Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
CORTICOTROPIN-RELEASING FACTOR INCREASES GABA
SYNAPTIC ACTIVITY AND INDUCES INWARD CURRENT IN 5-
HYDROXYTRYPTAMINE DORSAL RAPHE NEURONS
Lynn G. Kirby, Ph.D.1, Emily Freeman-Daniels, B.A.1, Julia C. Lemos, B.A.2, John D. Nunan,
M.Sc.1, Christophe Lamy, Ph.D.2, Adaure Akanwa, B.A.2, and Sheryl G. Beck, Ph.D.2
1Dept. of Anatomy and Cell Biology and Center for Substance Abuse Research, Temple University School of
Medicine, Philadelphia, PA 19140
2Dept. of Anesthesiology, University of Pennsylvania School of Medicine and Children’s Hospital of
Philadelphia, Philadelphia, PA 19104
Abstract
Stress-related psychiatric disorders such as anxiety and depression involve dysfunction of the
serotonin (5-hydroxytryptamine; 5-HT) system. Previous studies have found that the stress
neurohormone corticotropin-releasing factor (CRF) inhibits 5-HT neurons in the dorsal raphe nucleus
(DRN) in vivo. The goals of the present study were to characterize the CRF receptor subtypes (CRF-
R1 and R2) and cellular mechanisms underlying CRF-5-HT interactions. Visualized whole-cell patch
clamp recording techniques in brain slices were used to measure spontaneous or evoked GABA
synaptic activity in DRN neurons of rats and CRF effects on these measures. CRF-R1 and -R2-
selective agonists were bath applied alone or in combination with receptor-selective antagonists.
CRF increased presynaptic GABA release selectively onto 5-HT neurons, an effect mediated by the
CRF-R1 receptor. CRF increased postsynaptic GABA receptor sensitivity selectively in 5-HT
neurons, an effect to which both receptor subtypes contributed. CRF also had direct effects on DRN
neurons, eliciting an inward current in 5-HT neurons mediated by the CRF-R2 receptor and in non
5-HT neurons mediated by the CRF-R1 receptor. These results indicate that CRF has direct
membrane effects on 5-HT DRN neurons as well as indirect effects on GABAergic synaptic
transmission that are mediated by distinct receptor subtypes. The inhibition of 5-HT DRN neurons
by CRF in vivo may therefore be largely an indirect effect via stimulation of inhibitory GABA
synaptic transmission. These results regarding the cellular mechanisms underlying the complex
interaction between CRF, 5-HT and GABA systems could contribute to the development of novel
treatments for stress-related psychiatric disorders.
Keywords
serotonin; stress; urocortin II; antalarmin; antisauvagine-30; IPSC
INTRODUCTION
Stress-related psychiatric disorders such as depression and anxiety are frequently characterized
by dysfunctions of corticotropin-releasing factor (CRF) (Nemeroff, 1988;Gold et al.,
1995;Arborelius et al., 1999;Reul and Holsboer, 2002) and 5-hydroxytryptamine (5-HT)
Corresponding author: L. G. Kirby, Ph.D. Department of Anatomy and Cell Biology Temple University School of Medicine 3400
North Broad Street Philadelphia, PA 19140 Phone: 215-707-8556 Fax: 215-707-9468 Email: lkirby@temple.edu.
NIH Public Access
Author Manuscript
J Neurosci. Author manuscript; available in PMC 2009 May 26.
Published in final edited form as:
J Neurosci. 2008 November 26; 28(48): 12927–12937. doi:10.1523/JNEUROSCI.2887-08.2008.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
systems (Meltzer, 1990;Charney et al., 1990a;Charney et al., 1990b). Stress regulates the 5-
HT system in a complex stressor- and region-specific manner (Kirby et al., 1995;Adell et al.,
1997;Kirby et al., 1997). In vivo microdialysis and electrophysiology studies have shown that
a particular stressor, swim stress, inhibits 5-HT release in certain targets, and that this effect is
mediated by the inhibition of 5-HT neuronal activity in the serotonergic dorsal raphe nucleus
(DRN) by the endogenous release of CRF (Kirby et al, 1995; Price et al, 2002). Exogenous
administration of CRF alters 5-HT DRN neuronal activity and 5-HT release in forebrain targets
with a U-shaped dose-response function (Price et al., 1998;Kirby et al., 2000;Price and Lucki,
2001). Both CRF receptor subtypes (CRF-R1 and -R2) are found in the DRN (De Souza et al.,
1985;Potter et al., 1994;Chalmers et al., 1995) and both receptors contribute to CRF effects on
neuronal activity of 5-HT DRN neurons (Kirby et al., 2000;Pernar et al., 2004). CRF terminals
target both 5-HT and GABA neurons in the DRN (Waselus et al., 2005) and CRF receptors
are located on both GABA and 5-HT DRN neurons (Kirby et al., 2001;Roche et al., 2003;Day
et al., 2004). CRF is thus anatomically poised to regulate 5-HT neurotransmission in the DRN
either directly at 5-HT neurons or indirectly via GABAergic afferents to those neurons. The
goal of this study was to determine at the cellular level the receptors underlying these CRF-5-
HT interactions by examining the effects of receptor-selective CRF ligands on 5-HT and non
5-HT DRN neurons and their GABA synaptic activity in rats. The effect of the CRF-R1-
preferring agonist ovine CRF [oCRF; 8 times more potent at the CRF-R1 than -R2 receptor
(Lovenberg et al., 1995)] was compared to the CRF-R2 agonist urocortin II (UCN II). In
addition, oCRF effects were examined in the presence of the CRF-R1 antagonist antalarmin
or the CRF-R2 antagonist antisauvagine-30 (ASVG-30).
MATERIALS AND METHODS
Subjects
Male Sprague-Dawley rats (Taconic Farms, Germantown, NY), 4-5 weeks of age, were housed
2-3 per cage on a 12-h light schedule (lights on at 07:00 AM) in a temperature-controlled (20°
C) colony room. Rats were given access to standard rat chow and water ad libitum. Animal
protocols were approved by the Institutional Animal Care and Use Committee and were
conducted in accordance to the NIH Guide for the Care and Use of Laboratory Animals.
Slice Preparation
Rats were rapidly decapitated and the head placed in ice cold artificial cerebrospinal fluid
(ACSF) in which sucrose (248 mM) was substituted for NaCl. The brain was rapidly removed
and trimmed to isolate the brainstem region. Slices 200μm thick were cut throughout the rostro-
caudal extent of the DRN using a Leica microslicer (Leica, Allendale, NJ) or Vibratome 3000
Plus (Vibratome, St. Louis, MO) and placed in a holding vial containing ACSF with l-
tryptophan (50 μM) at 35°C bubbled with 95% O2/5% CO2 for one hour. Slices were then
maintained in room temperature ACSF bubbled with 95%O2/5%CO2. The composition of the
ACSF was (mM), NaCl 124, KCl 2.5, NaH2PO42, CaCl2 2.5, MgSO4 2, Dextrose 10 and
NaHCO3 26.
Electrophysiological Recording
Slices were transferred to a recording chamber (Warner Instruments, Hamden, CT) and
continuously perfused with ACSF at 1.5-2.0 ml/min at 32-34°C maintained by an in-line
solution heater (TC-324, Warner Instruments). Only one cell was recorded per brain slice.
Raphe neurons were visualized using a Nikon E600 upright microscope fitted with a 40X water-
immersion objective, differential interference contrast (DIC) and infrared filter (Optical
Apparatus, Ardmore, PA). The image from the microscope was enhanced using a CCD camera
and displayed on a computer monitor. Whole-cell recording pipettes were fashioned on a P-97
micropipette puller (Sutter Instruments, Novato, CA) using borosilicate glass capillary tubing
Kirby et al. Page 2
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
(1.2 mm OD, 0.69 mm ID; Warner Instruments). The resistance of the electrodes was 4-8
MΩ when filled with an intracellular solution of (in mM) Kgluconate 70, KCl 70, NaCl 2,
EGTA 4, HEPES 10, MgATP 4, Na2GTP 0.3, 0.1% Biocytin, pH 7.3.
Spontaneous inhibitory postsynaptic current (IPSC) recordings were made in cells located in
the dorsomedial and ventromedial subdivisions of the DRN at the mid-caudal levels
(corresponding to -7.6 to -8.3 mm caudal to bregma in Paxinos and Watson (1998)) that contain
dense clusters of 5-HT neurons. These DRN subdivisions were also chosen because earlier in
vivo studies of CRF effects on 5-HT DRN neuronal activity were conducted in dorsomedial/
ventromedial DRN 5-HT neurons (Kirby et al., 2000). A visualized cell was approached with
the electrode, a gigaohm seal established and the cell membrane ruptured to obtain a whole-
cell recording using either a Multiclamp 700B (Axon Instruments, Foster City, CA), Axopatch
200B (Axon Instruments) or HEKA patch clamp EPC-10 amplifier (HEKA Elecktronik, Pfalz,
Germany). Series resistance was monitored throughout the experiment. If the series resistance
was unstable or exceeded four times the electrode resistance, the cell was discarded. Once the
whole cell recording was obtained, IPSCs were recorded in voltage clamp mode (Vm =
−70mV). For the Axoclamp and Axopatch amplifiers, signals were digitized by Digidata 1320-
series analog-to-digital converters and stored on-line using pClamp 7/8/9 software (Axon
Instruments). For the HEKA amplifier, signals were stored on-line using Pulse software.
