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Vol. 281, Issue 1, 163-172, 1997
Department of Psychiatry, Allegheny University, Philadelphia, Pennsylvania
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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Corticotropin-releasing factor (CRF) administered
intracerebroventricularly (i.c.v.) activates noradrenergic locus
coeruleus (LC) neurons of halothane-anesthetized and unanesthetized
rats. This study used a technique for microinfusing CRF into the LC from calibrated micropipettes to characterize and quantify the effects
of locally administered CRF on LC discharge in halothane-anesthetized rats. CRF (3-100 ng) microinfusion into the LC increased discharge rate in a dose-dependent manner from 28 ± 8 to 105 ± 26%
above preinfusion discharge rates. The CRF dose-response curve
generated by local microinfusion was parallel to, and shifted
approximately 200-fold to the left, of that generated by i.c.v.
administration. Intracoerulear microinfusion of the CRF antagonist,
[DPhe12,Nle21,38,C
MeLeu37]r/hCRF(12-41),
greatly attenuated LC activation produced by a maximally effective dose
of i.c.v. administered CRF, suggesting that these effects are primarily
due to actions within the LC. In rats in which both LC discharge rate
and norepinephrine levels in prefrontal cortex were measured by
in vivo microdialysis, CRF microinfused into the LC
increased both endpoints. Finally, LC activation produced by CRF (60 ng) microinfusion into the LC was associated with cortical
electroencephalographic activation. Taken together with previous
anatomical and electrophysiological evidence for endogenous CRF
interactions in the LC, our results support the hypothesis that CRF
serves as an excitatory neurotransmitter in the LC, and suggest that
its actions on LC neurons are translated to enhanced norepinephrine
release and an impact on cortical targets.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The noradrenergic nucleus LC
gives rise to a divergent efferent system that provides the major
source of norepinephrine in the forebrain (Swanson and Hartman, 1976
).
The LC-noradrenergic projections have long been thought to play a role
in the stress response based on electrophysiological and neurochemical
indices of activation of this system by stressors. For example,
stressors increased norepinephrine turnover (Cassens et al.,
1981; Cassens et al., 1980; Korf et al., 1973;
Thierry et al., 1968) and release (Abercrombie et
al., 1988; Nisenbaum and Abercrombie, 1993; Nisenbaum et
al., 1991; Smagin et al., 1994) in forebrain targets of
the LC. Stress also increased tyrosine hydroxlase expression in LC neurons (Melia and Duman, 1991; Melia et al., 1992).
Finally, many of the same stressors that elicit neurochemical indices
of activation have also been demonstrated to increase discharge
activity of LC neurons (Abercrombie and Jacobs, 1987; Elam et
al., 1981, 1984, 1986; Morilak et al., 1987a, b).
Recently, anatomical and physiological studies designed to delineate
afferents to the LC that regulate its activity have begun to identify
potential mediators of LC activation by various stimuli, including
stressors (see for review, Aston-Jones et al., 1991). CRF,
the neurohormone that initiates adrenocorticotropin release from the
anterior pituitary during stress (Vale et al., 1981), has
been implicated as one potential mediator of LC activation. Thus, CRF
fibers (Sakanaka et al., 1987; Swanson et al.,
1983; Valentino et al., 1992) and binding sites (DeSouza,
1987; DeSouza et al., 1985) have been visualized in the LC.
More recent studies demonstrated synaptic contacts between
CRF-immunoreactive terminals and LC dendrites (Van Bockstaele et
al., 1996). CRF administered i.c.v. in doses that mimic other
aspects of the stress response increased LC discharge rates of both
anesthetized and unanesthetized rats (Valentino and Foote, 1987, 1988;
Valentino et al., 1983). Moreover, LC activation by
hypotensive stress (Curtis et al., 1994; Valentino et
al., 1991) and low magnitudes of colon distention (Lechner
et al., in press) were greatly attenuated by local
administration of CRF antagonists into the LC. Taken together, these
findings provide strong evidence that endogenous CRF may act as an
excitatory neurotransmitter within the LC to mediate its activation by
certain stimuli.
