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Articles in PresS. J Neurophysiol (March 20, 2003). 10.1152/jn.00228.2003
Excitable and Synaptic Properties of Neurons of Mouse Bed Nucleus of the Stria Terminalis:
Differential Dorsal and Ventral Distribution of Excitable Properties
Regula E. Egli1 and Danny G. Winder1,2,3
1
Department of Molecular Physiology and Biophysics
2
Center for Molecular Neuroscience
3
John F. Kennedy Center for Research on Human Development
Vanderbilt University School of Medicine
Nashville, TN 37232-0615
Running Head: Electrophysiological properties of the BNST
Contact Information:
Danny G. Winder, PhD
Department of Molecular Physiology and Biophysics
Vanderbilt University School of Medicine
724B Robinson Research Building
23rd and Pierce Ave.
Nashville, TN 37232-0615
Phone: (615) 322-1144
Fax: (615) 322-1462
Email: [email protected]
Copyright (c) 2003 by the American Physiological Society.
ABSTRACT
The bed nucleus of the stria terminalis (BNST) is a structure uniquely positioned to
integrate stress information and regulate both stress and reward systems. Consistent with this
arrangement, evidence suggests that the BNST, and in particular the noradrenergic input to this
structure, is a key component of affective responses to drugs of abuse. We have utilized an in
vitro slice preparation from adult mouse to determine synaptic and membrane properties of these
cells, focusing on the dorsal and ventral subdivisions of the anterolateral BNST (dBNST and
vBNST) because of the differential noradrenergic input to these two regions. We find that while
resting membrane potential and input resistance are comparable between these subdivisions,
excitable properties, including a low-threshold spike (LTS) likely mediated by T-type calcium
channels and an Ih dependent potential are differentially distributed. IPSPs and EPSPs are
readily evoked in both dBNST and vBNST. The fast IPSP is predominantly GABAA-receptor
mediated and is partially blocked by the AMPA/kainate-receptor antagonist CNQX. In the
presence of the GABAA-receptor antagonist picrotoxin, cells in dBNST but not vBNST are more
depolarized and have a higher input resistance, suggesting tonic GABAergic inhibition of these
cells. The EPSPs elicited in BNST are monosynaptic, exhibit paired pulse facilitation, and
contain both an AMPA- and an NMDA-receptor mediated component. These data support the
hypothesis that neurons of the dorsal and ventral BNST differentially integrate synaptic input,
which is likely of behavioral significance. The data also suggest mechanisms by which
information may flow through stress and reward circuits.
Electrophysiological properties of the BNST
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Introduction
The bed nucleus of the stria terminalis (BNST) is considered an integral regulator of the
hypothalamic-pituitary-adrenal stress axis (Cullinan et al. 1993; Dong et al. 2001a; Georges and
Aston-Jones 2001, 2002; Herman and Cullinan 1997; Herman et al. 2002; McDonald et al. 1999;
Moga et al. 1989; Saper and Loewy 1980; Weller and Smith 1982). The BNST receives
“processive” stressor input from regions of the brain involved in cognitive information
processing, such as the prefrontal cortex and the hippocampal formation. This information only
becomes a stressor once it has been processed and compared to prior events in one’s experience.
The BNST also receives “systemic” stressor information, i.e. hypotension or hemorrhage. This
input comes directly from visceral efferent pathways and elicits a stress response without any
need of higher order processing. Evidence suggests that the BNST acts as a key intermediary by
receiving these inputs and then sending its own projections to the paraventricular nucleus of the
hypothalamus (PVN), the key central regulator of peripheral glucocorticoid levels.
In addition to being interconnected with the stress axis, the BNST is a key anatomical
bridge in an assemblage of brain regions referred to as the extended amygdala, which also
includes the central nucleus of the amygdala and the shell of the nucleus accumbens (NAc)
(Alheid and Heimer 1988). Further, the BNST projects to and regulates the firing of
dopaminergic cells within the ventral tegmental area (VTA) (Georges and Aston-Jones 2001,
2002). Thus the BNST is uniquely positioned to receive stress axis information and integrate it
into reward/motivation circuitry. Consistent with these anatomical interconnections, recent data
demonstrate that the BNST plays a key role in mediating stress-induced relapse to cocaineseeking behavior (Erb et al. 1996; Shaham et al. 2000; Sinha et al. 1999), as well as in stressinduced maintenance and reinstatement of morphine-conditioned place preference (Wang et al.
Electrophysiological properties of the BNST
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2001). Further, Delfs et al., showed that the BNST plays a key role in morphine withdrawalinduced conditioned place aversion (Delfs et al. 2000).
The systemic stress input to the BNST consists primarily of input from the central
nucleus of the amygdala and noradrenergic input from the A1 and A2 cell groups of the caudal
medulla. Indeed the BNST is one of the heaviest sites of noradrenergic innervation in the CNS.
