Evidence for a role of GABA and Mas-allatotropin in photic entrainment of the circadian clock of the cockroach Leucophaea maderae
1 Institut für Zoologie/Biologie I, Universität Regensburg, 93040
Regensburg, Germany
2 Fachbereich Biologie, Tierphysiologie, Philipps-Universität Marburg,
35032 Marburg, Germany
* Author for correspondence at address 2 (e-mail: stengl{at}mailer.uni-marburg.de
Accepted 5 March 2002
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Summary |
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Key words: circadian rhythm, locomotor activity, light entrainment, cockroach, insect, Leucophaea maderae, Mas-allatotropin, -aminobutyric acid, GABA, neuropeptide
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Introduction |
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More recent evidence suggests that the accessory medulla, a small neuropil
in the region where the lesion studies had located the circadian pacemaker, is
the actual site of the internal clock in the cockroach and in other insects
(Homberg et al., 1991;
Stengl and Homberg, 1994
;
Helfrich-Förste, 1995
;
Frisch et al., 1996
;
Petri et al., 1997
;
Petri and Stengl, 1997
;
Kaneko et al., 1997
;
Reischig and Stengl, 1998
;
Helfrich-Förster et al.,
1998
). The organization of the accessory medulla of L.
maderae into noduli, an internodular region and loose neuropil around the
accessory medulla has been studied in detail
(Petri et al., 1995
;
Reischig and Stengl, 1996
).
Also, neuronal systems appropriate to form input and output pathways of the
circadian system have been identified
(Stengl and Homberg, 1994
;
Petri et al., 1995
;
Reischig and Stengl, 1996
;
Petri and Stengl, 1997
). One
of these pathways, the distal tract, is currently the best candidate for a
photic entrainment pathway because it appears to connect the distal medulla
and/or the lamina to the noduli of the accessory medulla
(Reischig and Stengl,
1996
).
The prominent arborizations of the distal tract in the noduli of the
accessory medulla suggest that photic information might be processed in the
noduli. Thus, in immunocytochemical studies, we searched for a
neurotransmitter candidate in the distal tract
(Petri et al., 1995). In
addition, we searched for neurotransmitter/neuropeptide candidates of neurons
with dense arborizations in the noduli of the accessory medulla, the
presumptive photic processing area. In a previously published report, we
showed that Mas-allatotropin-immunoreactive neurons densely innervate the
noduli (Petri et al., 1995
)
and, therefore, are candidates for processing photic information received from
the distal tract. In contrast, injection experiments and computer modelling
studies show that pigment-dispersing-hormone-immunoreactive neurons, which
arborize in the internodular neuropil, are involved in non-photic entrainment
of the clock (Petri and Stengl,
1997
,
2001
).
In the present account, we show that antibodies against GABA recognize the
distal tract and that immunostaining is particularly prominent in the noduli
of the accessory medulla. Immunostained axons of the distal tract connect the
lamina and medulla to the accessory medulla. In addition, we show that
injections of GABA and Mas-allatotropin into the vicinity of the accessory
medulla lead to biphasic phase shifts in circadian wheel-running activity
similar to those induced by light. This suggests that both transmitters are
involved in circuits relaying photic information to the clock. Preliminary
results of this study have appeared previously as abstracts (Petri et al.,
1997,
1999
;
Petri and Stengl, 1998
).
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Materials and methods |
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The free-running period and the induced phase shifts were estimated
by converting the raw data into ASCI format. They were then merged into 20 min
intervals and analyzed with Chrono II software (generously provided by Dr Till
Roenneberg; see Roenneberg and Morse,
1993
) on a Macintosh computer. Data were evaluated from 128 of the
172 animals used. The remaining 44 animals were excluded from further analysis
because they showed little activity after the injection or died within a week
following the operation.
The free-running periods () before and after the injection (see below)
were calculated by linear regression through daily activity onsets and by
2 periodogram analysis
(Sokolove and Bushell, 1978
;
Enright, 1965
). Changes in
(
=
after
before) were
calculated, with periods estimated by regression through activity onsets.
