Download Evidence that GABA augmentation of norepinephrine release is mediated by interneurons

Brain Research 790 Ž1998. 329–333
Short communication
Evidence that GABA augmentation of norepinephrine release is mediated by
interneurons
Jeannie M. Fiber, Anne M. Etgen
)
Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Accepted 20 January 1998
Abstract
GABA A receptor activation augments stimulated release of 3 H-norepinephrine ŽNE. in brain slices from female rats. This effect is not
blocked by acetazolamide or MK-801, indicating that permeability of the GABA A chloride channel to bicarbonate ions and NMDA
receptor activation do not mediate GABA-induced NE release. Furthermore, GABA augments 3 H-NE release from slices, but not from
isolated nerve terminals Žsynaptosomes., indicating that interneurons mediate GABA effects on 3 H-NE release. q 1998 Elsevier Science
B.V.
Keywords: Preoptic area; Hypothalamus; Frontal cortex; Brain slices; Synaptosome; NMDA
Both norepinephrine ŽNE. and GABA in the hypothalamus ŽHYP. and preoptic area ŽPOA. play critical roles in
the expression of female reproductive behavior and in
regulation of gonadotropins w2,8,11,15x. Previously, we
showed that the inhibitory amino acid neurotransmitter
GABA, acting through GABA A receptors, facilitates electrically evoked release of 3 H-NE in slices from the cortex,
HYP and POA of female rats w4x. The mechanism of this
paradoxical effect is unknown. Recently, it has been shown
that GABA can depolarize neurons because of the permeability of the GABA A chloride channel to bicarbonate
ions; the ensuing depolarization then facilitates NMDA
receptor stimulation w16x. These mechanisms could underlie GABA augmentation of 3 H-NE release w6x. Alternatively, because there are no NE cell bodies in these brain
regions, GABA could act directly on NE terminals to
augment release. However, it is also possible that GABA
acts on inhibitory interneurons to enhance NE release by
disinhibition. The present study determined whether any of
these mechanisms are responsible for GABA facilitation of
NE release from the cortex, HYP and POA of female rats.
Female Sprague–Dawley rats Ž150–175 g, Taconic
Farms, Germantown, NY. were bilaterally ovariohysterec)
Corresponding author. Neuroscience Department, Albert Einstein
College of Medicine, 1300 Morris Park Avenue, Forchheimer 113,
Bronx, NY 10461, USA. Fax: q 1-718-430-8654; E-mail:
[email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 0 1 - 2
tomized 10–14 days prior to killing. There is no effect of
gonadal steroids on GABA facilitation of electrically
evoked NE release w4x. Therefore, experiments reported
here were performed on ovariohysterectomized rats only.
After decapitation, brains were rapidly removed and placed
in ice-cold medium containing 124 mM NaCl, 5 mM KCl,
1.2 mM KH 2 PO4 , 1.3 mM MgSO4 , 2.4 mM CaCl 2 , 26.2
mM NaHCO 3 and 10.0 mM glucose w18x, previously saturated with O 2 :CO 2 Ž95:5. and at pH 7.4. For experiments
using acetazolamide, 10 mM HEPES ŽpH 7.4. replaced
NaHCO 3 , and the medium was saturated with 100% O 2
w16x. The HYP, POA and frontalrparietal cortex were
dissected over ice, removed as a block and 350 m m slices
were preloaded with 3 H-NE Ž0.1 m M; specific activity
58.1 Cirmmole. as previously described w4x.
Slices were placed into a Brandel SF2000 automated
superfusion apparatus and perfused for a 70-min washout
period at a flow rate of 1.5 mlr4.5 min. The medium
contained 1.0 m M desmethylimipramine ŽDMI; Sigma, St.
Louis, MO., a NE uptake inhibitor, and was saturated with
O 2 :CO 2 Ž95:5.. For electrical stimulation, 1 preconditioning stimulus Ž18 pulses, 18 mA, 0.3 Hz. was delivered to
slices with a Brandel electrical stimulator after 35 min.