This input is regionally compartmentalized within the BNST: NE input is much more dense to
the ventral BNST (vBNST) than to the dorsal BNST (dBNST) in the rat and primate brain
(Freedman and Shi 2001; Georges and Aston-Jones 2001; Woulfe et al. 1990). Interestingly, NE
levels increase in the BNST as a result of restraint stress (Pacak et al. 1995), and inhibiting α1
and/or β1-adrenergic receptors directly in the BNST can reduce anxiety-like behaviors in rats
(Cecchi et al. 2002), as well as the cocaine- and morphine-related behaviors described above, as
does lesioning the ventral noradrenergic bundle (Delfs et al. 2000; Wang et al. 2001). Moreover,
cocaine self-administration in subhuman primates decreases metabolic activity in BNST, as well
as upregulating norepinephrine transporter levels in this region (Macey et al. 2003).
Currently, little is known of the properties of BNST neurons. The BNST consists
primarily of neurons that stain positively for glutamic acid decarboxylase (GAD), the enzyme
responsible for the conversion of glutamate to γ-aminobutyric acid (GABA) (Sun and Cassell
1993), and also expresses a variety of neuropeptides (Ju et al. 1989). Despite a growing
literature on anatomical and neurochemical properties of the BNST, little is known of the
electrophysiological properties of neurons within this structure. In a recent initial study, Rainnie
recorded from rat dorsomedial and dorsolateral BNST cells via whole-cell patch methods,
demonstrating that these cells can be recorded from in vitro and that they are regulated by
serotonin (Rainnie 1999).
Electrophysiological properties of the BNST
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Here, we have confirmed and extended the findings of this initial report by
electrophysiologically characterizing neurons of the anterolateral BNST in vitro, comparing
dorsal versus ventral anterolateral BNST. We characterized several properties of excitability in
BNST, including a low-threshold spike, an Ih-dependent depolarization and inward rectification.
These properties of excitability are differentially distributed across BNST neurons along a
dorsal-ventral gradient, suggesting that common inputs to these two areas may evoke distinct
outputs that differentially regulate behavior. Moreover, local fiber stimulation evokes both an
AMPA- and NMDA-receptor mediated EPSP and a GABAA-receptor mediated IPSP. These
data allow us to suggest mechanisms by which information flows through stress and drug reward
pathways.
Materials and Methods
Animal Care
All animals were housed in the Vanderbilt Animal Care Facilities in groups of 2-5. Food
and water were available ad libitum.
Brain Slice Preparation
Male C57Bl/6J mice (5-10 weeks old, Jackson Laboratories) were decapitated under
anesthesia (Isoflurane). The brains were quickly removed and placed in ice-cold artificial
cerebro-spinal fluid (ACSF) (in mM: 124 NaCl, 4.4 KCl, 2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 10.0
glucose, and 26.0 NaHCO3). Slices 350 µm in thickness were prepared using a vibratome
(Pelco). Rostral slices containing anterior portions of BNST (Bregma 0.26 mm to 0.02 mm)
(Franklin and Paxinos 1997) were identified using the internal capsule, anterior commissure,
Electrophysiological properties of the BNST
4
fornix and stria terminalis as landmarks (Figure 1A,B). Slices were then transferred to an
interface recording chamber where they were perfused with heated (~28 °C), oxygenated (95%
O2/5% CO2) ACSF at a rate of about 1 to 1.5 mL per minute. Slices were allowed to equilibrate
in normal ACSF for one hour before experiments began. Increased divalent cation ACSF
consisted of in mM: 116 NaCl, 2.2 KCl, 7.0 CaCl2, 7.0 MgSO4, 1.0 NaH2PO4, 10.0 glucose, and
26.0 NaHCO3.
Intracellular Recording
Recordings from a total of 121 neurons in the BNST were utilized in the present study.
Recording electrodes were pulled on a Flaming-Brown Micropipette Puller (Sutter Instruments)
using thick-walled filament-containing borosilicate glass capillaries. Electrodes were filled with
2 M potassium acetate and were of approximately 120-170 MΩ resistance. A current/voltage
relationship from each cell was obtained by injecting a range of current via the recording
electrode. A bipolar nichrome wire stimulating electrode was placed locally within the BNST
~500 µm from the recording electrode. An input/output curve of synaptic responses was
generated by stimulating with a range of input voltages with stimulus durations from 50 to 100
µsec, starting at 3 V and increasing the stimulus in 2 V increments until the cell fired an action
potential (AP). If the cell did not fire, the maximum stimulus intensity administered was 35 V.
Input resistance (IR) was calculated by measuring the slope of the line created by plotting current
versus voltage from a series of current injections (-.25 nA to +.25 nA). IR was monitored
throughout the duration of the experiment by injecting a current pulse at the end of each stimulus
test pulse. AP amplitude was calculated by measuring the difference between the membrane
potential at which the AP fired in an all-or-none manner and the membrane potential at which the
Electrophysiological properties of the BNST
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AP reversed. AP amplitude was measured at the beginning and end of each experiment. If APs
did not consistently overshoot zero, the experiment was excluded from final analysis. AP
duration was calculated by measuring the width of the AP at the membrane potential
corresponding to 1/3 of total AP amplitude. The membrane time constant tau (τ) was calculated
using the standard exponential curve fitting function provided in pClamp9 (Axon Instruments,
Union City, CA). Synaptic transmission experiments were analyzed by measuring the peak
amplitude of the synaptic response which was normalized and averaged across experiments for
each manipulation using Excel.
