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α1-Adrenergic Receptor-Induced Heterosynaptic Long-Term
Depression in the Bed Nucleus of the Stria Terminalis Is
Disrupted in Mouse Models of Affective Disorders
Zoé A McElligott1 and Danny G Winder1,2,3,*
1Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA
2Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center,
Nashville, TN, USA
3Kennedy Center For Human Development, Vanderbilt University Medical Center, Nashville, TN,
USA
Abstract
The glutamatergic synapse in specific brain regions has been shown to be a site for convergence of
stress and addictive substances. The bed nucleus of the stria terminalis (BNST), a nucleus that
relays between higher order processing centers and classical reward and stress pathways, receives
dense noradrenergic inputs that are known to influence behavioral paradigms of both anxiety and
stress-induced relapse to drug seeking. α1-Adrenergic receptors (α1-ARs) within this region have
been implicated in modulation of the HPA axis and anxiety responses. We found that application
of an α1-AR agonist produced a long-term depression (LTD) of excitatory transmission in an acute
mouse BNST slice preparation. This effect was mimicked by a 20 min, but not a 10 min,
application of 100 µM norepinephrine (NE) in a prazosin-sensitive manner. This α1-AR LTD was
independent of N-methyl-D-aspartate receptor (NMDAR) function unlike previously described α1-
AR LTD in the hippocampus and visual cortex; however, it was dependent on the activation of L-
type voltage gated calcium channels (VGCCs). In addition, α1-AR LTD was induced
independently of the activation of mGluR5 which can also induce LTD in this region.
Furthermore, α1-AR LTD was intact in mice receiving an intraperitoneal injection of cocaine but
was disrupted in α2a-AR and NE transporter (NET) knockout (KO) mice. Thus a loss of this
plasticity at glutamatergic synapses in BNST could contribute to affective behavioral phenotypes
of these mice.
Keywords
synaptic plasticity; heterosynaptic neuromodulation; catecholamine; norepinephrine; anxiety;
depression
© 2008 Nature Publishing Group All rights reserved
*Correspondence: Dr DG Winder, Department of Molecular Physiology & Biophysics, Room 724B, RRB 1, Vanderbilt University
School of Medicine, Nashville, TN 37232-0615, USA, Tel: + 615 322 1144, Fax: + 615 343 0490, danny.winder@vanderbilt.edu.
DISCLOSURE/CONFLICT OF INTEREST
Ms McElligott declares that except for her graduate student stipend from Vanderbilt University she has not received financial support
or compensation from any individual or corporate entity for the past 4 years for research or professional service. Dr Winder has
received a distribution from Columbia University for the licensing of transgenic mouse technology to Memory Pharmaceuticals and
received a consultancy fee from MEDAcorp. Both authors declare that there are no personal financial holdings that could be perceived
as potential conflicts of interest.
NIH Public Access
Author Manuscript
Neuropsychopharmacology. Author manuscript; available in PMC 2011 March 1.
Published in final edited form as:
Neuropsychopharmacology
. 2008 September ; 33(10): 2313–2323. doi:10.1038/sj.npp.1301635.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
INTRODUCTION
Relapse to drug use after extended abstinence remains a troublesome aspect of addiction.
Clinical studies have implicated psychological stress as a major factor that induces relapse
behavior. Although the noradrenergic system has long been implicated in withdrawal
related-behaviors (Aston-Jones and Harris, 2004), recent evidence has highlighted its role in
addiction (Weinshenker and Schroeder, 2007), particularly with the stress-induced relapse
response (Shaham et al, 2000). For example, administration of an α2-AR agonist or
lesioning of the ventral noradrenergic bundle (VNAB) attenuates stress-induced
reinstatement to opiate seeking (Erb et al, 2000; Wang et al, 2001) and viral restoration of
dopamine β-hydroxylase (DBH, the enzyme that produces norepinephrine (NE)) in the
nucleus tractus solitarius (NTS) of mice lacking DBH restores conditioned place preference
(CPP) to morphine (Olson et al, 2006).
The bed nucleus of the stria terminalis (BNST), a component of the extended amygdala,
receives input from the VNAB, as well as glutamatergic inputs from cortical/limbic areas
and sends outputs to stress and reward centers (Forray and Gysling, 2004). The lateral
BNST (lBNST), and α2- and β-AR signaling therein, regulates stress-induced relapse
behaviors (Wang et al, 2001; Leri et al, 2002). Moreover, blockade of excitatory
transmission within this same region also disrupts anxiety-related behavior (Walker and
Davis, 1997). Due to these observations, our group characterized NE modulation of
glutamatergic signaling in the dorsal and ventral lBNST (dlBNST, vlBNST), focusing on α2-
and β-AR signaling (Egli et al, 2005). Studies, however, have also suggested a role for α1-
ARs in the BNST, demonstrating that blocking α1-ARs decreases anxiety responses
concurrent with reductions in hypothalamic-pituitary-adrenal (HPA) axis activation (Cecchi
et al, 2002). Further, activation of α1-ARs also increases spontaneous inhibitory
postsynaptic current (IPSC) frequency in the vlBNST of animals exposed to morphine
(Dumont and Williams, 2004).