Signals were filtered at 1 kHz and digitized at 10 kHz. The liquid junction potential was
approximately 9 mV between the pipette solution and the ACSF and was not subtracted from
the data obtained.
For evoked IPSC (eIPSC) experiments, tungsten stimulating electrodes (World Precision
Instruments, Sarasota, FL) were placed dorsolateral (100-200 μm distance) to the recorded cell
and stimuli delivered with an IsoFlex stimulus isolator (A.M.P.I., Jerusalem, Israel). For these
experiments, recorded cells were located in either the dorsomedial or ventromedial DRN. For
each cell, a stimulus response curve was generated and a stimulus intensity producing a half-
maximal response was used for subsequent paired pulse experiments. For paired pulse
experiments, the inter-pulse interval was 50 ms and the inter-pair interval was 10 s. The mean
stimulus intensity was 3.5 ± 0.6 mA with a range of 0.7 to 9.6 mA.
Experimental Protocols
GABAergic IPSCs were isolated by addition of the non-NMDA receptor antagonist 6-cyano-7-
nitroquinoxaline-2,3-dione (CNQX; 20 μM) or 6,7-dinitroquinoxaline-2,3(1H,4H)-dione
(DNQX; 20 μM). Spontaneous IPSCs (sIPSCs) were recorded for 6 min. Tetrodotoxin (TTX;
1 μM) was added to block action-potential dependent events and miniature spontaneous IPSCs
(mIPSCs) were recorded for 6-9 min. Ovine CRF or UCN II was then added to the perfusion
bath and recorded for 9 min. All statistically significant physiological effects of oCRF were
then tested for CRF receptor subtype contributions with the use of selective CRF antagonists.
For antagonist experiments, it was not possible to make within-cell comparisons of the effect
of oCRF before and after the antagonist since oCRF effects were so long lasting (see discussion
in Results section). Therefore, following baseline mIPSC recording, antalarmin or ASVG-30
was added and data collected for 6 min. Next, oCRF was added and data collected for 9 min.
For stimulation experiments, baseline paired-pulse data were collected for 10-20 min. Ovine
CRF was then added to the perfusion bath and paired pulse data collected for an additional
10-20 min. In some cells (both spontaneous and evoked IPSC experiments), the GABAA
receptor-mediation of IPSCs was verified at the end of the experiment with the addition of the
GABAA receptor antagonist bicuculline (20 μM). Under these conditions, bicuculline
eliminated all IPSC activity (for example, see Fig 2A).
Kirby et al. Page 3
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Immunohistochemistry
Standard immunofluorescence procedures were used to visualize the filled cell and
neurotransmitter content (Beck et al., 2004). Slices were postfixed in 4% paraformaldehyde
overnight. Sections were incubated in PBS containing 0.5% Triton and bovine serum albumin
(30 min). Sections were then incubated in rabbit α 5-HT antibody (1:2000; ImmunoStar,
Hudson, WI) for 1 week at 4°C or mouse α tryptophan hydroxylase antibody (1:500; Sigma-
Aldrich) overnight at 4°C. Subsequently, immunohistochemical labeling was visualized using
an Alexa 488-conjugated donkey α rabbit secondary antiserum (1:200; Molecular Probes,
Eugene, OR) or FITC-conjugated donkey α mouse secondary antiserum (1:200; Jackson
ImmunoResearch, West Grove PA) for 60 min at room temperature. The biocytin was
visualized using streptavidinconjugated Alexa 633 or 647 (1:100 or 1:200; Molecular Probes)
for 60 min at room temperature. Between incubations slices were rinsed with PB solutions (3
× 10 min). Sections were mounted with ProLong Antifade Kit (Molecular Probes) on
Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) and visualized and captured by digital
camera using a Leica DMIRE2 with Leica Confocal software (Leica Microsystems, Exton,
PA) or Olympus FV300 confocal microscope with FluoView software (Olympus America,
Mellville, NY). When using the confocal microscope sequential collection, i.e., Alexa 633/647
separately from FITC/Alexa 488, images of 0.6 μm thickness were acquired at the level of the
cell body of the biocytin-labeled neuron. The laser power and emission filters were adjusted
for both the red and green fluorophor so that there was minimal possibility of a false positive
result. No cell was included in the study if it could not be conclusively identified as either 5-
HT- or non 5-HT-containing by immunohistochemistry.
Data Analysis
MiniAnalysis software (Synaptosoft, Inc., Decatur, GA) was used to analyze mIPSC events
on the basis of amplitude, rate of rise, duration and area. Initially, noise analysis was conducted
for each cell and amplitude detection thresholds set to exceed noise values. Events were
automatically selected, analyzed for double peaks, then visually inspected and confirmed.
Event amplitude histograms were generated and compared to the noise histogram to ensure
that they did not overlap. Synaptic activity was analyzed for frequency, amplitude, baseline
holding current, rise time (calculated from 10-90% of peak amplitude) and decay time
(calculated by averaging 200 randomly selected events and fitting a double exponential
function from 10-90% of the decay phase). The double exponential function for the decay phase
generates an initial fast component and a subsequent slow component of the decay phase. Data
were collected in 1-min bins during the 9 min period following drug application, taking into
account a 2-min lag time from drug addition to initial drug effect due to recording chamber
volume and perfusion rate. The maximum post-drug steady-state value (1-min bin) was
reported as the drug effect for each cell, typically occurring 6-9 min following drug application.
Baseline synaptic activity data was compared between experimental groups by one-way
ANOVA (or Kruskal-Wallis one-way ANOVA on ranks for non-normally distributed data).
The effects of CRF agonists on synaptic event characteristics or holding current were analyzed
by paired Student’s t-tests (or Wilcoxan signed rank test for non-normally distributed data).
The effects of CRF antagonists on synaptic event characteristics or holding current were
presented either as a % change from baseline (Figures 2 and 5) or as raw data (Table 1). For
antagonist experiments in which two drugs were applied sequentially (antagonist + oCRF),
baseline for the antagonist was measured just prior to its application (the ‘no drug’ condition).
Baseline for the oCRF that followed was measured just prior to its application, when the
antagonist effect had reached steady state. Raw data for CRF antagonist studies (Table 1) were
analyzed by repeated-measures ANOVA (or Friedman repeated-measures ANOVA on ranks
for non-normally distributed data) with post-hoc Student-Newman-Keuls tests for pairwise
comparisons. In Figure 5, antagonist data presented as a % change from their baseline values
were analyzed by 2-way repeated measures ANOVA with post hoc Student-Newman-Keuls
Kirby et al. Page 4
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
tests for pairwise comparisons. In Figure 5, oCRF with antagonist pretreatment was compared
to oCRF with ‘ACSF pretreatment’. These ACSF control data represent a comparison of the
first to the sixth minute of baseline data during which cells were exposed only to ACSF. In
Figure 2, antagonist data presented as a % change from their baseline values were compared
to those baseline values by paired Student’s t-tests (or Wilcoxan signed rank test for non-
normally distributed data). In Figure 2, antagonists in combination with oCRF were also
compared to oCRF alone by unpaired Student’s t-tests (or Mann-Whitney rank sum test for
non-normally distributed data). To determine if pre- and postsynaptic effects of CRF agonists
occurred in the same cells, Pearson-Product Moment Correlation analysis was performed. The
frequency and amplitude of IPSC events in individual cells were also illustrated as cumulative
probability graphs for inter-event interval and amplitude and control vs. drug treatments
compared by the Kolmogorov-Smirnov test. A probability of p < 0.05 was considered
significant. Most data are reported as mean ± SEM. IPSC rise time, whose frequency
distributions were not normally distributed, was presented as median ± SEM.
Evoked IPSC amplitude was calculated by subtracting the peak current from the current
obtained during a 5 ms window immediately preceding the stimulus artifact. Baseline eIPSC
amplitude was averaged from at least 60 consecutive trials calculated over at least 10 min
before drug application. Stimulus pairs had a 50 ms inter-stimulus interval with a 10 s interval
between pairs. Paired pulse ratio was calculated as the amplitude of the second eIPSC divided
by the amplitude of the first eIPSC. Ovine CRF effects were determined from the average of
at least 60 consecutive trials following drug application. The effect of oCRF on eIPSC
amplitude or PPR was analyzed by comparing pre- and post-oCRF values by paired Student’s
t-test.