Pharmacology
All drugs were bath applied. Picrotoxin, CNQX, DL -2-Amino-5-phosphono-valeric acid
(DL-AP5) and nickel chloride hexahydrate were purchased from Sigma (St. Louis, MO). 4Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidium chloride (ZD7288) and [R-(R*,S*)]5-(6,8-Dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-1,3dioxolo[4,5-g]isoquinolinium bromide (bicuculline) were purchased from Tocris (Ellisville,
MO). DMSO (0.02% v/v) was the carrier for picrotoxin and CNQX.
Data Analysis
Electrophysiological data were collected using an Axoclamp 2B amplifier, digitized and
analyzed using pClamp 8.2 and 9.0 software. Appropriate statistical analyses (indicated within
figure legends) were performed using Prizm and InStat software (Graphpad).
Electrophysiological properties of the BNST
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Results
Basic Membrane Properties
Neurons were sampled within the dorsal and ventral lateral portions of the BNST in an
anterior section (Figure 1). We found that the resting membrane potential (RMP) recorded in
normal ACSF was comparable between dorsal and ventral BNST, as was the apparent input
resistance (IR) (Table 1). Action potential (AP) amplitudes, durations and thresholds were also
comparable between dBNST and vBNST. However, we found that the τ of cells in vBNST was
on average considerably faster than the τ of cells in dBNST. Table 1 compares these properties
between dBNST, vBNST and a neighboring brain region, the NAc core, since these regions
receive overlapping excitatory input. We recorded from 15 NAc core cells for analysis of basic
properties. We found that the RMP of NAc neurons was significantly more hyperpolarized than
the RMP of either dBNST or vBNST neurons, and the IR was significantly lower. AP amplitude
was significantly lower in NAc neurons than in dBNST and vBNST neurons. AP duration and
threshold were comparable between BNST and NAc neurons, but the τ for NAc neurons was
significantly lower than for either dBNST or vBNST neurons.
Depolarization Evokes Low-threshold Spiking in BNST Neurons
A substantial proportion of neurons in the BNST displayed a low-threshold spike (LTS)
in response to depolarizing current injections (Figure 1C, D). This LTS was observed as a
relatively rapidly activating and inactivating envelope lasting 63.5 ± 5.8 msec upon which were
frequently superimposed 1-5 action potentials. The first action potential on the LTS was similar
in amplitude and duration to APs elicited in the absence of a LTS (Table 2). Subsequent APs on
the LTS became progressively significantly smaller in amplitude and longer in duration than the
Electrophysiological properties of the BNST
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first AP on the LTS (Table 2), in contrast to groups of multiple APs observed upon non-LTS
inducing depolarizing steps. The LTS was not observable when the cell was held at -60mV via
constant current injection prior to the depolarizing step (Figure 1C, solid trace). However, upon
recovery from a hyperpolarizing pulse, the LTS was observable (Figure 1C, dotted trace). The
LTS seen upon depolarization from a hyperpolarized state could be reduced by bath application
of 200 µM Ni2+ (n=3) (Figure 1D) at a concentration known to selectively inhibit T- and R-type
calcium channels (Su et al. 2002). Application of NiCl2 had no apparent effect on the other basic
properties of these cells, i.e. membrane potential and input resistance remained constant during
nickel application. The LTS we observed had an activation threshold of approximately -60 mV,
which is consistent with being driven by T-type Ca2+ channels rather than R-type Ca2+ channels,
which have an activation threshold of -40 mV to -25 mV (Randall and Tsien 1997). The resting
state-dependence and activation threshold of the LTS coupled with its Ni2+ sensitivity suggest
that it is mediated by activation of T-type Ca2+ channels. Interestingly, the LTS also occurred on
EPSPs (data not shown), suggesting that cells demonstrating the LTS may be more prone to
synaptically-induced burst firing behavior.
BNST Neurons Display a Depolarizing Sag in Response to Hyperpolarization as well as Inward
Rectification Reminiscent of Medium Spiny Neurons
Another property we observed in BNST neurons was a depolarizing sag in response to a
hyperpolarizing current injection (Figure 2A). This sag was somewhat slowly developing, with
the time at which the depolarization began ranging from 13.8 to 120.8 msec, and it had an
amplitude ranging from 1 to 15 mV (measured from the peak hyperpolarization after initiation of
the current step to the final steady state hyperpolarization reached before current injection was
Electrophysiological properties of the BNST
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turned off). As this type of potential is often mediated by Ih, we tested this possibility via bath
application of the Ih channel blocker ZD7288 (n=3, 100 µM) (Figure 2B). While this drug had
no consistent effect on RMP, ZD7288 abolished the depolarizing sag. Inspection of the I-V
relationships in the absence and presence of ZD7288 suggests that this conductance contributes
to lowering the apparent input resistance in these cells (see inset in Figure 2B).
Finally, a number of cells displayed moderate inward rectification in response to current
injection (Figure 2C, D). While this property is reminiscent of medium spiny neurons of the
dorsal striatum and NAc, these BNST cells had substantially higher input resistances (113.3 ±
7.4 MΩ) and more depolarized resting membrane potentials (-66.3 ± 1.8 mV) than NAc core
medium spiny neurons.
