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Next Article 
The Journal of Neuroscience, May 15, 1998, 18(10):3489-3500
Galanin Receptor-Mediated Inhibition of Glutamate Release in the
Arcuate Nucleus of the Hypothalamus
Gregory A.
Kinney,
Paul J.
Emmerson, and
Richard J.
Miller
Department of Pharmacological and Physiological Sciences, The
University of Chicago, Chicago Illinois, 60637
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ABSTRACT |
It is thought that galanin, a 29 amino acid neuropeptide, is
involved in various neuronal functions, including the regulation of
food intake and hormone release. Consistent with this idea, galanin
receptors have been demonstrated throughout the brain, with high levels
being observed in the hypothalamus. However, little is known about the
mechanisms by which galanin elicits its actions in the brain.
Therefore, we studied the effects of galanin and its analogs on
synaptic transmission using an in vitro slice
preparation of rat hypothalamus. In arcuate nucleus neurons, application of galanin resulted in an inhibition of evoked
glutamatergic EPSCs and a decrease in paired-pulse depression,
indicating a presynaptic action. The fragments galanin 1-16 and 1-15
produced a robust depression of synaptic transmission, whereas the
fragment 3-29 produced a lesser degree of depression. The chimeric
peptides C7, M15, M32, and M40, which have been reported to antagonize some actions of galanin, all produced varying degrees of depression of
evoked EPSCs. In a minority of cases, C7, M15, and M40 antagonized the
actions of galanin. Analysis of mEPSCs in the presence of TTX and
Cd2+, or after application of  -latrotoxin,
indicated a site of action for galanin downstream of
Ca2+ entry. Thus, our data suggest that galanin acts
via several subtypes of presynaptic receptors to depress synaptic
transmission in the rat arcuate nucleus.
Key words:
galanin; galanin receptors; transmitter release; presynaptic; depression; EPSC; mEPSC
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INTRODUCTION |
Galanin, a 29 amino acid
neuroregulatory peptide, has been implicated in numerous neuronal
functions including feeding behavior (Crawley et al., 1990 ; Kyrkouli et
al., 1990) and regulation of hormone release (Bauer et al., 1986;
Bjorkstrand et al., 1993; Kondo et al., 1993; Sahu et al., 1994;
Rossmanith et al., 1996). These two effects of galanin are thought to
result from its actions within the hypothalamus. Galanin receptor
binding has been demonstrated to be high within the hypothalamus,
particularly within the arcuate and paraventricular nuclei (Skofitsch
and Jacobowitz, 1985; Melander et al., 1986; Vrontakis et al., 1991;
Merchenthaler et al., 1993). Thus, galanin very likely plays a
significant role in hypothalamic function, although the mechanisms
underlying its actions are unclear.
Two galanin receptors, GALR1 and GALR2, have been cloned to date
(Habert-Ortoli et al., 1994; Howard et al., 1997). Binding and other
studies using galanin analogs have indicated that additional subtypes
of galanin receptors are likely to exist. Chimeric peptides C7, M40,
M32, and M15 have been show to act as galanin antagonists in certain
systems, including feeding behavior (Bartfai et al., 1993; Corwin et
al., 1993; Crawley et al., 1993), stimulation (Pramanik and
Ögren, 1993) and inhibition (Bartfai et al., 1991, 1993; Lindskog
et al., 1992) of acetylcholine release in the striatum and hippocampus,
respectively, inhibition of glutamate and aspartate release in the
hippocampus (Zini et al., 1993), and stimulation of luteinizing hormone
release (Sahu et al., 1994). However, in other systems, such as
galanin-mediated inhibition of cAMP accumulation in RINm5F cells
(Bartfai et al., 1993), inhibition of glucose-induced insulin release
(Bartfai et al., 1993), facilitation of the spinal cord reflex (Bartfai
et al., 1993), mobilization of intracellular Ca2+
stores (Fridolf and Ahrén, 1993), and hyperpolarization of
magnocellular neurosecretory cells (Papas and Bourque, 1997), these
same compounds are inactive or act as partial or full agonists. In
addition, the galanin fragments 1-15 and 3-29 have been shown to
behave as full agonists in some preparations (Wynick et al., 1993;
Hedlund et al., 1994; Lorinet et al., 1994), whereas they have no
effect in others (Wynick et al., 1993; Lorinet et al., 1994), again
suggesting the existence of additional galanin receptor subtypes.