Drugs
Most chemicals for making the ACSF and electrolyte solution, as well as antalarmin and
ASVG-30, were obtained from Sigma-Aldrich (St. Louis, MO). DNQX was purchased from
Tocris (St. Louis, MO) and TTX was purchased from Calbiochem (San Diego, CA).
Antalarmin and DNQX were dissolved in DMSO (final DMSO concentration in bath 0.015%).
Ovine CRF and UCN II were generously supplied by Dr. Jean Rivier of the Clayton Foundation
Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA. Ovine CRF, UCN II, and
ASVG-30 were stored as 10 μg samples (−80°C) and dissolved in ACSF on the day of the
experiment to make a 1.0 mg/ml solution. The final drug concentrations were: oCRF = 10 nM,
UCN II = 10 nM, antalarmin = 300 nM and ASVG-30 = 100 nM.
RESULTS
Basal IPSC characteristics
Basal IPSC characteristics were examined in 5-HT and non 5-HT DRN neurons. Total
spontaneous IPSC (sIPSC) frequency (5-HT cells, 5.7 ± 1.3 Hz, N = 6; non 5-HT cells, 5.0 ±
1.1 Hz, N = 6) was not statistically different from miniature spontaneous IPSC (mIPSC)
frequency (5-HT cells, 5.9 ± 1.0 Hz, N = 6; non 5-HT cells, 5.5 ± 1.4 Hz, N = 6). This finding
indicates that the majority of spontaneous IPSCs in the DRN are non action potential-
dependent, representing spontaneous fusion of GABA vesicles with the presynaptic membrane.
As a consequence, sIPSC data are not shown in any of the figures. Basal mIPSC frequency,
amplitude, rise time, decay time and membrane holding current were also compared between
5-HT and non 5-HT neurons. For this comparison, baseline data from all experimental groups
(oCRF, UCN II, antalarmin, ASVG-30) were pooled since ANOVA demonstrated that there
were no significant differences in baseline values for mIPSC frequency, mIPSC amplitude,
mIPSC rise time, mIPSC fast or decay times or membrane holding current between these
experimental groups (5-HT cells: N = 79; non 5-HT cells: N = 41). The amplitude of mIPSCs
Kirby et al. Page 5
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
was significantly smaller in 5-HT than non 5-HT neurons (5-HT mIPSC amplitude: 17.5 ± 0.7
pA; non 5-HT mIPSC amplitude = 22.3 ± 1.8 pA, p < 0.01). Median rise time was greater in
5-HT than non 5-HT cells (5-HT: 1.21 ± 0.04; non 5-HT: 1.01 ± 0.05; p < 0.01). No other
mIPSC characteristics differed between the groups, indicating that resting membrane potential
and presynaptic GABA release are similar whereas postsynaptic GABA receptors and receptor
kinetics differ between the 5-HT and non-5-HT containing neurons in the basal state.
Ovine CRF, but not UCN II, has presynaptic actions on spontaneous GABA release:
increasing mIPSC frequency selectively in 5-HT DRN neurons
Ovine CRF effects on mIPSC frequency were examined in 5-HT and non 5-HT DRN neurons
(Figure 1). The majority of these recordings were in ventromedial DRN neurons. Data (basal
synaptic activity and oCRF responses) from the dorsomedial DRN neurons did not differ from
ventromedial DRN neurons by unpaired Student’s t-test, therefore, data from both subdivisions
were pooled. These effects were examined in 26 5-HT neurons recorded from 23 subjects and
in 13 non 5-HT neurons recorded from 13 subjects. Panels A and A’ show mIPSC activity
recordings and the effect of oCRF (10 nM) in two neurons that were subsequently identified
to be 5-HT-containing (tryptophan hydroxylase-IR positive, panel C) and non 5-HT-containing
(tryptophan hydroxylase-IR negative, panel C’) by immunohistochemistry. IPSC frequency
was increased from 6.7 to 8.45Hz (26%) in response to oCRF in the 5-HT but not the non 5-
HT neuron. Cumulative probability graphs of inter-event interval for each cell (B and B’)
illustrate a significant shift to the left in the 5-HT but not the non 5-HT neuron as IPSC
frequency was stimulated by oCRF (5-HT: Z = 2.45, p < 0.01; non 5-HT: Z = 0.89, n.s.). IPSCs
are mediated by GABAA receptors as mIPSC frequency was completely suppressed by the
GABAA receptor antagonist bicuculline (20 μM) under these conditions (data not shown).
UCN II did not alter mIPSC frequency in either 5-HT or non 5-HT containing neurons (N =
16 and 14 neurons recorded from 15 and 11 subjects respectively).
Data for all the cells are summarized in Table 1. There was a significant increase in mean
mIPSC frequency produced by oCRF selectively in 5-HT neurons (p < 0.01). These effects of
oCRF were produced by a 10 nM concentration. Most earlier studies with CRF in brain slices
have found effects with this and higher concentrations of CRF (0.01-1 μM; (Liu et al.,
2004;Nie et al., 2004;Tan et al., 2004;Kash and Winder, 2006)). We tested a higher
concentration of oCRF (100 nM) in 12 5-HT neurons recorded from 8 subjects and found that
it produced a small (4.0 to 5.0 Hz) but non-significant increase in mIPSC frequency. This higher
concentration also did not significantly affect any other aspects of GABA synaptic activity
examined (data not shown) and may reflect the U-shaped dose-effect function of oCRF on 5-
HT neurotransmission that has previously been observed in vivo. Whereas low concentrations
of oCRF significantly inhibit DRN neuronal activity and 5-HT release in forebrain, higher
concentrations produce only marginal or slightly excitatory effects (Price et al., 1998;Kirby et
al., 2000).
Ovine CRF has presynaptic actions on evoked GABA release: increasing eIPSC amplitude
and decreasing paired-pulse ratio selectively in 5-HT DRN neurons
The results from mIPSC data indicating an oCRF-induced increase in presynaptic GABA
release were confirmed with eIPSC experiments. Ovine CRF effects on eIPSC amplitude and
paired-pulse ratio were examined in 5-HT and non 5-HT DRN neurons (Figure 2). Evoked
IPSCs were recorded in 5-HT neurons in the dorsomedial (N = 14) as well as the ventromedial
subdivision of the DRN (N = 14). Basal eIPSC amplitude, basal paired pulse ratio (PPR), eIPSC
response to oCRF and PPR response to oCRF did not differ between cells in these two DRN
subdivisions, as determined by unpaired Student’s t-test. For this reason, data from all 5-HT
DRN cells were pooled (N = 28 neurons recorded from 17 subjects) in Figures 2A’ and 2B’.
Panel A shows eIPSCs averaged from 60 events before (control) and after oCRF administration
Kirby et al. Page 6
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
(29% increase) in a 5-HT DRN neuron. Panel A’ shows that oCRF significantly elevated mean
eIPSC amplitude in 5-HT DRN neurons for both the first and second eIPSCs in a pair (p <
0.01). In contrast, oCRF had no significant effect on mean eIPSC amplitude in non 5-HT DRN
neurons (N = 7 neurons recorded from 7 subjects; eIPSC1 control values: 157 ± 29 pA, eIPSC1
oCRF values: 179 ± 21 pA; eIPSC2 control values: 121 ± 27 pA, eIPSC2 oCRF values: 136 ±
23 pA). Bicuculline completely suppressed eIPSC amplitude, indicating that eIPSCs were
mediated by the GABAA receptor (panel A). Panel B shows paired pulse data averaged from
60 pairs before (control) and after oCRF administration in a 5-HT DRN neuron. Ovine CRF
decreased the PPR by 15% in this neuron. Panel B’ shows that oCRF significantly reduced
mean PPR in 5-HT DRN neurons (p < 0.01). In contrast, oCRF had no significant effect on
mean PPR in non 5-HT neurons (N = 7; control values: 0.75 ± 0.06, oCRF values: 0.74 ± 0.06).
Antagonism experiments (panel A”) demonstrated that while both oCRF and the CRF-R1
antagonist antalarmin individually produced significant elevations of eIPSC amplitude above
baseline (p < 0.01), oCRF’s effect was significantly diminished in the presence of antalarmin
(p < 0.01). Panel B” also shows that oCRF’s inhibition of eIPSC PPR in 5-HT DRN neurons
(p < 0.01) was blocked by antalarmin (p < 0.01). These data indicate that both of oCRF’s effects
on eIPSC amplitude and PPR were mediated by the CRF-R1 receptor subtype. We also tested
the washout of oCRF effects. In the majority of cells tested, oCRF effects failed to completely
reverse, persisting despite waiting as long as 60 min. We further tried to reverse oCRF’s effect
with a subsequent application of an antagonist, with only limited success. Similar difficulties
have been observed with bath application of CRF ligands onto brain slices in other studies
(Rainnie et al., 1992;Ungless et al., 2003;Jedema and Grace, 2004;Kash and Winder,
2006;Ugolini et al., 2008). The persistence of this CRF receptor-mediated effect has been
suggested to reflect slow kinetics of dissociation of CRF from its receptors in the brain slice
preparation (Ugolini et al., 2008).
