Royal Free Hospital School of Medicine, London NW3 2PF, United Kingdom
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ABSTRACT |
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Deuchars, Susan A.,
T. Trippenbach, and
K.
Michael Spyer.
Dorsal Column Nuclei Neurons Recorded in a Brain Stem-Spinal
Cord Preparation: Characteristics and Their Responses to Dorsal Root
Stimulation.
J. Neurophysiol. 84: 1361-1368, 2000.
Recordings were obtained from dorsal column
nucleus (DCN) neurons in a neonatal rat brain stemspinal cord
preparation to study their basic electrophysiological properties and
responses to stimulation of a dorsal root. Whole-cell patch-clamp
recordings were made from 21 neurons that responded to dorsal root
stimulation with a fast excitatory postsynaptic potential (EPSP). These
neurons were located lateral to, but at the level of, the area postrema at depths of 100-268 µm below the dorsal surface of the brain. The
neurons could be divided into groups according to the shape of their
action potentials or voltage responses to hyperpolarizing current
steps; however, the response profiles of the groups of neurons to
dorsal root stimulation were not significantly different and all
neurons were considered together. Dorsal root stimulation elicited
excitatory postsynaptic potentials (EPSPs) in all neurons with a very
low variability in onset latency and an ability to follow 100-Hz
stimulation, indicating that they were mediated by activation of a
monosynaptic pathway. The peak amplitude of the EPSP increased with
membrane hyperpolarization, and applications of the non-NMDA receptor
antagonists 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX)
and 6,7-dinitroquinoxaline-2,3-dione (DNQX) decreased the amplitude of the EPSP to 14.2% of the control response
(n = 6). The descending phase of the EPSP decreased
with membrane hyperpolarization and was reduced by the
N-methyl-D-aspartate (NMDA) receptor
antagonist AP-5 (n = 2). The EPSPs were also
reduced in amplitude by applications of the
-aminobutyric acid-B
(GABAB) receptor agonist baclofen, which had no effect on
membrane potential or input resistance. These results show that fast
EPSPs in DCN neurons elicited by dorsal root stimulation are mediated
by an excitatory amino acid acting at both non-NMDA and, to a lesser
extent, NMDA receptors. In addition, GABA acting at presynaptic
GABAB receptors can inhibit these responses.
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INTRODUCTION |
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The dorsal column nuclei (the
gracile and cuneate nuclei) are known to be major relays in the
transmission and processing of information from low-threshold
mechanoreceptors. Anterograde labeling studies have shown that primary
afferent inputs from the upper thoracic and cervical levels of the rat
spinal cord ascend to the dorsal column nuclei and terminate in the
cuneate and external cuneate nucleus (Basbaum and Hand
1973; Beck 1981
). The primary afferent input to
dorsal column nuclei (DCN) neurons may use glutamate as the main
neurotransmitter since ionophoretic applications of glutamate in the
vicinity of these neurons caused excitation (Galindo et al.
1967
), while 1-hydroxy-3-aminopyrrolid-2-one (HA-966, an
excitatory amino acid receptor antagonist) blocked excitatory synaptic
transmission within the cuneate nucleus (Davies and Watkins
1973
). In addition, glutamate immunoreactivity has been
demonstrated in identified primary afferent terminals in the cuneate
nucleus (De Biasi et al. 1994
), while AMPA receptors are
expressed by all neurons in the DCN (Propratiloff et al.
1997
). However, the exact nature of the fast response evoked by
dorsal root stimulation and the effect of selective excitatory amino acid receptor antagonists has yet to be determined. This was the first
aim of the study.
Inhibition of evoked activity in DCN neurons has been reported by many
groups (see Willis and Coggeshall 1991) and this may involve both a post- and a presynaptic GABAergic input onto these neurons (Lue et al. 1996
). The postsynaptic effect is
mostly of short duration, while the presynaptic effect may be important in the more prolonged inhibition observed in the DCN (Anderson et al. 1964
, 1970
; Jabbur and Banna 1968
, 1970
).