α1-ARs have been reported to modulate glutamatergic transmission in other brain regions.
α1-AR activation leads to depression of excitatory transmission that is long lasting in the
hippocampus and visual cortex (Kirkwood et al, 1999; Scheiderer et al, 2004) and transient
in the caudal NTS (Zhang and Mifflin, 2007; for review see Grueter et al, 2007.) In contrast,
in the paraventricular nucleus of the hypothalamus (PVN), α1-AR signaling enhances
excitatory transmission through both pre- and postsynaptic mechanisms (Gordon and Bains,
2003, 2005; Gordon et al, 2005).
Here we investigate the impact of α1-AR signaling on excitatory transmission in the lBNST.
We find an extended application of NE results in robust long-term depression (LTD), which
is dependent on α1-AR activation and that can be mimicked by α1-AR agonists. Intriguingly,
the LTD described here differs from the previously described α1-AR LTD in the
hippocampus and visual cortex in its induction characteristics. Additionally, because of the
relative importance of the BNST in relapse and anxiety paradigms, and adrenergic signaling
therein, we sought to determine if chronic alterations in adrenergic signaling would interfere
with the expression of α1-AR LTD. We found that BNST α1-AR LTD is intact in animals
acutely treated with cocaine, yet, is disrupted in both the α2A-AR knockout (KO) and the NE
transporter (NET) KO lines, suggesting that chronic, but not transient, alterations in
adrenergic signaling modulate the expression of α1-AR LTD.
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METHODS
Animal Care
Male C57BL/6j mice 5–10 weeks old (The Jackson Laboratories, Bar Harbor, ME) and α2A-
AR KOs and NET KOs which were both generated from in-house breeding on a C57BL/6
background were used in experiments. All animals were provided with food and water ad
libitum and housed in groups within the Vanderbilt Animal Care Facilities. Experiments
were performed under Vanderbilt Animal Care and Use committee approved guidelines.
Animals receiving cocaine were prehandled for 5 days, receiving saline injections for 4
days.
Brain Slice Preparations
Slicing methods were as previously described in Grueter et al (2006). Briefly, animals were
retrieved from the colony and allowed to rest in sound attenuating boxes for a minimum of 1
h after which they were anesthetized (isoflurane) and decapitated in a separate room. 300
µm coronal slices were made on a Leica VT1000S vibratome (Leica Microsystems,
Bannockburn, IL) in a 1–4°C, oxygenated (95% O2, 5% CO2), high-sucrose low Na+
artificial cerebral spinal fluid (ACSF in mM: 194 sucrose, 20 NaCl, 4.4 KCl, 2 CaCl2, 1
MgCl2, 1.2 NaH2PO4, 10 glucose, 26 NaHCO3).
Field Potential Recordings
After slicing, whole or hemisected slices were transferred immediately to interface chambers
where they rested for at least 30 min in a humidified and oxygenated environment while
continuously being perfused with oxygenated and heated (approximately 28–30°C) ACSF
(in mM: 124 NaCl, 4.4 KCl, 2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 10 glucose, 26 NaHCO3) at
a rate of 2 ml/min. Following this initial incubation, 25 µM picrotoxin was added to the bath
to block GABAA eceptors and slices were allowed to rest at least another 30 min prior to
recording, picrotoxin was included during the entirety of all experiments to isolate excitatory
transmission. This concentration has been shown by our group to sufficiently block all
inhibitory transmission via the GABAA receptor (Egli and Winder, 2003). Recording
electrodes of approximately 1MΩ resistance were pulled on a Flaming/Brown
microelectrode puller (Sutter Instruments, Novato, CA) and filled with ACSF. A bipolar
Nichrome (A-M Systems, Carlsborg, WA) stimulating electrode was placed dorsally to the
recording electrode within the dlBNST such that stimulation of the field resulted in two
distinguishable negative shifts in potential: N1 (the TTX sensitive fiber volley estimate) and
N2 (CNQX sensitive synaptic response) as previously reported (Weitlauf et al, 2004; Egli et
al, 2005; Grueter and Winder, 2005). The amplitude (voltage) of the N2 was measured at a
stimulation intensity that resulted in a voltage approximately 50% of the maximum N2
response. Slices were stimulated at a frequency of 0.05 Hz. Field potentials were recorded
using Clampex 8.2 (Molecular Devices, Sunnyvale, CA). All drugs were bath applied at
their final concentrations.
Whole Cell Recordings
Following slicing, hemisected slices were allowed to rest submerged in a holding chamber
filled with oxygenated and heated (28°C) ACSF for at least 30 min. After this incubation
time an individual slice was moved to the recording chamber where it was submerged in
oxygenated and heated (28°C) ACSF with added picrotoxin (25 µM included for the entirety
of all experiments as with the field recordings) to isolate currents evoked by glutamate
receptor activation at a rate of 2 ml/min. Stimulating electrodes were the same as for field
recordings in dorsal recordings and medial to the lBNST in ventral recordings. Patch
electrodes (3–6MΩ) were pulled on a Flaming/Brown microelectrode puller (Sutter
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Instruments) and filled with either Cs- or K-gluconate intracellular solution (in mM: Cs- or
K-gluconate 135, NaCl 5, MgCl2 2, HEPES 10, EGTA 0.6, Na2ATP 4, Na2GTP 0.4; there
was no observable difference with either intracellular solution on the LTD effect). In all
whole cell experiments, cells were clamped at −70 mV throughout and excitatory
postsynaptic currents (EPSCs) were recorded using Clampex 9.2 (Molecular Devices).