If CRF serves as an excitatory neurotransmitter within the LC,
intracoerulear administration should increase discharge rates of LC
neurons. The initial study that described the effects of i.c.v.
administered CRF on LC neuronal activity demonstrated excitatory effects of local CRF application onto LC neurons (Valentino et al., 1983). However, these effects were only examined on a small number of neurons and were not quantified. Moreover, a recent study
suggested that CRF injection into the LC decreases discharge rates of
LC neurons (Borsody and Weiss, 1996). Our study used calibrated double
barrel micropipettes to simultaneously record LC discharge and
microinfuse known concentrations and volumes of CRF into the LC to more
precisely characterize and quantify CRF effects on LC neurons.
Additionally, in some of these rats norepinephrine levels in prefrontal
cortex were measured by in vivo microdialysis, or cortical
EEG was recorded, to determine whether the response of LC neurons to
locally administered CRF was sufficient to impact on LC targets.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Surgery.
The procedures used for recording LC discharge of
halothane-anesthetized rats were similar to those previously reported
(Valentino et al., 1983). Male Sprague-Dawley rats (Taconic
Farms, Germantown, NY; approximately 300 g) were anesthetized with
a 1 to 1.5% halothane-in-air mixture administered through a nose cone.
Rats were positioned in a stereotaxic instrument using blunt ear bars,
and the head was oriented at a 15° angle to the horizontal plane
(nose down). Body temperature was maintained at 37 to 38°C with a
small heating pad. The skull was exposed, and a 3.0-mm diameter hole,
centered at 1.1 mm lateral to the midline and 3.5 to 3.7 mm caudal to
the lambda suture point, was drilled over the cerebellum for
approaching the LC. For experiments requiring i.c.v. administration of
CRF, a hole was drilled 1.0 mm caudal to bregma and 1.5 mm lateral to
midline for placement of a 26-gauge stainless steel cannula into the
lateral ventricle, 5.6 mm ventral to skull surface. For experiments in
which cortical EEG was recorded, a hole was drilled 3.0 mm rostral to
bregma and 1.5 mm lateral to the midline and two small holes were
drilled for skull screws. The dura was carefully removed using fine
forceps and iridectomy scissors. For five rats in which mean arterial
pressure was monitored, a catheter (PE 50) was inserted into the
femoral artery and secured prior to positioning the rat in the
stereotaxic.
Recording and microinfusion.
Double barrel glass
micropipettes were used to record single unit LC discharge and
simultaneously microinfuse peptides or artificial cerebrospinal fluid
(ACSF). This technique has been previously characterized and described
in detail (Akaoka et al., 1992). Double barrel micropipettes
consisted of a recording pipette glued using a photopolymerizing resin
(Silux, 3 M Dental Products, St. Paul, MN) next to an infusion pipette
(Fisher Scientific, Pittsburgh, PA). The recording pipette had a 2 to 4 µm diameter tip (4-7 MOhm) and was filled with 2% Potamine Sky Blue
(PSB) dye in 0.5 M sodium acetate. The infusion pipette (20-50 µm
diameter tip) was angled at approximately 30 to 45o with
its tip adjacent to the tip of the recording pipette but 100 to 150 µm dorsal. This was filled with a solution of either CRF (0.1-2.0
mg/ml), DPheCRF12-41 (0.33 mg/ml) or ACSF and connected by
PE tubing to a source of solenoid-activated pneumatic pressure
(Picospritzer, General Valve, Inc., Fairfield, NJ). This infusion
pipette was calibrated such that known volumes could be administered (1 mm displacement = 60 nl).
Microdialysis.
