Download Modulation of Responses of Feline Ventral Spinocerebellar Tract

THE JOURNAL OF COMPARATIVE NEUROLOGY 443:298 –309 (2002)
Modulation of Responses of Feline
Ventral Spinocerebellar Tract Neurons
by Monoamines
INGELA HAMMAR,1* BARBARA CHOJNICKA,2 AND ELZBIETA JANKOWSKA1
1
Department of Physiology, Göteborg University, 405 30 Göteborg, Sweden
2
Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland
ABSTRACT
Ventral spinocerebellar tract neurons located in laminae V–VII of cat lumbar spinal cord
were tested for the effects of ionophoretically applied monoamines and receptor selective
agonists. Extracellularly recorded responses, monosynaptically evoked by group I afferents in
a muscle nerve, were compared before, during, and after ionophoresis. They were analyzed
with respect to changes in the number of evoked spikes and in the latency. Both serotonin
(5-HT) and noradrenaline (NA) were found to facilitate responses of all neurons tested.
Ionophoresis of three serotonin subtype receptor agonists (5-carboxamidotryptamine maleate, 5 methoxytryptamine HCl, and alpha-methyl 5-hydroxytryptamine) and of two NA
receptor agonists (phenylephrine and isoproterenol) likewise had a facilitatory effect. However, three other 5-HT receptor agonists (8-hydroxy-dipropylaminotetraline hydrobromide),
2-methyl 5-hydroxytryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl
and two NA receptor agonists (tizanidine and clonidine) had the opposite effect because they
depressed responses of the tested neurons. These results show that information forwarded by
means of the ventral spinocerebellar tract may be modulated by monoamines and that
several receptor subtypes, located pre- or postsynaptically, may be involved. The results also
demonstrate that transmission by means of group I muscle afferents may not only be
facilitated by monoamines but also depressed by selective receptor subtype activation. J.
Comp. Neurol. 443:298 –309, 2002. © 2002 Wiley-Liss, Inc.
Indexing terms: spinal cord; serotonin; noradrenaline; ionophoresis; spinocerebellar tract
Monoamines have been shown to modulate synaptic
transmission from primary afferents to spinal ascending
tract neurons in a highly differentiated manner and may,
thus, assist in adjusting information forwarded to higher
centra according to the actual needs in a given situation.
Modulation by serotonin (5-HT) and noradrenaline (NA)
was first demonstrated in pathways between nociceptors
or other high threshold afferents and cells of the spinothalamic and other ascending tract neurons (Jordan et al.,
1978, 1979; Lundberg, 1982) where primarily depressive
actions were found. Modulation has been found also in the
case of responses evoked by group II muscle spindle afferents and low threshold cutaneous afferents in dorsal
spinocerebellar and other ascending tract neurons
(Jankowska et al., 1995, 1997); these responses were either depressed (mainly by NA) or facilitated (mainly by
5-HT). In contrast, responses evoked by group I muscle
afferents were hardly affected (Bras et al., 1989;
Jankowska et al., 1997) However, when more sensitive
tests were used in a study on spinal interneurons, both
5-HT and NA were found to facilitate activation of these
© 2002 WILEY-LISS, INC.
DOI 10.1002/cne.10135
neurons by group I afferents (Jankowska et al., 2000). The
aim of the present study was, therefore, using as sensitive
tests as possible on interneurons, to investigate whether
activation of the ventral spinocerebellar tract (VSCT) neurons by group I afferents might likewise be facilitated.
Neurons investigated in this study belonged to the subgroup of VSCT neurons located in the middle part of
Rexed laminae V–VII. The main monosynaptic input to
these neurons is provided by tendon organ (group Ib)
afferents (Eccles et al., 1961; Hubbard and Oscarsson,
Grant sponsor: Swedish Medical Research Council (MFR); Grant number: 05648; Grant sponsor: EU Tempus Grant Commission; Grant sponsor:
the Swedish Society For Medical Research.
*Correspondence to: Ingela Hammar, Department of Physiology and
Pharmacology, Box 432, Göteborg University, 405 30 Göteborg, Sweden.
E-mail: [email protected]
Received 14 March 2001; Revised 2 July 2001; Accepted 20 November
2001
MODULATORY ACTIONS OF MONOAMINES
1961, 1962; Oscarsson, 1965), in contrast to another subpopulation of VSCT neurons to which the main monosynaptic input comes from group Ia muscle spindles afferents
(Burke et al., 1971; Lundberg and Weight, 1971) and
which are located along the border between the gray and
white matter in lamina IX and in the lateral part of
lamina VII, and therefore, termed spinal border cells (Cooper and Sherrington, 1940; Matsushita et al., 1979; Bras
et al., 1988). Because VSCT neurons forward information
to the cerebellum on both muscle tension and on the
operation of spinal neuronal networks that process this
information (Lundberg, 1971), modulation of the effectiveness with which VSCT neurons are activated by group I
afferents must be of prime importance for the control of
spinal activity based on this information. A preliminary
report on some of these results has been published (Hammar Simonsberg et al., 1998).
METHODS
Preparation
Experiments were performed on 13 cats, 6 males and 7
females, each weighing 2.40 –3.9 kg and aged 6 – 8 months.
Anesthesia was induced by a single dose of pentobarbital
Natricum i.p. (45 mg/kg) and maintained by intermittent
doses of chloralose (Alpha Chloralose, Rhône Poulenc
Santé) up to 50 – 60 mg/kg i.v. A pneumothorax was performed bilaterally to avoid respiratory movements, and
the animals were paralyzed with pancuronium bromide
(Pavulon, Organon, 0.4 mg i.v.) supplemented at regular
intervals (0.2 mg i.v. every 2–3 hours). End expiratory
CO2 (EtCO2) was measured throughout the experiment,
and the respiratory rate and volume adjusted to keep the
EtCO2 at approximately 4.0%. The blood pressure was
measured continuously by means of an intra-arterial catheter and maintained above 90 mm Hg by adjusting the
parameters of the artificial respiration and the infusion
rate (1–2 ml/kg per hour) of a glucose buffer (5 g of glucose
and 0.84 g of Na HCO3 in 100 ml of H2O, pH 8.4; Haglund
and Lundgren, 1972). The heart rate was monitored by
means of two subcutaneous electrodes and ranged between 180 and 235 beats per minute. Urine was collected
by means of an indwelling catheter. The temperature was
monitored by an esophageal thermometer, and two infrared lamps were automatically switched off when the core
temperature rose above 38°C. The depth of anesthesia was
Abbreviations
2-me-5-HT
5-CT
5-HT
5-meT
8-OH-DPAT
␣-me-5-HT
DOI
FDL
GS
L4-L5
nA
NA
Q
PBST
SMAB
T
VSCT
2-methyl 5-HT
5-carboxamidotryptamine maleate
5-hydroxytryptamine; serotonin
5 methoxytryptamine HCl
8-hydroxy-dipropylaminotetraline hydrobromide
alpha-methyl 5-HT
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCL
flexor digitorum longus
gastrocnemius
lumbar segments in the spinal cord
nanoampere
noradrenaline
quadriceps
posterior biceps and semitendinosus
semimembranosus and anterior biceps
threshold
ventral spinocerebellar tract
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checked continuously during the experiments by verifying
either that there was no withdrawal reflex present or,
when the animals were paralyzed, that the size of the
pupils as well as the heart rate and blood pressure remained constant. The animals were killed after the experiments by a lethal dose of pentobarbital i.v. (30 mg/kg)
followed by air injection. Electrocardiograph monitoring
was discontinued only after cardiac arrest. The Animal
Ethics Committee of Göteborg University has approved of
the experimental procedures.