Series resistance was monitored throughout each experiment and a change greater than 20%
resulted in the exclusion of the experiment from the data set. EPSCs were evoked at a
frequency of 0.167 Hz and 100–400 pA EPSCs were recorded. Consistent with the field
experiments, drugs were bath applied at their final concentrations.
Analysis of Field Recordings
All recorded data were analyzed via Clampfit 9.2 (Molecular Devices). All field recordings
contain a 20 min baseline recording prior to agonist application and all data points were
normalized to the baseline 5 min prior to the agonist application. Plotted time courses for
field experiments are represented as 1 min averages. For the majority of LTD experimental
measurements, the LTD measurement was taken 55–60 min post agonist application. The
exceptions are the experiments with prazosin (Figure 1c and d), in which the LTD
measurement was the final 5 min of each recording.
Analysis of Whole Cell Recordings
A 5 min baseline prior was acquired prior to agonist application and all points were
normalized to minutes 3–5 within each experiment (with the exception of the low
concentration methoxamine experiments where a 10 min baseline was acquired). Points are
30 s averages on plotted time course. For the majority of LTD experiments the LTD
measurement is taken at 58–60 min within the experimental time course. The exceptions to
this rule are the dual agonist application experiments, where the first LTD measurement is at
28–30 min within the time course and the second measurement is at min 55–57 min within
the time course. The experiments with the lower concentration of methoxamine had
measurements taken in the last 2 min of recording due to the shorter duration of wash.
Statistics
All data points were reported as the mean ± SEM and significance (determined by paired
and unpaired Student’s t-test) is reported in the text and figure legends. Significant
differences were defined as having a p < 0.05.
Reagents
(2S)-3-(((1S)-1-(3,4-Dichlorophenyl)ethyl)amino-2-hydroxypropyl) (phenylmethyl)
phosphinic acid (CGP 55845, Tocris, Ellisville, MO), Cocaine (Sigma, St Louis, MO),
(RS)-3,5-Dihydroxyphenylglycine (DHPG, Tocris), DL-2-amino-5-phosphonopentanoic
acid (DL-APV, Sigma), methoxamine-HCl (Sigma), 2-methyl-6-(phenylethynyl) pyridine
hydrochloride (MPEP, Tocris), nimodipine (Tocris), picrotoxin (Tocris), prazosin (Tocris).
Dimethylsulfoxide (DMSO) was the solvent used for stock solutions of CGP 55845, MPEP,
nimodipine, picrotoxin, and prazosin where the maximum final concentration of DMSO was
0.02% by volume.
RESULTS
α1-AR Activation Produces LTD of Excitatory Responses in the BNST
Both dlBNST and vlBNST are activated in response to various stressors (footshock,
yohimbine, restraint, etc; Funk et al, 2006). Additionally electrical stimulation of dlBNST
and vlBNST produces behavior similar to that observed after experiencing a stressor
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(Casada and Dafny, 1991). We began our investigation into the modulation of excitatory
synapses via α1-AR in the BNST by recording extracellular excitatory potentials in the
dlBNST (Figure 1a). We first applied the α1-AR selective agonist methoxamine (100 µM)
for 20 min and observed a long lasting depression in excitatory transmission (79.5 ± 4.7%, p
< 0.01, N = 6; Figure 1b) that was absent in the presence of the α1-AR antagonist 10 µM
prazosin (101.9 ± 5.6%, N = 6; Figure 1c). To verify that this phenomenon was LTD and not
the result of a constitutively activated α1-AR, we applied prazosin (10 µM) to slices 60 min
after a 20 min application of methoxamine (100 µM) was terminated. This treatment failed
to reverse the depression observed after agonist application (67.3 ± 3.0%, p < 0.001, N = 5;
Figure 1d) suggesting that the 20 min activation of α1-ARs results in an LTD of excitatory
inputs. The BNST, however, is composed of a heterogeneous population of dendrites and
cell bodies and therefore extracellular recordings may potentially reflect effects on
excitability of the postsynaptic dendrites/cell. We therefore utilized whole-cell voltage
clamp recordings to measure isolated EPSCs in dlBNST and vlBNST neurons. Application
of methoxamine (10 or 100 µM) for 15 min produced a robust depression of the evoked
EPSC that persisted for at least 40 min after washout of agonist (dlBNST: 10 µM, 65.6 ±
6.3%, N = 5, p < 0.001, Figure 2a; 100 µM, 46.1 ± 9.8%, N = 6, p < 0.001, Figure 2b; 10 vs
100 µM was not statistically different p > 0.15; vlBNST 100 µM: 63.9 ± 8.3%, N = 9, p <
0.001, Figure 2c; representative experiment from the dlBNST 100 µM methoxamine
recording Figure 2d). We did not see changes in the input resistance (IR) following
application of methoxamine in either the dlBNST (10 and 100 µM) nor the vlBNST (100
µM) (97.3 ± 5.5%, p > 0.7, N = 19, Figure 2d representative trace). Additionally, we did not
observe a significant change to a second 15 min application of methoxamine in the dlBNST
(second methoxamine application vs first methoxamine application p > 0.05, N = 6; Figure
2e). This, along with the extent of the depression, suggests that the initial induction of α1-
ARLTD was saturated under our conditions although we cannot rule out that receptors may
have become desensitized to the first agonist application.