The procedures for measuring cortical
norepinephrine levels using microdialysis were similar to those
described in a previous study (Lehmann et al., 1992). For
experiments involving LC microinfusion and in vivo
microdialysis, rats were first implanted with microdialysis cannula
guides 1 wk before the experiment. Rats were anesthetized with
pentobarbital (50 mg/kg) and positioned in a stereotaxic instrument. A
hole centered at 3.4 mm rostral to bregma and 0.9 mm lateral to the
midline was drilled for positioning a 25-gauge stainless steel cannula
guide (Plastic Products, Roanoke, VA) in the prefrontal cortex. Two
additional holes were drilled for skull screws. The guide was
positioned 1.6 mm ventral to the skull surface and secured to the skull
and skull screws with cranioplastic cement. A dummy cannula whose tip
did not extend past the tip of the cannula guide was inserted. After
surgery, rats were housed individually and allowed to recover for at
least 1 wk before experimentation.
Histology.
After the experiment, the recording site was
marked by iontophoresis of PSB from the recording pipette (-15 µA, 10 min). In experiments in which CRF was administered i.c.v., 5 µl of
neutral red dye were injected through the i.c.v. cannula. Rats received injections with pentobarbital (100 mg/kg) and were perfused
transcardially with a 10% solution of paraformaldehyde. The brains
were cut to visualize i.c.v. administered neutral red. They were then
stored in the fixative. Frozen 40-µm sections were cut and mounted on glass gelatinized slides and stained with neutral red for visualization of the PSB spot and tract made by the dialysis probe. Data from rats
that had electrode placements outside of the LC, dialysis probe
placements outside of prefrontal cortex or no neutral red throughout
the ventricles were not used in the analysis (for example histology,
see Valentino et al., 1983).
Analysis. LC discharge was recorded on-line on either an Apple 2E computer or Cambridge Electronics Design 1401 data analysis system using Spike 2 software. The mean LC discharge rate determined over three 3-min intervals before microinfusion was taken as the mean basal discharge rate and discharge rates were expressed as a percentage of this mean. ED50s were calculated by log-probit analysis.
EEG activity was recorded on-line using the Cambridge Electronics Design 1401 data analysis system with Spike 2 software. EEG segments (120 sec) before and after microinfusion (at the time of the peak CRF effect on LC discharge) were subject to fast Fourier transform and power spectrum analysis. The mean amplitude in different frequency bands was compared before and after microinfusion using the Wilcoxon matched-pairs signed-ranks test for correlated samples. Dialysis data are plotted as a percentage of the mean baseline level in the presence of desipramine and analyzed using a repeated measures one-way analysis of variance with the Scheffe test for individual comparisons. The mean arterial pressure prior to CRF administration was calculated by averaging the pressure each min for 10 min immediately before CRF administration. A repeated measures analysis of variance was performed using this mean basal pressure and pressures determined each minute after CRF administration (from 0-15 min).Compounds. ovineCRF and DPheCRF12-41 were generously supplied by Dr. Jean Rivier of the Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA. The peptides were dissolved in water to make a 1 mg/ml solution. Aliquots (10 µl) of this solution were concentrated using a Savant Speed Vac concentrator. The 10-µg aliquots were stored at -70°C and dissolved in ACSF on the day of the experiment. Desipramine HCl was obtained from Sigma Chemical Co. (St. Louis, MO).
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effects of CRF on LC discharge rate.