A laminectomy was performed exposing the spinal cord
between the second and seventh lumbar segments (L2–
L7), and an additional laminectomy was made at a low
thoracic level, preferably Th 12–13. The dura mater was
kept intact, and only small openings were made in it at the
sites of micropipette insertion.
Peripheral nerves in the left hindleg were cut and dissected free. These nerves included the quadriceps (Q),
sartorius (Sart), gastrocnemius (GS), posterior biceps and
semitendinosus (PBST), anterior biceps and semimembranosus (SMAB), plantaris (Pl), flexor digitorum and hallucis longus (FDL, the interosseus nerve being removed),
superficial peroneus (SP), deep peroneus (DP), and suralis
(Sur). The Q and Sart nerves were mounted together in a
sub-cutaneously fastened cuff electrode. Other peripheral
nerves were mounted on pairs of silver wire electrodes in
a pool of paraffin oil kept at 36 –37°C. In two animals, a
craniotomy was performed exposing the caudal part of the
cerebellum. A tungsten electrode insulated except for its
very tip was inserted into the contralateral superior cerebellar peduncle (at an angle of 30 degrees from the vertical, at Horsley-Clarke coordinates P2, L3.5-4, H1-2).
Histologic control of the location of the
stimulating electrode in the superior
cerebellar peduncle
At the end of the experiment an electrolytic lesion was
made by passing 0.5 mA for 10 seconds through the tungsten electrode. The lesion was made at the depth at which
the threshold for antidromic activation of the first encountered VSCT neurons was at its lowest. The animal was
perfused with formaldehyde through the carotid artery.
The brain was removed and cut in 50-␮m-thick sections by
using a freezing microtome, and the sections were counterstained with cresyl violet. The electrode tracks were
reconstructed by superimposing consecutive camera lucida drawings made from all the sections in which the
tracks were seen. The site of the electrolytic lesion was
verified (arrow in the photomicrograph in Fig. 1) and
related to morphologic structures (diagram in Fig. 1). Images were taken by using Olympus DP50 digital camera.
Recording and stimulation
The neurons were recorded extracellularly by using
glass micropipettes filled with a 2 M NaCl solution and
with a tip diameter of 1.5–2.5 ␮m and a resistance of
0.8 –2.5 M⍀. The cord dorsum potentials evoked by afferent volleys were recorded through the dura by using a
silver ball electrode placed over the same segment. Rectangular electrical pulses of 0.1-msec duration were used to
stimulate the nerves. The stimulus intensity is given as
multiples of threshold (T) for the most sensitive afferents
in a given nerve. Pairs of stimuli, 2.5 or 3.3 msec apart,
were usually used to activate the neurons. The intensity of
the stimuli was then adjusted so that the first stimulus
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evoked an EPSP that was either above or just below
threshold for generating an action potential. If it was
subthreshold, the intensity of the stimuli was adjusted
according to whether any action potential was induced by
the second stimulus. Because the EPSP after the second
stimulus was superimposed on that after the first stimulus, the presence of an action potential after the second
stimulus indicated that the first stimulus was close to
threshold. In addition, in the cases of inhibitory modulatory actions, the presence of an action potential after the
second stimulus (in the absence of an action potential
after the first stimulus) provided evidence that the experimental conditions had not changed. When facilitatory
effects of the tested drugs were expected, the stimulus
intensity was adjusted to induce less than 5 of 20 possible
responses by the first stimulus in a series of 20 trials.
When depressive effects were expected, the stimulus intensity was adjusted to elicit 10 –15 of 20 possible responses. In the latter case, stimuli near maximal for group
I afferents (near 2T) were often used. Nevertheless, despite efforts to use standardized test responses, strictly
comparable numbers of responses could not be evoked by
the test stimuli in all the tested neurons. The facilitation
and the depression were expressed by an increase and a
decrease in the proportion of responses evoked by the first
stimulus, respectively, even if the total number of responses evoked by the pair of the stimuli remained unchanged. As the afterhyperpolarization after a spike elicited after the first stimulus will depress the EPSP after
the second stimulus, only responses after the first stimulus were taken into account. It will be noted that, because
of a large and long-lasting afterhyperpolarization after
action potentials of VSCT neurons (Eccles et al., 1961), the
2.5- to 3.3-msec intervals between the stimuli that were
optimal for the temporal facilitation of synaptic actions of
group I afferents were too short to allow the neurons to be
activated by the successive stimuli.
Sampling and cell identification
VSCT neurons were tracked for in the L4 –L5 segments
and were usually found in the area just ventral to that of
prominent intermediate zone group II field potentials in
middle parts of Rexed laminae V–VII where group I field
potentials from the dissected nerves appeared. They were
identified according to the following criteria: (1) The ascending axonal projection of all neurons was established
by antidromic invasion from the contralateral ventrolateral funiculus at the Th12 level where transdural stimulation by pulses of 0.2-msec duration was applied by a pair
of silver ball electrodes. The threshold was usually between 40 and 180 ␮A, and latency was in the range of
0.52–1.68 msec. In addition, in 12 neurons in two cats, the
identification of the neurons was further verified by antidromic activation from the cerebellum, evoked by 170 –360
␮A stimulation (Hubbard and Oscarsson, 1961). A collision test for antidromic invasion was performed routinely,
and only those neurons in which a collision was observed
at a critical interval (as illustrated in Fig. 1) were included
in the sample. (2) Monosynaptic group I input was defined
as a neuronal response to either single or double stimuli
not exceeding 1.8 times threshold (T) for the most sensitive fibers in a nerve (Jack, 1978), a minimal segmental
latency of up to 1.7 msec after near-maximal stimuli, or
both. (Oscarsson, 1957). The only exception was made for
stimuli applied to Q (a much thicker nerve and, in addi-
tion, stimulated in a cuff electrode) in the case of which
stimuli up to 2.2T were considered to be within the group
I range and subthreshold for group II afferents (Edgley
and Jankowska, 1987).
The reported results are based on records from 97 neurons. Their position in the spinal cord both regarding
depth (1.86 –3.48 mm as measured from the surface) and
segmental distribution (L4 and L5) corresponds to those in
previous reports (Hubbard and Oscarsson, 1961, 1962).