Ovine CRF and UCN II have postsynaptic actions on GABA receptors: increasing mIPSC
amplitude selectively in 5-HT DRN neurons
Ovine CRF and UCN II (10 nM) effects on mIPSC amplitude were examined in 5-HT and non
5-HT DRN neurons (Figure 3). Panels A and B are mIPSC traces averaged from 200 events
before (control) and at the maximal steady-state effect of oCRF in a 5-HT containing and a
non 5-HT containing DRN neuron. In agreement with the eIPSC results, oCRF elevated mIPSC
amplitude by 17% in the 5-HT but not the non 5-HT neuron. This effect is further illustrated
as a significant shift to the right of the cumulative probability graph of amplitude for the 5-HT
(panel A’; Z = 1.70, p < 0.01) but not the non 5-HT neuron (panel B’; Z = 0.89, n.s.). UCN II
effects were recorded in 16 5-HT neurons recorded from 15 subjects and in 14 non 5-HT
neurons recorded from 11 subjects. Panels C and D are mIPSC traces averaged from 200 events
before (control) and at the maximal steady-state effect of UCN II in a 5-HT containing (C) and
a non 5-HT containing DRN neuron (D). UCN II elevated mIPSC amplitude by 21% in the 5-
HT but not the non 5-HT neuron. This effect is further illustrated as a significant shift to the
right of the cumulative probability plot of amplitude for the 5-HT (panel C’; Z = 2.46, p < 0.01)
but not the non 5-HT neuron (panel D’; Z = 1.26, n.s.). The data for all of the cells recorded
with oCRF and UCN II are summarized in Table 1. The increase in amplitude produced by
oCRF and UCN II in 5-HT containing neurons was significant (p < 0.01) as shown in Table 1.
In contrast there was no significant change in mIPSC amplitude in non 5-HT containing
neurons.
A correlation analysis was performed in order to determine if the pre- and postsynaptic actions
of CRF agonists were observed in the same cells. There was a significant positive correlation
between oCRF effects on mIPSC frequency and amplitude in individual 5-HT DRN neurons
(R = 0.59, p < 0.01). Though UCN II did not have a significant effect on mIPSC frequency,
Kirby et al. Page 7
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
there was a significant positive correlation between UCN II effects on mIPSC frequency and
amplitude (R = 0.80, p < 0.01).
Ovine CRF and UCN II have direct postsynaptic effects on 5-HT neurons
Ovine CRF and UCN II effects on inward current were examined in 5-HT and non 5-HT DRN
neurons (Figure 4). Panel A shows oCRF (100 nM) increasing the inward current by 15 pA in
a 5-HT DRN neuron from an initial holding current of -9 pA to a maximal steady-state effect
of oCRF at -24 pA. Ovine CRF (10 nM) significantly increased inward current by 7-10 pA in
both 5-HT and non 5-HT neurons (p < 0.01), as shown in Table 1 and Fig. 4B. Figure 4B further
shows that the effect in 5-HT neurons is dose-dependent with a higher 100 nM dose producing
an increase of inward current (p < 0.05) of greater magnitude. Whereas oCRF produced an
inward current in all DRN cells, UCN II was more selective, significantly increasing inward
current in 5-HT (p < 0.01) but not non 5-HT neurons (Table 1). This is further illustrated in
Fig. 4C as UCN II produced a 10 pA mean inward current in 5-HT neurons (p < 0.01) as
compared to a 3 pA inward current in non 5-HT neurons (n.s.).
Ovine CRF and UCN II alter GABA receptor kinetics: increasing mIPSC fast decay time in 5-
HT DRN neurons
Ovine CRF and UCN II effects on mIPSC fast decay time were examined in 5-HT and non 5-
HT DRN neurons (Table 1). Both ligands produced a similar significant (p < 0.05) increase in
fast decay in 5-HT neurons (see examples in Figs 3A and 3C) but not in non 5-HT neurons
(see examples in Figs 3B and 3D). Rise time as well as the slow component of the mIPSC
decay time were unaffected by any drug treatments in 5-HT and non 5-HT DRN neurons (Table
1).
CRF-R1 and CRF-R2 antagonists selectively block the effects of oCRF
Ovine CRF effects on GABA synaptic activity [mIPSC frequency (panel A), amplitude (panel
B) and inward current (panel C)] were examined in the presence of the CRF-R1-selective
antagonist antalarmin (300 nM) or the CRF-R2-selective antagonist ASVG-30 (100 nM) in 5-
HT DRN neurons (Figure 5). Antagonist effects were also examined on mIPSC rise and decay
times (see Table 1). These concentrations of antagonist were approximately 100 times their
affinity for their respective receptors (Webster et al., 1996;Ruhmann et al., 1998;Higelin et al.,
2001). Antalarmin effects were examined in 20 5-HT neurons recorded from 13 subjects;
ASVG-30 effects were examined in 17 5-HT neurons recorded from 15 subjects.
In the absence of an antagonist, oCRF significantly elevated mIPSC frequency (panel 5A and
table 1; p < 0.01), amplitude (panel 5B and table 1, p < 0.01), fast decay time (Table 1, p <
0.05) and inward current (panel 5C and table 1, p < 0.01). Table 1 shows that antalarmin blocked
the effect of oCRF on mIPSC frequency, amplitude and fast decay time, but not on inward
current (X2(2)=18.9, p < 0.01). ASVG-30 blocked the oCRF effect on mIPSC amplitude, fast
decay time and inward current, but not on mIPSC frequency (X2(2)=8.94, p < 0.05). The data
are presented in a different manner as percentage change from baseline in Figure 5. Antalarmin
blocked oCRF’s effect on mIPSC frequency [panel A; significant main effect of antalarmin:
F(1,44)=5.47, p < 0.05 and significant interaction of antalarmin x oCRF: F(1,44)=4.40, p <
0.05; post hoc comparisons show oCRF significantly elevated mIPSC frequency in the absence
(p < 0.05) but not the presence of antalarmin and oCRF’s effect was significantly reduced in
the presence of antalarmin as compared to it’s effect in the presence of ACSF (p < 0.05)].
However, antalarmin did not block oCRF’s effect on inward current [panel C; significant main
effects of antalarmin: F(1,44)=10.08, p < 0.01 and oCRF: F(1,44)=8.24, p < 0.01 but no
interaction between these factors]. In contrast to antalarmin, ASVG-30 blocked oCRF’s effect
on inward current [panel C; significant interaction of ASVG-30 x oCRF: F(1,41)=5.73, p <
0.05; post hoc comparisons show that oCRF significantly elevated inward current in the
Kirby et al. Page 8
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
absence (p < 0.05) but not the presence of ASVG-30 and oCRF’s effect was significantly
reduced in the presence of ASVG-30 as compared to it’s effect in the presence of ACSF (p <
0.05)]. However, ASVG-30 did not block oCRF’s effect on mIPSC frequency [panel A; no
significant interaction between ASVG-30 and oCRF]. While in the presence of ACSF, oCRF
significantly elevated mIPSC amplitude (p < 0.05), in the presence of either antagonist, oCRF
was unable to significantly elevate mIPSC amplitude above its baseline, indicating that both
antagonists block this effect as well [panel B; significant interaction of antalarmin x oCRF: F
(1,44)=5.90, p < 0.05 and significant interaction of ASVG-30 x oCRF: F(1,41) = 7.72, p <
0.01]. Based on these data, we conclude that the actions of oCRF on mIPSC frequency are
mediated by CRF-R1, the actions on mIPSC amplitude and decay time by both CRF-R1 and
CRF-R2 and the actions on inward current by CRF-R2.
Interestingly, we found that both antagonists had intrinsic effects indicating the possibility that
these antagonists had partial agonist activity (see Discussion, Figure 5 and Table 1). Both
antalarmin and ASVG-30 increased mIPSC amplitude (Figure 5B, p < 0.05 by post hoc
Student-Newman-Keuls test) (Table 1: ANOVA, antalarmin: F(2,38)=8.29, p < 0.01 and
ASVG-30: F(2,32)=9.18, p < 0.01; p < 0.05 by post-hoc Student-Newman-Keuls test).