LC discharge rate
recorded from 65 rats ranged from 0.7 to 4.0 Hz with a mean of 1.7 ± 0.1 Hz, comparable to previous reports in halothane-anesthetized
rats (Curtis et al., 1994). Microinfusion of CRF (3-100 ng)
into the LC produced a dose-dependent increase in LC discharge rate
that usually began during (for 56% of neurons) or within 60 sec (for
24% of neurons) after the offset of the injection and peaked within 3 to 5 min (e.g., fig. 1). The latency of the
effect was somewhat longer (between 2-4 min) for the remainder of the
neurons and this longer latency was associated with the administration
of relatively small doses of CRF (3 and 10 ng). The latency of this
response was comparable to the latencies reported with microinfusion of
other agents into the LC using this technique (Ennis and Shipley, 1992;
Aston-Jones et al., 1992; Chiang and Aston-Jones, 1993). The
duration of activation was at least 30 min, although it gradually
declined during this time (fig. 1A). The increase in LC discharge rate
was not accompanied by a change in mean arterial pressure (fig. 1A). In
five rats in which mean arterial pressure was continuously monitored,
the mean pressure before CRF administration was 95 ± 2 mm Hg and
this was not significantly altered at any time up to 15 min after CRF
administration [F(15,79) = 0.9, P > .1]. In contrast to CRF,
ACSF (100 nl) microinfused into the LC did not alter discharge rate
(fig. 1B, open diamond).
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). However, i.c.v. administered CRF still produced a small, but statistically significant, increase in
LC discharge rate in rats pretreated with DPheCRF12-41 [for both the 3 and 10 µg dose, F(5,65) = 6.9, P < .0001 and
F(5,29) = 8, P < .0003, respectively; one-way analysis of
variance with repeated measures).
Effects of CRF on norepinephrine levels in prefrontal cortex.
The mean base-line norepinephrine level in prefrontal cortex before
desipramine was 2.5 ± 0.4 pg/20 µl (n = 10) and
this stabilized at 4.9 ± 0.9 pg/20 µl after desipramine. The
mean norepinephrine levels before microinfusion of CRF
(n = 7) or ACSF (n = 3) into the LC
were 5.3 ± 1.2 and 4.2 ± 0.4 pg/20 µl, respectively.
Microinfusion of CRF (60 ng in 60 nl) increased the norepinephrine
level by 37 ± 7% in the 20-min sample corresponding to the
microinfusion (fig. 3A). Norepinephrine levels in
subsequent samples were comparable to base-line samples. In three of
these rats in which LC recordings were sufficiently stable throughout
the time period of the sample, LC discharge rate was increased by
95 ± 17% during the CRF microinfusion (fig. 3B). In contrast to
CRF, microinfusion of ACSF into the LC did not alter norepinephrine
levels (fig. 3A).
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Effects of CRF on cortical EEG.
Cortical EEG was recorded
simultaneously with LC unit activity during CRF microinfusion in 7 rats. Figure 4 shows an example of the effects of
intracoerulear CRF (60 ng in 60 nl) microinfusion on raw cortical EEG
activity, the EEG power spectrum and LC discharge rate. CRF
microinfusion into the LC was associated with EEG activation as
indicated by a decrease in the power of low frequency activity (fig.
4A). This effect became apparent when the increase in LC discharge rate
had peaked, and was of a shorter duration than the increase in LC
discharge rate. The mean power in the 0 to 2 Hz and 2 to 4 Hz frequency
bands was significantly decreased by CRF microinfusion, and this effect
recovered by 10-12 min after the infusion (fig. 5).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Consistent with its effects after i.c.v. administration (Valentino
and Foote, 1988; Valentino et al., 1983), CRF increased LC
discharge rate after microinfusion into the LC in our study and was
approximately 200 times more potent when administered directly into the
LC compared to i.c.v. Moreover, microinfusion of a CRF antagonist into
the LC greatly attenuated LC activation by a maximally effective dose
of i.c.v. administered CRF. Taken together with recent ultrastructural
evidence for CRF synapses on LC dendrites (Van Bockstaele et
al., 1996), these results are consistent with the hypothesis that
CRF serves as an excitatory neurotransmitter within the LC. The finding
that increased LC discharge rate produced by CRF microinfusion was also
associated with increased norepinephrine levels in prefrontal cortex
and cortical EEG activation in the same rats indicates that LC
activation by local infusion of CRF is sufficient to impact on cortical
targets.