Following the washout period, up to 20 consecutive 4.5
min fractions were collected for each slice. After collection
of 3 baseline samples, slices were stimulated Ž72 pulses,
18 mA, 3 Hz. at 15 min ŽS1., 50 min ŽS2. and 75 min ŽS3.
from the start of the 100-min collection period. A mini-
330
J.M. Fiber, A.M. Etgen r Brain Research 790 (1998) 329–333
mum of 3 baseline samples were collected between each
electrical stimulation. In independent experiments, either
MK-801 Ž10 m M, dissolved in water; RBI, Natick, MA., a
specific NMDA antagonist, acetazolamide Ž10 m M, dissolved in dimethylsulfoxide; RBI, Natick, MA., a carbonic
anhydrase inhibitor, tetrodotoxin ŽTTX; 1–2 m M, dissolved in water; RBI, Natick, MA. or vehicle was introduced 10 min prior to S2. This was followed by the
addition of 100 m M GABA or vehicle simultaneously with
S2. All drugs were removed 5 min later. Calculations for
fractional release, S2:S1 ratios and S3:S1 ratios have been
previously described w4x.
For KCl stimulation, slices were perfused as described
above for a 40-min washout period at a flow rate of 1
mlr3 min. Following the washout period, up to 20 consecutive 3.0 min fractions were collected for each slice. After
collection of 3 baseline samples, 20 mM KCl with either
100 m M GABA or vehicle was introduced for 3 min ŽS1..
Following the first stimulation, 5 baseline samples were
collected, then a second 20-mM KCl stimulation ŽS2. was
delivered without GABA. At the end of the superfusion
period, each slice was dissolved in 1.0 M NaOH. The 3 H
content of each superfusion sample and 3 H remaining in
the slice were determined using a Beckman scintillation
counter. Fractional release in each sample for each slice
was determined after calculating the 3 H content of each
slice at the start of sample collection as described previously w4x. We have previously determined using high-pressure liquid chromatography that stimulated 3 H release
represents at least 65% authentic NE w9x.
Synaptoneurosomes Žcrude synaptosomes. were prepared by homogenization of each brain region from 1 rat
in 3 ml ice cold modified Krebs medium ŽpH 7.4. containing 120 mM NaCl, 5 mM KCl, 1.2 mM KH 2 PO4 , 1.3 mM
MgSO4 , 0.8 mM CaCl 2 , 26 mM NaHCO 3 and 10.0 mM
glucose, previously saturated with O 2 :CO 2 Ž95:5. with 20
strokes of a hand-held Teflon homogenizer. Homogenate
was passed through a 20-m m pore nylon mesh filter and
centrifuged at 20,000 = g for 20 min at 48C. Pellets containing synaptoneurosomes were resuspended in 3 ml Krebs
and equilibrated to 378C with shaking for 10 min. 3 H-NE
Ž0.1 m M. was added and allowed to incubate for 15 min,
followed by centrifugation at 20,000 = g for 20 min at
48C. Pellets were washed and resuspended 2 additional
times as previously described w5x. Aliquots of 200 m l were
then incubated in medium containing 5, 20 or 50 mM KCl,
with or without 100 m M GABA, at 308C for 10 min, or on
ice Žcontrol.. The suspension was centrifuged and medium
collected to determine 3 H-NE released from each pellet.
Each pellet was dissolved in 750 m l of 1 M NaOH and
counted. Using this preparation, 1 m M DMI blocks 60–
75% of 3 H-NE uptake, and 10 m M DMI blocks 77–81%
of 3 H-NE uptake into synaptoneurosomes, demonstrating
that 3 H-NE was taken up via the NE transporter and not
leaking in or out of the synaptoneurosomes. Percent release of 3 H-NE from synaptoneurosomes was determined
by dividing the dpm of 3 H-NE in the medium by the dpm
of 3 H-NE in the pellet plus medium and multiplying by
100. The percent of 3 H-NE released in the ice control
aliquots was subtracted from percent released in samples
incubated at 308C.
Differences between groups were determined using a
1-way analysis of variance ŽANOVA. for independent
samples. Each brain region was analyzed separately. For
experiments using synaptoneurosome preparations, differences between groups were determined using a 2-way
ANOVA, with GABA as one between-groups variable,
and KCl concentration as the second. Groups were considered to be different from each other if p - 0.05. Post-hoc
analysis was performed using a Student Newman–Keuls
test.