Conduction velocity was calculated over distances between recording sites in the spinal cord and either the
cerebellum (mean, 88.8 m/sec) or the thoracic segments
(mean, 79.8 m/sec). In these calculations, 0.2 msec were
subtracted from the latency of antidromic activation to
account for the latent period of initiation of action potentials by electrical stimuli (Jankowska and Roberts, 1972).
The results are comparable to previous data giving a conduction velocity of these neurons in the range of 70 –120
m/sec (Oscarsson, 1965). Of muscle nerves, GS was the
predominant source of input to the tested neurons (43
neurons) followed by Q (38 neurons), PBST (13 neurons),
FDL (8 neurons), and SMAB (3 neurons). Five neurons
were monosynaptically activated by two different nerves,
all in combinations with GS (with FDL in three cells, with
PBST and SMAB in one cell each).
Drug ionophoresis
A double micromanipulator consisting of two independently controlled microdrives, each holding a separate
glass micropipette was used (Engberg et al., 1972). The
tips of the micropipettes were positioned less than 10 ␮m
apart under microscopic control but manipulated separately so that the micropipettes could be inserted individually into the spinal cord (for detailed description see Bras
et al., 1989). The recording micropipette, filled with a 2 M
NaCl solution and with a tip diameter of 2.0 –2.5 ␮m, was
used for tracking for individual neurons. The second glass
micropipette (tip diameter 2.5–3.0 ␮m) was used for ionophoresis. It was filled with a monoamine or one of its
agonists (resistance, 6 –22 M⍀) and only inserted into the
spinal cord after the investigated cell had been properly
identified and control records obtained. Neither the shape
nor the amplitude of action potentials of the cells to be
accepted for a further analysis changed during this procedure more than marginally. A retaining current of 10 –20
nA was applied through the drug-containing electrode
during the positioning phase. The drugs were ejected by
ionophoresis by passing a positive constant current for 1–3
minutes. They were ejected with 20 nA, but the current
was reduced to 8 –10 nA if the electrodes showed a tendency to block. During the recovery period, the drugcontaining electrode was either withdrawn or, if the cell
was to be exposed to the same drug again after the recovery period, a retaining negative current of 20 – 40 nA was
passed.
From several more or less specific 5-HT and NA receptor
agonists available, the following were chosen as representative of their different classes, having been used previously in similar studies on monoaminergic modulatory
effects in the cat spinal cord (Bras et al., 1989) and as
being readily soluble in water for ionophoresis. These agonists included 5-hydroxytryptamine (5-HT; serotonin,
Sigma, 0.2 M, number of tests ⫽ 15, 13 neurons) and its
agonists alpha-methyl-5-HT (␣-me-5-HT, RBI, 0.1 M,
number of tests ⫽ 6, 6 neurons), 8-hydroxy-dipropyl-
MODULATORY ACTIONS OF MONOAMINES
aminotetraline hydrobromide (8-OH-DPAT, RBI, 0.15 M,
number of tests ⫽ 11, 11 neurons), 2-methyl-5-HT (2-me5-HT, Sandoz, 0.1 M, number of tests ⫽ 10, 10 neurons), 5
methoxytryptamine HCl (5-meT, RBI, 0.2 M, number of
tests ⫽ 6, 5 neurons), 1-(2,5-dimethoxy-4-iodophenyl)-2aminopropane HCl (DOI, RBI, 0.15 M, number of tests ⫽
10, 10 neurons), 5-carboxamidotryptamine maleate (5-CT,
Glaxo, 0.2 M, number of tests ⫽ 7, 4 neurons), noradrenaline (NA, Sigma, 0.2 M, number of tests ⫽ 16, 13 neurons), tizanidine (Sandoz, 0.2 M, number of tests ⫽ 9, 5
neurons), clonidine (Sigma, 0.1 M, number of tests ⫽ 4, 2
neurons), phenylephrine (Sigma, 0.2 M, number of tests ⫽
11, 7 neurons), and isoproterenol (Sigma, 0.2 M, number
of tests ⫽ 10, 5 neurons). These compounds were dissolved
in distilled water, and the pH was adjusted to 4.5 by
adding HCl.
Ten neurons were tested with more than one drug; this
was done by replacing the drug-containing micropipette
after completed ionophoresis with a new micropipette
filled with the new compound, different from the one previously used. This procedure involved withdrawing both
the recording and the drug-containing electrodes from the
spinal cord to adjust the microelectrode tips, while taking
precautions so that the recording electrode was not manipulated in any other direction. Upon re-entering the
spinal cord, the recording micropipette was advanced to
the same depth from the surface as before and the neuron
re-identified by antidromic activation and latency of responses, before the ionophoresis procedure was repeated
with the new drug-containing micropipette. With four
neurons, the same drug was applied twice by using the
same drug-containing electrode (5-HT twice, DOI and NA
once each) after a period of recovery.
For interpretation of the effects of ionophoresis, it is
important to verify that the effects seen are mediated by
the drug itself and cannot merely be ascribed to the effects
of H⫹ ions ejected by passing positive current (see Krnjevic and Phillis, 1963; Bras et al., 1989; Skydsgaard and
Hounsgaard, 1996, for discussion of this point). Therefore,
in a control experiment on eight neurons, an HCl solution
at pH 4.5 was used for ionophoresis instead of a monoamine solution. The number of responses of these neurons
remained unaffected (P ⫽ 0.3 after 1 minute), and the
latency remained consistent. This finding argues against
any significant direct action of H⫹ ions and indicates that
the effects seen in this experimental series are not merely
the result of pH changes. Further evidence in this direction are the opposite effects of different drugs ejected from
solutions with the same pH, in some instances even on the
same neuron, both in the present and in previous studies
(Jankowska et al., 1997).
As a further measure of control, the effect of ionophoretic application of some of the substances used in the
present series was tested on field potentials to ensure that
a drug solution was effective and that its actions were
consistent with those described previously (Bras et al.,
1989).
Analysis
The effect of monoamines was judged from changes in
the number of spikes evoked by the first stimulus, and in
their latency, even when a pair of stimuli was used (see
above). Records were analyzed on- and off-line by using a
software program designed for this purpose (Eide, Holmström and Pihlgren; see Fig. 1 in Jankowska et al., 1997).
301
The peristimulus time histograms (PSTH; bin width 20
␮sec) and cumulative sums of action potentials in response to 20 consecutive stimuli were made on line and
stored in parallel with the original records. Control
records were taken before the drug-containing electrode
was inserted into the spinal cord. These records were then
compared with records obtained immediately after the
insertion of the second, drug-containing, electrode and
before any drug was administered (placement records,
which should not differ from control records more than
marginally). During ionophoresis, records were sampled
every 15 to 30 seconds for up to 3 minutes and during the
following recovery period every 1–5 minutes up to 15
minutes. The effects of the drugs were evaluated taking
into account changes in responses evoked within a time
window of 1 msec from the afferent volleys. Data are
expressed as means ⫾ SEM. Statistical significance was
calculated by using the Wilcoxon signed rank test.
