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The Journal of Neuroscience, April 15, 2000, 20(8):2978-2987
Role of Brain-Derived Neurotrophic Factor in the Circadian
Regulation of the Suprachiasmatic Pacemaker by Light
Fong-Qi
Liang,
Gregg
Allen, and
David
Earnest
Texas A&M University Health Science Center, Department of Human
Anatomy and Medical Neurobiology, College of Medicine, College Station,
Texas 77843-1114
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ABSTRACT |
The central pacemaker located in the suprachiasmatic nucleus (SCN)
of the hypothalamus mediates the generation of mammalian circadian
rhythms, including an oscillation in pacemaker sensitivity to photic
signals conveyed by the retinohypothalamic tract. Because brain-derived
neurotrophic factor (BDNF) has been implicated in the functional
regulation of neural input to other targets of visual pathways, the
present study examined whether changes in BDNF expression or blockade
of its action in the SCN affect circadian pacemaker responses to light.
In rats receiving infusion of exogenous BDNF into the SCN, the
free-running rhythm of activity in constant darkness was characterized
by large phase advances in response to light exposure during the
midsubjective day, when the circadian pacemaker is normally insensitive
to photic perturbation. In contrast, SCN infusion of BDNF did not
potentiate either phase-delaying or phase-advancing effects of light on
the rat activity rhythm during the subjective night. In heterozygous
BDNF mutant mice, deficits and damped rhythmicity in SCN levels of this
neurotrophin were accompanied by marked decreases in the amplitude of
light-induced phase shifts during the subjective night. In agreement
with the effects of decreased BDNF expression, SCN infusion of the
tyrosine kinase inhibitor K252a blocked or strongly inhibited both the phase-delaying and -advancing effects of light during the subjective night. Collectively, these findings suggest that BDNF-mediated signaling may play an important role in the circadian regulation of SCN
pacemaker sensitivity to light.
Key words:
brain-derived neurotrophic factor; neurotrophins; growth
factors; tyrosine kinase receptors; circadian rhythms; suprachiasmatic
nucleus; photoentrainment
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INTRODUCTION |
In mammals, the suprachiasmatic
nucleus (SCN) functions as an internal biological clock governing the
generation and photic entrainment of circadian rhythms (Moore, 1983 ).
Entraining light signals are transmitted to the SCN by the
retinohypothalamic tract (RHT), a direct projection from a subset of
retinal ganglion cells that mainly terminates within the ventrolateral
SCN. Circadian photoentrainment occurs because the SCN pacemaker is
reset by light during the subjective night (i.e., active phase of a
nocturnal animal) but is insensitive to photic manipulation during the
subjective day (Takahashi et al., 1984). Recent evidence of
time-dependent changes in the effects of optic nerve stimulation on SCN
neuronal activity suggests that this rhythm of pacemaker sensitivity to light may involve the differential regulation of RHT photic input and/or SCN cell responses to such information (Cui and Dyball, 1996).
Specific members of the neurotrophin family that includes nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3,
and neurotrophin-4/5 have been implicated in the regulation of
circadian pacemaker function by localization of these neurotrophins or
their receptors in the SCN region (Sofroniew et al., 1989; Lehman et
al., 1996; Liang et al., 1998b). BDNF provided a central focus in the
present analysis because involvement of this neurotrophin and its
cognate receptor the TrkB tyrosine kinase in visual system function is
suggested by studies demonstrating the following: the localization of
TrkB receptors on retinal fibers near BDNF-expressing cells in their
target areas (Jelsma et al., 1993; Pearson et al., 1995), structural
and functional effects of BDNF on retinal ganglion cells and the visual
cortex (Cohen-Cory and Fraser, 1995; Akaneya et al., 1997), and the
photic induction of BDNF and trkB mRNA expression within the
visual cortex (Castren et al., 1992). A role for BDNF in the photic
regulation of the SCN is indirectly supported by our anatomical
observation that BDNF-expressing cells in the ventrolateral SCN are
closely apposed to TrkB-immunoreactive fibers emanating from the optic
chiasm (Liang et al., 1998b). Coupled with increasing evidence of BDNF function in the short-term control of neuronal signaling within the
brain (Kang and Schuman, 1995; Korte et al., 1995; Levine et al.,
1995), these findings raise the possibility that BDNF may be involved
in gating photic input to the SCN and its phase-shifting effects on
circadian rhythms. Consistent with its potential function in regulating
pacemaker responses to light, BDNF is rhythmically expressed within the
SCN such that levels are elevated during the subjective night, when
light shifts the phase of circadian rhythms, but remain low throughout
the subjective day, when the clock mechanism is insensitive to photic
perturbation (Liang et al., 1998a). If this relative amplification of
BDNF-mediated signaling during the subjective night is an important
process in the circadian sensitivity of the pacemaker mechanism to
photic signals, then alterations in BDNF expression or inhibition of
its action on TrkB receptors within the SCN should produce predictable
changes in the resetting effects of light on circadian rhythms. To test this hypothesis, three experiments were conducted to determine (1) whether exogenous BDNF administration enables light to
induce phase shifts during the subjective day and whether the resetting action of light during the subjective night is impaired in (2) heterozygous BDNF-deficient knock-out mice and (3) rats receiving the
tyrosine kinase inhibitor K252a. BDNF and K252a were infused into the
SCN because their short-lived biological activity in vivo
has warranted this approach in comparable studies (Altar et al., 1992;
Croll et al., 1994) and acute injections were ineffective in
preliminary analyses.