In cortical, HYP and POA slices, 100 m M GABA
introduced simultaneously with S2 increases electrically
stimulated 3 H-NE release above that of vehicle-perfused
controls ŽFig. 1.. The S2:S1 ratios are greater in tissue
perfused with 100 m M GABA than those of vehicle
controls Ž p - 0.0005; Fig. 1A.. In addition, GABA increases the duration of the 3 H-NE efflux during S2 Ž p 0.005; Fig. 1B.. GABA also has long-lasting effects on
3
H-NE release. GABA significantly increases the S3:S1
ratios, although GABA is washed out immediately following S2 Ž p - 0.0001; Fig. 1C.. Acetazolamide, a carbonic
anhydrase inhibitor, was used to test the role of bicarbonate regeneration in the augmentation of stimulated 3 H-NE
release by GABA. Acetazolamide alone Ž10 m M. has no
effect on the S2:S1 ratio, the duration of the 3 H-NE efflux
during S2, or the S3:S1 ratio. Furthermore, it does not
attenuate GABA enhancement of the S2:S1 ratio, the
duration of the S2 or the S3:S1 ratio ŽFig. 1.. The data in
Fig. 1 show results from slices perfused with 10 mM
HEPES-buffered medium. Acetazolamide also failed to
modify GABA facilitation of 3 H-NE release in slices
perfused with sodium bicarbonate-buffered medium Ždata
not shown..
MK-801, a specific NMDA antagonist, was introduced
10 min before S2 to determine whether GABA augments
electrically stimulated release of 3 H-NE by facilitating
NMDA receptor activity. The S2:S1 ratios are greater in
tissue perfused with 100 m M GABA than those of vehicle
controls Ž p - 0.01; Fig. 2A.. GABA also increases the
duration of the 3 H-NE efflux during S2 Ž p - 0.005; Fig.
2B. and increases the S3:S1 ratios Ž p - 0.05; Fig. 2C..
MK-801 alone has no effect on the S2:S1 ratio, S2 duration or S3:S1 ratio and fails to attenuate the GABA
augmentation of S2:S1 ratio, S2 duration or S3:S1 ratio
ŽFig. 2..
To determine whether GABA acts via an interneuron to
augment 3 H-NE release, TTX was introduced prior to and
during S2 using the electrical and KCl stimulation
paradigms. TTX inhibited electrically stimulated and KClstimulated release of 3 H-NE by more than 90% in both the
presence and absence of GABA Ždata not shown.. Other
J.M. Fiber, A.M. Etgen r Brain Research 790 (1998) 329–333
331
3B.. Raising the KCl concentration to 50 mM also increases 3 H-NE release compared to 5 mM KCl in all three
brain regions Ž p - 0.0005; data not shown..
These data show that GABA augments electrically and
KCl-stimulated 3 H-NE release from HYP, POA and frontal
cortex slices. However, permeability of the GABA A chloride channel to bicarbonate ions is not responsible for
GABA facilitation of stimulated 3 H-NE release, because
acetazolamide did not attenuate the GABA enhancement
of 3 H-NE release. GABA facilitation of NMDA receptor
activity following bicarbonate ion-induced depolarization
w16x is also not responsible for GABA facilitation of
Fig. 1. Acetazolamide ŽACET. has no effect on GABA enhancement of
electrically stimulated 3 H-NE release. Values are means"S.E.M. for
each group Ž ns 4 for cortex; ns 7–8 for hypothalamus wHYPx; ns 5–8
for preoptic area wPOAx.. HEPESs10 mM HEPES-buffered artificial
cerebrospinal fluid, pH 7.4. ŽA. ACET Ž10 m M. did not attenuate
enhancement of electrically stimulated 3 H-NE release ŽS2:S1. by 100
m M GABA. )Significantly greater than slices perfused without GABA,
p- 0.0005. ŽB. ACET did not attenuate GABA augmentation of the
duration of S2. )Significantly greater than slices perfused without GABA,
p- 0.005. ŽC. ACET had no effect on GABA enhancement of S3:S1
ratios. )Significantly greater than slices perfused without GABA, p0.0001.
researchers have also observed this effect of TTX in slice
preparations w3x. Thus, TTX could not be used to evaluate
the role of interneurons in GABA enhancement of 3 H-NE
release. Therefore, synaptoneurosomes were prepared from
cortex, HYP and POA and depolarized with KCl in order
to address this question. Additional experiments confirmed
that GABA augments KCl-stimulated release of 3 H-NE
from slices Ž p - 0.01; Fig. 3A.. In synaptosomes, however, GABA at 100 m M has no effect on basal Ž5 mM
KCl. or stimulated Ž20 mM KCl. release of 3 H-NE ŽFig.