Previous studies demonstrated that CRF, administered i.c.v. in doses
that mimic certain autonomic and behavioral responses to stress, also
increased LC discharge rates of halothane-anesthetized rats (Valentino
et al., 1983) and this effect was structurally specific, in
that CRF analogues that do not elicit ACTH release were ineffective
(Valentino and Foote, 1988; Valentino et al., 1983). The
finding that this also occurred in unanesthetized rats and that CRF was
somewhat more potent and efficacious in these rats indicated that this
effect was not some artifact of anesthesia (Valentino and Foote, 1988).
Taken with evidence for CRF innervation of the LC (Sakanaka et
al., 1987; Swanson et al., 1983; Valentino et
al., 1992; Van Bockstaele et al., 1996) and the
presence of CRF binding sites in LC (DeSouza, 1987; DeSouza et
al., 1985), these studies suggested that CRF might serve as a
neurotransmitter within the LC to activate this noradrenergic system
during stress. Since these initial studies, a number of other
electrophysiological and neurochemical studies have supported this
hypothesis. Thus, CRF microinfusion into the LC increased
norepinephrine turnover (Butler et al., 1990) or release
(Page and Abercrombie, 1995; Smagin et al., 1995; Schulz and
Lehnert, 1996) in forebrain, similar to the effects of stressors.
Stress-elicited increases in tyrosine hydroxylase expression in the LC
were prevented by local microinfusion of CRF antagonists (Melia and
Duman, 1991). Finally, LC activation by hypotensive stress (Curtis
et al., 1994), and more recently by colon distention (Florin
et al., 1995), was blocked or attenuated by microinfusion of
CRF antagonists into the LC. Nonetheless, a crucial test of this
hypothesis requires the demonstration that CRF administered into the LC
increases discharge rates of these neurons. In contrast to the
substantial data that support a direct excitatory action of CRF within
the LC, a recent report suggested that CRF directly inhibits LC
discharge (Borsody and Weiss, 1996).
Technical considerations.
A critical aspect of this study, and
one that may explain the discrepencies between these results and those
of Borsody and Weiss (1996), lies in the method of CRF microinfusion.
Our study used double-barrel micropipettes designed such that the
microinfusion barrel was never more than 150 µm from the recording
pipette, and compounds were infused by the application of small
pressure pulses of short (msec) duration to minimize damage to
parenchyma. This technique has been well characterized (Akaoka et
al., 1992) and used to examine the effects of various agents on LC
neurons, including parasympathomimetic agents (Ennis and Shipley,
1992), 2 adrenergic receptor antagonists (Aston-Jones
et al., 1992), norepinephrine (Akaoka et al.,
1992), excitatory amino acid agonists (Brun et al., 1993)
and antagonists (Akaoka and Aston-Jones, 1991; Page et al.,
1992) and GABA agonists and antagonists (Shiekhattar and Aston-Jones,
1992a). This technique has also been used to manipulate ion
concentrations (Ca2+ or Mg2+) within the LC
in vivo with resulting effects that were similar to those
observed in the in vitro LC slice preparation (Shiekhattar et al., 1991; Shiekhattar and Aston-Jones, 1992b). Other
studies involving discrete application of compounds to nuclei other
than the LC, including GABA agonists, GABA antagonists and dopamine agonists to substantia nigra neurons (Akaoka et al., 1992;
Akaoka et al., 1987) and lidocaine, GABA and synaptic
decouplers to neurons in the ventrolateral medulla (Chiang and
Aston-Jones, 1993) have also utilized this technique. Finally, using
this technique to microinfuse glutamate into the dorsal pons while
recording bladder pressure, pontine neurons involved in the micturition
reflex were mapped and found to be well localized to Barrington's
nucleus, as predicted by anatomical studies (Pavcovich and Valentino,
1995). In this study, relatively small changes in the position of the electrode (50-100 µm) could influence the bladder response to glutamate.