Electrophysiological Properties of BNST Neurons Are Differentially Distributed within Dorsal
and Ventral BNST
Interestingly, many of the basic properties we observed in the BNST were not
homogeneously expressed within the BNST, but rather were regionally differentiated. The τ of
cells in vBNST was on average considerably faster than in dBNST neurons (Table 1). The
percentage of neurons displaying a LTS was significantly greater in vBNST than in dBNST while a LTS could be evoked in 74.5% of cells in vBNST, it could only be evoked in 22.9% of
cells in dBNST (p < 0.0001, Figure 3A). As with the LTS, the Ih-dependent depolarizing sag
was differentially distributed between dorsal and ventral BNST; however, rather than being
found predominantly in vBNST this property was significantly more frequently observed in
dBNST (15.7 % and 48.6%, respectively, p = 0.0002, Figure 3A). Cells displaying inward
rectification were found only slightly more abundantly in dBNST (Figure 3A). Interestingly,
Electrophysiological properties of the BNST
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multiple cells from vBNST and dBNST showed 2 out of these 3 characteristics, and 2 cells we
recorded from, one in vBNST and one in dBNST, exhibited all three characteristics (Figure 3B).
Neurons in the Dorsal BNST Are Tonically Inhibited by GABA
The BNST receives strong GABAergic input from both intrinsic and extrinsic sources.
To begin to explore the role of GABAergic transmission in BNST physiology, we examined the
properties of dBNST and vBNST neurons in the presence and absence of the GABAA receptor
antagonist picrotoxin. RMP and IR of cells in vBNST appeared unaffected by the inhibition of
fast GABAergic transmission. In contrast, the RMP of cells in dBNST depolarized significantly,
from -69.1 ± 2.0 mV in normal ACSF to -64.1 ± 1.3 mV in picrotoxin (p = 0.0289) (Figure 4A),
suggesting tonic inhibition of these cells by GABA. Accordingly, the IR in dBNST cells also
increased significantly in the presence of picrotoxin, from 115.2 ± 10.8 MΩ to 143.2 ± 6.2 MΩ
(p = 0.0197) (Figure 4B). It should be noted that the Ih current was still active in the presence of
picrotoxin, so the increase in the apparent IR of dBNST neurons seen in the presence of
picrotoxin is not likely due to an inhibition of Ih current activation.
Inhibitory Synaptic Transmission
Local fiber stimulation in the BNST results in an excitatory post-synaptic potential
(EPSP) and an inhibitory post-synaptic potential (IPSP) in both dBNST and vBNST. Depending
on the membrane potential, the EPSP, the IPSP, or both are observable (Figure 4C). The IPSP
occurs with a latency of 2.39 ± 0.04 msec (measured in the presence of 10 µM CNQX, an
AMPA/kainate-receptor antagonist). The major component of the IPSP is mediated by the
GABAA-receptor as it can be blocked by application of 25 µM picrotoxin (Figure 4D) or, another
Electrophysiological properties of the BNST
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GABAA-receptor antagonist, 20 µM bicuculline (Figure 5E, trace 2). A small late
hyperpolarization was occasionally observed after the initial IPSP that could be a GABABmediated IPSP and/or a glycinergic IPSP (Figure 4D), but this was inconsistently seen and would
need to be analyzed pharmacologically to definitively identify its origins. To begin to determine
the source of the GABAA-mediated IPSP, we recorded under conditions of increased
concentrations of Mg2+ and Ca2+ (7 mM each) to increase AP firing threshold. An increase in
AP threshold would be predicted to reduce the probability of multisynaptic events relative to
monosynaptic events. Thus, these conditions allow one to begin to discriminate a monosynaptic
response (which should be unaffected under these conditions) from a polysynaptic response
(which should be reduced). As predicted, this manipulation substantially shifted the threshold
for somatic AP firing to the right, with APs only elicited upon greater amounts of depolarization,
i.e. APs fired at a membrane potential of ca. -40 mV, instead of -55 mV (Figure 4E, insets).
Under these conditions of high divalent cation concentrations, the IPSP was elicited with no
failures (Figure 4E), suggesting that at least a component of the IPSP is monosynaptic
GABAergic transmission. Curiously though, this IPSP was partially reduced by application of
CNQX (10 µM, Figure 4F), suggesting that the IPSP may have both mono- and polysynaptic
components that may be due to both direct GABAergic input stimulation as well as either
indirect low-threshold GABAergic inputs stimulated by AMPA/kainate-receptor mediated
circuits, or conceivably, GABAergic terminals presynaptically regulated by AMPA/kainate
receptors (Braga et al. 2003).
Electrophysiological properties of the BNST
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Excitatory Synaptic Transmission
In addition to an IPSP, synaptic stimulation evoked a substantial EPSP which, by raising
stimulus strength, was routinely of sufficient size to generate APs (Figure 5A). This EPSP
appeared to be monosynaptic in origin. First, the response was elicited with a very short latency
with very little variability (2.56 ± 0.04 msec, measured in the presence of 25 µM picrotoxin).