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MATERIALS AND METHODS |
Animals and housing conditions
Adult male Sprague Dawley rats (175-200 gm) were purchased from
Harlan Sprague Dawley (Indianapolis, IN). Adult male wild-type (C57/Bl6) and heterozygous BDNF null mutant (Conover et al.,
1995) mice were obtained from Regeneron Pharmaceuticals (Tarrytown, NY). All animals were housed individually in cages equipped with running wheels so that the circadian rhythm of wheel-running activity could be continuously recorded. Before experimentation, animals were
maintained under a standard 12 hr photoperiod [light/dark cycle of
12:12 hr (LD 12:12); lights on at 06:00 hr], and baseline activity behavior was recorded for 10-14 d. Periodic animal care and
experimental manipulations in constant darkness (DD) were accomplished
using an infrared viewer (FJW Optical Systems). Animals were provided
food and water ad libitum.
SCN cannulation for chronic infusion or acute injection
Sprague Dawley rats were anesthetized (ketamine, 87 mg/kg, and
xylazine, 1.3 mg/kg), and a stainless steel cannula with an osmotic
pump connector (28 gauge; Plastic One, Roanoke, VA) was stereotaxically
implanted near the ventral extent of the third ventricle to allow drug
infusion into the SCN. Empirical stereotaxic coordinates for the SCN
region (1.3 mm posterior to bregma and 7.5 mm ventral to the dura along
the midline) were derived from those established by Paxinos and Watson
(1997).
Three days after cannula placement surgery, a miniosmotic pump (model
2004; Alzet) was implanted subcutaneously in the intrascapular region
of anesthetized rats (ketamine, 87 mg/kg, and xylazine, 1.3 mg/kg) and
attached to the connector cannula via polyethylene 60 (PE60)
tubing. Osmotic pumps were filled with recombinant human BDNF (10 µg)
or K252a (467 ng) diluted in 200 µl of artificial CSF (aCSF),
pH = 7.4. On the basis of the operational specifications of this
osmotic pump (delivery rate = 0.25 µl/hr), 250 ng of BDNF or
56.04 ng of K252a was infused into the SCN region per day for a maximum
duration of 28 d. Control animals received osmotic pump infusion
of aCSF alone into the SCN region.
For acute injection of BDNF or K252a into the SCN, rats were implanted
with a stainless steel guide cannula (24 gauge; Plastic One).
Stereotaxic coordinates were similar to those for placement of the
osmotic pump cannula, except that the guide cannula was implanted (6.5 mm ventral to the dura) to allow extension of an internal injector
stylus (33 gauge) into the parenchyma (by 1 mm). A compatible dummy
stylet was inserted into each guide cannula to prevent debris entry.
Histological analysis
At the conclusion of behavioral analysis, anesthetized animals
(sodium pentobarbital, 3.0 mg/kg) were injected via the guide cannula
with fluorogold (2%; Fluorochrome, Englewood, CA) and killed by
transcardiac perfusion with 50 ml of 0.1 M phosphate buffer, pH = 7.3, containing heparin, followed by 200-250 ml of 4% paraformaldehyde. Immediately after perfusion, the brains were removed, post-fixed for 1-2 hr at 4°C, and stored overnight in cryoprotectant solution (15% sucrose in 0.15 M phosphate
buffer). The tissue was then frozen and sectioned in the coronal plane at 30 µm using a sliding microtome. Cannula placement was determined by fluorogold localization in mounted sections using fluorescence microscopy. Only data from animals with verified cannula placement in
the SCN region were used for analysis of treatment effects on circadian
wheel-running behavior.
Experimental protocols
After completion of all surgical procedures, animals were
exposed to DD. Animals were allowed to free-run in DD for at least 2 weeks before experimental manipulations. Light pulses (150-200 lux)
were delivered by transferring the home cages of individual animals to
a ventilated, light-proof chamber. After experimental treatment,
animals were allowed to free-run in DD without any further perturbation.
Experiment 1: effect of BDNF infusion into the SCN on
light-induced phase shifts of the rat activity rhythm. Effects of
BDNF on the resetting of the SCN pacemaker by light at different
circadian times (CT) were examined in male rats receiving an osmotic
pump infusion of either aCSF or BDNF (250 ng/d). Control
(n = 10) and BDNF-treated (n = 13)
animals were subjected to a 60 min light pulse ~6 hr before the onset
of activity (CT 6) because light exposure at this time is known to have
no phase-shifting effect on rodent circadian rhythms (Summer et
al., 1984; Takahashi et al., 1984; Schwartz and Zimmerman,
1990). Exclusion of two aCSF-infused and four BDNF-treated rats from
analysis was necessary because treatment was disrupted by cannula
failure or because arrhythmicity or irregular activity onsets
compromised the evaluation of phase shifts in the activity pattern. To
determine whether BDNF can potentiate light-induced phase shifts during
the subjective night, additional groups of animals receiving SCN
infusion of BDNF (n = 6) or aCSF (n = 5) were exposed to a 20 min light pulse at either 2 or 10 hr after the
onset of activity (CT 14 or 22) because large phase delays or advances
of the rat activity rhythm occur after light treatment at these
circadian times (Summer et al., 1984). Exclusion of one
BDNF-treated rat from analysis was necessary because arrhythmicity
compromised the evaluation of light-induced phase shifts in the
activity pattern at CT 22. All experimental and control animals
received light treatment only once during the 4 week period of infusion.