Prolonged Exposure to NE Results in α1-AR-Dependent LTD
NE application can elicit LTD of excitatory inputs in the visual cortex and the hippocampus
(Kirkwood et al, 1999; Scheiderer et al, 2004). Thus, we decided to test whether NE induces
similar LTD in the BNST, a nucleus heavily innervated by adrenergic fibers. Our group has
previously reported that a 10 min application of 100 µM NE results in a transient bimodal
response (an increase or decrease) in the extracellular field potential that is mediated by α2-
and β-ARs (Egli et al, 2005). This application, however, was insufficient to produce a
sustained depression in excitatory transmission (98.4 ± 3.4%; Figure 3a, N = 6, Egli et al,
2005) in field recordings. In the BNST and other regions, chronic stressors are thought to
promote lasting increases in extracellular NE levels by shifting the firing patterns of
noradrenergic cells from phasic firing to burst firing (Forray and Gysling, 2004). We found
that increasing the duration of application of NE to 20 min at the same concentration
produced a robust LTD of excitatory responses (67.1 ± 7.3%, p < 0.01, N = 6) that persisted
for over 60 min after agonist application (Figure 3a). Moreover, the experiments where we
applied 100 µM NE for 20 min were significantly different from the experiments where we
applied 100 µM NE for 10 min (p < 0.05) during the LTD phase of the recording. With the
extended application time we still observed the same bimodal effect to NE in our transient
responses. (Two of the six experiments resulted in an initial increase in synaptic efficacy and
four of six in an initial decrease in synaptic efficacy.) The sustained depression, however,
was observed irrespective of the polarity of the initial response to NE (Figure 3b). To test
whether the persistent depression induced by 100 µM NE was due to activation of α1-ARs
we applied the α1-AR specific antagonist prazosin (10 µM) to the slice prior to NE
application and throughout the experiment. The application of prazosin completely ablated
the ability of NE (20 min at 100 µM) to induce a long-lasting depression in excitatory
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responses (94.6 ± 4.5%, N = 5; Figure 3c); however, we still observed the initial bimodal
response (3 of 5 increases in synaptic efficacy, 2 of 5 decreases in synaptic efficacy).
α1-AR LTD in the BNST Is Not Dependent on NMDAR Activation or Concurrent Stimulation
of Presynaptic Fibers but Is Dependent on L-Type VGCCs
α1-AR LTD has been previously described in the visual cortex and most recently in the
hippocampus where it has been shown to require concurrent activation of presynaptic inputs
and N-methyl-D-aspartates (NMDARs) (Kirkwood et al, 1999; Scheiderer et al, 2004). We
found, however, that applying methoxamine in the absence of stimulation resulted in
significant LTD (82.3 ± 2.4%, p < 0.01, N = 6; Figure 4a) that was indistinguishable from
that observed with concurrent stimulation. The absence of concurrent stimulation itself,
however, had no effect on the amplitude of subsequent field potentials (97.4 ± 9.5%, N = 5;
inset, Figure 4a). To examine the influence of NMDAR activation on α1-AR LTD in the
BNST, we recorded EPSCs at −70 mV in the presence of 100 µM DL-APV. Again under
these conditions, a 15 min application of methoxamine still produced robust LTD (66.7 ±
13.3%, N = 5, p < 0.05, not significantly different from single methoxamine application p >
0.05; Figure 4b). LTD induced by group 1 mGluR receptors (that are coupled to Gq) in other
brain regions has been shown to involve L-type voltage gated calcium channels (VGCCs;
for review see Grueter et al, 2007). Thus, we hypothesized that α1-AR LTD might also
require L-type calcium signals. We applied methoxamine (100 µM) in the presence of the L-
type VGCC blocker nimodipine (10 µM) and found that, although there was a small early
depression (minutes 47–49, 86.6 ± 3.4%, p < 0.01), LTD was blocked (99.5 ± 4.8%, N = 7;
Figure 4c). Our group has previously described LTD in the dlBNST that can be induced by
activating the group 1 mGluR, mGluR5 (Grueter et al, 2006). To ensure that α1-ARLTD
was not the result of increased glutamate inducing mGluR5-LTD, we applied methoxamine
in the presence of the mGluR5 antagonist MPEP (10 µM) at a concentration that prevents
the induction of mGluR-LTD (Grueter et al, 2006). We found that MPEP had no effect on
α1-AR LTD (54.3 ± 6.7%, p < 0.005, N = 5; Figure 4d). These data thus suggest that α1-ARs
heterosynaptically induce LTD via a non-Hebbian mechanism in the dlBNST in an L-type
VGCC dependent manner.