RESULTS
Modulatory actions of 5-HT and NA
The main result of this study is that both 5-HT and NA
facilitate activation of VSCT neurons by group I afferents.
These facilitatory effects are illustrated in Figures 1 and 2
and summarized in Figures 6A and 7A.
5-HT. Figure 1 shows the facilitatory effects of 5-HT of
responses of a VSCT neuron, identified by antidromic
activation from the cerebellum (bottom records) and short
latency group I input from GS (top records). The facilitation is illustrated with PSTHs (left panel) and with simultaneously obtained cumulative sums of the responses
(right panel) evoked before (control), during and after
(recovery) application of 5-HT. The figure shows an increase in the effectiveness of the first stimuli in activating
the neuron during 5-HT ionophoresis, indicated by an
increase in the PSTHs and cumulative sums after the first
stimulus, by an increase in the total number of spikes
induced by the two stimuli and by a shortening in the
latency of the responses (with increasing proportions of
responses appearing earlier than originally, i.e., to the left
of the dotted lines).
Similar effects were seen in all 13 neurons tested. As
shown in Figure 6A, the mean number of responses to the
first stimulus increased after 1 minute of 5-HT ionophoresis from 2.27 ⫾ 1.0 in control records up to 10.5 ⫾ 2.0 (P ⫽
0.001). In the case of seven of these 13 neurons originally
responding only to the second stimulus, the effect was
even more marked because application of 5-HT made
these neurons respond to the first stimulus, and the number of spikes evoked by this stimulus after 2 minutes of
ionophoresis was increased from 0 to 8.7 ⫾ 2.5 (P ⫽ 0.02).
Records and data in Figure 1 are from such a neuron. The
mean latencies of responses of all neurons decreased correspondingly, by 0.32 msec for those originally induced by
the first stimulus and by up to 0.48 msec if the total
decrease is considered. The facilitation was typically evident already after 15–30 seconds of 5-HT ionophoresis and
remained unchanged until the ionophoresis was discontinued. During the first 10 minutes of the recovery period,
the number of spikes in all but two neurons returned to
values at or very close to the original control numbers.
When 5-HT was applied for a second time after a recovery
period after the first application (in two neurons, as illus-
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I. HAMMAR ET AL.
trated in Fig. 1), the facilitatory effects were reproduced
although not necessarily at the same time course. The
most likely reason for the later appearance of the facilitation during the second application was the use of the
retaining current during the recovery period and the re-
Fig. 2. Examples of facilitatory effects of NA. Top records show
responses of a VSCT neuron to the second stimulus applied to the GS
nerve and the simultaneous records from the cord dorsum. Peristimulus time histograms (PSTH and cumulative sums of responses evoked
before, during, and after NA ionophoresis are shown in the left and
right panels, as in Figure 1. All indications as in Figure 1. For
abbreviations, see list.
sulting lower concentration of 5-HT in the tip of the micropipette.
NA. Facilitatory effects of NA greatly resembled those
of 5-HT, both in individual neurons (as illustrated in Fig.
2) and in the total sample, even though the mean facilitation appeared to be somewhat weaker than that by 5-HT
(cf. Figs. 7A, 6A). The mean total number of spikes evoked
by the first stimulus increased in 12 of the 13 neurons
from 4.8 ⫾ 1.5 to 11.5 ⫾ 2 (P ⫽ 0.01) after 1 minute of
Fig. 1. Examples of records from VSCT neurons and of facilitatory
effects of 5-HT. A: Top records show responses of a VSCT neuron to
the second stimulus applied to the GS nerve, with the parallel records
from the cord dorsum. Arrows indicate incoming afferent volleys. The
photomicrograph shows an unstained section of the L4 segment of the
spinal cord. The enclosed area indicates the region within which the
recorded neurons were located. Peristimulus time histograms (left
panel) and cumulative sums (right panel) illustrate changes in the
timing and in the number of responses after 20 consecutive stimuli:
before 5-HT ionophoresis (control), after 1 and 2 minutes of ionophoresis, after 9 minutes of recovery during which a retaining current with
an opposite polarity was applied, and during a second period of ionophoresis. Amplitude calibration is for the cumulative sums only. Vertical dotted lines indicate the original minimal latencies. B: Records
illustrate antidromic activation of the neuron illustrated in A from the
superior peduncle and its collision by the preceding synaptic activation (by single stimulus at a higher intensity). The site of the application of the stimuli in the superior peduncle is indicated on the
corresponding plate from Figure 94 of Verhaart (1964). The photomicrograph shows the area enclosed in the box at a higher magnification.
The stimulation site at the level of the most lateral of the five electrode tracks 0.5 mm apart is indicated by an arrow. Asterisk, superior
peduncle. Thin vertical line, midline. Sup. Ped., superior cerebellar
peduncle For abbreviations, see list. Scale bars ⫽ 200 ␮m in A, 1 mm
in B.
MODULATORY ACTIONS OF MONOAMINES
303
ionophoresis. In seven of these neurons, the latency was
shortened by as much as 0.32 msec (0.18 ⫾ 0.02 msec after
1 minute of ionophoresis, P ⫽ 0.002), whereas in the
remaining five neurons the latencies were unchanged.
Only in one exceptional neuron were no changes found,
either in the number of spikes or in the latency after 3
minutes of ionophoresis. One neuron was tested twice, and
the effects of the second application of NA (after a 10minute-long recovery period) were consistent with the initial effect.
To further investigate the actions of 5-HT and NA and
to elucidate by means of which particular membrane receptors they might be evoked, effects of several 5-HT and
NA receptor agonists were compared. Facilitatory effects
of 5-HT and NA turned out to be replicated only by some
of their receptor agonists while others had depressive
actions. The effects of all the tested agonists, therefore,
will be reported separately.
Facilitatory actions of 5-HT agonists
Of the six 5-HT agonists tested, three had facilitatory
effects. Examples of these effects are shown in Figure 3
and the results for the whole sample are summarized in
Figure 6B–D. Effects almost as strong as those of 5-HT
were evoked by 5-meT and 5-CT, whereas the effects of
alpha-methyl-5-HT were somewhat weaker and seemed to
have a different time course. However, effects of each of
these agonists on all tested VSCT neurons were consistent. Furthermore, whenever they were tested on responses evoked from two different muscle nerves (stimulated approximately 40 msec apart), in one, three, and one
neurons, respectively, a similar degree of facilitation of
responses evoked from these nerves was found and its
time course was similar.