Antalarmin increased mIPSC fast decay time [Table 1, ANOVA: F(2,35)=6.48, p < 0.01, p <
0.05 by post-hoc Student-Newman-Keuls test]. Antalarmin also had an effect on inward current
(Table 1: (X2(2)=18.9, p < 0.01; p < 0.05 by post-hoc Student-Newman-Keuls test). The one
significant effect produced by oCRF in non 5-HT neurons was an increase in inward current
(Table 1, p < 0.01). In contrast to what was found in 5-HT neurons where the increase in inward
current was blocked by ASVG-30 and also produced by UCN II, this effect in non 5-HT neurons
was blocked by antalarmin (F(2,10)=0.97, n.s.) and not produced by UCN II, indicating that
the effect is mediated by CRF-R1 receptors.
DISCUSSION
CRF, a neuromodulator, regulates stress induced behaviors through participation in neural
circuitries that are still being defined. Our data provide important new information regarding
the effects of CRF in the DRN. We conclude, based on our data, that CRF modulates 5-HT
DRN neurons in a direct as well as indirect manner on GABAergic synaptic activity and that
it does so via different CRF receptor subtypes. Whereas CRF effects on presynaptic GABA
release were mediated by CRF-R1, its direct postsynaptic effects on inward current were
mediated by CRF-R2 and on GABA receptors were mediated by both receptor subtypes. In
contrast, CRF induced inward current by CRF-R1 receptor activation in non 5-HT neurons.
The selectivity of CRF effects further suggest that there are distinct GABAergic afferents
targeting different neurochemical populations of DRN neurons and that these afferents are
differentially responsive to CRF.
Direct effects of CRF on DRN neurons
Ovine CRF induced an increase in inward current in DRN neurons. Interestingly, this effect
was mediated by different CRF receptor subtypes on different neurochemical populations, i.e.,
5-HT neurons by the CRF-R2 receptor and non 5-HT neurons by the CRF-R1 receptor. In
support of our findings, anatomical data show CRF-R2 receptors at mid-levels of the DRN
located almost exclusively on 5-HT neurons (Day et al., 2004), the same area in which we
recorded. Unfortunately the neurochemical identity of the non 5-HT neurons is unknown.
Approximately half of DRN neurons are non 5-HT (Steinbusch et al., 1980;Descarries et al.,
1982;Kohler and Steinbusch, 1982;Jacobs and Azmitia, 1992;Baumgarten and Grozdanovic,
1997). One prominent population of non 5-HT DRN neurons are GABAergic (Allers and
Sharp, 2001;Allers and Sharp, 2003). These GABA neurons express CRF-R1 receptors (Roche
Kirby et al. Page 9
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
et al, 2003). If at least some of the recorded non 5-HT neurons were GABAergic, our data
indicate that postsynaptic CRF-R1 receptors increase inward current in GABA neurons.
Presynaptic effects of oCRF on GABA synaptic activity
Converging evidence from both spontaneous and evoked IPSC experiments indicate that oCRF
elevated presynaptic GABA release at 5-HT DRN neurons. Ovine CRF increased mIPSC
frequency, indicating enhanced probability of GABA release from presynaptic terminals.
There was large variance in mean basal mIPSC frequency of 5-HT neurons in different drug
experiments (Table 1). The lack of drug-induced increases of mIPSC frequency in the
antalarmin and UCN II experiments could theoretically be produced by a “ceiling effect” due
to their higher basal mIPSC frequencies. However, linear regression analysis, both within and
between groups, showed that there was no relationship between basal mIPSC frequency and
subsequent oCRF or UCN II response, making this interpretation unlikely. An additional
indication of enhanced presynaptic neurotransmitter release (Zucker, 1989;Dobrunz and
Stevens, 1997;Melis et al., 2002) was that oCRF also increased eIPSC amplitude while
decreasing eIPSC paired-pulse ratio.
An increase in GABA release by CRF-R1 presynaptic receptor activation would lead to a
decrease in DRN neuronal activity, an effect that is consistent with previous studies. In vivo
studies show that certain stressors inhibit 5-HT release via endogenous CRF (Price et al.,
2002) and activation of CRF-R1 by low doses of oCRF inhibits 5-HT DRN neuronal activity
(Kirby et al., 2000) and 5-HT release (Price et al., 1998;Price and Lucki, 2001). Ultrastructural
studies show greater contacts of CRF terminals with GABA-labeled than with 5-HT-labeled
dendrites and also CRF terminals in contact with GABA terminals (Waselus et al., 2005).
Functional neuroanatomy studies using c-fos demonstrate that swim stress engages a
population of CRF-R1 receptor-expressing GABAergic DRN neurons (Roche et al., 2003).
Our data provide electrophysiological evidence for this circuitry at a single-cell level: CRF-
R1 activation elevates presynaptic GABA release at 5-HT DRN neurons and increases inward
current in non 5-HT putative GABA neurons.
Postsynaptic effects of oCRF on GABA synaptic activity
In addition to increasing the release of GABA onto 5-HT DRN neurons, oCRF increased
mIPSC amplitude and fast decay time, indicating an increase in the sensitivity, number, or open
time of the activated GABAA receptor-ionophore. These effects were mediated by both CRF-
R1 and CRF-R2 subtypes. A correlation analysis demonstrated that the pre- and postsynaptic
actions of CRF agonists were observed in the same cells. Potentially, if mIPSC amplitude is
increased, it is more likely to be detected above noise levels, thus artificially elevating mIPSC
frequency. However, noise histograms and amplitude histograms were clearly separated for
each recorded cell and pre- and postsynaptic effects were mediated by different receptor
subtypes, making this technical argument unlikely. For example, ASVG-30 blocked oCRF-
induced elevation of mIPSC amplitude but spared the oCRF effect on mIPSC frequency in the
same cell population. Another potential interpretation is that CRF agonist-induced elevations
of mIPSC amplitude reflect an increase in neurotransmitter release, driven by the changes in
mIPSC frequency (Behrends and ten Bruggencate G., 1998). This would not explain the CRF-
R2 mediated increase in amplitude, but we cannot rule out this possibility for the CRF-R1
receptor mediated actions. Nonetheless, the net effect of either increased postsynaptic receptor
sensitivity or increased neurotransmitter release would enhance GABAergic inhibition of the
postsynaptic neuron. Therefore, our interpretation of mIPSC frequency and amplitude data is
that oCRF enhanced GABAergic inhibition of the target 5-HT DRN neuron by multiple
mechanisms. These data agree with studies conducted in other brain regions demonstrating
that CRF enhances GABAergic synaptic activity at the pre- or postsynaptic level and is
mediated by different CRF receptors (Nie et al., 2004;Kash and Winder, 2006). In sum, an
Kirby et al. Page 10
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
emerging theme is that CRF enhances GABA synaptic transmission by different CRF receptor
subtypes acting at pre- or postsynaptic sites, often in the same cell.
Intrinsic antagonist effects
Both CRF receptor antagonists had intrinsic effects on several physiological measures in this
preparation. Antalarmin increased mIPSC amplitude, mIPSC fast decay time and inward
current whereas ASVG-30 increased mIPSC amplitude. These intrinsic effects indicate that
the antagonists may have partial agonist activity. Minimal partial agonist activity has been
reported for these antagonists in other preparations (Ruhmann et al., 1998;Lawrence et al.,
2002).
Neurochemical selectivity of oCRF effects
All of oCRF’s effects on GABAergic synaptic activity occurred selectively in recordings from
5-HT, and not non 5-HT, DRN neurons. These data indicate that select GABA afferents target
distinct neurochemical populations of DRN neurons and these select afferents are differentially
responsive to oCRF. This finding compliments recent data from our laboratory demonstrating
distinct glutamatergic afferents to neurochemically distinct populations of DRN neurons that
are also differentially responsive to stress (Kirby et al., 2007). Therefore, the anatomical design
of the DRN enables stressors and stress hormones to selectively modulate distinct populations
of DRN neurons via both excitatory and inhibitory afferents. This arrangement may contribute
to the complex stressor-specific and region-specific responses of the 5-HT system to stress
(Kirby et al., 1995;Adell et al., 1997;Kirby et al., 1997).
Functional implications
These data provide functional evidence for the cellular mechanisms and circuitry underlying
the interactions between the stress neurohormone CRF and the 5-HT system. Previous studies
characterizing oCRF effects on 5-HT neurotransmission in vivo indicated a U-shaped dose-
response curve for oCRF: inhibiting 5-HT neuronal activity and release at low doses,
stimulating 5-HT neuronal activity and release at higher doses (Price et al., 1998;Kirby et al.,
2000;Price and Lucki, 2001). As oCRF is eight times more potent at CRF-R1 than CRF-R2
receptors (Lovenberg et al., 1995), low doses act primarily at CRF-R1 receptors while higher
doses activate CRF-R2 receptors. Our data supports a model (see Figure 6) whereby activation
of CRF-R1 receptors by low doses of oCRF stimulates presynaptic GABA release and enhances
postsynaptic GABA receptor sensitivity at 5-HT neurons, indirectly inhibiting their activity
and 5-HT release. Higher doses of oCRF activate CRF-R2 receptors on 5-HT neurons, resulting
in their depolarization, increased activity and release of 5-HT. This model is supported by our
finding that CRF-R1 receptor-mediated increases in presynaptic GABA release are observed
maximally at the lower dose of oCRF tested whereas the CRF-R2-receptor mediated inward
current is dose-dependent with the greatest effect produced by the higher dose of oCRF tested.