In parallel with this behavioral analysis, SCN content of BDNF was
examined by ELISA in auxiliary groups of control and experimental rats
to determine the effects of the infusion paradigm on BDNF levels and
their rhythmic pattern in the SCN. Male rats receiving an infusion of
either aCSF (n = 28) or BDNF (250 ng/d;
n = 28) for 14 d (i.e., midpoint of minipump
functional duration) under LD 12:12 conditions were exposed to DD
(beginning at CT 12) and 12 hr later killed at 4 hr intervals for 24 hr
by decapitation using an infrared viewer. SCN tissue was immediately
dissected as described previously (Earnest and Sladek, 1986) and stored at  80°C until subsequent assay of BDNF content.
To determine whether BDNF alone has any phase-shifting effect on the
circadian rhythm of wheel-running activity, acute injections (1 µl)
of BDNF (40 ng) or aCSF were administered at either CT 6, 14, or 22 into the SCN region of rats maintained in DD (n = 18).
These injections were delivered under light anesthesia (Metofane; Mallinckrodt Veterinary) via the guide cannula using a 10 µl
Hamilton syringe with connector tubing (PE80) and an internal injector stylus. The injection volume was delivered slowly (1-2 min interval), and then the internal cannula was left in place for an equivalent period of time to allow drug diffusion. The animals were then allowed
to free-run in DD for at least 17 d before receiving a second
treatment. Experiments used a crossover design such that each animal
received only two injections, one with BDNF and another with aCSF.
Experiment 2: effect of decreased BDNF expression on
light-induced phase shifts of the activity rhythm in heterozygous
mutant mice. To determine whether decreased BDNF expression within
the SCN will abate the phase-shifting effect of light, mice
heterozygous for the BDNF null mutation
(bdnf+/ ) were examined for
their circadian responses to light during the subjective night. In
bdnf+/ mice, both the levels of
BDNF protein and neuronal number in different sensory ganglia are
decreased by ~50%, but body mass, structural organization of the
CNS, and motor neuron development or function show no gross
abnormalities (Jones et al., 1994; Conover et al., 1995; Korte et al.,
1995; Bianchi et al., 1996). After recording baseline activity in LD
12:12 (lights on at 06:00 hr), wild-type mice (n = 28)
and bdnf+/ (n = 28) were maintained in DD. After establishment of a stable free-running
period in DD ( 2-3 weeks), animals were subjected to 20 min light
pulses at either CT 6, 14, or 22 because light exposure at these times
induces no shift, delays, or advances in the phase of the mouse
activity rhythm (Schwartz and Zimmerman, 1990). All wild-type and
mutant mice were only subjected to light treatment on two different
occasions during the course of this experiment. At the conclusion of
behavioral analysis, animals were first housed under LD 12:12
conditions for 14 d to provide for stable entrainment of the
activity rhythm and then exposed to DD (at 18:00 hr). Twelve hours
later, groups of wild-type and mutant mice (each
n = 4) were killed at 4 hr intervals for 24 hr by
decapitation using an infrared viewer. The SCN was immediately dissected, frozen in liquid nitrogen, and later extracted to recover total RNA and soluble proteins. BDNF content in recovered protein samples was determined using a sandwich ELISA protocol (Liang et al.,
1998a,b).
Experiment 3: effect of K252a on light-induced phase shifts of
the rat activity rhythm. Complementary to the analysis of
decreased BDNF expression in mutant mice, studies were performed to
determine whether the phase-shifting effects of light on the rat
activity rhythm are impaired by SCN infusion of K252a, an alkaloid
compound that inhibits the phosphorylation of all tyrosine kinase
receptors (Berg et al., 1992; Tapley et al., 1992). Use of K252a in the present context is appropriate because this tyrosine kinase receptor inhibitor has been shown to block BDNF-induced enhancement of neuronal
signaling within the brain (Kang and Schuman, 1995; Levine et al.,
1995; Akaneya et al., 1997). Adult male rats were implanted with an
indwelling cannula to provide for the chronic infusion of aCSF
(n = 12) or K252a (56.04 ng/d; n = 12)
via an osmotic minipump for 28 d. After establishment of a stable
free-running period in DD (2-3 weeks), animals were exposed to 30 min light pulses at either CT 14 or 22 because photic stimulation at
these circadian times is known to induce maximal phase delays or
advances (Summer et al., 1984), respectively. All animals
received light treatment only once during the experiment. Exclusion of
three aCSF-infused rats and one K252a-treated animal from analysis was necessary because treatment was disrupted by cannula failure or arrhythmicity or irregular activity onsets compromised the evaluation of phase shifts in the activity pattern.
To determine whether K252a alone has any phase-shifting effect on the
circadian rhythm of wheel-running activity, acute injections (1 µl)
of K252a (9.34 ng) or aCSF were administered at either CT 14 or 22 into
the SCN region of rats maintained in DD (n = 12). K252a
was injected using procedures described in Experiment 1 for BDNF injections.
Analysis of circadian behavior
Wheel-running activity was continuously recorded, summed, and
stored in 10 min bins using a computer running Dataquest IV data
acquisition software (Data Sciences). Graphical records of circadian
activity rhythms were generated and analyzed using Tau software. Light-
and drug-induced shifts in the circadian phase of free-running activity
rhythms in DD were assessed as described previously (Ellis et al.,
1982) using guide lines through steady-state activity onsets during
10-15 d of the pretreatment and post-treatment intervals for
reference. The phase-shift amplitude was derived from the difference
between the expected time of activity onset as projected using
pretreatment reference points and the actual time of activity onset
after post-treatment restoration of steady-state circadian period
( ). Shifts in the circadian phase of the activity rhythm were
independently assessed in this manner by two experienced individuals,
and reported values reflect the averages of their determinations. By
the use of the chi-square periodogram (Tau) and fast Fourier transform
analyses, steady-state of the activity rhythm in DD was also
determined in all animals to provide several comparisons:
before and after light treatment within individual animals
and between control and experimental groups during infusion treatment.