5-meT (5-HT2, 5-HT4 receptor agonist). Effects of
5-meT were studied on five neurons. Responses of four of
these neurons were potently facilitated and only in one
neuron were they mildly affected. As shown in Figure 6B,
the number of spikes evoked by the first stimulus increased from 0 in the control records to 5.8 ⫾ 1.8 after 1
minute of ionophoresis (P ⫽ 0.04) and up to 16.6 ⫾ 2.4
after 2 minutes (P ⫽ 0.04) and the effects appeared already after 15 seconds. The latencies decreased by up to
0.48 msec (mean, ⫺0.26 ⫾ 0.02, P ⫽ 0.04) after 1.5 minutes of ionophoresis, but changes in latencies were highly
variable, even in individual neurons.
5-CT (5-HT1A,1E,5-HT5, 5-HT7 receptor agonist).
5-CT had similarly strong facilitatory effects on responses
of all four neurons tested. The number of spikes increased
from 6.0 ⫾ 2.1 in control records to 18.3 ⫾ 1.7 (P ⫽ 0.02)
after 1 minute of ionophoresis, and facilitation was seen
already after 15 seconds (Fig. 6C). The changes in latency
(⫺0.31 ⫾ 0.07, P ⫽ 0.007) were likewise evident in all
neurons already after 15 seconds.
Alpha-me-5-HT (5-HT2B receptor agonist). Facilitatory effects of alpha-me-5-HT were seen on all six neurons
tested but appeared to be weaker than those of 5-HT and
to manifest themselves much later (cf. Fig. 6D). The total
increase in the number of responses (from 4.4 ⫾ 2.1 in
control records up to 9.9 ⫾ 2.9 after 2 minutes of ionophoresis; P ⫽ 0.03) was not much weaker than that found
after application of 5-HT. However, the facilitation developed slower than the 5-HT–induced facilitation; it was in
fact seen only after 1 minute (in four neurons) or even only
after 2 minutes (in two neurons). Furthermore, the facili-
Fig. 3. Examples of facilitatory effects of two 5-HT agonists, 5-meT
(left) and alpha-me-5-HT (right). The same format as in Figures 1 and
2. Note that the neurons originally responded either only (5-meT) or
mainly (alpha-me-5-HT) after the second stimulus, whereas during
application of the drugs, the responses appeared in increasing numbers after the first stimulus. Note also that the recovery was complete
after the ionophoresis of 5-meT had ceased, whereas a weaker recovery was seen for alpha-me-5-HT. For abbreviations, see list.
tatory effects continued to increase after the ionophoresis
had been discontinued and were longer lasting than those
of 5-HT. Despite recovery periods of up to 20 minutes, only
four neurons showed any signs of recovery and it was
noted only after between 6 and 20 minutes.
Depressive actions of 5-HT agonists
The three 5-HT receptor agonists with depressive actions included 8-OH-DPAT, 2-me-5-HT, and DOI. Effects
of 8-OH-DPAT and 2-me-5-HT were clear-cut, as shown in
Figure 4 and summarized in Figure 6E and F, whereas
those of DOI were inconsistent; individual neurons were
found to be either depressed by DOI or unaffected. Some of
the depressive effects of 8-OH-DPAT and 2-me-5-HT were
found on the same neurons (n ⫽ 8) on which facilitatory
effects were induced by either 5-HT or by one of the three
agonists described in the previous section. To this aim, the
technique of changing the drug-containing micropipette
described in the Materials and Methods section was used.
8-OH-DPAT (5-HT1A,1D, 5-HT7 receptor agonist).
Depressive effects of 8-OH-DPAT were evoked in all of the
neurons tested and with the highest statistical significance (Fig. 6E). The number of spikes decreased from
16.4 ⫾ 1.2 after the second stimulus in control records to
10.8 ⫾ 1.8 after 1 minute of ionophoresis (P ⫽ 0.005), but
the latencies of the responses were only minimally prolonged (by 0.09 ⫾ 0.02 msec after 1 minute), although this
change was statistically significant (P ⫽ 0.01). The effect
was evident already after 15 seconds of ionophoresis in
seven of the cells and in one cell a decrease in number of
304
I. HAMMAR ET AL.
spikes and a prolongation of the latency (by 0.22 msec)
was seen as soon as the drug-containing electrode was
placed near to it. In this latter cell, the inhibition increased during the ionophoresis, thus indicating that the
effect was drug related and not caused by other factors.
The depression was almost maximal after 1 minute (Figs.
4, 6E) and was maintained for as long as the ionophoresis
was continued. Upon withdrawing the drug-containing
electrode, a complete recovery to the original level of activity was noted within the first 5 minutes in three cells,
whereas the remaining eight cells showed only an incomplete recovery (two cells) or none at all after up to 18.5
minutes of observation.
2-me-5-HT (5-HT3 receptor agonist). This agonist
depressed responses of nine of ten neurons tested, the
remaining neuron being unaffected after up to 2 minutes
of ionophoresis. In those depressed, the number of spikes
was reduced from 14.0 ⫾ 2.1 to 6.0 ⫾ 1.5 (P ⫽ 0.03) after
1 minute of ionophoresis. Although changes in the latencies were variable, the overall effect was a prolongation by
0.14 ⫾ 0.05 msec after 1 minute of ionophoresis (P ⫽
0.008). The onset of the depressive actions of 2-me-5-HT
tended to be as rapid as of depression evoked by 8-OHDPAT. In the five neurons in which a recovery was investigated, a partial recovery was found after 6 –10 minutes.
DOI (5-HT2A,2B,2C receptor agonist). Depressive effects of DOI were seen in only 6 of 10 tested neurons. The
data in the affected and unaffected neurons, thus, are
shown separately (in Fig. 6G and H, respectively). When
present, the depression was found to be as effective as
after 2-me-5-HT application. The mean number of spikes
of these neurons decreased from 16.2 ⫾ 1.9 to 9.3 ⫾ 1.8
(P ⫽ 0.04) after 1 minute of ionophoresis, and the effect
was enhanced during the next minute. The latency was
prolonged by 0.31 ⫾ 0.16 msec after 1.5 minutes of ionophoresis, but this was not statistically significant. Responses of the four remaining neurons were practically
unchanged (see Fig. 6H). However, in two of these four
neurons, inhibitory effects were seen at the commencement of the ionophoresis, after which there was either a
return to the original control levels after 1 minute of
ionophoresis or, in one neuron, a slight facilitation. Although no technical problems were encountered while
passing current through the drug-containing electrodes, it
cannot be excluded that the lack of response in the four
unaffected neurons might nonetheless be ascribed to drugejecting problems.
Facilitatory actions of NA agonists
Of the four NA receptor agonists tested, the ␣1-adrenoceptor agonist phenylephrine and the ␤-adrenoceptor agonist isoproterenol were found to facilitate responses of VSCT
cells.