Additional support for this model comes from studies of the CRF-R2 selective agonist urocortin
II which, at higher doses, activates 5-HT DRN neuronal activity (Pernar et al., 2004) and 5-
HT release in terminal fields (Amat et al., 2004). Earlier studies identified inhibitory actions
of particular stressors on the 5-HT system (Kirby et al., 1995;Adell et al., 1997;Kirby et al.,
1997) that were mediated by CRF (Price et al., 2002). In this way, particular stressors initiate
the release of CRF in the DRN that stimulates both presynaptic GABA release and postsynaptic
GABA receptor sensitivity on 5-HT DRN neurons, resulting in inhibition of 5-HT neuronal
activity and forebrain 5-HT release. This raphe circuitry provides a framework for
understanding how exposure to stress can lead to 5-HT dysfunction, potentially contributing
to stress-related psychopathology such as anxiety and depression. The findings from our studies
add important information to the understanding of the complex neural circuitries underlying
stress regulation and stress mediated mood disorders. Novel compounds directed at the CRF
Kirby et al. Page 11
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
or GABA receptors identified to be key within this neural circuitry may be useful in the
treatment of mood and anxiety disorders.
ACKNOWLEDGEMENTS
We thank Dr. Jean Rivier of the Clayton Foundation Laboratories for Peptide Biology at The Salk Institute for his
generous donations of oCRF and UCN II for use in these studies. We thank Dr. Yu-Zhen Pan for her technical assistance
and contributions to electrophysiological studies. We also thank Dr. Rita Valentino, Division of Stress Neurobiology
at Children’s Hospital of Philadelphia for her valuable input and advice for this project and all of our earlier studies
with CRF. This work was supported by a Young Investigator Award from the National Alliance for Research on
Schizophrenia and Depression (NARSAD), the National Institute of Mental Health (MH 63301) and National Institute
on Drug Abuse (DA 20126) grants issued to Dr. Kirby and by National Institute of Mental Health (MH 60773) and
the Office of Naval Research (N00014-03-1-0311) grants issued to Dr. Beck.
REFERENCES
Adell A, Casanovas JM, Artigas F. Comparative study in the rat of the actions of different types of stress
on the release of 5-HT in raphe nuclei and forebrain areas. Neuropharmacol 1997;36:735–741.
Allers KA, Sharp T. Identification of 5-HT containing neurones in the dorsal raphe nucleus (DRN) using
juxtacellular labeling methods in vivo. Soc Neurosci Abs 2001;27:806.13.
Allers KA, Sharp T. Neurochemical and anatomical identification of fast- and slow-firing neurones in
the rat dorsal raphe nucleus using juxtacellular labelling methods in vivo. Neurosci 2003;122:193–
204.
Amat J, Tamblyn JP, Paul ED, Bland ST, Amat P, Foster AC, Watkins LR, Maier SF. Microinjection of
urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular
serotonin in the basolateral amygdala. Neurosci 2004;129:509–519.
Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB. The role of corticotropin-releasing factor in
depression and anxiety disorders. J Endocrinol 1999;160:1–12. [PubMed: 9854171]
Baumgarten, GG.; Grozdanovic, Z. Anatomy of central serotonergic projection systems. In: Baumgarten,
HG.; Gothert, M., editors. Serotonergic Neurons and 5-HT Receptors in the Central Nervous System.
Springer; Berlin: 1997. p. 41-89.
Beck SG, Pan YZ, Akanwa AC, Kirby LG. Median and dorsal raphe neurons are not
electrophysiologically identical. J Neurophysiol 2004;91:994–1005. [PubMed: 14573555]
Behrends JC, ten Bruggencate G. Changes in quantal size distributions upon experimental variations in
the probability of release at striatal inhibitory synapses. J Neurophysiol 1998;79:2999–3011. [PubMed:
9636103]
Chalmers DT, Lovenberg TW, De Souza EB. Localization of novel corticotropin-releasing factor receptor
(CRF2) mRNA expression to specific subcortical nuclei in rat brain: comparison with CRF1 receptor
mRNA expression. J Neurosci 1995;15:6340–6350. [PubMed: 7472399]
Charney DS, Krystal JH, Delgado PL, Heninger GR. Serotonin-specific drugs for anxiety and depressive
disorders. Annu Rev Med 1990a;41:437–446. [PubMed: 2139556]
Charney DS, Woods SW, Krystal JH, Heninger GR. Serotonin function and human anxiety disorders.
Ann N Y Acad Sci 1990b;600:558–572. [PubMed: 2252335]
Day HE, Greenwood BN, Hammack SE, Watkins LR, Fleshner M, Maier SF, Campeau S. Differential
expression of 5HT-1A, alpha 1b adrenergic, CRF-R1, and CRF-R2 receptor mRNA in serotonergic,
gamma-aminobutyric acidergic, and catecholaminergic cells of the rat dorsal raphe nucleus. J Comp
Neurol 2004;474:364–378. [PubMed: 15174080]
De Souza EB, Insel TR, Perrin MH, Rivier J, Vale WW, Kuhar MJ. Corticotropin-releasing factor
receptors are widely distributed within the rat central nervous system: an autoradiographic study. J
Neurosci 1985;5:3189–3203. [PubMed: 3001239]
Descarries L, Watkins KC, Garcia S, Beaudet A. The serotonin neurons in nucleus raphe dorsalis of adult
rat: a light and electron microscope radioautographic study. J Comp Neurol 1982;207:239–254.
[PubMed: 7107985]
Dobrunz LE, Stevens CF. Heterogeneity of release probability, facilitation, and depletion at central
synapses. Neuron 1997;18:995–1008. [PubMed: 9208866]
Kirby et al. Page 12
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Gold PW, Licinio J, Wong ML, Chrousos GP. Corticotropin releasing hormone in the pathophysiology
of melancholic and atypical depression and in the mechanism of action of antidepressant drugs. Ann
N Y Acad Sci 1995;771:716–729. [PubMed: 8597444]
Higelin J, Py-Lang G, Paternoster C, Ellis GJ, Patel A, Dautzenberg FM. 125I-Antisauvagine-30: a novel
and specific high-affinity radioligand for the characterization of corticotropin-releasing factor type
2 receptors. Neuropharmacol 2001;40:114–122.
Jacobs BL, Azmitia EC. Structure and function of the brain serotonin system. Physiol Rev 1992;72:165–
229. [PubMed: 1731370]
Jedema HP, Grace AA. Corticotropin-releasing hormone directly activates noradrenergic neurons of the
locus ceruleus recorded in vitro. J Neurosci 2004;24:9703–9713. [PubMed: 15509759]
Kash TL, Winder DG. Neuropeptide Y and corticotropin-releasing factor bi-directionally modulate
inhibitory synaptic transmission in the bed nucleus of the stria terminalis. Neuropharmacol
2006;51:1013–1022.
Kirby LG, Allen AR, Lucki I. Regional differences in the effects of forced swimming on extracellular
levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. Brain Res 1995;682:189–196.
[PubMed: 7552310]
Kirby LG, Chou-Green JM, Davis K, Lucki I. The effects of different stressors on extracellular 5-
hydroxytryptamine and 5-hydroxyindoleacetic acid. Brain Res 1997;760:218–230. [PubMed:
9237538]
Kirby LG, Cryan JF, Page ME, Bello NT, Kung M-P, Kung HF, Lucki I, Valentino RJ. Substantial
reduction in corticotropin-releasing factor (CRF) binding sites in the dorsal raphe nucleus (DRN) of
rats administered 5,7-dihydroxytryptamine (5,7-DHT). Soc Neurosci Abs 2001;27:414.9.
Kirby LG, Pan YZ, Freeman-Daniels E, Rani S, Nunan JD, Akanwa A, Beck SG. Cellular effects of swim
stress in the dorsal raphe nucleus. Psychoneuroendocrinology 2007;32:712–723. [PubMed:
17602840]
Kirby LG, Rice KC, Valentino RJ. Effects of corticotropin-releasing factor on neuronal activity in the
serotonergic dorsal raphe nucleus. Neuropsychopharmacol 2000;22:148–162.
Kohler C, Steinbusch H. Identification of serotonin and non-serotonin-containing neurons of the mid-
brain raphe projecting to the entorhinal area and the hippocampal formation. A combined
immunohistochemical and fluorescent retrograde tracing study in the rat brain. Neurosci 1982;7:951–
975.