Animals showing highly irregular onsets of activity, arrhythmicity, or
cannula failure were excluded from analysis. Statistical analyses were
performed on the raw data using a pooled t test to determine
the significance of treatment effects on phase shifts induced by light
and of the activity rhythm (see Figs. 1B,
2, 4B, 6B).
BDNF and K252a
Recombinant human (rh) BDNF (PeproTech) was diluted to 41.7 ng/µl in aCSF containing 0.1% BSA before loading into osmotic minipumps (Alzet). Previous reports on the stability of BDNF during osmotic pump delivery at 37°C indicate that this neurotrophin retains
at least 80% of its bioassayable activity in comparison with stock
solutions that had been stored at 4°C (Altar et al., 1992). The
tyrosine kinase inhibitor K252a (Calbiochem, La Jolla, CA) was diluted
to 9.34 ng/µl (20 µM) in dimethylsulfoxide (DMSO; Sigma, St. Louis, MO) and then loaded into minipumps.
BDNF ELISA
For analysis of BDNF content by ELISA, SCN tissue from
individual animals was homogenized in TRIzol reagent (Life
Technologies, Gaithersburg, MD). Soluble proteins were recovered
from the organic phase by sequential precipitation
(Chomczynski, 1993) and dissolved in 1% SDS. Before ELISA analysis,
the protein content in each sample was determined by the bicinchoninic
acid method (Micro BCA; Pierce, Rockford, IL).
BDNF content in recovered protein samples was determined using a
sandwich ELISA protocol. Ninety-six-well microplates (Nunc, Naperville,
IL) were coated overnight at 4°C with 100 µl of rabbit anti-BDNF
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted (1:250) in
0.025 M carbonate-bicarbonate buffer, pH 9.7. With
interceding washes [50 mM Tris-buffered saline (TBS), pH 7.4], the plates were subjected to sequential 2 hr incubations at room
temperature with blocking solution (1% bovine serum albumin in TBS),
triplicate aliquots of tissue protein samples or BDNF standards
(0.1-100 ng/well), chicken anti-BDNF antibody (1:500; Promega,
Madison, WI), and alkaline phosphatase-conjugated goat anti-chicken
IgY (1:1000; Promega). Alkaline phosphatase activity was
detected using p-nitrophenyl phosphate (Pierce) dissolved in
0.1 M bicarbonate buffer, pH 10, as the color
substrate. After color development (40-50 min), the absorbance at 405 nm was measured using a plate reader (Bio-Tech). By the use of serial
dilutions of known amounts of rhBDNF, this color reaction yielded a
linear standard curve from 0.5 to 100 ng/ml. The intra-assay and
interassay coefficients of variation were <5 and 10%, respectively.
BDNF content in SCN lysates was quantified within the linear range of a
standard curve and normalized for the soluble protein (micrograms) that
was assayed in each sample. The rabbit and chicken anti-BDNF antibodies
used in this assay show <2% cross-reactivity with NGF, neurotrophin-3, or neurotrophin-4/5 (at concentrations as high as 10 µg/ml). In addition, the specificity of these antibodies for BDNF has
been verified previously by Western blot analysis (Liang et al.,
1998b).
Within a treatment group, the mean values for time-point determinations
of BDNF protein levels in the SCN were evaluated for evidence of
temporal variation using one-way ANOVA. Paired comparisons of
BDNF levels at specific circadian times were analyzed post hoc for statistical differences using the Newman-Keuls sequential range test. In addition, statistical analysis was performed using a
pooled t test to determine whether BDNF levels at each time point were significantly different between control and experimental groups (see Figs. 3, 5).
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RESULTS |
Experiment 1: effect of BDNF infusion into the SCN on light-induced
phase shifts of the rat activity rhythm
During the 28 d infusion interval, both aCSF-
(n = 8) and BDNF-treated (n = 9) rats
showed clear free-running activity rhythms in DD with similar
periodicities (mean = 24.00 ± 0.04 vs 24.06 ± 0.07 hr, respectively). Although BDNF had no significant effect on the
steady-state circadian period (p > 0.05), SCN
infusion of this neurotrophin did alter the effect of light on the
activity rhythm during the midsubjective day. Exposure to a 1 hr light pulse at CT 6 induced large phase advances (mean   = +2.28 ± 0.35 hr) of the activity rhythm in BDNF-infused rats but
had little or no phase-shifting effect (mean = +0.25 ± 0.12 hr) on the circadian behavior of animals receiving aCSF (Fig.
1). Light-induced phase shifts at CT 6 in
BDNF-treated rats were significantly greater (p < 0.01) than those observed in control animals. These advancing shifts
in BDNF-treated animals were often distinguished by a large number of
transient cycles (7-20) before a new steady state was reached. At the
conclusion of these transients, the steady-state period of the activity
rhythm in BDNF-infused rats was similar to that expressed before light
exposure (mean = 24.06 ± 0.07 vs 24.05 ± 0.09 hr,
respectively), indicating that this effect truly reflects a phase
shift, not a long-term change in circadian period, in response to
light. Importantly, acute injection of BDNF alone (40 ng in 1 µl)
into the SCN at CT 6 without exposure to light had no significant
effect (p > 0.05) on the circadian phase of the
rat activity rhythm (mean = +0.33 ± 0.20 hr) relative to that of matched aCSF controls (mean = +0.23 ± 0.10 hr).