After the injections, activity onsets varied transiently until the animals
resumed stable activity rhythms. These transients were excluded from
calculations of phase and period. Phase shifts (
) were determined
as time differences between the regression lines before and after the
injection extrapolated to the day of treatment. The phase shifts were then
normalized with respect to
before the treatment. Phase delays were
plotted as negative values and phase advances as positive values. Time on the
x-axis of the resulting phase response curve is shown as circadian
time (CT), with CT12:00h=activity onset=beginning of the subjective night.
Daily activity onsets were determined using Chrono II
(Roenneberg and Morse,
1993
).
The microinjection data were merged into 2h time intervals, and the mean, the standard error of the mean (S.E.M.) and the standard deviation (S.D.) were calculated. Changes in phase and period in a given time interval were considered to be significantly different from zero when the calculated 95 % confidence interval (95 % CI, Table 1, values superscripted b) of the respective time interval did not contain the value zero. The phase response curves were analyzed by one-way analysis of variance (ANOVA) with a Scheffé multiple-range test. Significance in all cases was taken as P<0.05. Statistical analyses were performed with SPSS (Superior Performing Software Systems; SPSS Inc.) on a personal computer. To analyze the statistical significance of the phase-dependence (Table 1, values superscripted c, e, g) and to test whether transmitter-dependent phase shifts were significantly different from control values (Table 1, values superscripted a, see asterisks in Fig. 5), we used one-way ANOVA in combination with a Scheffé multiple-range test. Smoothed phase response curves were produced with Excel.
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Operation and injection
All manipulations were performed in dim red light at 25 °C with a
microinjector (Microinjector 5242, Eppendorf, Hamburg, Germany). Experimental
animals were removed from the running wheels and mounted in metal tubes at
different times during the circadian cycle. After anaesthetization with
CO2, a small window was cut in the head capsule of the cockroach,
and the optic lobe was revealed by moving the trachea, ocellus and fat body
carefully to one side. The neurolemma of the optic lobe was penetrated with a
borosilicate glass capillary whose tip was broken to give a tip diameter of
2-3 µm (Clark, Pangbourne Reading, UK). Great care was taken to
pressure-inject, under visual control, Masallatotropin or GABA very close to
the accessory medulla into one medulla. After injection, the removed piece of
cuticle was waxed back and the animal was returned to the running wheel. A
treatment typically lasted 6-12 min. Only one optic lobe was injected; the
other one was left intact. All experimental animals were kept under constant
conditions (DD) before, during (red light=DD) the injection.
Injections (N=128) consisted of 10-4 to 10-2
moll-1 GABA (Sigma) and 10-4 moll-1 synthetic
Mas-allatotropin (Sigma). The injected volume ranged from 0.5 to 2 nl
(1.50.6 nl; mean ± S.D.). Thus, injected doses ranged from 0.15 to
15 pmol. These doses were chosen because similar doses were effective in
previous experiments (Petri and Stengl,
1997
). The solutions contained 10 % aqueous blue food dye
(McCormic, Baltimore, USA) to visualize the exact site and spread of the
injection without the need for further neuroanatomical processing of the
brain. Each micropipette was calibrated by measuring the injected volume.
Before and after the injection, a test pulse was injected into mineral oil to
control for changes in tip diameter during penetration of the neurolemma.
Control injections consisted of 10 % aqueous blue food dye without neuroactive
substances.
Immunocytochemistry
Immunocytochemistry of GABA was performed on free-floating Vibratome
sections and on paraffin sections by means of the indirect
peroxidase/antiperoxidase (PAP) technique
(Sternberger, 1979). Brains
were dissected out of the head capsule under glutaraldehyde fixative
(Boer et al., 1979
), fixed for
another 2 h and rinsed in phosphate buffer. For immunostaining of paraffin
sections, brains were subsequently dehydrated through a graded series of
aqueous ethanol solutions and toluene and embedded in Paraplast Plus (Monoject
Scientific, St Louis, MO, USA). Sections at 8 µm were cut with a rotary
microtome and processed for GABA immunoreactivity as described previously
(Homberg et al., 1987
). For
immunostaining of Vibratome sections, brains were fixed as described above and
embedded in gelatin/albumin. Sections of 25 µm thickness were made with a
Vibratome (Technical Products, St Louis, MO, USA).