Phenylephrine (␣1 receptor agonist). Phenylephrine increased the number of spikes evoked by the first
stimulus in all of the neurons tested, from 3.8 ⫾ 1.1 to
10.1 ⫾ 2.4 (P ⫽ 0.01) after 1 minute of ionophoresis (see
Fig. 7B). In individual neurons, the effect was often as
dramatic as the effects of NA, as in the case of the neuron
illustrated in Figure 5, which displayed an increase from
2 responses after the first stimulus in control records up to
the maximal number of 20 responses after 1 minute of
ionophoresis. The onset of the facilitation was likewise
very early, in a few instances already during the placement but mainly during the first 15–30 seconds of iono-
Fig. 4. Examples of depressive effects of two 5-HT agonists, 8-OHDPAT (left) and 2-me-5-HT (right). The same format as in Figures 1
and 2. Note that the number of spikes after the second stimulus was
reduced during ionophoresis of 8-OH-DPAT and that the responses
evoked by the first stimulus disappeared after the ionophoresis of
2-me-5-HT; they could then only be evoked after the second stimulus.
In both neurons the recovery was evident already 1.5 minutes after
the ionophoresis was discontinued. Note also that the depressive
effects were reflected in the reduction in the number of spikes,
whereas the changes in the latency were seen only during the recovery period. For abbreviations, see list.
phoresis. The main difference with effects of NA appeared
to be the longer duration of effects of phenylephrine. A
tendency for the neurons to recover was noted in three of
the neurons within 5 minutes, but in the remaining four
neurons, the degree of the facilitation even increased after
the end of ionophoresis and no recovery was seen up to 19
minutes. In contrast to the consistent increase in the
number of spikes, the latencies were only inconsistently
decreased.
Isoproterenol (␤ receptor agonist). Effects of isoproterenol tended to be facilitatory but much weaker and less
consistent than those of either NA or phenylephrine. However, after 1 minute of ionophoresis, the number of spikes
had increased in four of the five neurons tested, from 9.6 ⫾
2.5 to 16.8 ⫾ 3.0 (P ⫽ 0.04). When present, the facilitatory
effect appeared at the commencement of the ionophoresis
and remained for the duration of the drug application.
Any changes in the latency were inconsistent.
Depressive actions of NA agonists
Depression of transmission between group I afferents
and VSCT neurons by NA agonists is illustrated in Figure
5. It was evoked by tizanidine and clonidine, both acting
on ␣2 receptors, although tizanidine is considered more
specific than clonidine (Coward, 1994).
Tizanidine (␣(1) ␣2 receptor agonist). The number
of spikes evoked by the first of two stimuli in all neurons
tested were reduced, from 12.9 ⫾ 2.0 in control records to
6.3 ⫾ 2.5 after 1 minute of ionophoresis (P ⫽ 0.01; see Fig.
7D). The depressive actions appeared 15–30 seconds after
MODULATORY ACTIONS OF MONOAMINES
305
Fig. 5. Examples of facilitatory and depressive actions of NA receptor agonists, phenylephrine (left) and tizanidine (right). Format as
in Figures 1 and 2. Note that, during ionophoresis of phenylephrine,
all the responses appeared after the first stimulus, whereas in the
control recordings, almost all appeared only after the second stimulus.
Note also that, after 13 minutes, the recovery was only partial. During
ionophoresis of tizanidine, almost all responses appeared only after
the second stimulus, whereas both before and after the ionophoresis
the first stimulus was effective. For abbreviations, see list.
ionophoresis had commenced and lasted throughout the
drug application. A partial recovery was found in four of
the five neurons 5 minutes after ionophoresis had ceased.
The latencies were slightly longer during application of
tizanidine in three of the neurons, by up to 0.2 ⫾ 0.06 after
1 minute, but this prolongation was not statistically significant (P ⫽ 0.06).
Clonidine (␣(1) ␣2 receptor agonist). The modulatory effects of clonidine were very similar to those of tizanidine. The number of spikes was reduced from 10.2 ⫾ 4.2
in control records to 5.3 ⫾ 4.9 after 2 minutes of ionophoresis (Fig. 7E), and a small increase in the latency
(0.05 ⫾ 0.03 msec) was observed after 2 minutes of ionophoresis.
Fig. 6. Comparison of modulatory effects of 5-HT and 5-HT agonists (with the main affinities of these agonists indicated) on VSCT
neurons. The effects are indicated by changes in the number of responses evoked by 20 consecutive stimuli. Ordinate, the mean numbers of responses with SEM. These means were calculated for the
indicated periods: before (control) and just after the placement of the
drug-containing pipette close to the tip of the recording microelectrode, during 15–30 seconds, 45– 60 seconds, and 1.5–2.0 minutes of
ionophoresis of the indicated compounds and during the first 5 minutes of recovery. The data are for all VSCT neurons tested for effects
of a given compound (indicated in parentheses), unless stated otherwise. Statistically significant changes (compared with control data)
are indicated: single asterisks, P ⬍ 0.05; double asterisks, P ⬍ 0.01.
The lighter rightmost bar in F is for five of nine neurons only. For
abbreviations, see list.
DISCUSSION
The results of this study extend evidence (see Introduction section) that monoamines modulate transmission in
spinal pathways not only from nociceptors, other high
threshold muscle and skin afferents or group II muscle
afferents, but also transmission from group I afferents. A
previous study revealed facilitatory effects of monoamines
on transmission from group I afferents to interneurons
(Jankowska et al., 2000). In the present study, the evidence for such facilitatory effects has been extended to
VSCT neurons. In addition, evidence has now been provided that transmission from group I afferents may be not
only facilitated but also depressed by monoamines, because some 5-HT and NA receptor agonists used in this
study induced depression rather than facilitation. Thus,
these results show that monoamines may either assist or
counteract transfer of information from group I afferents
to the cerebellum. They show also that some of the membrane receptors involved in the modulation of responses of
VSCT neurons by 5-HT and NA might contribute to effects
that oppose the overall facilitatory effects of monoamines
on these neurons.
Mechanisms of modulatory actions of
monoamines on VSCT neurons
To relate the facilitatory and depressive effects of monoamines to the various membrane receptors seems easier in
306
Fig. 7. Comparison of modulatory effects of NA and NA agonists
on VSCT neurons. The same format as in Figure 6. For abbreviations,
see list.
the case of effects of NA than of 5-HT. The much clearer
facilitation by the ␣1-adrenoceptor agonist phenylephrine
than by the ␤-adrenoceptor agonist isoproterenol and the
potent depression by both ␣2-adrenoceptor agonists
tested, tizanidine and clonidine, indicate that the facilitation is mediated primarily by ␣1 receptors and the depression by ␣2 receptors. Because both tizanidine and
clonidine display some affinity to ␣1 receptors (Davies et
al., 1984; Coward, 1994) and clonidine has been reported
to act as an ␣1 receptor agonist under certain circumstances (e.g., Anden et al., 1970; Austin et al., 1976; Kehne
et al., 1985), the depressive modulatory effects evoked by
means of ␣2-adrenoceptors might, thus, be even stronger
than suggested by the present observations. However,
because the overall effects of NA are facilitatory, the contribution of the ␣2 receptors to these effects must be effectively counteracted by effects of activation of the ␣1 receptors. Thus, it may be noted that the facilitatory effects of
phenylephrine were in fact both stronger and longer lasting than those of noradrenaline. However, which of the
several subtypes of ␣1 receptors (for review, see Nicoll et
al., 1990; Zhong and Minneman, 1999) might be involved
remains to be established. There are indications that ␣1Aadrenoceptors may be functionally predominant in the
facilitation of rat spinal motoneuron activity (Wada et al.,
1997).