). The likelhood of damage to the parenchyma,
including dendrites or terminals of afferents that may be the targets
of CRF within the LC (see below), is much greater with this technique
compared to that used in our study. Such damage can also contribute to
spread of peptide to regions outside of the nucleus.
Other methodological aspects that could contribute to the discrepancies
between our study and that of Borsody and Weiss (1996) are the volumes
of peptide microinfused and the use of cumulative vs.
single-dose testing. In our study peptides were usually microinfused in
a 30 nl volume, with the exception of supramaximally effective doses,
and the volumes never exceeded 100 nl. The previous study used
cumulative dosing, with total volumes in the range of 200 to 500 nl for
CRF administration and 300 nl for administration of the CRF antagonist
(see below). Evidence for the spread of peptide outside of the LC was
suggested by the finding that similar effects on LC discharge were
obtained with injections into the parabrachial nucleus. Although the
magnitude of the inhibition was smaller when administered into the
parabrachial nucleus vs. the LC, it is highly possible that
such volumes of CRF spread to affect other nearby pontine nuclei that
provide inhibitory input to the LC.
In our study each determination was based on the effects of a single
dose on an individual cell in an individual rat. This was initially
done because pilot data had been obtained suggesting that tachyphalaxis
occurred with cumulative dosing. These pilot data were extended in our
study and confirmed the phenomenon of tachyphalaxis with cumulative
dosing. LC desensitization to CRF has also been observed in rats
administered a single dose of CRF (3 µg, i.c.v.) and challenged with
a second dose of CRF 24 to 96 hr later (Conti and Foote, 1995).
Consistent with this, a single 30-min session of shock shifted the CRF
dose response curve for LC activation to the right (Curtis et
al., 1995). Desensitization that may occur with cumulative dosing
could explain the decreased efficacy of i.c.v. administered CRF
observed in a previous study (Borsody and Weiss, 1996) compared to our
study. Although, this cannot explain the inhibitory effects observed
after local administration of CRF, perhaps coupled with other factors
such as parenchymal damage and spread of large volumes, desensitization
could play an additional role.
Although the technique of CRF administration is the most obvious cause
of discrepancies between the previous and current studies, other
differences that may appear minor should not be discounted. These
include the source of CRF used (Dr. Jean Rivier for our study
vs. Bachem) and the source of rats (Taconic Farms for our study vs. rats bred at an institutional facility). That a
difference in rat supplier may have an impact was recently shown in a
study where LC projections to spinal cord in the Sprague Dawley rat were found to have very different termination patterns in Harlan vs. Sasco Sprague-Dawley rats (Clark and Proudfit, 1992).
Site of action of i.c.v. administered CRF.
Although the
effects of i.c.v. administered CRF were qualitatively similar to those
observed with intracoerulear administration, it was possible that they
were mediated by mechanisms other than, or in addition to, direct
actions within the LC. In a previous study, it was determined that
local microinfusion of a CRF antagonist prevented LC activation by 3 µg CRF administered i.c.v. (Curtis et al., 1994). This
finding was extended in our study by the demonstration that a maximally
effective dose (10 µg) of i.c.v. administered CRF could be
antagonized by intracoerulear administration of a CRF antagonist.