Recent work has identified various cellular effects, typical of
G-protein-linked receptors, through which galanin might regulate neuronal function. Galanin is capable of opening K+
channels and hyperpolarizing neurons (Ahrén et al., 1989; Dunne et al., 1989; Konopka et al., 1989; Parsons and Merriam, 1992; Papas
and Bourque, 1997), inhibiting adenylate cyclase activity (Nishibori
et al., 1988; Chen et al., 1992), inhibiting voltage-gated Ca2+ channels (Merriam and Parsons, 1995),
inhibiting phosphoinositide turnover (Palazzi et al., 1988), and
regulating the release of dopamine (Nordström et al., 1987),
noradrenaline (Tsuda et al., 1989), acetylcholine (Fisone et al., 1987;
Ögren and Pramanik, 1991; Ögren et al., 1993), and
glutamate (Zini et al., 1993). The regulation of transmitter release is
an important mechanism by which galanin could potentially modulate
neuronal function. However, few studies to date have directly
investigated these actions of galanin. We have now investigated the
ability of galanin to modulate synaptic transmission within the arcuate
nucleus of the hypothalamus. Our results indicate that galanin acts
through multiple presynaptic receptors to inhibit the release
process.
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MATERIALS AND METHODS |
Preparation of brain slices. The methods for the
preparation of thin hypothalamic brain slices were similar to those
described previously (Glaum et al., 1994). Experiments were conducted
on Wistar rats of either sex, age 10-20 d postnatal. Animals were anesthetized with ether by inhalation, and killed by decapitation using
a guillotine. The brain was removed rapidly by dissection and placed in
chilled (0-6°C) extracellular solution of the following composition
(in mM): NaCl 126, KCl 3, CaCl2 2.5, MgSO4 1.3, NaH2PO4 1.25, NaHCO3 26, and D-glucose 10 (gassed with 95%
O2/5% CO2, pH 7.4;
osmolarity = 310 mOsm). Thin (200- to 220-µm-thick) coronal slices of the arcuate nucleus of the hypothalamus were cut using a
vibrating tissue chopper (Vibratome). Slices were maintained at
30-32°C until needed for recording.
For recording, slices were transferred to a submersion chamber mounted
on the stage of an upright microscope (Leitz Laborlux) and viewed with
a Zeiss 40× water immersion objective with Hoffman Contrast Optics.
The slices were perfused continuously throughout the experiment with
extracellular solution at room temperature (20-25°C). All recordings
were made from visually identified neurons located in the arcuate
nucleus. The arcuate nucleus is rich in galanin-positive neuronal cell
bodies and may be an important structure in the regulation of
reproduction and food intake (Landry et al., 1995).
Patch-clamp recording, synaptic stimulation, and data
analysis. Patch-clamp recording pipettes were made from
thin-walled borosilicate glass capillaries (DC resistance = 5-10
M when filled with internal solution) using a Flaming-Brown
horizontal pipette puller (Sutter Instruments). In all experiments,
electrodes were filled with internal solution of the following
composition (in mM): potassium gluconate 145, MgCl2 2, K2ATP 5, EGTA 1.1, CaCl2 0.1, and HEPES 5, pH 7.2; osmolarity adjusted to 280-290 mOsm. Patch
recording pipettes were mounted in the headstage attached to a
stage-mounted three-way hydraulic micromanipulator (Narashige) and
positioned over the somas of neurons under visual control. Conventional
methods for obtaining whole-cell recordings from thin slices (Hamill et
al., 1981; Edwards et al., 1989) were used. After the attainment of
cell access, transmembrane voltage and current were recorded using an
Axoclamp 2B (Axon Instruments, Foster City, CA) amplifier in the
discontinuous voltage-clamp mode (filtered at 5 kHz and acquired at 20 kHz), stored on a Pentium computer (Quantex) and via chart recorder
(Gould), and analyzed using pClamp 6.0.1 (Axon Instruments).
Bipolar tungsten stimulating electrodes were placed lateral to the
arcuate nucleus to activate inputs to arcuate nucleus neurons. In all
experiments, stimuli of between 50 and 500 µsec were used to elicit
synaptic responses and were maintained at 10 sec intervals to record
the time-dependent effects of drug perfusion. Paired-pulse stimuli were
recorded at a 20 sec interval using a 30 msec interpulse interval. In
normal saline, the paired-pulse ratio was a random mixture of both
paired-pulse facilitation and depression that likely resulted from the
polysynaptic inputs recorded in arcuate neurons. Therefore, studies of
paired stimuli were recorded in high Ca (5 mM) and low Mg
(0.5 mM) saline solution, which increases the probability
of neurotransmitter release in response to the initial stimulus and
favors paired-pulse depression (Khazipov et al., 1995; Dobrunz and
Stevens, 1997). These recording conditions significantly improved
the reproducibility of the paired-pulse ratio recorded from arcuate
neurons. For the purposes of data analysis, 2-3 min of evoked EPSCs
were averaged (12-18 EPSCs), and the peak of the averaged EPSC was
measured. Cells that responded to drug application with a 15% or
greater reduction (a change greater than the 99% confidence
limits of the control window mean) were considered to have responded
positively. Data are expressed in mean ± SEM.