The immunocytochemical staining protocol was performed as described by
Homberg (1991). Secondary
antiserum (goat-anti rabbit; Sigma, Deisenhofen, Germany) was used at a
dilution of 1:40 and rabbit PAP (Dako, Hamburg, Germany) at a dilution of
1:300. The anti-GABA antiserum was provided by Dr T. G. Kingan (University of
Arizona, Tuscon, AZ, USA). The antiserum was raised in rabbit against a
conjugate of GABAglutaraldehydekeyhole limpet haemocyanin. Its
specificity has been characterized by Hoskins et al.
(1986
). The antiserum was used
at a dilution of 1:40 000 on Vibratome sections and at 1:1500 on paraffin
sections.
Fibre tracts and neuropil structures immunostained for GABA were reconstructed from serially stained sections with the aid of a camera lucida attachment on a Leica compound microscope. Photographic images were captured using a Polaroid DMC digital camera linked to a Pentium II computer. Images were processed using Adobe Photoshop and Corel Draw software. Fig. 1A,B are overlaid images from adjacent areas of the same section (Fig. 1A) and from the same area of two consecutive sections (Fig. 1B). Images were printed with an Epson Stylus Photo ink jet printer.
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Results |
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GABA immunostaining
GABA immunostaining was detected in a large number of neurons in the optic
lobe of L. maderae, and immunostained somata were distributed
throughout the cortex of the optic lobe
(Fig. 1). All three optic
neuropils were innervated by GABA-immunoreactive (GABA-ir) processes. A median
layer of the medulla exhibited particularly intense immunostaining
(Fig. 1A,C). Approximately 25
small immunostained cell bodies in the vicinity of the accessory medulla sent
primary neurites into the accessory medulla (Figs
1B,C,
2A). The nodular neuropil of
the accessory medulla showed strong granular immunostaining. Two bundles
composed of at least 20 densely packed GABA-ir axons left the accessory
medulla, joined and entered the distal tract along the distal surface of the
medulla towards the lamina (Figs
1B,C,
2A). Along the surface of the
medulla, GABA-ir processes gradually left the distal tract in several
fascicles that entered the medulla neuropil at right angles
(Fig. 1C). Because of the large
number of GABA-ir neurons in the optic lobe, the branching pattern of these
processes in the medulla could not be elucidated.
|
In addition to small cell bodies, a large GABA-ir neuron in the accessory
medulla could be reconstructed individually
(Fig. 2B). From a cell body
adjacent to the accessory medulla, its primary neurite projected through the
accessory medulla into the medulla, but not via the distal tract. One
set of branches innervated the medulla, another set of six neuronal fibres
projected over the frontal surface of the medulla and entered the posterior
face of the lamina. Varicose processes throughout the lamina also extended to
and branched within small neuropil structures adjacent to the posterior lamina
termed accessory laminae (Loesel and
Homberg, 2001). A side branch of the neuron also invaded the
accessory medulla, but the full extension of these ramifications could not be
distinguished from other GABA-ir processes.