Various studies have investigated the role of
-aminobutyric acid-A
(GABAA) -mediated primary afferent depolarization
in the presynaptic inhibition of DCN neurons (see Willis and
Coggeshall 1991
). However, it is likely that activation of
presynaptic
-aminobutyric acid-B (GABAB) receptors which decrease synaptic activity may be the important mechanism involved in the prolonged inhibition observed in the dorsal
column nuclei. Indeed, Newberry and Simmonds (1984)
revealed a bicuculline resistant component of the negative wave of the field potential recorded in response to dorsal column stimulation in
the gracile nucleus. Therefore, the second aim of this study was to
explore the role of the GABAB receptor in the DCN.
We have developed a neonatal rat brain stem-spinal cord preparation
for recording from sympathetic preganglionic neurons (SPNs) to study
the pharmacology of inputs onto these neurons (Deuchars et al.
1995a,b
). The preparation is highly suitable for the study of
dorsal root inputs onto neurons within the dorsal column nuclei since
this long pathway is also maintained and dorsal roots can be stimulated
to determine the responses of individual DCN neurons. Using whole-cell
patch-clamp recordings from DCN neurons, the changes in membrane
potential of these neurons evoked by stimulation of the dorsal roots
can be determined and the effects of drugs on these responses can be
resolved. This study examined 1) the basic membrane
properties of neurons within the cuneate nucleus with an aim to
grouping the neurons according to these characteristics, 2)
their responses to dorsal root stimulation and the effects of
excitatory amino acid antagonists, and 3) the effects of
applications of baclofen and the selective GABAB
antagonist CGP35348 on the evoked responses to dorsal root stimulation
to determine whether there is a presynaptic GABAB
receptor-mediated inhibition of these responses. These results have
been presented in abstract form (Deuchars et al. 1997
).
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METHODS |
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Neonatal rats (2-5 days) were anesthetized initially with
isoflurane. They were then placed on ice to maintain anesthesia by
hypothermia as described previously (Deuchars et al.
1995a). A high decerebration was performed rapidly and the
brain stem and spinal cord were isolated in artificial cerebrospinal
fluid (aCSF) equilibrated with 95% O2-5%
CO2 (see Deuchars et al. 1995b
). The brain stem-spinal cords were then pinned onto Sylguard in a
recording chamber with the dorsal surface of the brain stem uppermost.
The spinal cords were twisted at the upper cervical level to allow
simultaneous access to dorsal and ventral roots from the lower cervical
levels. The preparations were superfused at a rate of 5 ml/min with
aCSF composed of (in mM) 128 NaCl; 3 KCl; 0.5 NaH2PO4 · H2O;
1.5 CaCl2 · 2H2O; 1 MgSO4; 23.5 NaHCO3; 30 glucose, and 2 mannitol equilibrated with 95%
O2-5% CO2 and maintained
at 26-27°C. The dura mater was removed to increase the diffusion of
O2 to the tissue. Suction electrodes were placed on 1) an upper thoracic ventral root to record ongoing
activity and thus determine the viability of the preparation and
2) a dorsal root from the lower cervical or upper thoracic
levels for stimulation purposes (see Fig.
1A). The responses of the
ventral roots to stimulation of the dorsal roots were monitored at the
beginning of each experiment. Dorsal root stimulation (single- or
twin-pulse 100 Hz; 0.5-ms pulse width; 25-50 µA) elicited a
short-latency response on the ventral root activity as observed
previously (Deuchars et al. 1995b
).
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Whole-cell patch-clamp recordings were made from neurons in the dorsal
column nuclei by approaching the neurons from the dorsal surface of the
brain stem, lateral to, but at the level of the area postrema (see Fig.