LTD can be maintained at synapses via either a pre- or postsynaptic mechanism. To begin to
address questions of the synaptic locus of α1-AR LTD we conducted paired pulse ratio
(PPR) analysis. Increases observed in the PPR associated with a decrease in EPSC
amplitude are suggestive of a presynaptic mechanism. Evoked EPSCs to two paired stimuli
with a 50ms inter-stimulus interval were acquired during whole cell recordings and we
analyzed the ratio of the second response to the first response. In the dlBNST we did not
observe a change in the PPR upon application of methoxamine (10 and 100 µM) nor at the
LTD time point (N = 11, p > 0.15; Figure 4e).
α1-AR LTD Is Disrupted in Mice with Aberrant Noradrenergic Signaling
Alterations in noradrenergic signaling may underlie several affective disease states (Raskind
et al, 2000; Stone et al, 2007). A single administration of cocaine elicits a transient increase
in extracellular NE, while depression, anxiety and alcoholism are thought to involve more
chronic alterations in adrenergic tone. Noradrenergic signaling in the BNST has been
implicated in anxiety, depression, and drug abuse (Shaham et al, 2000; Forray and Gysling,
2004; Morilak et al, 2005) and synaptic plasticity is altered by multiple substances of abuse
and stress in reward nuclei (Saal et al, 2003). We therefore chose to examine α1-AR LTD in
the BNST in animals treated with cocaine (20 mg/kg) 30 min prior to slicing, and two
animal models of affective disorders, α2A-AR and NET KOs. Both of these KO lines of
mice have altered adrenergic systems and behavior (Bohn et al, 2000; Xu et al, 2000;
Schramm et al, 2001; Lahdesmaki et al, 2002; Dziedzicka-Wasylewska et al, 2006; Keller et
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al, 2006). Animals receiving cocaine 30 min prior to slicing still showed robust α1-AR LTD
(54.1 ± 9.4%, p < 0.005, N = 5; Figure 5a). In both KO animal models, α1-AR LTD was not
observed upon application of methoxamine (α2A-AR KO: 96.0 ± 2.8%, p > 0.05, N = 5;
NET KO: 104.2 ± 6.0%, p > 0.05, N = 7; Figure 5b and d); however, application of the
mGluR5 agonist DHPG still resulted in robust depression in the α2A-AR KO mouse
indicating that LTD via Gαq coupled mechanisms is still intact (57.6 ± 3.7%, p < 0.001, N =
5; Figure 5c). This is additionally intriguing because induction of α1-AR LTD occludes
further depression of EPSCs in response to subsequent application of DHPG (100 µM) (p >
0.05, N = 5; Figure 5c inset).
DISCUSSION
Previously, our group reported on noradrenergic modulation of excitatory synapses within
the dlBNST, finding that both α2- and β-ARs contributed to effects observed with a 10 min
application of 100 µM NE and that α2-ARs contributed to effects in the vlBNST (Egli et al,
2005). Egli et al found that in the dlBNST, 100 µM NE resulted in either a transient increase
or decrease in excitatory transmission, while in the vlBNST it resulted in a transient
decrease in excitatory transmission. Here we investigated the possibility that α1-ARs
modulate glutamatergic synapses in this region as well. We found that a 20 min, but not a 10
min, 100 µM NE application resulted in an LTD of excitatory transmission that was
mediated by the α1-AR and L-type VGCCs, however, this LTD was independent of
NMDAR, and mGluR5 activation, or concurrent stimulation. Finally, we found that α1-AR
LTD in the dlBNST was disrupted in two KO mice that have genetically manipulated
adrenergic systems, and exhibit altered anxiety, depression and reward phenotypes; but, not
in animals that received a transient alteration of their adrenergic system—a single
intraperitoneal (i.p.) injection of cocaine 30 min prior to slicing.
NE Induces LTD in a Time-Dependent Manner
Our group has shown that while a 10 min application of NE in the dlBNST results in a
bimodal transient regulation of the glutamatergic field potential (Egli et al, 2005), it fails to
induce LTD via the α1-AR. Doubling the duration of NE application, however, results in α1-
AR LTD. This duration-dependent result was an intriguing finding. It is clear that in the 10
min NE application experiments agonist wash-in has occurred and GPCRs are being
activated based on data from our group (Egli et al, 2005) and the data shown here.