Recording and analysis of mEPSCs. Miniature EPSCs (mEPSCs)
were recorded from arcuate nucleus neurons at a holding potential of
 60 to 70 mV in the presence of 1 µM TTX, 40 µM 7-chlorokynurenic acid, 10 µM
bicuculline, and, unless noted otherwise, 100 µM cadmium chloride (Cd2+). Currents were filtered at 1-2 kHz,
sampled at 8 kHz, and acquired to disk using pClamp software. Cells
were monitored periodically for changes in access resistance, and cells
that exhibited any significant (>15%) changes during the recording
period were rejected.
mEPSCs were analyzed using pClamp software or software written by Drs.
Andreas Kyrozis, Romain Girod, and Daniel McGehee in Axobasic
specifically for the analysis of mEPSCs. This program allows for the
automatic screening of events based on amplitude or 10-90% rise time
or both. All events were examined visually and accepted or rejected on
the basis of subjective visual criteria as well as the objective
criteria of amplitude, rise time, and decay time. Events that had an
amplitude of >4 pA, rise times of between 300 µsec and 3 msec, and
decay times of between 3 and 30 msec were included in the analysis.
Analyzed data from a 3-5 min recording period (100-1000 events) were
examined and further analyzed using Prism (Graphpad) and Statmost
(Datamost). Cumulative probability plots were constructed to visually
examine the effects of galanin on the amplitude and interval
distributions of mEPSCs, whereas amplitude and interval distributions
were compared statistically using a Kolmogorov-Smirnov test or a
Student's unpaired t test with Welch's correction.
Differences in distributions were considered significant if
p < 0.05. Data are expressed in mean ± SEM.
Application of drugs. Drugs were dissolved in distilled
water or DMSO ( 0.1% final concentration in artificial CSF) and
applied by bath perfusion. The following compounds were used:
bicuculline methobromide (Sigma, St. Louis, MO), 7-chlorokynurenic acid
(Tocris Cookson), D,L-2-amino-5-phosphonpentanoic acid
(D,L-AP5) (Tocris Cookson), TTX (Sigma), -latrotoxin
(Alomone Labs), (rat) galanin (American Peptides), galanin 1-16
(Bachem, Torrance, CA), galanin 1-15, 3-29, and M32 (gifts of Mary
Walker, Synaptic Pharmaceuticals), galantide (M15) (American Peptides),
M40 (American Peptides), and C7 (American Peptides). All drugs, with
the exception of AMPA and -latrotoxin, were applied for between 5 and 10 min to obtain a steady-state bath concentration. AMPA was
applied 2-3 times in succession (25 µM for a period of
40 sec at 10 min intervals) to achieve a series of rapid inward
currents (60 to 150 pA), which were then averaged. In the case of
-latrotoxin, it was found that the time required for action of the
toxin in an in vitro slice preparation was unfeasible; thus,
slices were incubated for 2 hr in 2 nM -latrotoxin and
then transferred to the recording chamber, where the slice was then
perfused with standard extracellular Ringer's solution. The effects of
-latrotoxin are likely irreversible within the time course of this
experiment (Wanke et al., 1986; Capogna et al., 1996a,b), and thus
there should be no significant "washout" of its effects during the
recording period. Such extensive exposure to -latrotoxin could
result in some depletion of transmitter stores (Tzeng et al., 1978;
Hurlbut et al., 1990; McMahon et al., 1990; Storchak et al., 1994), as
well as some loss of synaptic morphology (Tzeng et al., 1978). Under
these conditions, however, a much greater percentage of cells (~80%)
was observed than under control conditions (~25%), which displayed
an mEPSC frequency adequate for analysis (>0.5 Hz) (see Results).
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RESULTS |
Whole-cell patch recordings were obtained from 110 neurons in 65 preparations of 200-220 µM thick slices of rat arcuate
nucleus. Unless noted otherwise, all experiments were performed at a
cell holding potential of between 60 and 70 mV in the presence of bicuculline (10 µM), 7-chlorokynurenic acid (40 µM), and 1.5 mM external
Mg2+ to pharmacologically isolate the AMPA
receptor-mediated EPSCs. The mean cellular input resistance was
1200 ± 74 M (n = 71). The mean 10-90% rise
time was 2.14 ± 0.09 msec (n = 50), and the mean
10-90% decay was 12.77 ± 0.49 msec (n = 50). No
significant differences in values were observed between control and
drug application, so the values were pooled. The figure for the
10-90% rise time indicates that many of the synaptic responses
recorded were the result of multiple asynchronous inputs.