Second, in ACSF containing increased divalent ion concentrations, the EPSP was still reliably
elicited and comparable in amplitude to EPSPs recorded in normal ACSF. In addition, there was
no increase in failure rate during low frequency stimulation (Figure 5B). The EPSP recorded in
25 µM picrotoxin ACSF was consistently elicited during relatively high frequency (400 msec of
25 Hz) stimulation (Figure 5C), and could be almost entirely blocked by 10 µM CNQX (Figure
5D). An NMDAR-mediated EPSP could be revealed after blocking an IPSP recorded in the
presence of 10 µM CNQX (Figure 5E, trace 1) with 20 µM bicuculline (Figure 5E, trace 2). This
remaining component of the EPSP could be blocked by the NMDAR antagonist, DL-AP5 (100
µM, Figure 5E, trace 3).
In the presence of picrotoxin, EPSPs in cells in both vBNST and dBNST showed paired
pulse facilitation (Figure 5F, G) across a range of interpulse intervals, with the most robust
facilitation at the shorter time points. The presence of paired pulse facilitation indicates that
these cells can undergo at least short term forms of plasticity.
Discussion
The BNST plays a key role in integrating information from stress input pathways and in
turn regulating stress output and reward pathways. In the present study, we have performed an
initial characterization of the electrophysiological properties of neurons within this brain region
Electrophysiological properties of the BNST
12
in the adult male C57Bl/6J mouse and have demonstrated that while several basic membrane
properties are conserved between cells of dorsal and ventral BNST, many properties of
excitability are differentially distributed within these two regions in a manner that parallels their
differential catecholaminergic innervation. These data provide us with information with which
we can begin to make hypotheses about the way the BNST participates in processing information
in stress and reward circuits.
Excitable Properties
From a circuit perspective, it is useful to compare the properties of BNST neurons to a
population of cells receiving heavily overlapping afferent input. When compared to medium
spiny neurons in the core of the NAc, a neighboring population of neurons that receives many of
the same excitatory inputs, we find that neurons of the BNST are much more excitable, having
more depolarized resting membrane potentials, much higher input resistances, longer membrane
time constants, and slightly larger AP amplitudes. In addition, we report three voltage-dependent
properties present in a large percentage of BNST neurons. First, we found that these cells
express a Ni2+-sensitive low-threshold spike. The LTS is most likely mediated by T-type
calcium channels, as its activation properties were consistent with those of a T-type calcium
channel (Randall and Tsien 1997), and it could be blocked by the T- and R-type calcium channel
blocker Ni2+. Second, we found that many cells exhibited a depolarizing sag in response to
hyperpolarizing current injection that was sensitive to the Ih blocker ZD7288. The excitable
properties we report here in mouse brain slices with sharp microelectrodes are very similar to
those reported by Rainnie in rat brain slices (Rainnie 1999). We have here further extended his
results by pharmacologically probing these properties. Third, we found that many cells exhibited
Electrophysiological properties of the BNST
13
inward rectification in response to increasing current injection steps that was reminiscent of that
observed in medium spiny neurons of the dorsal striatum and the NAc (Nisenbaum and Wilson
1995; O'Donnell and Grace 1993).
Inhibitory Transmission
We have found that upon synaptic stimulation, both a GABAA-receptor mediated IPSP
and an AMPA/kainate and NMDA-receptor-mediated EPSP could be elicited in BNST neurons.
The IPSPs elicited in these cells via modest distal stimulation (approximately 500 µm away from
the recording electrode) appeared to be at least partially monosynaptic, as they could be elicited
in the presence of high divalent cation concentrations and had a very short, relatively invariant
latency. These IPSPs likely originated from both a large GABAergic projection from the central
nucleus of the amygdala as well as from intra-BNST projections of GABAergic BNST neurons.
However, even in the presence of high divalent cation concentrations we found that the IPSP
could be partially reduced by application of 10 µM CNQX. These IPSPs are likely due to highly
excitable local circuit interneurons, although it is possible that GABAergic transmission in this
brain region may be modulated by either axonal or presynaptic terminal localized ionotropic
glutamate receptors (Braga et al. 2003). In future studies, analysis of spontaneous transmission
will be needed to resolve this issue.
In addition to activity-dependent fast GABAergic inhibition, we found that cells in
dBNST were tonically inhibited by GABAergic transmission. In the presence of 25 µM
picrotoxin, the RMP of cells in dBNST was significantly depolarized relative to dBNST cells in
normal ACSF. Consistent with relief of tonic inhibition, the IR of dBNST cells also increased
significantly when picrotoxin was present. This change in IR does not appear to have been
Electrophysiological properties of the BNST
14
driven by an alteration in Ih activation as Ih persisted in the presence of picrotoxin. Tonic
inhibition by GABA has been demonstrated in hippocampus (Yeung et al. 2003) and is
correlated with the presence of specific subunits of the GABAA receptor. The δ subunit of the
GABAA receptor has been clearly demonstrated to have an extrasynaptic localization, and
heteromeric assemblies of this subunit produce inactivating and non-desensitizing GABAA
receptors (Bianchi et al. 2002). The δ subunit is present in the BNST, particularly in neuronal
processes, though it is not clear whether there is a dorsal versus ventral distribution (Pirker et al.
2000). It is also possible that this basal inhibition of cells in dBNST may be the result of a
steady stream of spontaneous inhibitory transmission, as opposed to tonic presence of GABA.
However, we have no evidence that there is an exceptionally high level of spontaneous
neurotransmitter release (either GABA or glutamate) that could contribute to tonic regulation of
membrane potential or input resistance, although our experiments have not been performed
under conditions which would optimize the detection of these events.
