°C for heat and <10°C for
cooling). Timings of stimuli applications were marked on recordings
with a foot switch. A strip chart recorder (TA-2000; Gould, Valley
View, Ohio) was used to monitor long-term fluctuations in
Vm and
RN, the latter using 600 ms negative
pulses (0.05 nA) through the pipette.
Drug application
In some experiments, the selective µ-opioid receptor agonist
DAMGO, (free base, MW = 513.6; Sigma) was applied by placing 0.5 µl of a 1 µg/µl solution directly into the ASCF reservoir overlying the spinal cord (ASCF bath volume ~150 µl). The final concentration in the reservoir was estimated to be ~5 µM. Drugs could not be removed from the reservoir so only cumulative applications of drugs were performed. The opioid receptor antagonist, naloxone hydrochloride (FW = 363.8; Sigma) was applied in a similar fashion (0.5 µl of 0.1 µg/µl) for an estimated final concentration of ~0.5 µM. The drug application was later modified and applied to the
reservoir by continuous flow similar to the in vitro studies of
Schneider et al. (1998). Flow application had the
advantage of allowing application of known concentrations of drugs that could be removed quickly. This allowed before and after drug
comparisons and applications of several different drug concentrations.
Because we could not determine the actual drug concentration at the
membrane, which is affected by clearance of the drug via tissue
dilution and the vascular uptake, the final concentration is not known. However, it was always less than the maximum applied concentration of 5 µM. In addition, because of the close proximity of the recorded cells
to the spinal cord surface, the concentration at the membrane was
probably relatively close to that in the bath.
Histochemistry
After the neurons were characterized, the animals were perfused
through the heart with 0.01 M phosphate buffered saline (pH 7.4)
followed by cold 4% paraformaldehyde and 10% sucrose in 0.1 M
phosphate buffered saline (pH 7.4). The lumbar spinal cord was removed,
postfixed (4% paraformaldehyde with 30% sucrose), and sectioned on a
cryostat at 60 µm. Free-floating sections were collected, rinsed in
0.05 M tris-buffer (pH 7.6) with 0.3% Triton-X and 2.7% NaCl (TBS/TX
buffer, pH 7.6), and pretreated with graded alcohols to eliminate red
blood cells. The sections were rinsed with TBS/TX buffer and 2% normal
goat serum, and incubated in avidin-biotin HRP complex (Vectastain
Elite ABC Kit, PK-6100; Vector Laboratories, Burlingame, CA) for 1 h. After rinsing with TBS/TX, the tissue was incubated in
diaminobenzidine (DAB, 0.2%) and nickel ammonium sulfate (0.14%) with
7 µl of 30% hydrogen peroxide. The sections were rinsed with TBS/TX,
mounted, air-dried, and coverslipped with DPX. On examination, biocytin
labeled neurons were revealed as densely stained, gray-black cell
bodies with heavy to diffuse stained dendrites and axons. Cells were
magnified (×400-1000), photographed, and reconstructed using a Nikon
Optiphot microscope equipped with a drawing tube. The laminar locations were designated based on the relative density and orientation of
myelinated fibers (Light 1992) and verified using dark
field illumination.
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RESULTS |
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The data presented are from 73 neurons. Recording times varied
from 10 to 90 min with half being 30 min. The input impedances ranged
from 100 to 700 M
[332 ± 132 (SD) M
, n = 60]. The membrane time constants were 4-25 ms (13.1 ± 4.8 ms,
n = 29). The resting membrane potentials were between
24 and
80 mV (
52 ± 12 ms, n = 65). The size
of action potentials ranged 15-80 mV (52 ± 17 mV,
n = 40). Small action potentials were not related to
less negative resting membrane potentials, lower inputs impedances, or
spontaneous action potentials, suggesting that they were not the result
of injury. Furthermore, when action potentials were less than the
membrane potential (not overshooting), the PSPs were often quite large
(5-15 mV). The lack of signs of injury and the nonovershooting action
potentials combined with anecdotal evidence from biocytin labeling
suggested that these recordings were from dendrites at a distance from
the action potential initiation site. Furthermore, these data indicate
that the dendrites of some neurons recorded here did not back-propagate
action potentials to the dendritic recording sites (see Johnston
et al. 1996
for review of this topic).