Galanin receptor-mediated synaptic depression
To investigate the effects of galanin on synaptic transmission in
the hypothalamus, a stimulating electrode was placed ventrolateral to
the arcuate nucleus, and synaptic inputs were stimulated at a frequency
of 0.1 Hz while recording from arcuate neurons in the discontinuous
voltage-clamp mode. In the presence of 7-chlorokynurenic acid and
bicuculline, stimulation of inputs resulted in exclusively AMPA
receptor-mediated EPSCs (Glaum et al., 1996). Under such conditions,
application of galanin (galanin 1-29, 100 nM) caused a
strong reduction of synaptic transmission in 19 of 26 cases examined
(55.54 ± 4.03%; n = 19) (Figs.
1A,
2). In cases in which a sufficient
washout period was recorded, there was some variability in the degree
of recovery observed. In some cells the effect of galanin readily
reversed during washout (Fig. 1A). However, in six cells there was a slow and incomplete recovery observed after a
long (+40 min) washout period (data not shown). Such variability was
observed with galanin 1-15 and galanin 1-16 as well (see below). These observations are consistent with previous reports of long-lasting actions of other peptides in the hypothalamus (Van den Pol et al.,
1996; Rhim et al., 1997)
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Figure 1.
Application of galanin caused a depression of AMPA
receptor-mediated synaptic transmission in the arcuate nucleus.
A, Application of 100 nM galanin caused a
depression in the evoked EPSC, as recorded from an arcuate nucleus
neuron. After washout of the drug, the EPSC recovered to control levels
within 20 min. For A, a represents the
time course of the effects of galanin on synaptic transmission, whereas
b-d are averaged EPSCs taken from the respective cells
showing EPSCs before drug application (b), during
drug application (c), and after washout of the
drug (d). B, Dose-response curve
for galanin. Application of progressively higher doses of galanin
(0.1-100 nM) resulted in a dose-dependent decrease in the
amplitude of evoked synaptic currents (n = 5), as
recorded from arcuate nucleus neurons. Data are mean ± SEM.
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Figure 2.
Histogram representing the degree of inhibition of
the evoked EPSC by the different galanin analogs. Vertical
bars represent the absolute magnitude of inhibition produced by
each of the compounds tested in this study, as marked below each bar.
The numbers of observations for each compound are as follows (number of
positive trials per total number of trials): galanin (19 of 26),
galanin 1-16 (7 of 9), galanin 1-15 (4 of 10), galanin 3-29 (4 of
11), C7 (7 of 15), M40 (10 of 15), M15 (9 of 12), M32 (3 of 3). Data
are mean ± SEM.
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The dose-response relationship for the effect of galanin on synaptic
transmission was investigated by bath applying the peptide in
increasing doses to slices while evoked EPSCs were recorded. The
concentration producing a maximal effect was determined to be ~100
nM, with an EC50 of 0.85 nM
(n = 5) (Fig. 1B).
To investigate the types of galanin receptors mediating inhibition of
the EPSC, various galanin analogs were tested. Galanin (1-15) and
(1-16) are N-terminal fragments of galanin, whereas galanin 3-29 is a
galanin fragment truncated at the N terminus. Application of both
galanin 1-15 (100 nM) and galanin 1-16 (100 nM) resulted in a depression of evoked EPSCs (Figs. 2,
3A,B). Although both fragments
caused a similar degree of inhibition (1-15: 56.56 ± 7.28%,
n = 4; 1-16: 51.84 ± 7.60%, n = 7), galanin 1-16 acted on a larger group of cells (seven of nine) than
galanin 1-15 (4/10). Strikingly, galanin 1-16 was effective on some
cells in which galanin 1-15 had no effect (four of five cells) (Fig. 3B). Galanin 3-29 (100 nM to 1 µM) was much less potent, causing a moderate inhibition
in only 4 of 11 cells (21.28 ± 1.37%) (Figs. 2, 3C).
In one cell on which galanin was also applied, the degree of inhibition
mediated by galanin was much greater. In addition, galanin caused
inhibition in each of the cells that did not respond to galanin 3-29
(Fig. 3C).
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Figure 3.
Application of galanin 1-15, 1-16, and 3-29
caused a depression of AMPA receptor-mediated synaptic transmission in
the arcuate nucleus. A, Application of 100 nM galanin 1-15 caused a depression in the evoked EPSC, as
recorded from an arcuate nucleus neuron. After washout of the drug, the
EPSC partially recovered. Application of 100 nM galanin
1-16 after washout resulted in a nearly complete depression of the
synaptic response. B, Application of 100 nM
galanin 1-15 to another cell failed to elicit a response. However,
application of galanin 1-16 after washout resulted in a significant
depression of transmission. C, In another cell,
application of 100 nM galanin 3-29 produced only a small
effect on transmission. After washout of this peptide fragment,
application of 100 nM galanin resulted in a significantly
larger depression of transmission. For A-C,
a-e are the same as in Figure
1A.