1A). This area corresponds to the region of axonal labeling
observed by Maslany et al. (1992) after injections of
transganglionic tracer into the rat forepaw. Positive pressure was
maintained in the patch electrode to depths of 20 µm to prevent attachment to loose material. Electrodes were advanced into the brain
stem in 1.2-µm steps until a change in resistance was observed. Gentle suction was applied until a high-resistance (1-5 G
) seal was
made; then, brief pulses of negative pressure caused rupture of the
membrane. The electrodes were filled with (in mM) 145 KGluconate; 2 MgCl2; 5 HEPES; 1.1 EGTA; 0.1 CaCl2; and 5 K2ATP (pH 7.2;
osmolality 310 mOsmol/kg H2O). This filling
solution also contained 0.5-1% biocytin for labeling neurons.
Recordings were made using an Axoclamp 2A amplifier (Axon Instruments,
3-kHz bandwidth) in bridge balance mode. During the search for a
neuron, current pulses of 50-1250 pA (10-30 ms pulse width) were
applied at a rate of 2 Hz. To determine the input resistance and the
current-voltage relationship of a neuron, rectangular currents pulses
of +60 to 110 pA (0.5-1 s pulse width) were applied and the
resulting voltage deflections measured. The effects of stimulation of
the dorsal roots (single-pulse stimulation, 0.1-30 V, 0.5-1 ms pulse
width) were determined at a range of membrane potentials (
40 to
90
mV). Once stable responses had been obtained and various manipulations
carried out, drugs were applied to determine their effects on the
evoked postsynaptic potentials. All drugs were dissolved in aCSF at
known concentrations and applied to the superfusing medium at a rate of
5 ml/min and as such were subject to a dead space of around 30 ml due
to the volume of the bath and superfusion tube. This meant that both
the effect of the drug and the washout after switching to control
medium took time to occur. The following drugs were applied: the
non-NMDA receptor antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX; Research Biochemicals International) and
6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX, Tocris
Cookson); the NMDA receptor antagonist AP-5; the GABAB-receptor agonist ± baclofen (both
Research Biochemicals International); and the
GABAB-receptor antagonist CGP 35348 (a gift from
Novartis). All drug concentrations given in RESULTS are the final concentrations in the perfusing medium.
Data analysis
Membrane potentials, current injections, whole-nerve recordings,
and trigger pulses were stored via an interface (Instrutech; 11 kHz/channel sampling rate) on videotape for analysis. All data analysis
was carried out using an IBM-compatible microcomputer (interface and
software supplied by Cambridge Electronic Design UK). Spike durations
and amplitudes were measured as the time and amplitude from the start
of the rising phase of the action potential to the start of the
afterhyperpolarization, respectively. Input resistance was measured at
60 mV and taken as the voltage value at the beginning of a response
to a 50-pA current step. Conduction distance was measured and the
axonal conduction velocity calculated for the afferent input onto each
neuron. The fluctuations in onset latency of the EPSPs for each DCN
neuron were determined by measuring the onset latencies of 20 single-sweep EPSPs. The mean amount by which these values varied from
the mean onset latency was then calculated for each neuron (average
absolute deviation). The amplitudes of the evoked postsynaptic
potentials were measured as the greatest voltage deflection from the
membrane potential and were calculated for each membrane potential. The
voltage relationships of the fast EPSPs evoked by dorsal root
stimulation were evaluated by plotting the percentage change in EPSP
peak amplitude against membrane potential, taking the EPSP amplitude at
60 mV as 100%. Comparisons of the amplitudes and latencies of action
potentials, input resistances, depths of neurons,
afterhyperpolarizations, and voltage deflections due to activation of
an IH current were made using the
Mann-Whitney U test, as were comparisons of input resistances before and after baclofen applications. All values are
given as mean ± SE unless stated.