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Figure 1.
Effects of BDNF infusion on the responses of the
rat activity rhythm to light at CT 6. A, Representative
activity records of two adult male rats receiving infusion of either
aCSF or BDNF into the SCN during exposure to DD. Actograms are
double-plotted over a 48 hr period, and shaded
circles on each record indicate the day and time (CT 6)
during which animals were exposed to a 1 hr light pulse
(LP). A thin shaded bar has been
fitted through the activity onsets for 5-6 d before the light pulse to
facilitate visualization of phase shifts () in the activity
rhythm. B, Mean (±SEM) phase advance (+) in hours
of the activity rhythm induced by a 1 hr light pulse at CT 6 in rats
receiving SCN infusion of aCSF (aCSF + LP) or BDNF
(BDNF + LP) or by a single injection of BDNF (40 ng)
alone at CT 6 without exposure to light (BDNF Inject).
For each treatment group, the sample size is designated in
parentheses on the bar. Light-induced
phase shifts were significantly greater (*p < 0.01) in BDNF-infused rats relative to those in animals receiving aCSF
infusion or BDNF injection alone.
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In contrast to its modulatory effects on circadian responses to light
at CT 6, BDNF infusion did not alter light-induced phase shifts of the
rat activity rhythm at CT 14 or 22. In both BDNF- (n = 11) and aCSF-treated (n = 10) animals, a 20 min light
exposure induced phase delays at CT 14 (Fig.
2; mean = 2.42 ± 0.26 vs 2.65 ± 0.27 hr, respectively) and advances at CT 22 of
comparable amplitude (mean = +2.14 ± 0.2 vs
+2.26 ± 0.46 hr, respectively). No significant differences in the
phase-shifting responses to light at either time
(p > 0.05) were detected between BDNF- and aCSF-infused rats. For both experimental and control groups, phase delays of the activity rhythm were complete within one cycle, whereas
advancing shifts required four to six transient cycles before
steady-state was re-established.
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Figure 2.
Effects of BDNF infusion on the responses of the
rat activity rhythm to light at CT 14 and 22. Mean (±SEM) phase shift
() in hours of the activity rhythm induced by a 20 min light
pulse (LP) at CT 14 or 22 in rats receiving SCN infusion
of aCSF (aCSF + LP) or BDNF (BDNF + LP)
or by a single injection of BDNF (40 ng) alone at CT 14 or 22 without
exposure to light (BDNF Inject). For each treatment
group, the sample size is designated in parentheses.
Phase delays are indicated by negative
values, and advances are denoted by
positive values.
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Effects of the infusion paradigm on BDNF levels and their rhythmic
pattern in the SCN were analyzed in parallel groups of animals. Control
rats receiving infusion of aCSF exhibited a rhythmic pattern of BDNF
content in the SCN similar to that described previously for normal
intact rats (Liang et al., 1998a). SCN levels of BDNF protein reached
peak values during the subjective night and were low
throughout the subjective day (Fig. 3).
In contrast, BDNF-infused rats showed no evidence of rhythmic
fluctuation in SCN content of BDNF. Instead, BDNF levels in the SCN of
these animals constantly remained at higher levels than
those observed in aCSF-treated control rats. BDNF levels in
the SCN at times throughout the subjective day (CT 0, 4, and 8)
were significantly greater (p < 0.05) in BDNF-infused rats than in control animals. During the subjective day,
experimental and control animals were distinguished by a four-fold to
fivefold difference in SCN content of BDNF, primarily because of the
rhythmic decline occurring only in aCSF-treated animals.
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Figure 3.
Temporal pattern of BDNF content in the SCN of
rats receiving infusion of aCSF or BDNF. Symbols
represent the mean values (±SEM) of BDNF protein expression in the SCN
of rats that were infused with aCSF or BDNF (250 ng/d) for 14 d
and then killed at 4 hr intervals (n = 4) in DD. At
CT 12, 16, and 20, BDNF levels in aCSF-infused rats were significantly
greater (*p < 0.05) than those observed at all
other times. Significant differences in the SCN content of BDNF
( p < 0.05) were observed between aCSF- and
BDNF-infused rats at CT 0, 4, and 8.
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Experiment 2: effect of decreased BDNF expression on light-induced
phase shifts of the activity rhythm in heterozygous mutant mice
The absolute levels and rhythmic patterns of wheel-running
activity (Fig. 4) were comparable in
wild-type (bdnf+/+) and heterozygous
BDNF knock-out (bdnf+/) mice.