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Various putative galanin antagonists have been reported (Bartfai et
al., 1991, 1993; Lindskog et al., 1992; Pramanik and Ögren, 1992;
Corwin et al., 1993; Crawley et al., 1993; Zini et al., 1993; Sahu et
al., 1994). C7, M40, M15, and M32 all proved to have agonist effects in
our experiments. Application of C7 (100 nM) caused a
reduction (32.27 ± 3.52%) in synaptic transmission in 7 of 15 cases examined, as did M40 (100 nM) (35.8 ± 6.15%; 10 of 15), M15 (100 nM) (33.22 ± 4.47%; 9 of 12),
and M32 (100-500 nM) (35.59 ± 9.34%;
n = 3) (Fig. 2). Thus, in the majority of experiments,
application of these "antagonist" peptides resulted in a depression
of synaptic transmission, although the magnitude of the depression was
less than that observed with galanin. This may indicate partial agonist
activity or selective activation of limited subtypes of galanin
receptors. In many cases, application of galanin after a response to
one of the antagonist peptides resulted in a further depression of
synaptic transmission. The application of galanin after C7 produced a
46.07 ± 9.12% depression (relative to C7; four of five) (Fig.
4A), and similar
effects were seen with M15 (39.88 ± 8.06%; four of six) (Fig.
4B), and M40 (37.00 ± 17.09%; three of five)
(data not shown).
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Figure 4.
C7 and M15 have partial agonist-antagonist
actions on galanin receptors within the arcuate nucleus.
A, Application of 100 nM C7 caused a
reduction in synaptic transmission. Application of galanin (100 nM) resulted in a further reduction in transmission. During
washout of C7, there was no further depression of transmission induced
by galanin. B, Application of M15 to another cell also
caused a moderate depression in synaptic transmission. Concurrent
application of galanin resulted in a further depression of the
response. C, Application of C7 had no effect on synaptic
transmission. Galanin applied in the presence of C7 was also
ineffective. After washout of C7, however, galanin caused a significant
reduction in the synaptically evoked current. For A-C,
a-e are the same as in Figure
1A.
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Finally, in a minority of experiments, there was convincing evidence
for some antagonist activity associated with the putative galanin
antagonists. In three of seven cases, washout of C7 while in the
presence of galanin resulted in a reduction in the evoked current
(43.33 ± 10.09%) (Fig. 4C). M15 and M40 were less
active as antagonists, similarly antagonizing the actions of galanin in
only one of three and one of five cases, respectively (36.1% and
18.5%) (data not shown). In some experiments, application of galanin
in the presence of one of the antagonist peptides produced no
additional effect. It is unclear, however, whether this was caused by
occlusion or antagonism of the galanin response.
Mechanisms of action of galanin
A series of experiments was performed to determine the mechanism
by which galanin produced synaptic inhibition in the arcuate nucleus.
Activation of K+ channels in the presynaptic
terminal could serve to shunt the incoming action potential, reducing
Ca2+ channel activation and Ca2+
influx, thereby reducing transmitter release (Segev, 1990; Gage, 1992;
Graham and Redman, 1994; Miller, 1997). Although no outward currents
were observed in any of the cells from which we recorded, it is
possible that K+ channel activation in the
presynaptic terminal might still occur. However, application of galanin
in the presence of 100 µM Ba2+, which
blocks G-protein-activated K+ channels (Ransom and
Sontheimer, 1995; Wang and McKinnon, 1996; Rhim et al., 1997), still
depressed synaptic transmission to the same extent (n = 4; data not shown), indicating that K+ channel
activation was not involved in the actions of galanin in this case. The
effects of galanin were also examined using a paired-pulse stimulus
protocol (Baskys and Malenka, 1991). Application of galanin (100 nM) was found to depress the initial evoked EPSC, P1, in 6 of 11 cells examined (57.5 ± 4.7%; n = 6) (Fig.
6A). In cases in which a depression of the initial
EPSC was observed, the paired-pulse ratio was strongly increased in
galanin (P2/P1: 1.27 ± 0.17;
n = 6) versus control
(P2/P1: 0.61 ± 0.05;
n = 6) (Fig. 5). In
contrast, in the cells in which no inhibition of the initial EPSC was
observed, the paired-pulse ratio in galanin (100 nM)
(P2/P1: 1.10 ± 0.25) was not
significantly different from the control period (1.01 ± 0.07;
p = 0.740, unpaired t test; n = 5; data not shown). Finally, direct postsynaptic
modulation of the receptor was examined using a series of short bath
applications of AMPA (25 µM for 40 sec). Simultaneous
application of galanin had no effect on the whole-cell currents
recorded under these conditions (107.7 ± 6.5% of control;
n = 6) (data not shown). These effects of galanin
support a presynaptic mechanism of action.
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Figure 5.