Histology
During the recording period, biocytin, which is contained within the intracellular medium, diffused into the neurons to fill them. At the end of the experiment, brain stems were fixed in 10% formal saline for up to 1 week. Eighty-micron-thick sections of brain stem were cut and placed in 1% hydrogen peroxide to inactivate peroxidase activity of blood. After washing, the sections were then incubated overnight in avidin-biotinylated horseradish peroxidase complex (ABC, Vector Labs, Peterborough, UK), washed, and reacted using a diaminobenzidine tetrachloride reaction with nickel intensification. The sections were counterstained with hematoxylin and viewed at the light-microscopic level and filled neurons, where recovered, were photographed or reconstructed using a drawing tube.
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RESULTS |
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Twenty-one neurons responded to dorsal root stimulation with an EPSP that occurred with a very low variation in latency (see The monosynaptic nature of the EPSP) and it is these neurons that are studied further in this report. Neurons recorded outwith the DCN either did not respond to dorsal root stimulation or responded with a polysynaptic EPSP.
Location of neurons
DCN neurons were found lateral to the area postrema at depths of
100-268 µm below the dorsal surface of the brain stem. Neurons filled with biocytin (n = 5) and recovered were located
in dorsal aspect of the cuneate nucleus (according to the atlas of
Paxinos and Watson 1986, see Fig. 1, B and
C). Since only five filled neurons were recovered, it was
not possible to correlate the anatomy of these neurons with their
electrophysiological properties and their responses to dorsal root stimulation.
Electrophysiological properties of the neurons
Neurons that responded to dorsal root stimulation with a
monosynaptic EPSP (n = 21) had a mean resting membrane
potential of 42.2 ±1.1 mV. The spontaneous action potentials of
these neurons had a mean amplitude of 61.2 ± 1.7 mV at their
resting membrane potential and a mean duration of 8.1 ± 0.4 ms.
The input resistance of the neurons (measured at
60 mV) was 597 ± 70 M
. Current-voltage relationships were determined in 17 of the
DCN neurons and in 12 of these neurons, a sag in the voltage responses
to current steps was revealed at membrane potentials of more than
80
mV (see Fig. 2). This
sag (measured as the difference between the instantaneous and
steady-state voltage values) had a mean amplitude of 9.2 ± 1.3 mV
(measured at
100 mV). The responses of these neurons to dorsal root
stimulation were not different from those neurons not expressing the
sag.
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Positive current steps were applied to 14 neurons to reach firing
threshold and differences in the shape of the action potentials could
be distinguished. Six of the 14 neurons tested had action potentials
that were followed by afterhyperpolarizations of mean amplitude at
firing threshold of 13 ± 1.2 mV and duration 268 ± 26 ms
[see Fig. 2A(ii)]. The remaining eight neurons
displayed action potentials with the repolarizing phase characterized
by a transient afterdepolarization which often also reached the
threshold for firing [see Fig. 2, B(ii) and
C]. Where the neurons fired again in short succession, the
second action potential showed a less prominent afterdepolarization
(shown in Fig. 2C). This transient depolarization may
involve activation of a low-threshold calcium current, as described by
Jahnsen and Llinas (1984) in the thalamus and recently
by Canedo et al. (1998)
in the DCN of the cat. The basic
characteristics of these two groups of neurons did not differ
significantly with regards to the amplitude and duration of the action
potentials and the presence or absence of a depolarizing sag. There
were no significant differences in the input resistance of the two
groups of neurons and the depths at which they were recorded. Once
more, there were no significant differences in the responses of the two
groups of neuron to dorsal root stimulation with respect to EPSP onset
latency and amplitude at
60 mV [see Fig. 2,
A(i) and B(i)]. Therefore,
all neurons were considered together for the remainder of this paper.