Under LD 12:12 conditions, bdnf+/
mice and their wild-type counterparts showed similar patterns of
entrainment such that the daily onset of activity occurred shortly
after lights off (data not shown). In DD, the free-running period of
the activity rhythm in the bdnf+/
mice was significantly shorter (p < 0.01) than
that observed in wild-type mice (mean = 23.23 ± 0.08 and
23.69 ± 0.10 hr, respectively). Mutant
bdnf+/ mice were also
distinguished by abated phase-shifting responses to light. As expected,
20 min light pulses induced clear phase delays at CT 14 (mean
= 2.66 ± 0.57 hr) and large advances at CT 22 (mean
= +4.76 ± 0.30 hr) of the activity rhythm in all
wild-type animals. In bdnf+/ mice,
the same light stimulus produced only small phase delays at CT 14 (mean
= 1.26 ± 0.13 hr) and had little or no
phase-shifting effect at CT 22 (mean = +0.53 ± 0.35 hr). The mean phase delay at CT 14 and advance at CT 22 in
bdnf+/ mice were significantly
decreased (p < 0.05 and p < 0.01, respectively) relative to the light-induced shifts observed in
wild-type mice at these circadian times (Fig. 4). Light exposure at CT
6 had a negligible effect on the phase of the activity rhythm in both wild-type and bdnf+/ mice (mean
= +0.20 ± 0.13 and +0.17 ± 0.11 hr,
respectively).
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Figure 4.
Effects of decreased BDNF expression in the mouse
SCN on light-induced phase shifts () of the rhythm in
wheel-running activity. A, Representative activity
records in DD of wild-type mice (WT:
bdnf+/+) and mice heterozygous for
the BDNF null mutation (HET:
bdnf+/) exposed to a 20 min light
pulse (LP) at CT 14 (top) or 22 (bottom). Actograms are double-plotted over a 48 hr
period, and shaded circles on each record
indicate the day and time during which light pulses were administered.
A thin shaded bar has been fitted through the activity
onsets for 6 d before each light pulse to facilitate visualization
of the phase shifts in the activity rhythm. B, Mean
(±SEM) phase shift () in hours of the activity rhythm induced by
light pulses at CT 6, 14, and 22 in wild-type and
bdnf+/ mice. For each treatment
group, the sample size is designated in parentheses.
Phase delays are indicated by negative
values, and advances are denoted by
positive values. Light-induced phase
delays at CT 14 (*p < 0.05) and advances at CT 22 (**p < 0.01) were significantly decreased in
mutant bdnf+/ mice compared with
those in wild-type mice.
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Wild-type and bdnf+/ mice were
also marked by differences in the relative levels and the temporal
profile of BDNF content in the SCN. In wild-type mice, the SCN content
of BDNF oscillated in a rhythmic manner similar to that reported
previously in rats (see Liang et al., 1998a), with peak values during
the subjective night (Fig. 5). One-way
ANOVA indicated that determinations of BDNF protein levels in the SCN
at 4 hr intervals throughout the circadian cycle were significantly
different (F = 15.81; p < 0.01). Mean
levels of BDNF protein in the SCN during the subjective night at CT 16 and 20 were significantly greater (p < 0.05)
than those observed at all times throughout the subjective day (CT 0, 4, and 8). The wild-type rhythm in the SCN expression of BDNF protein was characterized by a twofold difference between peak and minimum values. In bdnf+/ mice, the
temporal pattern of BDNF expression in the SCN followed a similar trend
in which levels were higher during the subjective night than during the
subjective day. However, no significant variation was observed between
time-based determinations of BDNF levels in the SCN of mutant mice
(F = 1.15; p > 0.05). At all circadian
times, the SCN content of BDNF in
bdnf+/ mice was significantly less
(p < 0.05) than that found in wild-type mice,
although this phenotypic difference was more pronounced during the
subjective night. In accord with previous comparisons for sensory
neurons (Bianchi et al., 1996), the SCN of mutant mice showed an
overall decrease in BDNF protein expression of ~50% in relation to
that found in wild-type animals.
View larger version (25K):
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[in a new window]
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Figure 5.
Temporal patterns of BDNF content in the SCN of
wild-type and heterozygous BDNF knock-out mice. Symbols
represent the mean values (±SEM) of BDNF protein expression in the SCN
of wild-type mice (WT:
bdnf+/+) and BDNF knock-out mice
(HET: bdnf+/) killed
at 4 hr intervals (n = 4) in DD. Circadian times
during which BDNF levels in bdnf+/+
mice were significantly greater (*p < 0.05) in
comparison with those observed at CT 0, 4, and 8 are indicated.
Comparisons of bdnf+/+ and
bdnf+/ mice revealed significant
differences (p < 0.05) in SCN content of BDNF
at all times.
|
|
Experiment 3: effect of K252a on light-induced phase shifts of the
rat activity rhythm
Throughout the 28 d treatment interval, rats receiving SCN
infusion of K252a (n = 11) or aCSF (n = 9) showed clear free-running activity rhythms in DD with similar
periodicities (mean = 24.05 ± 0.07 and 24.03 ± 0.06 hr, respectively). Although K252a infusion had no significant
effect on the steady-state circadian period (p > 0.05), treatment with this tyrosine kinase inhibitor blocked or
abated the phase-shifting effect of light on the activity rhythm during
the subjective night (Fig. 6). In control
animals receiving infusion of aCSF, light exposure induced large phase
delays at CT 14 (mean = 2.84 ± 0.23 hr) and
advances at CT 22 (mean = +2.68 ± 0.46 hr) in the
circadian rhythm of activity. In contrast, 6 out of 11 K252a-infused
rats showed no phase shifts of the activity rhythm in response to light
stimulation at CT 14 or 22. The average phase delays at CT 14 (mean
= 0.67 ± 0.25 hr) and advances at CT 22 (mean
= 0.66 ± 0.17 hr) in K252a-infused rats were
significantly less (p < 0.01) than the light-induced shifts observed in aCSF-infused animals at these circadian times. Acute injection of K252a alone (9.34 ng in 1 µl)
into the SCN at CT 14 or 22 without exposure to light had little effect
on the circadian phase of the rat activity rhythm (mean = 0.37 ± 0.18 and +0.35 ± 0.11 hr, respectively).