Application of galanin reduced paired-pulse
depression in the arcuate nucleus. A, Application of 100 nM galanin produced a reversible decrease in the amplitude
of the initial first evoked EPSC, P1, and an
increase in the paired-pulse ratio
P2/P1. A,a represents the
time course of the effects of galanin on the ratio of the amplitude of
the evoked EPSCs, P2/P1. For
A, b-d are the same as in Figure
1A. Interpulse interval was 30 msec.
B, Histogram representing the effect of galanin (100 nM) on the mean paired-pulse depression from six cells. The
pooled data show a significant increase in the paired-pulse ratio
(p = 0.01; unpaired t
test).
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Mechanisms underlying modulation of synaptic transmission can also be
studied by recording the spontaneous release of vesicles from the
presynaptic terminal. Alterations in the frequency or amplitude
distribution of miniature events are indicative of a presynaptic or
postsynaptic site of action, respectively. mEPSCs were recorded from
arcuate nucleus neurons in the presence of 7-chlorokynurenic acid (40 µM) or D,L-AP5 (100 µM),
bicuculline (10 µM), TTX (1 µM), and
Cd2+ (100 µM) to isolate
Ca2+ entry-independent spontaneous AMPA
receptor-mediated events. To confirm that Cd2+ was
adequately blocking Ca2+ entry through voltage-gated
Ca2+ channels, 100 µM
Cd2+ was applied to the preparation while evoked
EPSCs were recorded. In four of four cases, application of 100 µM Cd2+ completely abolished synaptic
transmission (data not shown). Under these conditions, spontaneous
mEPSCs were observed in approximately one of four cells recorded from
at a frequency adequate for analysis (>0.5 Hz). Application of galanin
(100 nM) resulted in a 54.07 ± 3.45% reduction in
the frequency of recorded mEPSCs but had no effect on the amplitude
distribution (95.83 ± 2.06%) (Fig. 6A-F)
(n = 7).
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Figure 6.
Galanin caused a reduction in the frequency of
mEPSCs with no effect on the amplitude distribution. A,
B, Samples of mEPSCs recorded from arcuate nucleus neurons. In
both A and B, traces represent a
continuous recording period of ~3 sec for control
(A) and in the presence of 100 nM
galanin (B). C, D, Cumulative
probability plots of the distribution of mEPSC amplitudes
(C) and intervals (D) for a
single cell, showing a significant change in the interval distribution
(p < 0.0001; unpaired t test
with Welch's correction). E, F, Effect of galanin on
the mean amplitude (E) and mean interval
(F) for data pooled from six cells. The pooled
data showed a significant change in the interval
(p < 0.0001; unpaired t test
with Welch's correction), whereas no significant change in the mean
amplitude was observed. All data were acquired in the presence of 1 µM TTX, 40 µM 7-chlorokynurenic acid, 10 µM bicuculline, and 100 µM
Cd2+.
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-latrotoxin binds to at least two types of presynaptic receptors and
enhances transmitter release in both a
Ca2+-dependent and -independent manner (Misler and
Hurlbut, 1979; Grasso et al., 1980; Nicholls et al., 1982; Meldolesi et
al., 1984; Rosenthal et al., 1990; Capogna et al., 1996a; Krasnoperov et al., 1997). When galanin was applied to slices that had been incubated in 2 nM -latrotoxin, there was a 47.76 ± 6.33% reduction in the frequency of mEPSCs (Fig.
7A-F)
(n = 7). The amplitude distribution remained unchanged
(97.78 ± 2.70% of control; n = 7) (Fig.
7A-F). These results suggest that galanin can
inhibit transmitter release at a site "downstream" of
Ca2+ entry through voltage-dependent
Ca2+ channels and may act at a site very close to
the final step in the release process.
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Figure 7.
Galanin caused a reduction in the frequency of
mEPSCs with no effect on the amplitude distribution in slices exposed
to -latrotoxin. A, B, Samples of mEPSCs recorded from
arcuate nucleus neurons in slices exposed to -latrotoxin for >2 hr.
In both A and B, traces represent a
continuous recording period of ~4 sec total for control
(A) and in the presence of 100 nM
galanin (B). C, D, Cumulative
probability plots of the distribution of mEPSC amplitudes
(A) and intervals (B) for a
single cell, showing a significant change in the interval distribution
(p < 0.0001; unpaired t test
with Welch's correction). C, D, Effect of galanin on
the mean amplitude (C) and mean interval
(D) for data pooled from seven cells. The pooled
data showed a significant change in the interval
(p < 0.0001; unpaired t test
with Welch's correction), whereas no significant change in the mean
amplitude was observed. All data were acquired in the presence of 1 µM TTX, 100 µM D,L-AP5, 10 µM bicuculline, and 100 µM
Cd2+ in slices exposed to 2 nM
-latrotoxin for 2 hr before recording.
|
|
| |
DISCUSSION |
Galanin acts in the hypothalamus to stimulate feeding behavior
(Crawley et al., 1990; Kyrkouli et al., 1990; Leibowitz and Kim, 1992;
Corwin et al., 1993) and to regulate hormone secretion (Bauer et al.,
1986; Bjorkstrand et al., 1993; Kondo et al., 1993; Landry et al.,
1995) and the reproductive cycle (Grafstein-Dunn et al., 1994; Sahu et
al., 1994; Rossmanith et al., 1996). In addition, galanin has been
shown to exert numerous effects elsewhere in the brain, including
effects on learning and memory (Sundström et al., 1988;
Ögren and Pramanik, 1991; Crawley et al., 1993).