Responses of DCN neurons to dorsal root stimulation
Dorsal roots were stimulated with a single pulse and the fast
EPSPs elicited were examined (see Fig. 3A). EPSPs were
similar in amplitude and onset latency regardless of the level at which the dorsal roots were stimulated (from C6 to T3). When trains of up to
eight stimuli (at frequencies of up to 100 Hz) were applied, EPSPs were
observed with no failures of transmission (see Fig. 3B). At stimulus strengths
just above threshold, large EPSPs could be observed often reaching the
threshold for firing action potentials (see Fig. 3C). The
EPSPs showed large deflections on both the rising and falling phases,
suggesting activation of afferents with different conduction velocities
(see Fig. 2). Indeed, as the stimulus intensity was increased further
above threshold, the EPSPs increased in amplitude and the shape changed
with respect to the duration and decay phase as possibly more fibers
were recruited with different conduction velocities and/or some
polysynaptic pathways were activated. Due to the long duration of the
EPSPs, it was difficult to tell the effects of high-frequency
stimulation on the later phases of the EPSP since summation of these
phases occurred. The CNS is largely unmyelinated in rats of this age (Davison and Dobbing 1966), although some immature
myelinated afferents are present at birth (Fitzgerald
1985
) and these increase in number from days 0 to 6, the ages
of the preparations used here. Thus, the intensity-related increases in
EPSP amplitude are probably due to recruitment of myelinated and
unmyelinated axons with different axonal diameters.
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The monosynaptic nature of the EPSP
The average latency to onset of the EPSP was 14.7 ± 1.1 ms for all neurons tested (n = 21) and the conduction velocity calculated was 0.74 ± 0.07 m/s. For each neuron, a small variability in onset latency was observed stimulating at 2 times threshold for each neuron. The difference in onset latency from the mean onset latency was calculated for 20 single sweeps and an average was calculated for the set of neurons. This absolute average deviation was 0.17 ± 0.02 ms for the first phase of each EPSP, which is extremely low considering the length of the pathway being stimulated (up to 1.5 cm). This constant latency to onset of the EPSP and its ability to follow high- (up to 100 Hz) frequency stimulation suggested that at least the initial part of the EPSP was mediated by activation of a monosynaptic pathway.
The effect of membrane hyperpolarization on EPSP amplitude was determined. With hyperpolarization, the peak amplitude of the EPSPs increased in a linear fashion in all 21 of the neurons (Fig. 4A). In 11 of the neurons in this study, the descending phase of the EPSP showed a distinct deflection at more depolarized potentials, which decreased as the membrane was hyperpolarized (see Fig. 5A).
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The chemical nature of the EPSP
The effects of applications of the excitatory amino acid
antagonists DNQX or NBQX, which are selective for the non-NMDA
receptor, were determined on six occasions. For all these DCN neurons,
the dorsal root evoked EPSPs were reversibly reduced in amplitude from
14.6 ± 2.8 to 2.3 ± 1 mV (14.2 ± 5.7% of the control
response; n = 6) by DNQX (10 µM; see Fig.
4B) or NBQX (5 µM). Recovery from this antagonism took up
to 30 min to occur after return to control medium. Neither DNQX nor
NBQX had any significant effect on the membrane potential or input
resistance of the neurons. On all occasions, DNQX applications left a
residual EPSP as observed in Fig. 4B. Therefore, on two
occasions, the NMDA receptor antagonist AP-5 was also applied to look
at the effect on the control response. These two neurons both exhibited
an inflection on the falling phase of the EPSP (see Fig. 5A
at 60 mV) that decreased with membrane hyperpolarization as described
above. On both occasions, the later part of the EPSP was reduced in
amplitude by applications of AP-5 and the residual EPSP was abolished
by applications of DNQX or NBQX (see Fig. 5B).
Effects of activating GABAB receptors on the evoked response to dorsal root stimulation
The GABAB receptor agonist baclofen was superfused at a concentration in the bath of 1 µM onto four DCN neurons. Baclofen decreased the amplitude or abolished the EPSP evoked by dorsal root stimulation on all occasions tested. The EPSP amplitude was decreased from 16.0 ± 4.7 to 1.6 ± 0.6 mV [11.7 ± 3.3% of the control response (see Fig. 6)]. Baclofen had no significant effect on the membrane potential or input resistance of the neurons. The GABAB receptor antagonist CGP 35348 blocked the effects of baclofen and the evoked EPSP returned to control levels within 10 min of changing to aCSF with CGP35348 (see Fig. 6).