View larger version (29K):
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|
Figure 6.
Effects of K252a on light-induced phase shifts of
the rat activity rhythm at CT 14 and 22. A,
Representative activity records of adult male rats receiving infusion
of either aCSF or K252a into the SCN for 28 d during exposure to
DD. Actograms are double-plotted over a 48 hr period, and
shaded circles on each record indicate
the days and times (top, CT 14; bottom,
CT 22) during which animals were exposed to a 30 min light pulse
(LP). A thin shaded bar has been fitted
through the activity onsets for 5-6 d before each light pulse to
facilitate visualization of the phase shifts in the activity rhythm.
B, Mean (±SEM) phase shift () in hours of the
activity rhythm induced by light pulses at CT 14 and 22 in rats
receiving SCN infusion of aCSF (aCSF + LP) or K252a
(K252a + LP) or by a single injection of K252a (9.34 ng)
alone at CT 14 or 22 without exposure to light (K252a
Inject). Phase delays are indicated by negative
values, and advances are denoted by
positive values. For each treatment
group, the sample size is designated in parentheses.
Light-induced phase delays at CT 14 and advances at CT 22 were
significantly decreased (*p < 0.01) in
K252a-treated rats relative to those in aCSF-infused controls.
|
|
| |
DISCUSSION |
The present study provides multiple lines of evidence
demonstrating that changes in BDNF expression or inhibition of its
cognate receptor TrkB within the SCN alter the phase-shifting effects of light on circadian rhythms. First, administration of exogenous BDNF
into the rat SCN transformed normal pacemaker responses to light,
enabling the photic induction of large phase advances in the activity
rhythm during the midsubjective day. Second, mutant bdnf+/ mice exhibited deficits and
damped rhythmicity in SCN expression of BDNF that were accompanied by
corresponding decreases in the amplitude of light-induced phase shifts
during the subjective night. In agreement with this observation, SCN
infusion of the tyrosine kinase inhibitor K252a blocked or abated both
phase delays and advances of the rat activity rhythm in response to
light during the subjective night. In conjunction with previous
evidence of the close association of BDNF-expressing cells in the
ventral SCN with TrkB-immunoreactive fibers emanating from the optic
chiasm and of the circadian expression of BDNF within the SCN (Liang et
al., 1998a,b), these findings suggest that BDNF may play an important
role in the time-dependent regulation of circadian rhythms by light.
Although "nonphotic" stimuli such as dark pulses, induced activity,
social cues, and neuropeptide Y are known to induce phase advances of
rodent rhythms directly during the subjective day (Boulos and Rusak,
1982; Albers and Ferris, 1984; Mrosovsky, 1988; Reebs and Mrosovsky,
1989), BDNF infusion in the present study is distinguished by its
effect in engendering large phase shifts in response to light during
this portion of the circadian cycle. Importantly, acute treatment with
BDNF alone during the midsubjective day had no phase-resetting action
on the rat activity rhythm, indicating that this neurotrophin did not
induce the observed phase advances at this circadian time by unmasking
the influence of nonphotic inputs to the SCN. Comparison of SCN
profiles of BDNF content in control and experimental animals may have
some implications for the effect of BDNF infusion on light-induced phase shifts during the subjective day. In particular, the infusion paradigm does not appear to deliver pharmacological doses of BDNF to
the SCN but instead maintains levels consistently within the range of
those found in control animals during the subjective night. This
infusion-related increase in BDNF levels during the subjective day may
effectively gate photic input to the SCN and its phase-shifting action
on circadian rhythms by achieving a critical threshold that normally is
attained only during the subjective night via endogenous rhythmic
amplification of BDNF-mediated signaling. Despite its effects on photic
regulation of the pacemaker during the subjective day, BDNF infusion
did not potentiate the phase-shifting effect of light during the
subjective night perhaps because TrkB receptors in the SCN region are
already saturated by the peak expression of endogenous ligand that
occurs over this interval (Liang et al., 1998a). Alternatively, this
treatment, combined with SCN expression of BDNF, may downregulate TrkB
receptor expression in a manner similar to that observed within the
midbrain and olfactory bulb (Venero et al., 1994; Frank et al., 1997),
thereby obstructing further augmentation of light-induced phase delays
or advances during the subjective night.
The converse analysis using BDNF-deficient mutant mice yielded
supportive observations on the functional relation between this
neurotrophin and photic regulation of the SCN pacemaker. In
bdnf+/ mice, phase-shifting
responses to light during the subjective night were diminished in
association with abated BDNF levels and rhythmicity in the SCN. It is
noteworthy that this distinction in SCN circadian function of
bdnf+/ mice was marked by a
greater reduction in light-induced phase advances than delays. In
accord with previous observations on the relationship between phase
response curves to light and circadian period (Daan and Pittendrigh,
1976), this pattern of smaller advances than delays in response to
light was correlated with the shorter free-running period of activity
rhythm in mutant mice. Although the influence of developmental
abnormalities associated with BDNF deficits cannot be excluded, it is
important to note that no gross alterations in the histological
organization, ganglion cell number, and synaptogenesis of the neural
retina or in the structural organization of the CNS have been observed
in homozygous BDNF knock-out mice (Jones et al., 1994; Rickman et al.,
1998). Moreover, our preliminary analysis has produced accordant
observations indicating that basic anatomical and histochemical
organization of the SCN are indistinguishable between wild-type and
bdnf+/ mice. Therefore, it seems
unlikely that the decreased phase-shifting responses to light in
bdnf+/ mice are merely
attributable to developmental defects in visual system function or SCN organization.