In total, our data suggest at least three different forms of fast GABAergic inhibition in the
BNST: 1) glutamatergic transmission-independent forms of GABAergic transmission, possibly
arising from the central nucleus of the amygdala and/or local GABAergic transmission, 2)
glutamatergic transmission-dependent GABAergic transmission which may reflect local circuit
interactions, and/or presynaptic actions of glutamate on GABAergic terminals, and 3) tonic
GABAergic tone in dBNST.
Excitatory Transmission
In addition to an IPSP, upon local stimulation in the BNST, an EPSP could also be
elicited. The ventral subiculum and cortical areas provide the predominant known glutamatergic
Electrophysiological properties of the BNST
15
input into the BNST (Herman et al. 2002), and since the vast majority of cells intrinsic to the
BNST are GABAergic, are likely responsible for the EPSPs recorded here. The EPSP was
largely mediated by non-NMDA ionotropic glutamate receptors, but also had a clear NMDAreceptor dependent component. The EPSP was most likely monosynaptic, as it could be elicited
in high divalent cation concentrations, followed relatively high frequency stimulation with no
failures, and had a very short and invariant latency. EPSPs recorded from cells in dBNST and
vBNST exhibited paired pulse facilitation, a form of short-term plasticity. The amount of
facilitation at longer interpulse intervals was not pronounced, suggesting that the release
probability at these synapses under the present conditions may be relatively high.
It is also worth noting that many of the glutamatergic inputs to the BNST also project to
the NAc core, a neighboring population of neurons also implicated in the drug-reward pathway.
Based on our comparison of properties of these cells, it would be predicted that neurons of the
BNST would be much more responsive to these inputs than NAc neurons, suggesting that BNST
neurons may be activated by glutamatergic stimuli from these regions that would not activate
NAc neurons, which may be of functional significance when the stress and drug reward
pathways are recruited.
Dorsal and ventral BNST neurons display divergent properties
The differing distribution of excitable properties between dBNST and vBNST may
confer different characteristics of synaptic processing upon these two regions. The lowthreshold spike that predominates in the vBNST promotes burst firing, which may alter
transmitter release from these cells. For example, in dopaminergic cells burst firing results in a
more efficient release of DA from terminals (Suaud-Chagny et al. 1992), and in many systems is
Electrophysiological properties of the BNST
16
correlated with release of peptidergic transmitters from dense core vesicles. Efferents from the
BNST contain many different types of neuropeptides, including corticotrophin releasing factor,
neurotensin, galanin, substance P and cholecystokinin (Ju et al. 1989), which may be released in
a similar manner from BNST burst firing cells. Moreover, cells in vBNST also tended to have
relatively shorter τs compared to cells in dBNST. The combination of the shorter τ with the LTS
could serve to increase signal-to-noise ratios by dampening temporal summation of weak inputs
but rewarding larger inputs with burst firing evoked by the LTS.
In contrast to cells of the vBNST, neurons within the dBNST tended not to express a
LTS, were under tonic GABAergic inhibition and had Ih activity. The lack of the LTS and the
presence of tonic GABAergic inhibition suggest that these cells may be more quiescent in the
native state and may require greater input in order for efferent connections to be activated.
However, it is interesting to note that the τs for these neurons were significantly longer than for
cells in the vBNST, despite the presence of Ih activity. This suggests that either these cells are
significantly larger in size than cells of the vBNST, that compensatory effects with other channel
types are employed to compensate for Ih activity, or a combination of these two.
Potential functional significance
It has been suggested that the BNST acts as an intermediary for regions of the brain
involved in perceiving stress and regions of the brain producing responses to stress (Herman and
Cullinan 1997). The ventral subiculum and cortical areas that transmit “processive” stressor
information, and the noradrenergic projections from the nucleus of the solitary tract that transmit
“systemic” stressor information project to the PVN via a relay in the BNST, as well as via direct
projections to the hypothalamus. Interestingly, the BNST also projects to brain systems involved
Electrophysiological properties of the BNST
17
in reward responses, including the VTA and the NAc. The interactions between the BNST, PVN
and VTA are complex, but one prediction would be that activation of the BNST by processive
stressors (i.e. ventral subiculum and cortical areas) would result in increased output from BNST
to the NAc, VTA, PVN and/or the hypothalamic area surrounding the PVN (PVN surround).
However, it is as yet unclear whether this would have a net excitatory or inhibitory effect on
these regions. For example, while the majority of the neurons of the BNST appear to be
GABAergic, strong electrophysiological evidence suggests an excitatory projection between the
BNST and the VTA (Georges and Aston-Jones 2002) which could result in increased dopamine
release in the NAc as a result of BNST activation.
It is important to note that although there are similarities in the organization of dBNST
and vBNST, these regions do have anatomically distinct inputs and outputs. For example, while
the dBNST may send a stronger projection to the NAc (Dong et al. 2001b), vBNST acts as a
GABAergic relay for glutamatergic ventral subicular inputs to the PVN (Cullinan et al. 1993)
and has also been shown to project directly to the VTA via an excitatory projection (Georges and
Aston-Jones 2002). There also appears to be differential expression of various neuropeptides in
dBNST and vBNST (Ju et al. 1989; Moga et al. 1989), which may also be of functional
consequence in integrating their respective inputs and outputs. Additionally, Rainnie described
differences in input resistance and τ between cells in dorsomedial and dorsolateral BNST,
providing more evidence in support of functional differences between subregions of the BNST
(Rainnie 1999).