Lawrence AJ, Krstew EV, Dautzenberg FM, Ruhmann A. The highly selective CRF(2) receptor
antagonist K41498 binds to presynaptic CRF(2) receptors in rat brain. Br J Pharmacol 2002;136:896–
904. [PubMed: 12110614]
Liu J, Yu B, Neugebauer V, Grigoriadis DE, Rivier J, Vale WW, Shinnick-Gallagher P, Gallagher JP.
Corticotropin-releasing factor and Urocortin I modulate excitatory glutamatergic synaptic
transmission. J Neurosci 2004;24:4020–4029. [PubMed: 15102917]
Lovenberg TW, Liaw CW, Grigoriadis DE, Clevenger W, Chalmers DT, De Souza EB, Oltersdorf T.
Cloning and characterization of a functionally distinct corticotropin- releasing factor receptor subtype
from rat brain. Proc Natl Acad Sci USA 1995;92:836–840. [PubMed: 7846062]
Melis M, Camarini R, Ungless MA, Bonci A. Long-lasting potentiation of GABAergic synapses in
dopamine neurons after a single in vivo ethanol exposure. J Neurosci 2002;22:2074–2082. [PubMed:
11896147]
Meltzer HY. Role of serotonin in depression. Ann N Y Acad Sci 1990;600:486–499. [PubMed: 2252328]
Nemeroff CB. The role of corticotropin-releasing factor in the pathogenesis of major depression.
Pharmacopsychiatry 1988;21:76–82. [PubMed: 3293091]
Nie Z, Schweitzer P, Roberts AJ, Madamba SG, Moore SD, Siggins GR. Ethanol augments GABAergic
transmission in the central amygdala via CRF1 receptors. Science 2004;303:1512–1514. [PubMed:
15001778]
Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates. Academic Press; New York: 1998.
Pernar L, Curtis AL, Vale WW, Rivier JE, Valentino RJ. Selective activation of corticotropin-releasing
factor-2 receptors on neurochemically identified neurons in the rat dorsal raphe nucleus reveals dual
actions. J Neurosci 2004;24:1305–1311. [PubMed: 14960601]
Kirby et al. Page 13
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Potter E, Sutton S, Donaldson C, Chen R, Perrin M, Lewis K, Sawchenko PE, Vale W. Distribution of
corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. Proc Natl
Acad Sci U S A 1994;91:8777–8781. [PubMed: 8090722]
Price ML, Curtis AL, Kirby LG, Valentino RJ, Lucki I. Effects of corticotropin-releasing factor on brain
serotonergic activity. Neuropsychopharmacol 1998;18:492–502.
Price ML, Kirby LG, Valentino RJ, Lucki I. Evidence for corticotropin-releasing factor regulation of
serotonin in the lateral septum during acute swim stress: adaptation produced by repeated swimming.
Psychopharmacology (Berl) 2002;162:406–414. [PubMed: 12172694]
Price ML, Lucki I. Regulation of serotonin release in the lateral septum and striatum by corticotropin-
releasing factor. J Neurosci 2001;21:2833–2841. [PubMed: 11306635]
Rainnie DG, Fernhout BJ, Shinnick-Gallagher P. Differential actions of corticotropin releasing factor on
basolateral and central amygdaloid neurones, in vitro. J Pharmacol Exp Ther 1992;263:846–858.
[PubMed: 1331417]
Reul JM, Holsboer F. Corticotropin-releasing factor receptors 1 and 2 in anxiety and depression. Curr
Opin Pharmacol 2002;2:23–33. [PubMed: 11786305]
Roche M, Commons KG, Peoples A, Valentino RJ. Circuitry underlying regulation of the serotonergic
system by swim stress. J Neurosci 2003;23:970–977. [PubMed: 12574426]
Ruhmann A, Bonk I, Lin CR, Rosenfeld MG, Spiess J. Structural requirements for peptidic antagonists
of the corticotropin-releasing factor receptor (CRFR): development of CRFR2beta-selective
antisauvagine-30. Proc Natl Acad Sci U S A 1998;95:15264–15269. [PubMed: 9860957]
Steinbusch H, Van der Kooy D, Verhofstad A, Pellegrino A. Serotonergic and non-serotonergic
projections from the nucleus raphe dorsalis to the caudateputamen complex in the rat, studied by a
combined immunofluorescence and fluorescent retrograde axonal labeling technique. Neurosci Lett
1980;19:137–142. [PubMed: 6302595]
Tan H, Zhong P, Yan Z. Corticotropin-releasing factor and acute stress prolongs serotonergic regulation
of GABA transmission in prefrontal cortical pyramidal neurons. J Neurosci 2004;24:5000–5008.
[PubMed: 15163692]
Ugolini A, Sokal DM, Arban R, Large CH. CRF1 receptor activation increases the response of neurons
in the basolateral nucleus of the amygdala to afferent stimulation. Front Behav Neurosci 2008;2:1–
7. [PubMed: 18958191]
Ungless MA, Singh V, Crowder TL, Yaka R, Ron D, Bonci A. Corticotropin-releasing factor requires
CRF binding protein to potentiate NMDA receptors via CRF receptor 2 in dopamine neurons. Neuron
2003;39:401–407. [PubMed: 12895416]
Waselus M, Valentino RJ, Van Bockstaele EJ. Ultrastructural evidence for a role of gamma-aminobutyric
acid in mediating the effects of corticotropin-releasing factor on the rat dorsal raphe serotonin system.
J Comp Neurol 2005;482:155–165. [PubMed: 15611993]
Webster EL, Lewis DB, Torpy DJ, Zachman EK, Rice KC, Chrousos GP. In vivo and in vitro
characterization of antalarmin, a nonpeptide corticotropin-releasing hormone (CRH) receptor
antagonist: suppression of pituitary ACTH release and peripheral inflammation. Endocrinology
1996;137:5747–5750. [PubMed: 8940412]
Zucker RS. Short-term synaptic plasticity. Annu Rev Neurosci 1989;12:13–31. [PubMed: 2648947]
Kirby et al. Page 14
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 1. Ovine CRF enhanced GABAergic mIPSC frequency selectively in 5-HT DRN neurons
Panels A and A’ show raw traces of mIPSCs (recorded in the presence of TTX, 1 μM) and
mIPSCs + oCRF (10 nM) recorded from a 5-HT (A) and non 5-HT (A’) cell. Ovine CRF
elevated mIPSC frequency from 6.7 to 8.45 Hz in the 5-HT cell but had no effect in the non
5-HT cell. The cumulative inter-event interval graph for the 5-HT cell (panel B), but not for
the non 5-HT cell (panel B’), illustrates a significant shift to the left (by the Kolmogorov-
Smirnov test, P < 0.01) as mIPSC frequency was stimulated by oCRF. Panels C and C’ are
fluorescent photomicrographs demonstrating the neurochemical identity of the recorded cells
as 5-HT (C) and non 5-HT (C’). The recorded biocytin-filled cells are shown in red, tryptophan
hydroxylase-IR is shown in green and the merged panels demonstrate either a double labeled
Kirby et al. Page 15
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
(yellow; panel C) or non double-labeled cell (red; panel C’). Miniature IPSCs were mediated
by GABAA receptors as they were completely suppressed by the GABAA receptor antagonist
bicuculline (20 μM) under these conditions (data not shown).
Kirby et al. Page 16
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 2. Ovine CRF selectively increased amplitude of evoked IPSCs and decreased paired pulse
ratio in 5-HT DRN neurons
Panel A illustrates three traces of evoked IPSCs (averaged from 60 eIPSCs under each
condition: control, oCRF and bicuculline) from a 5-HT neuron. Ovine CRF (10 nM) elevated
eIPSC amplitude in this cell from 163 pA (control) to 210 pA. Addition of GABAA antagonist
bicuculline (20 μM) abolished eIPSC amplitude. Panel A’ illustrates the finding that oCRF
significantly elevated mean eIPSC amplitude above control values for both the first and second
eIPSCs in a pair (N = 28; p < 0.01). Panel A” shows that both oCRF and the CRF-R1 antagonist
antalarmin (300 nM) elevated eIPSC amplitude above baseline (p < 0.01) but oCRF’s effect
was significantly diminished in the presence of antalarmin (p < 0.01) (N = 15). Panel B shows
Kirby et al. Page 17
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
paired-pulse traces from a 5-HT neuron (averaged from 60 pairs under each condition: control
and oCRF). Ovine CRF decreased PPR from 0.68 to 0.58. Panel B’ shows that oCRF
significantly decreased the mean PPR in 5-HT DRN neurons below baseline values (N = 28,
p<0.01). Panel B” shows that oCRF’s inhibition of eIPSC PPR in 5-HT DRN neurons (p <
0.01) was blocked by antalarmin (p < 0.01) (N = 15). Neither eIPSC amplitude nor PPR was
affected by oCRF in non 5-HT DRN neurons (data not shown). For antagonist experiments
(A” and B”), in which the antagonist and oCRF were applied sequentially, a separate baseline
was calculated for each drug from measures taken just prior to the drug’s application. The
asterisks indicate a significant difference from baseline values by paired t-test (** p < 0.01)
and the pound signs indicate a significant difference from oCRF by unpaired t-test (## p <
0.01). Data are expressed as mean ± SEM.