Involvement of tyrosine kinase receptors in the photic regulation of
SCN circadian function is supported by the present finding that
light-induced phase shifts of the rat activity rhythm during the
subjective night are completely blocked or attenuated by K252a infusion. TrkB, the cognate receptor for BDNF, is a likely target for
K252a action in suppressing phase-shifting responses to light because
our anatomical studies demonstrate the localization of TrkB
immunoreactivity in the ventral SCN on both fibers emanating from the
optic chiasm and cells bordering the chiasm (Liang et al., 1998b).
Because this TrkB expression appears to be derived from RHT fibers and
SCN cells receiving RHT input, K252a may act to inhibit the peak
activity of BDNF during the subjective night on these TrkB receptors
and thereby block the endogenous gate for light to induce phase shifts.
Importantly, this postulated mechanism of K252a inhibition of photic
input and its resetting action on the SCN pacemaker is compatible with
recent evidence indicating that this compound blocks BDNF-mediated
potentiation of synaptic transmission within other brain regions (Kang
and Schuman, 1995; Levine et al., 1995; Akaneya et al., 1997;
Carmignoto et al., 1997).
Although these findings establish a role for BDNF in the photic control
of circadian rhythms, the mechanism by which this neurotrophin mediates
the gating of light input and its phase-shifting effects on the SCN
pacemaker is unclear. In the visual cortex and hippocampus, BDNF
appears to potentiate neuronal signaling by enhancing transmitter
release from presynaptic neurons or the sensitivity of postsynaptic
receptors (Levine et al., 1995; Carmignoto et al., 1997; Tanaka et
al., 1997). Thus, BDNF regulation of circadian pacemaker responses to
light signals may involve similar mechanisms of action on specific
neurotransmitter systems that are critical for SCN circadian function.
Because glutamate is the primary neurotransmitter of the RHT and mimics
the phase-shifting effects of light (Liou et al., 1986; van den Pol,
1993; Shirakawa and Moore, 1994), BDNF could modulate pacemaker
responses to photic input via presynaptic enhancement of glutamate
release from RHT fibers or postsynaptic modulation of glutamate
receptor responsiveness on SCN cells receiving RHT input. This possible
action of BDNF in potentiating glutamatergic input to the SCN is
consistent with evidence of BDNF enhancement of AMPA and NMDA
receptor-mediated synaptic transmission in hippocampal and visual
cortex neurons (Le mann et al., 1994; Carmignoto et al., 1997). Our
reported observations on the close association between BDNF-expressing
cells within the ventral SCN and TrkB receptors on fibers emerging from
the optic chiasm and on neighboring cells (Liang et al., 1998b)
indicate that the primary ligand and receptor components for BDNF
signal transduction are distributed in the appropriate anatomical
relationship. Moreover, BDNF-mediated potentiation of glutamatergic
neurotransmission would provide an applicable explanation of its
observed effect in enabling light to induce phase advances during the
subjective day because glutamate administration into the SCN at this
circadian time similarly produces comparable advancing shifts of the
hamster activity rhythm (Meijer et al., 1988). GABA is another suitable
target for the potential action of BDNF in regulating the
neurotransmission of photic signals and their effects on the SCN
pacemaker because (1) BDNF has been shown to reduce IPSCs
markedly and to attenuate GABAA receptor-mediated responses to applied GABA in rat hippocampal neurons (Tanaka et al.,
1997). (2) Also GABA is the predominant neurotransmitter in the SCN
(Moore and Speh, 1993), and GABAergic agents alter the phase-shifting
effects of light on circadian rhythms (Ralph and Menaker, 1986, 1989;
Golombek and Ralph, 1994). Consequently, BDNF could act via multiple
transmitter systems to regulate the circadian resetting of the SCN
pacemaker by light.
An alternative possibility is that BDNF could indirectly regulate the
phase-shifting effects of light by modulating local cellular
interactions or coupling between SCN cells. Throughout the medial SCN,
most BDNF-expressing cells are located adjacent to TrkB-immunopositive
perikarya (Liang et al., 1998b). Because the SCN contains many cells
capable of independent pacemaker function (Welsh et al., 1995), this
finding suggests that local paracrine interactions between BDNF and
TrkB receptors could provide an endogenous signaling pathway for the
intercellular regulation of cellular and metabolic activity within the
SCN and thus for pacemaker cell synchronization. Importantly,
BDNF-mediated changes in pacemaker cell synchronization would be
expected to alter and hence modify responses to light. Possible
involvement of BDNF in the coupling of SCN pacemaker cells is
compatible with the present finding that diminished BDNF expression and
rhythmicity in the SCN of heterozygous BDNF knock-out mice are
accompanied by decreases in and light-induced phase shifts relative
to that of wild-type mice.
| |
FOOTNOTES |
Received Nov. 30, 1999; revised Jan. 18, 2000; accepted Feb. 1, 2000.
We thank Rodney Walline for excellent technical assistance.
Correspondence should be addressed to Dr. David J. Earnest, Texas A&M
University Health Science Center, Department of Human Anatomy and
Medical Neurobiology, 238 Reynolds Medical Building, College Station,
TX 77843-1114. E-mail: dearnest{at}tamu.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