Importantly, the dose of DPheCRF12-41 used in our study
was previously demonstrated to be selective, in that it did not alter
LC activation by excitatory amino acid inputs to the LC (Curtis
et al., 1994). For the most part, these results are in
agreement with those of Borsody and Weiss (1996), using -helical
CRF9-41, a CRF antagonist with partial agonist activity
(Rainnie et al., 1992). However, in that study it was found
that microinfusion of the antagonist into the ipsilateral parabrachial
nucleus was somewhat more effective than microinfusion into the LC, and
microinfusion into the contralateral parabrachial nucleus also
attenuated LC activation by i.c.v. administered CRF. The dose (360 ng)
and volume (300 nl) of helical CRF9-41 used in that
study could be considered excessive. Other studies have shown that 100 ng of this antagonist administered into the LC is sufficient to
antagonize CRF-mediated effects in the LC and that when administered
intracoerulearly, -helical CRF9-41 is only
approximately 10 times less potent than D-PheCRF12-41 (Curtis et al., 1994; Page et al., 1993;
Valentino et al., 1991). Considering the partial agonist
activity of this antagonist and the doses and volumes used, the
previous results need to be interpreted with caution. For example, it
is possible that high concentrations of -helical
CRF9-41 achieved in the LC with intracoerulear injections
had partial agonist activity (as suggested by the significant increase
in LC discharge rate produced by these injections). In contrast,
injections outside of the LC (parabrachial nucleus), would result in
lower concentrations of the antagonist within the LC that may be below
those having agonist activity. Evidence for this was suggested by the
lack of an increase in LC discharge after antagonist infusion in the
parabrachial nucleus and more effective antagonism (Borsody and Weiss,
1996).
Cellular targets of CRF action in the LC.
Despite the evidence
suggesting that CRF has excitatory effects within the LC, the cellular
site of action has yet to be determined. A recent ultrastructural study
examining morphological interactions between CRF-immunoreactive
terminals and tyrosine hydroxylase-immunoreactive dendrites in the
rostral ventromedial dendritic zone of the LC found evidence for both
direct and indirect actions of CRF on LC neurons. Thus,
CRF-immunoreactive terminals were observed to make synaptic contacts
with tyrosine-hydroxylase-immunoreactive dendrites, and the majority of
these synapses was asymmetric, consistent with an excitatory effect of
CRF on LC neurons (Van Bockstaele et al., 1996). CRF
terminals also contacted unlabeled dendrites, as well as unlabeled
terminals that synapse with LC dendrites in this region (Van Bockstaele
et al., 1996). CRF terminals were also apposed to glia that
surrounded LC dendrites. Finally, CRF-immunoreactive large dense core
vesicles were observed to be colocalized in terminals with small clear
vesicles, indicative of colocalization with classical
neurotransmitters. Based upon these morphological interactions, there
are a number of possible mechanisms by which CRF could affect LC
discharge, including a number of indirect mechanisms. The inability to
detect mRNA for CRF binding sites in LC neurons (Potter et
al., 1994) argues for indirect actions of CRF, perhaps on
terminals of LC afferents whose cell bodies are distant from the LC.
However, these studies too, should be interpreted with caution and
regard to the technical limitations of detection.
Impact of LC activation by CRF on cortical targets.
Our study
indicated that LC activation by CRF microinfusions is translated to
increased norepinephrine release in forebrain. These findings support
previous studies which demonstrated that CRF microinfusion into the LC
increases norepinephrine levels in prefrontal cortex (Smagin et
al., 1995; Schulz and Lehnert, 1996) and hippocampus (Page and
Abercrombie, 1995). The study of hippocampal norepinephrine release
showed dose-dependent effects of CRF microinfusion into the LC of
unanesthetized rats with doses as low as 2 ng. The previous studies
measuring cortical norepinephrine release used doses somewhat higher
than our study. Importantly, the study by Smagin et al.
(1995), demonstrated that in contrast to LC microinfusion,
microinfusion of CRF into the nearby parabrachial nucleus did not
increase norepinephrine levels in cortex, arguing against the
possibility that diffusion to this nucleus is responsible for CRF
activation of LC discharge. In our study, norepinephrine release was
elevated only during the first 20-min sample after CRF administration,
i.e. the peak of LC activation, but was not apparent in the
next sample, although LC activity was still elevated. This may be a
reflection of activation of only a limited number of LC neurons by the
volume of CRF administered in this study.