3B.. Stimulated release of 3 H-NE Ž20 mM KCl. is greater
than basal Ž5 mM KCl. release of 3 H-NE Ž p - 0.005; Fig.
Fig. 2. MK-801 has no effect on GABA enhancement of electrically
stimulated 3 H-NE release. Values are means"S.E.M. for each group
Ž ns 4 for cortex; ns 5–8 for hypothalamus wHYPx and preoptic area
wPOAx.. ACSF sartificial cerebrospinal fluid, pH 7.4. ŽA. MK-801 Ž10
m M. did not attenuate enhancement of electrically stimulated 3 H–NE
release ŽS2:S1. by 100 m M GABA. )Significantly greater than slices
perfused without GABA, p- 0.01. ŽB. MK-801 did not attenuate GABA
augmentation of the duration of S2. )Significantly greater than slices
perfused without GABA, p- 0.005. ŽC. MK-801 had no effect on
GABA enhancement of S3:S1 ratios. )Significantly greater than slices
perfused without GABA, p- 0.05.
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J.M. Fiber, A.M. Etgen r Brain Research 790 (1998) 329–333
Fig. 3. Effects of GABA on KCl-stimulated 3 H-NE release from slices and synaptoneurosomes from the cortex, hypothalamus ŽHYP. and preoptic area
ŽPOA.. ŽA. GABA Ž100 m M, solid bar. increases 20 mM KCl-stimulated 3 H-NE release in cortex, HYP and POA slices. Values are means" S.E.M. for
each group Ž n s 4–5 for cortex; n s 6 for HYP; n s 7 for POA.. )Significantly greater than slices perfused without GABA, p - 0.01. ŽB. GABA has no
effect on basal Ž5 mM KCl. or stimulated Ž20 mM KCl. 3 H-NE release from synaptoneurosomes prepared from cortex, HYP and POA. Values are
means " S.E.M. for each group Ž n s 4.. )Significantly greater than synaptoneurosomes incubated with 5 mM KCl, p - 0.0005.
stimulated 3 H-NE release from slices as evidenced by the
failure of MK-801 to block GABA-augmented 3 H-NE
release. In addition, GABA augments KCl-stimulated release of 3 H-NE from slices, but not from synaptoneurosomes, indicating that GABA is acting on an interneuron
and not on the noradrenergic terminals in these brain
regions.
GABA augmentation of stimulated 3 H-NE release has
previously been shown to be mediated through the GABA A
receptor w4,13x. Because the present study shows that the
facilitatory action of GABA is not mediated by bicarbonate ion-induced depolarization, it is likely that GABA is
acting through inhibitory GABA A chloride channels located on interneurons. This interpretation is supported by
the finding that GABA fails to modify basal or evoked
3
H-NE release from isolated nerve terminals Žsynaptoneurosomes.. Thus, GABA indirectly augments stimulated 3 H-NE release through GABA A receptors on interneurons.
It is unknown what inhibitory neurotransmitters or neuromodulators might be localized within these GABA-receptive interneurons; however, there is anatomical and
neurochemical evidence that enkephalin and beta-endorphin are inhibited by GABA in other brain regions
w10,17x. These opioid peptides inhibit stimulated 3 H-NE
release in the POA w1x. Furthermore, GABA can reverse
inhibitory effects of morphine on 3 H-NE release from
frontal cortex slices w14x. Hence, it is possible that GABA
disinhibits 3 H-NE release via modulation of endogenous
opioids. Alternatively, GABA may disinhibit a tonic
GABAergic inhibition of NE release. GABA–GABA disinhibitory circuits have been proposed as a mechanism for
other neural functions w7,12x. Further investigation is nec-
essary to determine what neurotransmitters might be regulated by GABA to influence NE release in these brain
regions.
Acknowledgements
Supported by DHHS Grants MH 41414 and RSDA MH
00636 to AME and NRSA MH 10956 to JMF and by the
Department of Neuroscience, Albert Einstein College of
Medicine.
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