Activating stimuli
Receptive fields were most often located on the lateral portion of
the proximal hindlimb (see examples in Fig.
1A). Using natural stimuli, we
classified neurons (n = 73) according to action potential firing. Thirty-nine neurons were nociceptive. More
specifically, the nociceptive neurons were fairly evenly divided by
response such that six responded only to noxious pinch; five to pinch
and rapid cooling; eight to pinch, cold, and noxious heat; and seven to
pinch, cold, and heat, but demonstrating some responses to innocuous
brush as well. Five neurons responded to noxious heat and pinch; three
to brush and pinch; and two to brush, pinch, and cooling, but not to
heat. Three nociceptive neurons were not held long enough to complete
their characterization in detail. Six neurons were innocuous cooling
cells, 20 responded maximally to brush, however in 8 neurons we could
not find excitatory receptive fields. Whereas this was tested in all
cases, only one unit, held briefly and not characterized fully enough
to be included in this sample, appeared to respond to innocuous
warming. The types of input to these neurons are very similar to the
percentages and types of neurons found in spinal laminae I and II in
cats and monkeys (e.g., Jones et al. 1990; Light
and Kavookjian 1988
; Light et al. 1979
;
Réthelyi et al. 1989
), and similar to the
description of rat neurons recorded intracellularly (Woolf and
Fitzgerald 1983
). Examples of some of the responses of these
neurons are shown in Figs. 2, 5, and 6.
PSPs evoked by natural stimuli are shown in Fig. 7.
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The high resolution of the recordings (caused by very high-input
impedance and very low noise of patch-clamp electrodes) allowed us to
observe PSPs evoked by stimulating various types of afferent input (see
Figs. 2 and 7). These potentials varied in size from 0.5 mV to 8 mV,
depending on the input impedance and intrinsic properties of the
neuron. In many instances, both spontaneous and evoked PSPs appeared to
have a unitary nature, i.e., a PSP of a constant size and shape that
decayed exponentially appeared to sum with others of the same size to
produce larger events (see Fig. 7). Similar, apparent unitary events
have been observed in other patch clamp experiments in vitro
(McQuiston and Colmers 1996) and have also been observed
in the spinal cord (Bao et al. 1998
). However, we
interpret most if not all of our observations of unitary PSPs in vivo
as representing summed PSPs from the activation of primary afferent
axons or spinal interneurons (much like those observed in motoneurons
with spike triggered averaging from Ia axons e.g., Mendell and
Henneman 1971
) unlike the "true" miniature events observed
in in vitro studies.
Biocytin labeling
The locations of 15 recording sites were marked with biocytin (see
Fig. 1B). The two neurons responding only to innocuous cooling were found in lamina I and in Lissauer's tract. The one well
labeled cooling cell (Fig.
3E), may represent a pyramidal lamina I cell as suggested for cooling cells in other species (Han et al. 1998). Neurons responding to brush were
found in lamina I, the overlying white matter and lamina
IIi (Fig. 3, F and G, and
4). Innocuous mechanoreceptive neurons in
lamina IIi were both islet and stalked cell types
(Fig. 3G and 4). Neurons responding to noxious inputs were
found in both lamina I and lamina IIo. Nociceptive lamina I neurons were either fusiform (Fig. 3B)
or multipolar in shape (Fig. 3, C and D).
Nociceptive lamina II neurons were either similar to islet cells (Fig.
3A) or to stalked cells. Examples of several of these
neurons are reconstructed in the parasagittal plane and shown in Fig.
3. Photomicrographs of a labeled cell that responded to brush are seen
in Fig. 4, A and B.