How does galanin produce its effects? Cloning studies have indicated
that the actions of galanin are mediated through at least two subtypes
of galanin receptors, GALR1 and GALR2 (Habert-Ortoli et al., 1994;
Howard et al., 1997). In addition, the existence of other galanin
receptors has been inferred from binding and functional studies (Wynick
et al., 1993; Gu et al., 1995; Valkna et al., 1995). The effects of
galanin we have observed would be expected to involve at least two
different receptors. Both GALR1 and GALR2 have been shown to be present
within the hypothalamus (Habert-Ortoli et al., 1994; Gustafson et al.,
1996; Howard et al., 1997; Wang et al., 1997) and might be responsible
for much of the activity observed here using galanin agonists.
Furthermore, these receptors also interact with the putative galanin
antagonists we have used. The majority of our observations are
consistent with the idea that these peptides act as partial agonists,
as has been found in several other investigations (Fridolf and
Ahrén, 1993; Mulvaney et al., 1995; Papas and Bourque, 1997).
Additionally, we have made at least two other interesting observations.
The first of these concerns the activity of galanin 3-29. This analog does not interact significantly with either GALR1 or GALR2
(Habert-Ortoli et al., 1994; Parker et al., 1995; Howard et al., 1997;
Sullivan et al., 1997). However, Wynick et al. (1993) reported that the anterior pituitary and hypothalamus do contain a receptor that is
activated by N-terminal truncated analogs such as galanin 3-29. The
modest effects we have observed may reflect actions at such a receptor.
Second, it is striking that we have observed groups of cells that
responded to both galanin 1-16 and 1-15 or only to the former
peptide. It is clear that GALR1 and GALR2 both respond to galanin 1-16
(Habert-Ortoli et al., 1994; Parker et al., 1995; Howard et al., 1997;
Sullivan et al., 1997). However, previous studies have generally
indicated that the potency of galanin analogs decreases substantially
after the removal of the amino acid in the 16th position (Lagny-Pourmir
et al., 1989; Land et al., 1991). Thus, the robust activity of galanin
1-15 observed in some cells is somewhat surprising. However, there is
a report of a galanin 1-15 selective receptor in the hippocampus
(Hedlund et al., 1994), and intracerebroventricular injection of
galanin 1-15 but not 1-29 modulates baroreflex sensitivity in the rat
(Diaz et al., 1996). Thus, it is conceivable that this receptor also
occurs in the hypothalamus.
Further evidence for the presence multiple subtypes of galanin
receptors in the arcuate nucleus was obtained in experiments with the
chimeric peptides M40, M15, M32, and C7. In the majority of cases,
these compounds behaved as agonists. However, the degree of inhibition
was notably less than that caused by galanin alone. This may reflect
partial agonist actions of these compounds. Alternatively, galanin may
act simultaneously at several different subtypes of receptors, whereas
the chimeric peptides may be a more selective group of full agonists.
Antagonism by the chimeric peptides was observed only in a minority of
cases examined. This contrasts with other reports on the actions of
these peptides in which they have been shown to block galanin-induced
feeding behavior (Leibowitz and Kim, 1992; Corwin et al., 1993; Crawley
et al., 1993), stimulation of ACh release (Pramanik and Ögren,
1993), and the actions of galanin in the hippocampus, locus coeruleus,
and spinal cord (Bartfai et al., 1991, 1993). However, other reports
have suggested partial or full agonist actions of these compounds,
including on the mobilization of intracellular Ca2+
stores (Fridolf and Ahrén, 1993), the activation of
K+ currents in magnocellular neurosecretory cells
(Papas and Bourque, 1997), and in the inhibition of voltage-dependent
Ba2+ currents (Mulvaney et al., 1995). It appears
then that the actions of these compounds are not restricted to
antagonist effects and that they may even prove to be selective
agonists at specific subtypes of galanin receptors. In summary,
therefore, our results suggest the existence of multiple types of
galanin receptors within the arcuate nucleus. Nevertheless, it is
possible that some of the galanin analogs that we are using interact
with other receptor populations as well. Additional pharmacological
characterization is required before any definitive statements can be
made regarding the actions of these compounds.