Additionally, Dumont and Williams (2004) demonstrated that a brief (<2 min) application of
100 µM NE (in a submerged recording chamber) activated α1-ARs in vlBNST to transiently
increase spontaneous IPSCs, thus, the α1-AR is also presumably activated in our recordings,
but at a level below threshold for LTD. The duration-dependence may play a role
physiologically, as it may be disadvantageous to induce this plasticity with transient NE
increases in the BNST. Therefore this LTD may be activated when the animal experiences a
lasting stressor. Microdialysis studies have demonstrated that NE levels remain elevated
above the baseline in the BNST over an hour after restraint stress and blocking α1-ARs
attenuates stress-induced rises in adrenocorticotropic hormone (ACTH) (Pacak et al, 1995;
Cecchi et al, 2002). Moreover, Banihashemi and Rinaman (2006) showed that ablating
BNST NE inputs prevents increases in corticosterone to i.p. yohimbine 90 min post
injection. Therefore, under stress-producing conditions, α1-AR LTD may be recruited to
alter the engagement of the HPA stress axis by the BNST over an hour after the initial stress
insult. Thus, this α1-AR LTD may specifically participate in stress responses generated by
long-term increases in NE in normal subjects and may be dysregulated in addictive states
such as alcoholism and anxiety disorders like post traumatic stress disorder (PTSD).
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α1-AR LTD in the BNST Is a Heterosynaptic Form of Plasticity
LTD mediated via group I mGluRs remains the best characterized Gαq-coupled receptor
LTD and has been described in the cerebellum, hippocampus, cortex, dorsal and ventral
striatum, ventral tegmental area, and BNST (Ito, 2001; Robbe et al, 2002; Malenka and
Bear, 2004; Bellone and Luscher, 2005; Grueter et al, 2006, for review see Grueter et al,
2007). An interesting aspect exhibited by group I mGluR-LTD is a degree of promiscuity of
mechanism depending on the synapse where the LTD is expressed. This notion can now be
extended to the less characterized α1-AR LTD. Unlike in the hippocampus and visual cortex
(Kirkwood et al, 1999; Scheiderer et al, 2004), α1-AR LTD within BNST is independent of
the activation of NMDARs (in both the induction phase and the maintenance phase of the
LTD) and concurrent presynaptic stimulation. Intriguingly, we found that α1-AR LTD
expression in the BNST is dependent on L-type VGCC activity. L-type VGCC activity
(particularly CaV1.3) is required in the dorsal striatum to induce corticostriatal group I
mGluR-LTD (Wang et al, 2006). Furthermore, it has recently been demonstrated in the
hippocampus that signaling via phospholipase C can increase the conductance of CaV1.3 at
negative potentials (Gao et al, 2006). Consistent with the role of the L-type VGCC in the
induction of group 1 mGluR-LTD in the dorsal striatum, postsynaptic cells must be
depolarized to −50 mV (Adermark and Lovinger, 2007). In contrast, however, we were able
to induce α1-AR LTD holding the cell at −70 mV and in the absence of concurrent
stimulation; although, subsequent stimulation, following the activation of α1-AR, may
activate L-type channels in the maintenance phase of the LTD. Norepinephrine can
modulate cell excitability within BNST, causing depolarization especially in nonprojection
cells (Dumont and Williams, 2004) and, therefore, it is possible that the application of
methoxamine directly depolarized the cell sufficiently to activate L-type VGCCs.
Additionally, the concentrations of our extracellular and intracellular recording solutions
may have produced sufficiently depolarizing conditions. These seem unlikely given previous
sharp microelectrode studies under identical conditions that indicated resting membrane
potentials of −64 to –66 mV (Egli and Winder, 2003) coupled with a lack of change in the
input resistance. Another possibility was that α1-AR LTD may be dependent on signaling
via mGluR5 downstream of α1-AR activation. We found, however, that blockade of
mGluR5 during the induction of α1-AR LTD does not impact the expression of this LTD.
This suggests that in this region adrenergic afferents may influence plasticity independently
of descending glutamatergic inputs to the BNST from areas like the limbic cortex,
hippocampus, and amygdala.
The postulated heterosynaptic mechanism of this α1-AR LTD could be of behavioral
significance. A hallmark of some affective disorders is the inherent inability to overcome the
disorder by reasoning (ie feelings/urges are beyond the cognitive control of the patient)
(DSM-IV). α1-ARs in the BNST modulate ACTH levels in animals who have been exposed
to a stressor (Cecchi et al, 2002). Moreover it has recently been shown that the same axon
collaterals from the nucleus tractus solitarius that project to the BNST also may project to
the PVN (Banihashemi and Rinaman, 2006). NE may therefore modulate this circuitry
regardless of input from cortical structures. It is intriguing to think that this dissociation of
induction of α1-AR LTD from the glutamatergic input of cognitive centers innervating the
BNST potentially contributes to alterations in behavior in a disease state such as generalized
anxiety or addiction. Clearly, however, additional work would be needed to provide support
for this notion.
α1-AR LTD Is not Observed in Mice with Chronically Altered Adrenergic Signaling
Previously our group reported that a 10 min application of 100 µM NE failed to alter
glutamatergic transmission in α2A-AR KOs, mice with altered anxiety phenotypes. It was
concluded that the α2A-AR may gate responses to NE within BNST via its interactions with
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other receptors (Egli et al, 2005). Due to the shorter duration of agonist application,
however, contributions by the α1-AR in the α2A-AR KOs may not have been observed.