). Cortical EEG activation associated with LC
activation by CRF was not surprising, as other studies using direct
chemical manipulation of LC discharge have shown that changes in LC
discharge rate are sufficient to affect cortical EEG. Thus,
intracoerulear microinfusion of drugs that increase LC discharge rate,
e.g., muscarinic agonists (Berridge and Foote, 1991),
excitatory amino acids (Rispoli et al., 1994) and
2 adrenergic antagonists (De Sarro et al.,
1987, 1988) activated the cortical EEG, and inhibitory agents
(e.g., clonidine) had the opposite effect (Berridge et
al., 1993; De Sarro et al., 1988). The possibility that
LC activation by endogenous CRF during stress plays a role in
stress-induced EEG activation, and thereby arousal, was suggested by
the finding that EEG activation produced by hypotensive stress was
prevented by microinfusion of clonidine or CRF antagonists into the LC
(Page et al., 1993). As with cortical norepinephrine release, the effects on EEG in our study were not long lasting, and
this may also be a reflection of activation of only a limited number of
neurons by the volume of CRF administered in this study.
Functional implications.
The putative neurotransmitter actions
of CRF have been implicated in many autonomic and behavioral aspects of
the stress response (for review see Dunn and Berridge, 1990; Owens and
Nemeroff, 1991). In addition to the LC, physiological studies
demonstrating that CRF alters activity of neurons in amygdala (Rainnie
et al., 1992), hippocampus (Aldenhoff et al.,
1983), complex of the solitary tract (Siggins, 1990), hypothalamus,
cortex, thalamus and lateral septal area (Eberly et al.,
1983) suggest that some of these effects may be mediated by
neurotransmitter actions of CRF in these regions (for review see
Siggins, 1990). Studies using microinjection of CRF or CRF antagonists
into the LC have implicated CRF actions in the LC in some aspects of
the stress response (for review see Valentino et al., 1993).
Our own studies suggest that CRF effects on LC neurons may be important
in maintaining or increasing arousal during stress (Page et
al., 1993). Other studies have implicated CRF-LC interactions in
anxiogenic behaviors (Butler et al., 1990; Swiergel et
al., 1992). Findings from independent laboratories demonstrating
that CRF microinfusion into the LC suppresses peripheral blood and
spleen T lymphocyte mitogenic activity, suggest that LC activation by
CRF mediates certain immunological responses to stress (Caroleo
et al., 1993; Rassnick et al., 1994). Other reported consequences of CRF microinfusion into the LC include activation of the hypothalamic-pituitary axis (Butler et
al., 1990; Rassnick et al., 1994) and increases in
colonic motility (Monnikes et al., 1992). Thus, there is
evidence for a role of CRF-LC interactions in endocrine, autonomic,
behavioral and immunological responses to stressors. More systematic
studies are required to determine whether the numerous effects
discussed above are the result of direct CRF activation of LC neurons,
and the overall importance of the LC-noradrenergic system in the
constellation of responses to different stressors.
| |
Acknowledgments |
|---|
The authors thank Dr. Jean Rivier for the generous gifts of CRF and DPheCRF12-41. The expert technical assistance of Mr. Bowen Kang and Ms. Wei Ping Pu are gratefully acknowledged.
| |
Footnotes |
|---|
Accepted for publication December 24, 1996.
Received for publication April 24, 1996.
1 This work was supported by USPHS Grants MH40008 and MH00840 (an RSDA award to R.J.V.).
Send reprint requests to: Dr. Rita J. Valentino, Department of Psychiatry, Allegheny University, Broad and Vine Sts., Philadelphia, PA 19102-1192.
| |
Abbreviations |
|---|
ACSF, artificial cerebrospinal fluid;
CRF, corticotropin-releasing factor;
DPheCRF12-41, [DPhe12,Nle21,38,C
MeLeu37]r/hCRF(12-41);
EEG, electroencephalographic activity;
i.c.v., intracerebroventricular;
LC, locus coeruleus;
PSA, power spectrum analysis;
PSB, Pontamine sky
blue.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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