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DAMGO effects
Fifteen of 19 neurons were affected by adding DAMGO to the recording reservoir (Table 1). In 7 of 19 tested neurons, DAMGO caused a hyperpolarization of 7.0-25 mV (11.9 ± 6.5 mV) accompanied by a significant (P < 0.05) conductance increase (7-38%, 23.3 ± 11.2%; see Figs. 5 and 6). The locations of five of these neurons are shown with filled symbols in Fig. 1. All but one of these neurons were found in lamina II. Naloxone was applied to four of these seven neurons. In all four, naloxone clearly reversed the hyperpolarization and conductance increases evoked by DAMGO.
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In addition, DAMGO decreased the frequency of some evoked and spontaneous PSPs in seven of the hyperpolarized neurons and in seven additional neurons in which hyperpolarization or significant conductance increases were not observed. In some cases, it appeared that larger PSPs were lost following DAMGO application, but there was an increase in very small PSPs. The locations of three of these latter neurons are shown with stars inside open symbols in Fig. 1B. Naloxone restored the spontaneous PSPs and evoked action potential responses toward pre-DAMGO levels (see Fig. 6). The reduction by DAMGO of responses to noxious cooling and heating is demonstrated in Fig. 7, C and D. The reductions in evoked action potentials shown in Figs. 5 and 6 resulted both from hyperpolarization of the recorded neuron and reductions in the frequency of spontaneous and evoked PSPs.
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Of the 19 tested neurons, 1 was nociceptive and 5 were hyperpolarized by DAMGO. All of these five responded to noxious thermal stimuli. The morphology of one of these is shown in Fig. 3A. Of the nine nociceptive neurons that were not hyperpolarized, five demonstrated pronounced reductions in spontaneous postsynaptic potentials because of DAMGO application, whereas three showed no effect and one was depolarized by DAMGO application and demonstrated a 28% conductance decrease (located in lamina I with an asterisk in Fig. 1B and shown morphologically in Fig. 3D). The depolarization and conductance decreases were blocked by the coadministration of naloxone with DAMGO (this neuron was tested with the circulating drug system described in the METHODS section). The three nociceptive neurons that demonstrated no effect did not respond to noxious thermal stimuli and the five that demonstrated only effects on PSPs demonstrated either responses to noxious thermal stimuli (3) or only responded to noxious mechanical stimuli (2).
Of the five nonnociceptive neurons tested, four were mechanoreceptive. Of these four neurons, two were hyperpolarized by DAMGO (shown morphologically in Fig. 3F and 4), one showed reductions in spontaneous PSPs because of DAMGO, and one showed no effect of DAMGO application. The remaining tested neuron responded only to innocuous cooling and demonstrated no membrane effect but demonstrated a reduction in spontaneous PSPs when DAMGO was applied.
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DISCUSSION |
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The present study used whole cell, tight-seal in vivo
recordings in laminae I and II neurons in the spinal cord and examined the response properties of these neurons in the presence of
opioids. In vivo recordings of this type have been obtained
successfully from the visual cortex of kittens (Nelson et al.
1994), the somatosensory cortex of rats (Moore and
Nelson 1998
), and the inferior colliculus of the bat
(Covey et al. 1996
). However, this is the first
demonstration of the application of this technology in the spinal cord.
Moreover, this study 1) documents the classes of neurons in
laminae I and II in the rat in vivo, both physiologically and
anatomically, using whole cell recording techniques; 2)
documents the hyperpolarization of identified nociceptive lamina I and
II neurons by DAMGO in vivo in the rat; 3) demonstrates the
decrease in frequency of PSPs evoked by noxious thermal stimuli by
DAMGO in lamina I and II neurons in the rat; and 4) compares
some of the biophysical properties of lamina I and II neurons recorded
by patch clamp in vivo with those in vitro using the same recording procedures.