Therefore, we decided to examine α1-AR LTD in the α2A-AR KOs. Surprisingly, α1-AR
LTD could not be induced via a 20 min application of methoxamine. This implied that
perhaps the lack of LTD was due to functional desensitization of α1-AR or in vivo induction
of α1-AR LTD as a result of increased extracellular concentration of NE. These ideas are
supported by increased metabolite/transmitter ratios within several brain regions in the α2A-
AR KOs (Lahdesmaki et al, 2002), although these ratios can also be interpreted as increases
in catabolism and therefore have caveats (Commissiong, 1985). To support our data with the
α2A-AR KOs we next used the NET KOs, another mouse model with altered behavioral
phenotypes and adrenergic transmission. Fast cyclic voltammetry experiments in the BNST
in the NET KOs have demonstrated that NE clearance rates are over six times slower in the
KOs as compared to wild-type controls (Xu et al, 2000). As was observed with the α2A-AR
KOs, the NET KOs also failed to express LTD after a 20 min exposure to methoxamine.
Interestingly, the α2A-AR KOs express mGluR5-LTD after an application of DHPG,
demonstrating that signaling via GPCRs, and more specifically those coupled to Gαq, are
still intact. Autoradiography data in the NET KOs shows a reduction in the cell surface
expression of α1-ARs in several brain regions (Bohn et al, 2000; Xu et al, 2000; Dziedzicka-
Wasylewska et al, 2006) but an upregulation of α2A/C-AR within the BNST (Gilsbach et al,
2006). One possibility of our results, taken together with this data, suggest that within these
mouse models α1-ARs and/or their signaling pathways are desensitized, preventing
induction of α1-AR LTD. An intriguing observation was that α1-AR LTD can occlude
mGluR5-LTD in the BNST. This suggests that the two LTDs share a common mechanism as
they do in the visual cortex (Choi et al, 2005) There is evidence, however, in dopaminergic
cells in the VTA that α1-ARs desensitize group I mGluRs (Paladini et al, 2001). The L-type
VGCC experiments provide indirect support for the desensitized receptor hypothesis as well.
In these experiments there is a significant transient depression in response to the application
of methoxamine that is not observed in either of the mouse models. One possibility for this
transient depression would be GABAB receptor activation due to the activation of α1-ARs
on interneurons (Dumont and Williams, 2004). This transient depression is not observed in
either KO animal suggesting that there may be a tonic reduction in α1-ARs. Additional
experiments will need to be conducted to confirm these hypotheses.
Interestingly, both of these KO mice have altered anxiety/depression phenotypes (Schramm
et al, 2001; Lahdesmaki et al, 2002; Dziedzicka-Wasylewska et al, 2006; Keller et al, 2006)
including increased anxiety-like behavior in the elevated plus maze (α2A-AR KOs),
increased response to injection stress (α2A-AR KOs), decreased struggling/mobility in the
forced swim and tail suspension tests (NET KOs) and bradycardia to stressful stimuli (NET
KOs). In addition the NET KO animals have heightened sensitivity to psychostimulants,
enhanced conditioned place preference to cocaine, and increased analgesia to opiates (Bohn
et al, 2000; Xu et al, 2000; Hall et al, 2002). Furthermore it has been shown that the BNST
sends monosynaptic projections to dopaminergic VTA neurons to modulate reward
(Georges and Aston-Jones, 2002). During withdrawal from morphine there is an inhibition
of the firing of these dopaminergic cells that can not only be reversed with the α2-AR
agonist clonidine, but potentiated by its administration (Georges and Aston-Jones, 2003). It
is an interesting notion that the NET KO animals lack a mechanism (α1-AR LTD) that may
contribute to the inhibition of the dopaminergic cells in the withdrawal state while
simultaneously demonstrating increased behavioral sensitization and reward mediated
behaviors to drugs of abuse. A lack of functioning α1-ARs and/or their signaling pathways
may impact such behavior. Although α1-AR activation can affect the excitability of cells in
various ways, the lack of a long term change in cell function, like synaptic plasticity induced
by the α1-ARs, in these animals could have implications for their exhibited behavior.
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Clinical data have highlighted the α1-AR as a therapeutic target for anxiety disorders.
Raskind and colleagues reported that α1-AR antagonists are efficacious for patients with
combat/noncombat induced post-traumatic stress disorder (Raskind et al, 2000, 2002; Taylor
and Raskind, 2002; Peskind et al, 2003; Taylor et al, 2006). Additionally, α1-AR antagonists
are used to treat ailments like benign prostatic hypertrophy and hypertension and thus
specific pharmacological agents are available for further investigation into their benefits in
the treatment of affective disorders. Furthermore, the notion that alterations in adrenergic
mediated synaptic plasticity within nuclei like the BNST, and others implicated in stress and
anxiety, may contribute to the pathological learning (learned fear) that may mediate PTSD
and similar disorders remains an interesting possibility.
Previous work has highlighted the role of adrenergic modulation in BNST in behavioral
paradigms of stress-induced relapse to drug seeking and anxiety. Our findings showed that
NE modulates excitatory synapses in the dlBNST by inducing an LTD that is dependent on
the α1-AR and L-type VGCC activation and independent of the NMDAR and stimulation
from presynaptic inputs. A crucial element to α1-AR LTD is that the induction depends on
the length of exposure to NE, preventing transient increases in NE to elicit plasticity.