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DISCUSSION |
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This study has determined the responses of DCN neurons to
stimulation of a dorsal root in a neonatal rat brain stem-spinal cord
preparation. Characterization of the basic electrophysiological properties of DCN neurons revealed that these cells could be subdivided according to the shape of their action potential or the presence of a
depolarizing sag in the voltage response to hyperpolarizing current
pulses. However, the neurons were not grouped according to these
characteristics in this study since the responses of the neurons to
stimulation of a dorsal root were not significantly different. All DCN
neurons in this study responded to stimulation of a dorsal root with a
fast EPSP that was mediated at least in part by activation of a
monosynaptic pathway. The average conduction velocity of this pathway
was 0.74 m/s, which is relatively slow in comparison to adult
myelinated axons. Fitzgerald (1985) reported the
presence of some immature myelinated fibers at birth and the conduction
velocity of afferent fibers increased from days 0-6 as the degree of
myelination increased. Therefore, it is likely that the fast EPSPs
observed are due to activation of both immature myelinated and
unmyelinated axons. Study of the pharmacology of these inputs revealed
sensitivity to both non-NMDA and NMDA receptor antagonists. The fast
EPSP could also be reduced by applications of the
GABAB agonist baclofen, an effect that could be
antagonized by the GABAB receptor antagonist CGP
35348. Neither of these drugs had an effect on the membrane potential
or input resistance of the neurons, suggesting that the effect was due
to an action at presynaptic GABAB receptors since
activation of a postsynaptic site would be expected to cause membrane
hyperpolarization and a change in input resistance associated with an
opening of potassium channels (see Newberry and Nicoll
1984
).
The characteristics of EPSPs elicited by dorsal root stimulation
Detailed analysis of the onset latency of the EPSP revealed
a very low fluctuation in latency from sweep to sweep (average absolute
deviation was 0.17 ms). This indicates that stimulation of a dorsal
root activates afferent fibers that impinge directly onto DCN neurons.
In addition, DCN neurons were capable of responding to dorsal root
stimulation at frequencies of up to 100 Hz, which is also indicative of
activation of a monosynaptic pathway (see Inokuchi et al.
1992). Neurons responding to dorsal root stimulation in this
way could be subdivided according to their action potential shape or
the presence or absence of an IH; however,
the response profiles of the dorsal root evoked EPSPs were not
significantly different. This indicates that all neurons, regardless of
their basic electrophysiological characteristics, receive a
monosynaptic input from primary afferent fibers. This confirms the
observations from other studies in rat and cat (see wiring diagram in
Andersen et al. 1964
).
The strength of the synaptic security between individual afferent
fibers and DCN neurons appears to be very high (Ferrington et
al. 1986, 1987
). This is further supported in this study by the
observation that at stimulus strengths around threshold level the EPSP
produced at threshold was often large and did not fail from sweep to
sweep. As the stimulus intensity was increased, other fibers may have
been recruited which caused deflections on the rising and falling
phases of the EPSP at different latencies. The possibility of
activating polysynaptic pathways also cannot be ruled out.
The chemical nature of the EPSP
The dorsal root evoked EPSPs could be reduced in amplitude by
applications of both non-NMDA and NMDA receptor antagonists. All
neurons tested responded with fast EPSPs, where the initial phase of
the EPSP increased in amplitude as the membrane was hyperpolarized, consistent with the idea that the NMDA receptor plays a minimal role in
this part of the EPSP. In addition, on the six occasions tested, the
EPSP was reduced in amplitude by applications of the non-NMDA receptor
antagonists NBQX and DNQX. This indicated that these receptors were
always involved in mediating responses from primary afferent inputs.