Kirby et al. Page 18
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 3. Ovine CRF and UCN II enhanced GABAergic mIPSC amplitude selectively in 5-HT DRN
neurons
Panels A and B show averaged mIPSCs (from 200 events) recorded in a 5-HT (A) and non 5-
HT (B) cell: the grey line represents mIPSCs before and the black line represents mIPSCs after
oCRF administration. Ovine CRF (10 nM) increased mIPSC amplitude from 19.9 pA to 23.3
pA in the 5-HT cell but had no effect in the non 5-HT cell. The cumulative probability plot of
amplitude was shifted to the right by oCRF administration for the 5-HT cell (panel A’;
Kolmogorov-Smirnov test, p < 0.01) but not for the non 5-HT cell (panel B’). Panels C and D
show averaged mIPSCs (from 200 events) recorded in a 5-HT (C) and non 5-HT (D) cell: the
grey line represents mIPSCs before and the black line represents mIPSCs after UCN II
Kirby et al. Page 19
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
administration. UCN II (10 nM) increased mIPSC amplitude from 24 pA to 27 pA in the 5-HT
cell but had no effect in the non 5-HT cell. The cumulative probability graph of amplitude was
shifted to the right by UCN II administration for the 5-HT cell (panel C’; Kolmogorov-Smirnov
test, p < 0.01) but not for the non 5-HT cell (panel D’).
Kirby et al. Page 20
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 4. Ovine CRF and UCN II enhanced inward current in DRN neurons
Panel A shows that 100 nM oCRF increased the inward current by 15 pA in a 5-HT DRN
neuron from an initial holding current of −9 pA to a maximum of −24 pA. Panel B shows that
oCRF dose-dependently increased mean inward current in 5-HT neurons (10 nM: 7 pA, N =
26, 100 nM: 13 pA, N = 12) and 10 nM oCRF also increased mean inward current in non-5-
HT neurons (10 pA; N = 13). In contrast UCN II (10 nM) only increased inward current in 5-
HT containing neurons. Panel C shows that UCN II significantly increased mean inward current
by 10 pA in 5-HT containing neurons (N = 16) as compared to 3 pA in non 5-HT cells (N =
14). In panel A, the time break (double bar) represents 2 min and the scale bars represent 20
Kirby et al. Page 21
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
pA (vertical) and 10 s (horizontal). The asterisks indicate a significant inward current (* p <
0.05, ** p < 0.01). Data are expressed as mean ± SEM.
Kirby et al. Page 22
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 5. Ovine CRF effects on GABA synaptic activity and membrane characteristics in 5-HT
DRN neurons were differentially mediated by CRF-R1 and CRF-R2 receptor subtypes
Panel A illustrates oCRF effects (10 nM) on mean mIPSC frequency (expressed as a percent
of baseline) in the presence of ACSF or the CRFR1-selective antagonist antalarmin (300 nM)
or the CRF-R2-selective antagonist ASVG-30 (100 nM). The significant elevation of mIPSC
frequency by oCRF (panel A) was blocked by antalarmin but not by ASVG-30, indicating that
this effect was mediated by the CRF-R1 receptor subtype. Panel B illustrates oCRF effects on
mean mIPSC amplitude (expressed as a percent of baseline) in the presence of ACSF or the
receptor-selective antagonists. The significant elevation of mIPSC amplitude by oCRF (panel
B) was blocked by both antalarmin and ASVG-30, indicating that this effect was mediated by
Kirby et al. Page 23
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
both the CRF-R1 and CRF-R2 receptor subtypes. Panel C illustrates oCRF effects on mean
inward current in the presence of ACSF or the receptor-selective antagonists. The significant
change in mean inward current by oCRF (panel C) was blocked by ASVG-30 but not by
antalarmin, indicating that this effect was mediated by the CRF-R2 receptor subtype. Intrinsic
effects of antalarmin and ASVG-30 on mIPSC amplitude are also demonstrated. For these
experiments in which the antagonist and oCRF were applied sequentially, a separate baseline
was calculated for each drug from measures taken just prior to the drug’s application. Post hoc
Student-Newman-Keuls tests show significant group differences indicated by asterisk (* vs.
ACSF) or pound sign (# vs. ACSF + oCRF) (p < 0.05). Data are expressed as mean ± SEM.
Kirby et al. Page 24
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 6. Model of CRF actions in the DRN
This schematic models the effects of low and high doses of CRF on 5-HT, non 5-HT and GABA
neurons in the DRN. At 5-HT neurons, low doses of CRF activate CRF-R1 receptors, enhancing
presynaptic GABA release and postsynaptic GABA receptor sensitivity which result in the net
inhibition of 5-HT neurons. High doses of CRF act also at CRF-R2 receptors on 5-HT neurons,
resulting in the net excitation of 5-HT neurons. CRF also increases the excitability of non 5-
HT neurons via CRF-R1 receptors.
Kirby et al. Page 25
J Neurosci. Author manuscript; available in PMC 2009 May 26.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Kirby et al. Page 26
Table 1
Effect of CRF and CRF receptor-selective agonists and antagonists on mIPSC characteristics and inward current in DRN neurons
Data are expressed as mean (frequency, amplitude, decay time and inward current) or median (rise time) ± SEM. Asterisks indicate a
significant difference from mIPSC controls
mIPSC
Frequency
(Hz)
mIPSC
Amplitude
(pA)
mIPSC
Rise Time
(ms)
mIPSC Fast
Decay Time
(ms)
mIPSC Slow
Decay Time
(ms)
Inward Current
(pA)
5-HT Non
5-HT 5-HT Non
5-HT 5-HT Non
5-HT 5-HT Non
5-HT 5-HT Non
5-HT 5-HT Non
5-HT
Control
+ oCRF
(N=26,13)
6.7±0.6
8.7±0.8
**
6.0±1.0
5.8±1.1 16.2±0.8
18.6±1.3
**
29.3±3.8
29.0±4.1 1.3±0.1
1.4±0.1 1.0±0.1
1.1±0.9 3.6±0.2
4.4±0.5
*
3.8±0.4
4.2±0.5 30.4±11.7
36.7±16.1 23.5±6.3
42.9±18.0 -30.9±3.8
-36.9±3.9
**
-23.2±8.7
-33.0±9.1
**
Control
+ UCN II
(N=16,14)
8.3±0.9
8.9±1.0 4.7±0.7
5.6±0.7 18.5±1.2
22.9±1.6
**
19.0±2.0
20.8±1.7 1.1±0.1
1.0±0.1 1.0±0.1
1.1±0.1 3.3±0.2
4.1±0.3
*
3.5±0.4
3.8±0.4 30.2±8.3
32.6±7.3 14.9±2.4
17.9±3.0 -18.8±2.7
-28.4±4.1
**
-23.6±5.0
-26.5±6.4
Control
+ Antalarmin
+ oCRF
(N=20,6)
9.4±1.0
9.8±1.1
9.5±1.0
18.0±2.2
20.3±2.4
(#)
21.2±2.1
(#)
1.3±0.1
1.3±0.1
1.3±0.1
3.6±0.2
4.4±0.3
(#)
4.4±0.4
(#)
15.8±2.0
50.0±20.1
37.0±9.3
-27.6±4.3
-34.4±3.9(#)
-45.5±4.1
(#), (†)
-13.4±14.9
-22.1±18.9
-27.4±17.6
Control
+ ASVG-30
+ oCRF
(N = 17)
6.3±1.0
7.4±1.1
8.8±1.5
(#), (†)
18.1±1.4
20.6±1.8(#)
21.7±1.7
(#)
1.1±0.1
1.1±0.1
1.1±0.1
3.5±0.2
3.6±0.2
3.8±0.3
17.1±4.0
15.9±3.0
17.0±3.6
-25.8±4.1
-27.8±4.0
-27.4±3.7
*p < 0.05
**p < 0.01 by paired Student’s t-test. Antagonist experiments were analyzed by repeated measures ANOVA.
(#)The pound sign indicates a significant difference from mIPSC controls
(†)and the dagger indicates a significant difference from the antagonist baseline by post-hoc Student-Newman-Keuls test (p < 0.05).
J Neurosci. Author manuscript; available in PMC 2009 May 26.