Furthermore, a lack of this plasticity in animal models of affective disorders may impact
their behavioral phenotypes. Together, our results demonstrate a mechanism by which NE
may modulate BNST functional output under conditions of psychological stress.
Acknowledgments
We thank Thomas Kash and Amanda Vanhoose for critical comments on the manuscript. We also thank David
Robertson and Mark Caron for supplying norepinephrine transporter knockout mice. This work was supported by
NIDA (DGW) and NIAAA (DGW and ZAM).
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Figure 1.
α1-AR activation induces long-term depression (LTD) in bed nucleus of the stria terminalis
(BNST). (a) BNST schematic adapted from Paxinos and Franklin (2001). Gray shading
represents the lateral BNST, above anterior commissure dorsal lateral BNST, below anterior
commissure ventral lateral BNST. (b) Application of the α1-AR selective agonist
methoxamine (100 µM) induces a depression of extracellularly recorded excitatory
responses that persists for over 60 min post wash (N = 6). (Inset) Representative traces of N1
and N2, 5 min average of baseline (black) and LTD (gray) (scale bars 0.2 mV by 0.5 ms). (c)
The α1-AR antagonist prazosin (10 µM) blocks induction of the depression by methoxamine
(100 µM; (N = 6). (d) Prazosin (10 µM) cannot reverse the depression induced by
methoxamine (100 µM) when applied in wash (N = 5).
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Figure 2.
Excitatory postsynaptic currents (EPSCs) are depressed by α1-AR agonist application. (a)
Whole cell voltage clamp (−70 mV holding potential) experiments were performed to assess
long-term depression (LTD) in the dorsal lateral BNST (dlBNST). Methoxamine (100 µM)
was applied for 15 min and washed out for 40 min resulting in LTD (N = 6). (b) Under the
same conditions 100 µM methoxamine also induced LTD in the ventral lBNST (vlBNST).
(N = 8) (c) A lower concentration of methoxamine (10 µM) applied for the same duration
also can induce significant LTD in the dlBNST. (d) A representative experiment in the
dlBNST: EPSC (pA) = EPSC amplitude, HI (pA) = holding current, RA (MΩ) = Access
Resistance, and RI (GΩ) = Input resistance. (d inset) Representative traces of EPSCs, each a
2 min average of baseline (black line) and LTD (gray line; scale bar 40 pA by 5 ms). (e)
Applying 100 µM methoxamine after previous induction of LTD fails to further depress
EPSCs (N = 6).
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Figure 3.
Norepinephrine (NE) induces α1-AR LTD via a time-dependent mechanism. (a) Expressed
as percent of baseline, a 20 min application of NE (100 µM) results in a sustained
depression of extracellularly recorded excitatory response that lasts for over 60 min post
drug application (open symbols; N = 6). However, responses following a 10 min application
of NE (100 µM) fail to induce a sustained depression of the excitatory response (closed
symbols; N = 6). (b) Two representative experiments with 20 min applications of NE (100
µM) emonstrate the transient bimodal effect, open symbols: increase in EPSP response
followed by LTD, closed symbols: decrease in EPSP response followed by LTD. (c) 10 µM
of the α1-AR specific antagonist prazosin blocks the sustained depression of the excitatory
field potential (open symbols; N = 5).
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Figure 4.
α1-AR LTD (as measured in the dlBNST) is induced independently of evoked glutamatergic
synaptic activity but dependent on L-type voltage gated calcium channels (VGCCs). (a) To
address the involvement of presynaptic stimulation the stimulus was turned off prior to 100
µM methoxamine application and turned back on 2 min post α1-AR agonist removal. This
did not disrupt LTD expression. (N = 7). (a inset) To control for the lack of stimulation
interleaved experiments were run without the presence of agonist (N = 5). (b) To assess the
role of N-methyl-D-aspartate receptor (NMDAR) activation α1-AR LTD experiments were
performed in whole cell voltage clamp (−70 mV holding potential) and DL-APV (100 µM)
was included throughout the duration of the experiment (N = 5). (c) The L-type calcium
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channel blocker nimodipine (10 µM) prevented the induction of α1-AR LTD by 100 µM
methoxamine, however it did not prevent a significant transient depression. (N = 7) (d) To
verify that α1-AR LTD does not require mGluR5 signaling we applied 100 µM
methoxamine in the mGluR5 antagonist MPEP (10 µM; N = 5). (e) Paired pulse ratios do
not change in response to methoxamine (10 or 100 µM) in dlBNST (N = 11).
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Figure 5.
α1-AR is disrupted in animal models of affective disorders. (a) Cocaine (20 mg/kg) injected
30 min prior to slicing does not prevent the induction/expression of α1-AR LTD (N = 5). (b)
A 20 min application of methoxamine (100 µM) fails to induce α1-AR LTD in α2A-AR KO
mice (N = 5). (c) DHPG (100 µM) induces mGluR5-LTD in α2A-AR KO mice (N = 5). (d)
Methoxamine (100 µM) fails to induce α1-AR LTD in NET KO mice (N = 7).
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