This observation is supported by the fact that both projecting and
interneurons in the dorsal column nuclei of the rat express
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors, although there is heterogeneity in the subtypes expressed in
the different neurons (Propratiloff et al. 1997
). In 11 of the 21 neurons, a distinct inflection on the falling phase of the
EPSP was observed, which was reduced in amplitude as the membrane was
hyperpolarized. This reduction in EPSP amplitude at hyperpolarized
potentials may be due to voltage-dependent blockade by
Mg2+ of the NMDA receptor-activated channel (see
Nowak et al. 1984
). The later part of the EPSP was also
reduced on the two occasions tested by applications of the NMDA
receptor antagonist AP-5. Although the NMDA receptor antagonist was
only applied twice, this observation, when taken together with the
voltage relationship of the EPSP and the fact that the non-NMDA
receptor antagonists did not block the EPSP fully, indicate a role for
both non-NMDA and NMDA receptors in mediating the dorsal root evoked
responses in some DCN neurons. However, this study does not
definitively prove that NMDA receptors are located on all
DCN neurons postsynaptic to primary afferent terminals.
Inhibition of dorsal root evoked responses by baclofen
Fast EPSPs could be reduced in amplitude by applications of the
GABAB receptor agonist baclofen that had no
effect on the postsynaptic membrane potential or input resistance of
the neuron suggesting that the site of action was presynaptic. This is
of interest since until now, observations have been concentrated mainly
on the effects of activation of presynaptic GABAA
receptors. It is known that primary afferents innervating DCN neurons
are inhibited by GABA acting presynaptically at bicuculline sensitive sites to cause primary afferent depolarization (Davidson and
Southwick 1971; Simmonds 1978
). However, the
role of the GABAB receptor, which can also be
located presynaptically, has not been studied. Newberry and
Simmonds (1984)
reported a bicuculline-insensitive component of
the slow negative wave in their field potential recordings from gracile
nucleus when stimulating the dorsal columns that may have been due to
activation of GABAB receptors. In addition, activation of GABAB receptors decreases the size
of the receptive field of neurons in the DCN (Schwark et al.
1999
). It is likely that GABA may have a two-fold effect on
presynaptic release of excitatory neurotransmitter: 1) via
the ligand-gated GABAA receptors to depolarize
the terminal (Andersen et al. 1970
), and 2)
via GABAB receptors to prolong the presynaptic
inhibitory effect since this is mediated by activation of a second
messenger system (Malcangio and Bowery 1996
).
This double action of GABA has been observed on primary afferent input
to the dorsal horn where the two GABA receptors coexist on the membrane
of slowly conducting primary afferents (Desarmenien et al.
1984
). The GABAA receptor activation produces primary afferent depolarizations (Curtis et al.
1971
), and the GABAB receptor decreases
calcium conductance presynaptically to decrease neurotransmitter
release (see Malcangio and Bowery 1996
).
Baclofen reduced the responses of all the DCN neurons tested to dorsal root stimulation without affecting the intrinsic properties of the postsynaptic membrane, suggesting that GABAB receptors are found on presynaptic primary afferent terminals impinging onto all neurons (see Types of DCN neuron).