Effects of GABA and Mas-allatotropin injections on the phase of the
circadian locomotor activity rhythm
To investigate whether GABA and Mas-allatotropin are input signals to the
circadian clock, we examined whether they phase-shift the rhythm of locomotor
acitivity of the cockroach. Both substances were injected into the vicinity of
the accessory medulla, and the locomotor activity of the animals was recorded
before and after the injections. Control injections of 10 % aqueous blue food
dye (N=43) did not cause significant phase changes in circadian
locomotor rhythms irrespective of the circadian time of the injections
(P=0.69, one-way ANOVA) (Table
1). Injections of GABA (N=35) and synthetic
Mas-allatotropin (N=36) into the medulla resulted in time-dependent
phase shifts in the circadian activity rhythm of L. maderae (Figs
3,4,5,6;
Table 1). Maximal phase delays
occurred when GABA (-4.2 h) and Mas-allatotropin (Mas-At -4.9 h) were applied
early in the subjective night (GABA, CT14:50 h; Mas-At, CT14:05 h), and
maximal phase advances (GABA, 3.05 h; Mas-At, 3.2 h) occurred in response to
injections in the middle of the subjective night (GABA, CT16:50 h; Mas-At,
CT17:09 h). Examination of the 95 % confidence interval (CI, see Materials and
methods) for the phase shifts in 2 h bins indicates that significant GABA- and
Mas-allatotropin-dependent phase delays occurred at CT08:00-16:00 h, while
significant phase advances occured at CT18:00-22:00 h (values labelled b in
Table 1; asterisks in
Fig. 5). No statistically
significant phase shifts occurred during the rest of the cycle. The
phase-dependence was statistically significant (P<0.00005, one-way
ANOVA) since GABA- and Mas-allatotropin-dependent phase delays at
CT12:00-14:00 h (c in Table 1)
and CT14:00-16:00 h (e in Table
1) as well as phase advances at CT18:00-20:00 h (g in
Table 1) were significantly
different from GABA- and Mas-allatotropin-dependent phase shifts during other
times of the circadian cycle (d, f, h, respectively in
Table 1). In addition, phase
shifts induced by GABA at CT12:00-16:00 h and CT18:00-20:00 h and
Mas-allatotropin injections at CT14:00-16:00 h and CT18:00-20:00 h were
significantly different from control phase shifts
(Table 1). Effects of control
injections at different times were in no case significantly different from
those at 0 h (Table 1). In
nearly all experiments, the new phase relationship was reached after 14 days
of transients with variable periods of apparent unstable activity onset
(Fig. 3A,C), and no differences
in transition times were observed for phase delays and phase advances.
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Effects of GABA and Mas-allatotropin injections on the period of the
circadian locomotor activity rhythm
After the injection of saline, GABA or Mas-allatotropin, the free-running
period of individual cockroaches did not change significantly
(Fig. 3;
Table 2). Observed changes in
period were always small, included both lengthening (maximally -0.41 h) and
shortening (maximally 0.48 h) of the period, and were independent of the time
of injection in the circadian cycle. Thus, the mean period of
23.46±0.18 h (mean ± S.D.; N=124) was not altered
(Table 2) by any treatment.
|
Dose-dependency of GABA-dependent phase shifts
GABA-induced phase delays in circadian wheel-running activity at
CT12:00-16:00 h were positively correlated with the dose injected. Injections
of 0.15 pmol of GABA caused phase delays of -1.5±0.21 h (95 % CI=-2.00
to -1.11 h, N=10), while injections of 15 pmol of GABA resulted in
phase delays of -2.4±0.33 h (95 % CI=-3.10 to -1.67 h, N=11).
Only phase delays induced by 15 pmol of GABA had a significantly different
effect (P<0.05, one-way ANOVA) from control injections
(-0.49±0.22h, 95 % CI=-0.99 to 0.01 h, N=9) at the same
circadian time (P=0.85, one-way ANOVA, Scheffé multiple-range
test). Both 15 pmol of GABA and 150 fmol of Mas-allatotropin caused phase
delays that were significantly different from control injections at the same
circadian time (P<0.05, one-way ANOVA, Scheffé
multiple-range test). A 100-fold higher dose of GABA compared with
Mas-allatotropin was required to induce the same phase-shift.
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Discussion |
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Specificity of Mas-allatotropin and GABA-dependent phase shifts
The phase shifts of locomotor activity rhythm in the subjective night are
specifically dependent on GABA and Masallatotropin because they are
dose-dependent and significantly different from phase shifts in response to
control injections. In addition, the phase response curves obtained differ
from curves produced by pigment-dispersing hormone (PDH) or serotonin
injections (Page, 1987;
Petri and Stengl, 1997
). Thus,
the type of phase response curve obtained depends on the neuroactive substance
injected. The 100-fold difference in sensitivity of the circadian system for
Mas-allatotropin and PDH injections versus GABA injections might be a
consequence of the longer half-life of neuropeptides compared with GABA.
Alternatively, the difference in sensitivity might reflect differences in
affinity between GABA and neuropeptide receptors for their ligands.