The actions of galanin on fast glutamatergic transmission observed in
this study are analogous to those shown in other reports, such as its
effects on slow cholinergic transmission in the hippocampus (Dutar et
al., 1989). However, no studies have investigated directly the
mechanisms underlying such actions. Galanin inhibited evoked L-glutamate release in the hippocampus, an effect that was
blocked by glibenclamide, an ATP-sensitive K+
channel blocker (Zini et al., 1993). In many other systems, galanin activates a K+ conductance and hyperpolarizes
neurons (Ahrén et al., 1989; Dunne et al., 1989; Konopka et al.,
1989; Seutin et al., 1989; Papas and Bourque, 1997), an effect that
could produce inhibition of transmitter release (Segev, 1990; Gage,
1992; Graham and Redman, 1994; Miller, 1997). However, in our
studies no outward currents were observed with galanin. Furthermore,
application of 100 µM Ba2+ had no
effect on galanin receptor-mediated synaptic depression (Scanziani et
al., 1995; Miller, 1997).
There are also reports of galanin inhibiting voltage-gated
Ca2+ channels in neurons (Merriam and Parsons, 1995;
Mulvaney et al., 1995), and such an action could contribute to the
effects on synaptic transmission that we have observed. We have clearly
demonstrated that galanin acts presynaptically to directly decrease
transmitter release independent of Ca2+ entry
through voltage-sensitive Ca2+ channels. This
mechanism of presynaptic inhibition has now been widely reported in
slices and in culture (Scholz and Miller, 1992; Scanziani et al., 1995;
Tyler and Lovinger, 1995; Trudeau et al., 1996; Dittman et al.,
1996;), although its underlying molecular mechanisms are unclear. It is
conceivable, however, that the mechanism of release for mEPSCs versus
evoked currents is different, and consequently the mechanism of action
of galanin may not be the same for these two processes (but see Capogna
et al., 1996a). Interestingly, the magnitude of the effect of galanin
on mEPSCs in the presence of Cd2+ (54.07 ± 3.45% reduction in frequency) was similar to the effect of galanin on
synaptic transmission (55.54 ± 4.03% reduction in amplitude),
indicative of a common mechanism. Significant, however, was the
observation that galanin decreased the magnitude of paired-pulse depression, often reversing it to paired-pulse facilitation, indicative of a presynaptic action on evoked release (Baskys and Malenka, 1991).
It is still possible, however, that galanin is acting on a
Ca2+-dependent mechanism. Residual
Ca2+ inside the cell, or Ca2+
leak into the cell, might contribute to mEPSC frequency even in the
presence of Cd2+. However, -latrotoxin acts at
the presynaptic terminal to induce a partially
Ca2+-independent release of transmitter (Misler and
Hurlbut, 1979; Meldolesi et al., 1984; Rosenthal et al., 1990; Capogna
et al., 1996a; Krasnoperov et al., 1997). Exposure of the slices to
-latrotoxin would activate this mechanism, and thus a significant
portion of the resultant mEPSCs would be
Ca2+-independent. Under these conditions,
application of galanin resulted in inhibition of mEPSCs of a magnitude
comparable to that observed in the absence of -latrotoxin
(47.76 ± 6.33% reduction in frequency). These results are
similar to those reported for the actions of GABA in the hippocampus in
the presence of -latrotoxin (Capogna et al., 1996a) and suggest that
galanin has a powerful depressant effect on the release process at a
site close to the final vesicle-release step. There are numerous
potential sites within the presynaptic machinery at which it could act.
Krasnoperov et al. (1997) reported recently that -latrotoxin
stimulates Ca2+-independent exocytosis through its
interaction with a G-protein-coupled receptor (the CIRL-receptor), and
that this receptor copurifies with syntaxin. Furthermore, Linial et al.
(1997) reported that presynaptic muscarinic receptors could also
directly interact with syntaxin. Thus, the galanin receptor might
interact with, or signal to, a site within the release machinery and so
directly reduce exocytosis.
| |
FOOTNOTES |
Received Oct. 14, 1997; revised Feb. 18, 1998; accepted Feb. 25, 1998.
This work was supported by Public Health Service Grants DA02121,
DA02575, MH40165, NS33502, DK42086, and DK44840. We thank Drs. Daniel
McGehee and Aaron Fox for their technical expertise.
Correspondence should be addressed to Dr. Richard J. Miller, Department
of Pharmacological and Physiological Sciences, 947 E. 58th Street, MC
0926, Chicago, IL 60637.
Dr. Kinney's present address: Department of Physiology and Biophysics,
Veterans Affairs Medical Center, Seattle, WA
98108-1597.
| |
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Copyright © 1998 Society for Neuroscience 0270-6474/98/18103489-12$05.00/0
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Top
Abstract
Introduction