Use of the neonatal preparation: Relevance to the adult
These data have been obtained from a neonatal rat brain
stem-spinal cord preparation that enables in vitro study of the long afferent pathway onto DCN neurons. However, these preparations are
taken from rats of 2-5 days in age; is the pharmacology of these
inputs likely to be similar to the adult? Excitatory amino acids are
known to be important in the processing of primary afferent inputs onto
DCN neurons in adults (see INTRODUCTION). Furthermore, studies of the AMPA receptor distribution in adult rats show expression of AMPA subtypes in both projection neurons and interneurons, indicating that a large part of transmission in the DCN in adult involves activation of non-NMDA receptors (Propratiloff et al. 1997). This fits in well with the data presented here. The role of NMDA receptors in the DCN in adult is less well documented. In the
adult mouse brain stem, the DCN expressed high levels of two NMDA
receptor channel subunit mRNAs, indicative of a role for NMDA receptors
in these nuclei (Watanabe et al. 1994
), but to date the
role of these NMDA receptors in the adult DCN is unknown. In other
brain areas, e.g., the rat neocortex, NMDA receptor-mediated responses
in younger animals were longer and comprised a larger component of the
evoked responses than those observed in older animals (Burgard
and Hablitz 1993
). Lo Turco et al. (1991)
observed that in the neocortex, NMDA receptors were blocked in a
voltage-dependent manner by magnesium in rats at birth, although a
lesser degree of voltage dependency was reported in the hippocampus
(Ben-Ari et al. 1988
). Our observations indicate that in
the DCN of neonatal rats, NMDA receptors do play a role in the
transmission of afferent inputs onto neurons and that a degree of
voltage dependency is present at this age. However, it may be that with
maturation, the NMDA component of the evoked response to afferent nerve
stimulation decreases in the DCN.
GABA is known to be an important neurotransmitter in the DCN in the
adult rat (see INTRODUCTION), although the role of
GABAB receptors is less well documented. Early
studies by Newberry and Simmonds (1984) showed that a
component of the slow negative wave of the evoked field potential
recorded in the gracile nucleus was resistant to bicuculline. In
addition, GABAB receptors have recently been
shown to influence the size of the receptive field of neurons in the
dorsal column nuclei of rats, suggesting that these receptors play an
important role in the adult DCN (Schwark et al. 1999
).
In the hippocampus, it has been shown that the presynaptic GABAB-mediated inhibition is well developed at
birth (Gaiarsa et al. 1994
), although the postsynaptic
GABAB-mediated inhibition is poorly developed.
Therefore, it is likely that the role of GABAB
receptors in the presynaptic inhibition of neurotransmitter release is
similar in the neonate and the adult.
Types of DCN neuron
DCN neurons showed differing characteristics according to the
shape of their action potential and the presence or absence of a
depolarizing sag in response to application of hyperpolarizing current
pulses (indicative of activation of IH).
The different electrophysiological characteristics may be due to
recording from the two main types of neuron found in the DCN, the
projection neurons and interneurons (Anderson et al.
1964; Propratiloff et al. 1997
), and may
therefore prove to be a useful tool for identification of neurons in in
vitro preparations. Indeed, in a recent in vivo study, projection
neurons in the DCN displayed similar depolarizing sags while presumed
interneurons did not (Canedo et al. 1998
), although
evidence suggested the presence of a low-threshold calcium conductance
in both groups of neurons. It was not possible to distinguish between
the response profiles of the different types of neuron to dorsal root
stimulation, either from the shape of the EPSPs or the chemistry of the
responses. However, it may be that neuromodulators other than GABA
acting at GABAB receptors will affect the DCN
neurons differently.
In conclusion, these data have shown that in the neonatal rat brain stem-spinal cord preparation, fast EPSPs elicited in DCN neurons by stimulation of dorsal roots 1) have a monosynaptic component, 2) are mediated by excitatory amino acids acting on both non-NMDA and NMDA receptors, and 3) can be modulated by activation of GABAB receptors located presynaptically. The neonatal rat brain stem-spinal cord preparation seems very suitable for the study of the pharmacology of afferent transmission onto DCN neurons.
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ACKNOWLEDGMENTS |
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We thank Novartis for a generous gift of CGP35348. T. Trippenbach was a visiting Professor from McGill University, Montreal, Canada.
This work was supported by the British Heart Foundation.
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FOOTNOTES |
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Present address and address for reprint requests: S. A. Deuchars, Dept. of Physiology, University of Leeds, Worsley Medical and Dental Building, Leeds LS2 9NQ, UK (E-mail: phssad{at}leeds.ac.uk).
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 22 March 2000; accepted in final form 31 May 2000.
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REFERENCES |
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