In addition to the differences described above, a major distinction between these regions
is the differential noradrenergic innervation of BNST, with the ventral portions receiving
substantially more noradrenergic innervation by the caudal medulla than the dorsal regions
Electrophysiological properties of the BNST
18
(Woulfe et al. 1990). This NE input to vBNST is of key functional relevance given the
dependence on adrenergic receptor activity for many of the roles the BNST plays in drug related
behaviors (Delfs et al. 2000; Wang et al. 2001). Indeed, one possibility to be investigated in
future studies is whether the differential expression of excitable properties we find reflects
differential states of noradrenergic neuromodulation within BNST. Thus, while at present it is
difficult to predict the net effects of activation of BNST cells on target brain regions, these
studies coupled with more detailed understanding of the connectivity of these regions will
greatly aid in our understanding of the integration of the stress and reward axes.
Electrophysiological properties of the BNST
19
Acknowledgements
The authors would like to thank David Lovinger, Ph.D. and Robert Matthews, Ph.D. for helpful
comments on a previous version of this manuscript. This work was supported by NIAAA and
the Whitehall Foundation.
Electrophysiological properties of the BNST
20
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26
Figure Legends:
Figure 1. Representative slice and electrode placement and T-type calcium channel dependence
of the low-threshold spike
A. Schematic from Franklin and Paxinos rat brain atlas. dBNST is highlighted in blue, vBNST
is highlighted in purple. Landmarks used to identify slices containing BNST are labeled, anterior
commissure (AC), internal capsule (ic) and the stria terminalis (St).
B. Corresponding slice from adult male C57Bl/6J mouse. Stimulus electrode and recording
electrode are shown as they would be placed for recording from the vBNST with local
stimulation.
C. vBNST cell resting at -60 mV does not display the low-threshold spike upon injection of a
depolarizing step (solid trace), but does display the spike upon repolarization after a
hyperpolarizing step current injection (dotted trace).
D. Loss of low-threshold spike after application of 200 µM NiCl2. The same cell as
in part C held via DC current injection at -80 mV before and during application of 200 µM
NiCl2. Representative current injection traces shown below voltage traces are the same both preand post- NiCl2 (range -0.25 nA to + 0.15 nA, in increments of 0.1 nA). Scale bars: 50 msec, 20
mV.
Figure 2. Ih current and inward rectification
A. Current-voltage relationship of a dBNST cell displaying a depolarizing sag. Cell held via DC
current injection in both (A) and (B).
B. Current-voltage relationship of same cell after administration of 100 µM ZD7288, an Ihcurrent blocker. Inset: Overlaid I-V plots from cell at time points shown in (A) and (B), values
Electrophysiological properties of the BNST
27
used for calculating I-V plot taken immediately prior to initiation of current injection and ~20
msec into current injection . Scale bars for (A) and (B): 50 msec, 20 mV.
C. A different dBNST cell displaying inward rectification. Scale bars: 25 msec, 20 mV.
D. I-V plot of the cell shown in (C).
Figure 3. Differential distribution of properties within dorsal and ventral BNST
A. Percent of cells in dBNST versus vBNST displaying either the low-threshold spike, the Ih
current or inward rectification. Actual n’s are inside the appropriate bars
B. Percent of cells in dBNST versus vBNST that display combinations of the three properties
shown in part (A). p-values in (A) and (B) calculated using a Fisher’s exact test. *indicates
statistically significant.
Figure 4. Properties of inhibitory transmission in BNST
A. Average RMP of cells in normal ACSF does not differ significantly in cells from dBNST
(n=22) vs. vBNST (n=27). RMP of cells in dBNST depolarizes significantly in the presence of
picrotoxin (n=44), but not in vBNST (n=24) (unpaired t-test, p = 0.0289).
B. Average input resistance of cells in normal ACSF does not differ significantly in cells from
dBNST (n=22) vs. vBNST (n=27). IR of cells in dBNST increases significantly in the presence
of picrotoxin (n=44), but not in vBNST (n=24) (unpaired t-test, p = 0.0197).
C. An EPSP and an IPSP recorded from the same cell by holding the cell at different membrane
potentials. Scale bars: 25 msec, 5 mV.
D. 25 µM picrotoxin blocks IPSPs recorded from BNST neurons. Cell held at -65 mV using DC
current injection. Scale bars: 50 msec, 5 mV.
Electrophysiological properties of the BNST
28
E. An IPSP can be consistently elicited following low-frequency stimulation (0.1 Hz) with no
failures (7 overlaid traces) in the presence of high concentrations of Mg2+ and Ca2+ (7 mM each).
Scale bars: 25 msec, 5 mV. Insets show representative I/V plots of cells in high divalent cation
ACSF (left) and in normal ACSF (right). Note the higher threshold of AP firing in the cell in
high divalent cation ACSF. Scale bars: 25 msec, 20 mV.