Tizanidine and clonidine have been extensively used in
electrophysiological and clinical studies (for review, see
Nance, 1994; Coward, 1994; Jankowska et al., 1998). They
have been found to be very effective in modulating several
spinal reflexes, in particular, flexor reflexes and locomotion (Anden et al., 1970; Forssberg and Grillner, 1973;
I. HAMMAR ET AL.
Chen et al., 1987; Chau et al., 1998) but also reflexes from
nociceptors (Davies and Johnston, 1984; Wigdor and Wilcox, 1987) and synaptic actions of group II afferents (Bras
et al., 1990; Jankowska et al., 1998). The inhibitory effects
found in the present study are, thus, in line with depressive actions previously demonstrated. This is, however,
the first report of tizanidine- or clonidine-mediated depression of synaptic actions of group I afferents, which
was not previously detected in acute experiments in animals (Bras et al., 1990; Skoog, 1996) or in clinical studies
(Delwaide and Pennisi, 1994).
The identity of 5-HT receptors involved in the facilitation and depression of synaptic actions of group I afferents
on VSCT neurons, and most likely also on other neurons,
will require further studies because neither the three
5-HT receptor agonists with the facilitatory actions nor
the three agonists with the depressive actions seem to
show affinity to the same receptors.
However, in the case of the tested 5-HT2 receptor agonists differences in their profiles might at least to some
extent explain their different effects. Alpha-me-5-HT is
considered to act mainly on 5-HT2B receptors, whereas
5-meT is less specific (Barnes and Sharp, 1999). The third
5-HT2 agonist, DOI, is considered to display affinity for
5-HT2A, 5-HT2B, and 5-HT2C receptors (Barnes and
Sharp, 1999), although the involvement of the 5-HT2C
receptors would be unlikely in view of the indications that
they are not present in the cat spinal cord (Helton et al.,
1994). The distribution of 5-HT2B receptors seems limited
in the CNS, but there are reports on the presence of these
receptors in the spinal cord (Helton et al., 1994). One
possible explanation for the differences in the effects of
these agonists on VSCT neurons, thus, could be that the
5-HT2B receptors are responsible for the facilitatory actions (accounting for effects of alpha-me 5-HT), whereas
5-HT2A receptors are involved in depressive actions. If
effects of DOI are mediated by means of both 5-HT2A and
5-HT2B receptors, but the former dominate, it might explain the inhibitory actions of this compound. If, in addition, the 5-HT2B and 5-HT2A receptor subtypes are distributed at different locations, densities, or both, along the
soma and dendrites, they might contribute to a different
degree to the final effects of the ionophoresed drugs. The
inconsistent actions of DOI might also be explained by the
involvement of actions evoked by means of some local
interneurons, in addition to those on the VSCT neurons
themselves. If DOI excites local inhibitory interneurons,
the depressive effects mediated by these interneurons
would depend on the distribution of such interneurons,
their terminals on VSCT neurons, or both, and the distance over which DOI has to diffuse to affect them. Whatever is the reason for the observed differences, these results indicate that 5-HT2 receptors, and particularly the
5-HT2B receptors, may be involved in the modulatory actions of 5-HT and that more than one 5-HT2-receptor
subtype may mediate the serotoninergic modulatory actions on VSCT neurons.
As 5-HT2 receptors are considered to be positioned
postsynaptically (Aghajanian, 1995) the effects of ionophoresis of both alpha-methyl 5-HT and 5-meT are likely
to be evoked at the level of VSCT neurons and their
long-lasting character (see Fig. 6B,D) might indicate
postsynaptic actions induced by metabotropic receptors.
The third 5-HT receptor agonist found to have facilitatory
actions, 5-CT, is considered to be a nonselective 5-HT
MODULATORY ACTIONS OF MONOAMINES
agonist (Barnes and Sharp, 1999), which binds to 5HT1A,
5HT1D, 5HT1E, as well as to 5HT5 and 5HT7 receptors (see
Clarke et al., 1997, for discussion of this point). Of these,
5HT1A receptors might possibly be eliminated as involved
in facilitation, because they most likely mediate depressive effects of 8-OH-DPAT, a selective 5-HT1A agonist
(Hjorth et al., 1982). The remaining 5-HT1 receptor subtypes, thus, could contribute to the facilitatory effects of
5-CT. However, in view of 5-HT1 receptors being negatively coupled to adenylate cyclase, and thus more likely to
depress neuronal activity (Barnes and Sharp, 1999), their
involvement would not explain the observed facilitation.
If 5-CT acts mainly on 5-HT1D receptors, which are
considered to be located presynaptically, the facilitatory
effect of 5-CT (Fig. 6C) might be related to presynaptic
rather than to postsynaptic actions. However, 5-CT also
has affinity to 5-HT5 and 5-HT7-receptors, presumed to
act postsynaptically (Barnes and Sharp, 1999). Although
little is known about their presence or physiological role in
the spinal cord, their contribution to facilitatory effects of
5-CT on VSCT neurons cannot be excluded. In fact, there
are indications that 5-HT7-receptors are involved in the
enhancement of spinal reflexes in the rabbit (Ogilvie et al.,
1999). Taken together, these results suggest that the facilitatory effects of 5-HT might be mediated both pre- and
postsynaptically and by different subtypes of receptors.
With respect to the depressive actions, those of 8-OHDPAT may be expected to be evoked mainly by means of
5-HT1A receptors, because previously reported 8-OHDPAT-mediated effects in the spinal cord could be antagonized by the specific 5-HT1A-receptor antagonist Way 100
635 (Aggelopoulos et al., 1996; Fletcher et al., 1996). If
5-HT1A receptors are present on primary afferents as has
been suggested (Daval et al., 1987), the depressive actions
of 8-OH-DPAT might be evoked by modulation of transmitter release from primary afferent fibers rather than by
acting directly on the VSCT neurons. As 5-HT1A receptors
induce hyperpolarization by means of G-protein–mediated
opening of K-channels (for review, see Aghajanian, 1995)
their activation would reduce the probability of transmitter release. 8-OH-DPAT might also act postsynaptically
on VSCT neurons because 5-HT1A receptors have been
found to be located on at least some neurons (Pompeiano
et al., 1992; Kia et al., 1996). Thus, it remains to be
established which of the previously reported effects of
8-OH-DPAT on spinal cord reflexes (see, for example, Nagano et al., 1988; Crick and Wallis, 1991; Bervoets et al.,
1993; Clarke et al., 1997), as well as on VSCT neurons, are
mediated by means of presynaptic and which by means of
postsynaptic 5-HT1A receptors. Although 8-OH-DPAT is
one of the most selective 5-HT1A agonists (Hjorth et al.,
1982), it has also been shown to display affinity for the
5-HT1D (Pauwels and Colpaert, 1996) and the 5-HT7 receptors (Lovenberg et al., 1993) as well as the for ␣2- and
dopamine receptors (Barnes and Sharp, 1999). In addition, it has been shown recently that 8-OH-DPAT–
mediated enhancement of spinal reflexes in a rabbit preparation is not antagonized by WAY 100 635 and is
suggested to be mediated by means of the 5-HT7 receptors
(Ogilvie et al., 1999). The contribution of other than
5-HT1A receptors to the depressive effects of 8-OH-DPAT
on VSCT neurons, thus, cannot be excluded.