Mas-allatotropin has been sequenced from the sphinx moth Manduca
sexta (Kataoka et al.,
1989), and a related peptide, termed Lom-AG-myotropin-I, has been
identified in the locust Locusta migratoria
(Paemen et al., 1991
) and in
the beetle Leptinotarsa decemlineata
(Spittaels et al., 1996
).
These peptides are probably members of a larger insect family of
allatotropin-related peptides, and their wide distribution throughout the
nervous systems of different insect species, including L. maderae,
has been suggested by immunocytochemistry
(Paemen et al., 1992
;
it
an et al.,
1993
; Veenstra and Hagedorn,
1993
; Würden and Homberg,
1995
; Petri et al.,
1995
). The phase response curve obtained with Mas-allatotropin in
L. maderae strongly suggests that a related peptide is released by
neurons in the circadian system of this insect. Because the only
Mas-allatotropin-ir neurons that innervate the accessory medulla are local
neurons with arborizations restricted to the noduli of the accessory medulla,
we hypothesize that these neurons release a Mas-allatotropin-related peptide
in response to light. Thus, we assume that light input reaches the accessory
medulla via pathways that arborize in the noduli.
Neuronal pathways for light entrainment of the circadian pacemaker of
the cockroach
Evidence is increasing that the accessory medulla with associated
PDH-immunoreactive (PDH-ir) neurons is the site of the circadian clock in the
cockroach as well as in Drosophila melanogaster
(Homberg et al., 1991;
Stengl and Homberg, 1994
;
Helfrich-Förster, 1995
;
Frisch et al., 1996
;
Petri et al., 1997
;
Petri and Stengl, 1997
;
Kaneko et al., 1997
;
Reischig and Stengl, 1998
;
Helfrich-Förster et al.,
1998
). In D. melanogaster, PDH-ir neurons contain the
clock molecules PERIOD and TIMELESS and are circadian pacemaker candidates
(Helfrich-Förster, 1995
;
Kaneko and Hall, 2000
;
Kaneko et al., 1997
). In the
cockroach L. maderae, we also found PERIOD-immunoreactivity in cells
next to the accessory medulla, at the location of PDH-ir neurons
(Stengl et al., 2001
).
However, it is not known how light entrainment information reaches the PDH-ir
pacemaker candidates in the cockroach, while several parallel pathways are
known in the fruit fly
(Helfrich-Förster et al.,
2001
). Photoreceptors of the circadian system of L.
maderae occur in or near the compound eyes, but the light entrainment
pathway to the pacemaker has not been identified
(Roberts, 1965
;
Nishiitsutsuji-Uwo and Pittendrigh,
1968b
; Wiedenmann,
1977b
; Page,
1978
). Page (1978
,
1983b
) proposed inputs into
the pacemaker by ipsi- and contralateral light-entrainment pathways as well as
input of phase information by the contralateral pacemaker via a
coupling pathway. Recent backfill experiments strongly suggest that some of
the PDH-ir neurons form a direct circadian coupling pathway
(Reischig and Stengl, 2001
).
Because of this apparently direct coupling pathway in the cockroach, a single
injection of neurotransmitter and neuropeptide into one pacemaker region may
well cause transient changes in activity onsets over several days before a
stable, new phase relationship between both pacemaker centres is reached.
In addition to the circadian coupling pathways relaying phase information
from the contralateral pacemaker, cells projecting via the posterior
optic tract with projections in the ipsi- and contralateral accessory medulla
and ipsi- and contralateral medulla might bring contralateral light input into
the clock (Reischig and Stengl,
2001; Loesel and Homberg,
2001
). A recent study on histamine immunostaining by Loesel and
Homberg (1999
) demonstrated
that photoreceptor axons of the compound eye do not enter the accessory
medulla of L. maderae directly, but terminate in the lamina and in
distal layers of the medulla. This indicates that there is no direct
photoreceptor input to the clock. Our data presented here suggest that
GABAergic neurons in the distal tract might be the missing link between
compound eye photoreceptors and the accessory medulla neurons. Although this
hypothesis is the most straightforward interpretation for both the
immunocytochemical and injection data, we cannot, at present, rule out the
additional possibility that GABA, like Masallatotropin, acts in local
circuits, which would affect an unknown photic input to the clock. A second
photic input pathway to the clock, distinct from the distal tract, could be
provided by the single large-field GABA-ir neuron
(Fig. 2B). This neuron appears
to connect the medulla to the accessory medulla, the lamina and the accessory
laminae. Intracellular recordings showed that neurons with close similarity to
this cell respond strongly to light, as would be expected for neurons involved
in photic entrainment of the clock (Loesel
and Homberg, 2001
).