F. Time course of reduction of IPSP by 10 µM CNQX in high divalent cation ACSF. Data
shown as average ± SEM. Insets show representative traces pre- and post-CNQX application.
Scale bars: 20 msec, 5 mV.
Figure 5. Properties of excitatory transmission in BNST.
A. Representative stimulus input/output curve ranging from 3 V to 11 V at 50 µsec duration.
Action potential has been truncated. Scale bars: 10 msec, 5 mV.
B. An EPSP can be consistently elicited during low frequency stimulation (0.1 Hz) even in the
presence of high concentrations of Mg2+ and Ca2+ (7 mM each). Scale bars: 25 msec, 10 mV.
C. The EPSP consistently follows relatively high frequency stimulation (400 msec of 25 Hz) in
25 µM picrotoxin. Scale bars: 25 msec, 5 mV.
D. 10 µM CNQX almost completely blocks an EPSP recorded in the presence of 25 µM
picrotoxin. See panel (E) for appropriate scale bars.
E. Synaptic response recorded from a cell in the presence of 10 µM CNQX (1), 10 µM CNQX +
20 µM bicuculline (2), and 10 µM CNQX + 20 µM bicuculline + 100 µM DL-AP5 (3). Scale
bars: 25 msec, 5 mV.
F. Quantification of paired pulse facilitation at interpulse durations of 20 msec, 50 msec, 100
msec, 150 msec and 300 msec in dBNST (n=9). Inset: Overlaid representative traces of EPSPs
Electrophysiological properties of the BNST
29
from a dBNST cell at paired pulse intervals of 20 msec, 50 msec, 100 msec, 150 msec and 300
msec.
G. Quantification of paired pulse facilitation at interpulse durations of 20 msec, 50 msec, 100
msec, 150 msec and 300 msec in vBNST (n=9).
All cells shown are maintained at indicated membrane potential using DC current injection.
Electrophysiological properties of the BNST
30
Table 1: Comparison of basic properties of cells in dBNST, vBNST and the NAc core
RMP (mV)
dBNST
vBNST
NAc core
Avg. ± SEM (range)
Avg. ± SEM (range)
Avg. ± SEM (range)
-69.1 ± 2.0 (-50 to -90)
-68.6 ± 1.7 (-52 to -82)
-89.6 ± 1.7 (-75 to -91)*
p < 0.001
Input Resistance (MΩ)
115.2 ± 10.8 (26 to 167) 127.9 ± 10.9 (42 to 250)
35.1 ± 3.9 (12 to 67)*
p < 0.001
Action potential amplitude, mV
61.5 ± 1.9 (52 to 71)
62.1 ± 1.5 (50 to 70)
p < 0.05
(peak-threshold)
Action potential width, msec
54.7 ± 1.4 (44 to 64)*
1.4 ± 0.1 (1.0 to 2.7)
1.2 ± 0.1 (0.8 to 1.5)
1.3 ± 0.04 (1.0 to 1.6)
Action potential threshold, mV
-50.7 ± 1.1 (-42 to -56)
-51.8 ± 1.3 (-41 to -58)
-47.8 ± 2.3 (-27 to -59)
Tau (msec)
17.4 ± 3.4 (6.6 to 41.5)
(at 1/3 total amplitude)
n
22
9.7 ± 1.0 (4.7 to 18.5)†
4.0 ± 0.4 (2.1 to 6.6)*
p < 0.01
p < 0.05
27
15
One-way ANOVA followed by Bonferroni’s Multiple Comparison Test: * indicates statistically
significant difference with dBNST and vBNST, † indicates statistically significant difference
with dBNST alone.
Electrophysiological properties of the BNST
31
Table 2: Comparison of values for action potential amplitude and duration between action
potentials elicited from depolarizing steps and action potentials generated off of low-threshold
spikes
AP induced by
1st AP on LTS
2nd AP on LTS
3rd AP on LTS
depolarizing step
Avg. ± SEM (range)
Avg. ± SEM (range)
Avg. ± SEM (range)
Avg. ± SEM (range)
Amplitude (mV)
62.8 ± 0.8 (50 to 73)
64.4 ± 0.9 (55 to 75)
48.6 ± 2.2 (16 to 61)* †
45.8 ± 3.0 (26 to 65)* †
Duration (msec)
1.3 ± 0.04 (0.8 to 2.7)
1.1 ± 0.03 (0.8 to 1.4)
1.4 ± 0.05 (0.8 to 2.1)*
1.7 ± 0.08 (1.2 to 2.2)* †^
One-way ANOVA followed by Tukey’s Multiple Comparison Test: * statistically significant
difference versus 1st AP on the LTS (p < 0.01), † statistically significant difference versus AP
induced by depolarizing step (p < 0.001), ^ statistically significant difference versus 2nd AP on
LTS (p < 0.05).
Electrophysiological properties of the BNST
32
Figure 1
A.
B.
ic
St
ic
AC
AC
C.
-60 mV
D.
-80 mV
+ NiCl2
-80 mV
Electrophysiological properties of the BNST
Figure 2
33
Electrophysiological properties of the BNST
Figure 3
34
Electrophysiological properties of the BNST
Figure 4
35
Electrophysiological properties of the BNST
Figure 5
36