Methodological considerations
Whole cell, tight-seal recordings have several advantages over
sharp electrode, intracellular recordings. The pipettes have very low
impedances, which allows for very low noise recordings that are much
more stable. Whereas we and others have been able to stabilize cat and
monkey spinal cord sufficiently to obtain satisfactory sharp electrode
recordings (Iggo et al. 1988; Jones et al.
1990
; Light et al. 1979
, 1986
), we have been
largely unsuccessful in obtaining stable recordings from the small
cells of laminae I and II in previous attempts to record with sharp
electrodes in rats.
The quality of the in vivo recordings was very good. The cell input
impedances corresponded well with those obtained from similar cells
recorded in vitro (Schneider et al. 1998). Both ongoing
and evoked PSPs were observed with high resolution. The enhanced
resolution allowed for analyses of the characteristics of evoked PSPs
and consequent changes induced by opioid drugs. Conversely, these data
help to verify results from in vitro studies, demonstrating similar
properties (Schneider et al. 1998
). In previous in vitro
studies in rat, the input impedance of neurons recorded in the whole
cell mode was considerably higher than that reported in sharp electrode
studies (North and Yoshimura 1984
; Yoshimura and
North 1983
). These values are much higher than sharp electrode studies conducted on laminae I and II neurons in vivo in the cat (Iggo et al. 1988
; Jones et al. 1990
;
Light et al. 1979
, 1986
). This study makes it clear that
the high values of input impedances are not a result of preparing
slices for in vitro recording or bathing solutions, but rather a
property of the whole cell recording configuration which presumably has
less current shunt than sharp electrode recording techniques.
Physiological properties of lamina I and II neurons
The present results demonstrate that the organization of the rat
spinal cord marginal zone and substantia gelatinosa is quite similar to
that of the monkey spinal cord (Kumazawa and Perl 1978; Light et al. 1979
). Marginal zone neurons had synaptic
inputs and fired action potentials in a manner consistent with
nociceptors or innocuous cooling afferent inputs. Occasionally, slow
brush inputs were observed in neurons in the overlying white matter similar to previous observations by others in the rat (Woolf and Fitzgerald 1983
) or deeper in the substantia gelatinosa as
previously found in the rat, the cat, and the monkey (Light et
al. 1979
; Réthelyi et al. 1989
;
Woolf and Fitzgerald 1983
). Neurons in the outer
substantia gelatinosa appeared to receive inputs from both
mechanoreceptive and polymodal nociceptors; however, cells with input
from innocuous mechanoreceptors were located in the inner SG. These
recordings confirm the importance of the substantia gelatinosa in
nociceptive processing and demonstrate its similarity to the same
region in cats and monkeys. This study indicates that most of the
neurons in the inner substantia gelatinosa in all three species are
nonnociceptive. These neurons appear to have inputs dominated by
primary afferents that respond best to gentle, slowly moving stimuli.
The function of these neurons is unknown, but they may play a role in
modulating neurons in other laminae as we have observed that at least
some of these neurons have axons with terminal collaterals in laminae
I, III, and IV (Light and Kavookjian 1988
). Either by
affecting neurons in other laminae or by alternative projections to
higher centers, some of these neurons may be involved in sensations
such as "tickle" (Zotterman 1939
).
Anatomic appearance of recorded neurons
In addition to noting the location of some of the recorded
neurons, biocytin labeling allowed us to determine the types of laminae
I and II neurons recorded with the patch-clamp technique. A few neurons
lying in Lissauer's tract, immediately above lamina I and some lamina
I neurons were innocuous cooling input cells. One well-labeled cooling
cell may be of the same category (pyramidal) as cooling neurons
described from the cat dorsal horn (Han et al. 1998).
Other lamina I neurons fall into the categories of fusiform or
multipolar, as suggested by these same authors, and all were
nociceptive. The lamina II neurons labeled here can be described as
islet cells, stalked cells, or stellate cells. All morphological types
were nociceptive as well as innocuous mechanoreceptive with the only
distinction being that the innocuous mechanoreceptive cells were
located in IIi or overlying white matter, similar
to the findings of Woolf and Fitzgerald (1983)
.