The 5HT3 receptors involved in depressive actions of
2-me-5-HT are ionotropic, unlike all the other known
5-HT receptors. There are indications that 2-me-5-HT, the
307
5-HT3 receptor agonists used in the present experiments,
acts on postsynaptic neurons and depolarizes them, as
well as on presynaptic terminals (Derkach et al., 1989;
Barnes and Sharp, 1999). If the depolarization of terminals is associated with a decrease in the transmitter release, this process could explain the PAD (primary afferent depolarisation) evoked by 5-HT receptor agonists
(Khasabov, 1999) and the associated presynaptic inhibition. Other depressive actions mediated by 2-me-5-HT in
the spinal cord (in nociceptive pathways; Glaum et al.,
1988; Alhaider et al., 1991) have been suggested to be
mediated by an increase in the activity of inhibitory interneurons (Alhaider et al., 1991) in the same way as in the
hippocampus (Morales et al., 1996). Thus, it is an open
question whether depressive effects of 2-me-5-HT on
VSCT neurons are mediated by its direct actions on VSCT
neurons (which would be unlikely if it depolarizes them)
on terminals of group I afferents synapsing on VSCT neurons or are secondary to diffusion to some local, which
might in turn inhibit VSCT neurons.
The likely sites of modulatory actions of monoamines
found in this study will be discussed in the accompanying
paper (Hammar et al., 2002). Here, it will only be mentioned that the records from VSCT neurons investigated
in this study indicate that both the recording and the
drug-containing micropipettes were close to the initial
segment and soma (Gustafsson and Jankowska, 1976).
The slower developing facilitatory effects of alpha-methyl
5-HT, thus, might be interpreted as evoked by means of
receptors located more distally, to which diffusion of the
ejected molecules would take longer time. Rapidly occurring facilitation, like that evoked by NA or phenylephrine,
or depression, such as that evoked by 8-OH-DPAT, might
on the other hand be compatible with an effect mediated
by receptors located primarily on soma and proximal dendrites. This finding would reflect a situation similar to
that in the case of turtle motoneurons, for which some
effects of 5-HT are specifically evoked either at a distance
from the soma or at the soma level (Delgado-Lezama et al.,
1999). However, the differences in the temporal characteristics of the effects of various 5-HT agonists might also
depend on other factors. For instance, agonists activating
intracellular cascades of pathways by means of metabotropic receptors could induce slower developing but longer
lasting effects. However, metabotropic receptors could
also induce faster actions, if these were mediated by rapid
membrane-delimited pathways, and agonists acting on
ionotropic receptors could induce even more rapid modulatory actions. The observed differences might also depend
on whether a given agonist activates the intracellular
pathways (with a resulting facilitation, by means of
5-HT2, 7, ␤ receptors) or if it is negatively coupled to these
pathways (with a resulting depression, by means of
5-HT1A, ␣2 receptors). Secondary effects of diffusion of the
drugs to terminals of axons of other neurons synapsing on
the VSCT neurons may also contribute to the overall effect.
Functional considerations
In view of the very complex input to VSCT neurons, it
has been proposed that the functional role of these neurons is to integrate and mediate information on stages of
movement, or position of a limb, rather than on individual
muscles (Oscarsson, 1965). Comparison of peripheral and
descending input to VSCT neurons, ␣-motoneurons, and
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I. HAMMAR ET AL.
interneurons in spinal reflex pathways subsequently has
revealed great similarities in input to these neurons (for
ref. see Baldissera et al., 1981). This finding led to the
hypothesis that the main function of the VSCT is to monitor transmission in interneuronal pathways to motoneurons (Lundberg, 1971). According to this hypothesis, a
considerable proportion of VSCT neurons are of the inputoutput comparator type. Some of their fractions measure,
e.g., output from last-order inhibitory interneurons
against their excitatory input from group I afferents,
whereas other fractions compare input from group I afferents and from other sources, either with interneurons or
with motoneurons (Oscarsson, 1957; Lundberg and
Weight, 1971). Selective facilitation of transmission from
group I afferents to VSCT neurons by monoamines might,
thus, change the balance between the excitatory input
from group I afferents and either excitatory or inhibitory
input from other sources to these neurons. Thus, it might
interfere with the VSCT comparator function, because the
cerebellum might then be supplied with false information,
indicating insufficient activation of interneurons with input from group I afferents. However, we have shown recently (Jankowska et al., 2000) that transmission between
group I afferents and interneurons (between Ia afferents
and Ia inhibitory interneurons and between Ia and Ib
afferents and Ib interneurons) is also facilitated by monoamines. If descending monoaminergic tracts facilitate responses of the interneurons and of VSCT neurons in parallel, and to the same degree, this would allow VSCT
neurons to continue to reflect activity in the interneuronal
networks in a faithful way even when the efficacy of their
activation by group I afferents is enhanced. We also have
evidence that activation of Renshaw cells, which mediate
recurrent inhibition of not only ␣-motoneurons and interneurons mediating Ia reciprocal inhibition (Renshaw,
1946; Hultborn et al., 1971) but also of VSCT neurons
(Gustafsson and Lindstrom, 1973), may be facilitated by
monoamines (Hammar Simonsberg and Bichler, 1999a,b).
Any weakening by Renshaw cells of responses of Ia inhibitory interneurons providing inhibitory input to some
VSCT neurons, thus, might be paralleled by stronger inhibitory actions of Renshaw cells on either the same or other
VSCT neurons. It might be also considered that facilitation
of activation of VSCT neurons by Ib afferents might in some
instances favor forwarding of information on dynamic
changes in muscle tension to the cerebellum. This mechanism might, for example, be important in cases of complicated limb movements and/or when information forwarded
by means of other spinocerebellar tract neurons should be
supported by information by means of VSCT neurons.
ACKNOWLEDGMENTS
The authors thank R. Larsson for excellent technical
assistance. The authors also thank Prof. Bengt Gustafsson
for comments on the manuscript. Tizanidine was a gift to
E.J. from Sandoz. E.J. was funded by the Swedish Medical
Research Council, B.C. was funded by the EU Tempus
Grant Commission, and I.H. was funded by the Swedish
Society for Medical Research.
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