In contrast to GABA, Mas-allatotropin immunoreactivity was found in 20-30
intrinsic neurons of the accessory medulla
(Petri et al., 1995). These
local neurons extend a dense network of varicose terminals throughout the
noduli of the accessory medulla, which overlaps with GABA immunostaining
(Petri et al., 1995
). We
cannot at present rule out the possibility that GABA and Mas-allatotropin are
co-localized in some local neurons. However, striking differences in the
morphological appearance of the two staining patterns, in particular the large
number of GABA-ir axons in the distal tract and the fasciculated
(Mas-allatotropin) versus non-fasciculated (GABA) entry of primary
neurites into the accessory medulla, make it unlikely that the GABA- and
Mas-allatotropin-immunoreactive neurons are identical. Thus, only
immuno-electronmicroscopic studies will allow us to distinguish whether most
of the GABA-ir neurons that form an input into the accessory medulla originate
from the distal tract or from local GABA-ir neurons. Finally, Fleissner et al.
(2001
) recently described two
putative extraocular photoreceptor organs in the cockroach optic lobe, the
lamina and lobula organs, and proposed a role for these organs in light
entrainment of the circadian clock. At present, however, physiological
evidence for a photoreceptor role for these organs and for neuronal
connections to the clock have not been demonstrated.
Since injections of low doses of GABA and Mas-allatotropin were directed towards the vicinity of the accessory medulla, it is likely that the resulting phase shifts were caused by neurons postsynaptic to the GABA- and Mas-allatotropin-immunoreactive networks in the noduli of the accessory medulla. But it cannot be excluded that, in addition to neurons innervating the noduli of the accessory medulla, other unknown neurons elsewhere in the optic lobes with connections to the accessory medulla might contribute to the phase shifts observed.
The similarity of the phase response curves obtained for light pulses and
for GABA and Mas-allatotropin injections
(Fig. 6) suggests that both
substances are released in response to light
(Petri and Stengl, 2001),
possibly within the noduli of the accessory medulla. In contrast, injections
of PDH, a presumptive circadian coupling signal, revealed a monophasic
non-photic phase response curve (Petri and
Stengl, 1997
). Computer modelling of a molecular oscillator shows
that biphasic and monophasic phase response curves result from disturbance to
different, specific variables in the model molecular clock
(Petri and Stengl, 2001
). Our
oscillator model confirms that substances that produce light-like phase
response curves are likely to be involved in circuits relaying photic
information to the clock (Petri and
Stengl, 2001
). Therefore, our complementary analysis of GABA and
Mas-allatotropin immunostaining associated with the presumptive circadian
pacemaker centre provides important information about the neuronal pathways
through which light acts to reset the circadian clock in the cockroach. It
still remains to be shown which neurons convey light information from the
noduli of the accessory medulla to the internodular neuropil of the accessory
medulla, where PDH-ir neurons, the presumptive circadian pacemaker candidates
and circadian outputs arborize.
This study suggests that the light entrainment pathway of L.
maderae might be as complex as that for light entrainment in mammals
(Ralph and Menaker, 1985,
1986
,
1989
;
Hastings et al., 1991
;
Aronson et al., 1993
;
Ding et al., 1994
;
Gillespie et al., 1997
;
Wagner et al., 1997
;
van Esseveldt et al., 2000
).
As in the mammalian system, light entrainment involves more than one
neurotransmitter and consists of more than direct, unmodulated transmission of
photic information from photoreceptors to the pacemaker in the accessory
medulla. Future studies will examine the nature of circadian photoreceptors,
parallel entrainment pathways and the types of receptor involved in the
transmission of photic information to the circadian pacemaker in the
cockroach. In addition, we have begun an immuno-electronmicroscopic analysis
of the neuronal network of the accessory medulla.
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Acknowledgments |
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References |
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