Response to opioids
Our data indicate that evoked action potentials in a subset of nociceptive neurons are reduced by opioids both by hyperpolarization and by reductions in spontaneous and evoked PSPs. This results in a powerful overall inhibition of transmission from these neurons.
DAMGO, a selective µ-opioid receptor agonist, hyperpolarized (with a
conductance increase) about 37% of the neurons to which it was applied
in these experiments. This percentage is somewhat lower than found in
sharp electrode in vitro experiments (Grudt and Williams
1994; Jeftinija 1988
; Miletic and Randic
1981
; Yoshimura and North 1983
), but about the
same as similar in vitro patch-clamp experiments (Schneider et
al. 1998
). As in previous in vitro experiments, the predominant
postsynaptic effect of DAMGO application in vivo was hyperpolarization
with a conductance increase. In addition, we have shown that many of
these neurons could be classified as nociceptive cells. However, one
neuron was depolarized and appeared to be excited and demonstrated a
28% decrease in conductance. This is not entirely consistent with
previous in vivo experiments in cats in which we used sharp electrode
recordings. In these studies many neurons were excited by morphine, a
µ-opioid agonist (Jones et al. 1990
). It also is not
entirely consistent with other results in the cat (Craig and
Hunsley 1991
; Craig and Serrano 1994
) that
demonstrated that innocuous cooling neurons in lamina I were excited by
morphine. The reasons for these discrepancies are unclear, but may be
caused by a species difference, because behavioral responses to opioid
administration are different in cats compared with rats. However,
others have observed excitatory effects in rat spinal cord with
extracellular recording techniques both in vitro (Magnuson and
Dickenson 1991
) and in vivo (Sastry and Goh
1983
, Woolf and Fitzgerald 1981
).
The majority of neurons hyperpolarized by DAMGO were nociceptive
neurons that appeared to receive inputs from both mechanical and
thermal nociceptors. DAMGO also provoked a profound decrease in the
frequency of PSPs evoked by noxious heat and cooling after the
application of DAMGO in many nociceptive neurons. PSPs evoked by
noxious mechanical stimulation were affected to a lesser degree. Previous studies using whole cell recording methods have consistently reported that opioids caused decreases in the frequency of PSPs of
unknown origin (Glaum et al. 1994; Hori et al.
1992
; Schneider et al. 1998
). This study
suggests that many of these PSPs are from nociceptive primary afferent
neurons or interneurons.
All but one of the labeled neurons that were hyperpolarized by DAMGO were found in lamina II, with neurons demonstrating only presynaptic responses being found mostly in lamina I. The small numbers reported here make the significance of this separation unknown, but could reflect the presumed axonal projection of lamina II cells to lamina I.
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SUMMARY |
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This study documents the physiological and anatomic types of neurons using whole cell, tight-seal recordings in lamina I and II of the lumbar spinal cord of the adult rat in vivo. It also documents the relative selectivity of µ-opioid effects on C fiber evoked nociceptive inputs, both presynaptically and postsynaptically. Furthermore, it validates in vitro studies in neurons of lamina I and II in the rat for recording the biophysical properties and effects of opioids.
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ACKNOWLEDGMENTS |
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The authors acknowledge K. McNaughton for expertise with the histological processing, B. Taylor-Blake and M. Roberts for technical assistance, and T. Grudt and P. Dougherty for reading earlier versions of this manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grants R01-NS-16433 and P01-NS-14899.
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FOOTNOTES |
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Address for reprint requests: A. R. Light, Dept. of Cell and Molecular Physiology, CB#7545 Medical Sciences Research Building, University of North Carolina, Chapel Hill, NC 27599-7545.
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 24 May 1999; accepted in final form 17 August 1999.
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REFERENCES |
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