Ectopic transplantation of the accessory medulla restores circadian locomotor rhythms in arrhythmic cockroaches (Leucophaea maderae)
Biology, Animal Physiology, Philipps Universität Marburg, Karl von Frisch Str., D-35041 Marburg, Germany
* Author for correspondence (e-mail: stengl{at}staff.uni-marburg.de)
Accepted 11 March 2003
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Summary |
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Key words: circadian rhythm, accessory medulla, locomotor activity rhythm, pigment-dispersing hormone neuron, pacemaker, cockroach, Leucophaea maderae
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Introduction |
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Materials and methods |
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Surgery
All operations were performed on male cockroaches under steady
CO2 anaesthesia. Cell culture medium (L 15; GIBCO, Eggenstein,
Germany) containing penicillin and streptomycin was used to rinse the wounds.
For the initial left optic lobe (OL) section, a triangular cuticular flap was
cut into the head capsule to expose the OL. With an iridectomy scissor, the
optic nerves and the optic stalks were cut, and the OL was removed; the
cuticle was flapped back in place and sealed with wax.
For transplantation of AMe tissue into the right antennal lobe (AL) of a host cockroach (Fig. 1B), its right brain hemisphere was exposed. With a razorblade fragment, a pocket was cut into the right AL. Then, the donor animal was decapitated, its brain exposed, and the perineurium of one OL was removed. According to external markers, tissue containing the AMe with its adjacent PDH-ir cells (in controls: tissue out of the adjacent medulla) was excised from the donors brain with a fine glass pipette (tip-Ø, 150250 µm). The tip of the pipette was stuck into the AL of the host animal, the graft tissue was carefully blown out and occasionally its position was corrected with an eyebrow hair. Then, the right OL was removed; the cuticle was flapped back in place and sealed with wax. In the OL-to-OL transplantations, animals were donors and host at the same time (Fig. 1A) and the AMe graft was implanted into the location of the host's removed AMe.
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Activity analysis
Locomotor activity was monitored in running-wheels in constant darkness at
26°C as described previously (Stengl
and Homberg, 1994). Activity was visualized with double-plot
activity histograms; the heights of the bars represent the number of
revolutions per 5 min, truncated at 30 revs min1
(Stengl and Homberg, 1994
). To
distinguish rhythmic from arrhythmic locomotor activity (Figs
2,
3) we used
2-periodograms and mass entropy spectral analysis (MESA;
Dowse and Ringo, 1989
) and
averaged locomotor activity plots per circadian day to scrutinize rhythmic
data obtained. For a more objective, automated judgement of rhythmicity, we
developed a new software in Visual Basic for Applications (VBA); the `scan
periodogram analysis with Rhythm-Detector' allows distinction between rhythmic
and arrhythmic episodes in long data sets. Raw data were merged into 30-min
intervals and converted into Excel 97 format.
2-periodograms
were calculated with VBA according to the algorithms of Sokolove and Bushell
(1978
). The scan periodogram
analysis was performed as follows. Over a defined single periodogram length
(s.p.l.) of at least 8 days, the program calculated a periodogram from day 1
to day X, then from day 2 to day X+1, until the last day of
the recording. For every periodogram, the software determined the maximum
(Qp = peak height), the period
, the
2 for P=0.01 at
, and the width of the peak [at
the intersections with the Sokolove significance line (SSL;
2
for P=0.01)]. To normalize Qp against
2, the quotient Qp/
2 was
calculated and plotted against the number of the starting day of the
respective periodogram on the data record
(Qp/
2 curve in
Fig. 3C). The according
normalised
2 value is 1 and was plotted as well
(
2 for P=0.01 in
Fig. 3C).
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To distinguish `rhythmic' from `arrhythmic' periodogram peaks, we averaged
peak values from optic lobe-less cockroaches with no recognizable
periodicities (see next paragraph). This analysis revealed 26.6% of arrhythmic
periodogram peaks exceeding the SSL. These peaks had low peak heights
(9.2±7.8%, mean ± S.D., with 0%=2 and
100%=2x
2) and narrow widths (0.3±0.1 h) as
compared with periodogram peaks calculated over rhythmic activity phases.
Therefore, only peaks with heights of
20% and widths of
0.7 h, well
above the values of arrhythmic periodogram peaks, were chosen to indicate
rhythmicity. In this case, an arbitrary value of 0.5 was assigned to the
`rhythmicity' curve of the Rhythm-Detector plot; otherwise the value was set
to zero (Fig. 3C). Hence, a
locomotor record was judged as rhythmic if the postoperative scan periodogram
analysis revealed at least one peak with a peak height of
20% together
with a peak width of
0.7 h for at least two consecutive days
(Fig. 3C). With MESA
(Dowse and Ringo, 1989
; with
use of a demo version of El Temps 1.172, a chronobiological evaluation program
written by Antoni Díez-Noguera, Barcelona, Spain) and averaged
locomotor activity plots per circadian day, we confirmed rhythmicity in data
sets obtained with our new evaluation software. Thus, we obtained a reliable,
new, automated analysis method for long data records with objective measures
of short episodes of rhythmicity, which avoided subjective selection of data
sets and misjudgement of randomly generated activity peaks.
For the averaged activity plots, the circadian period lengths of the
activity episodes in question were determined. The activity values were
processed into a matrix using the same algorithms as for periodogram analysis
(Sokolove and Bushell, 1978),
with the determined period lengths as the test period. The means ± S.D.
of activity amounts for every 30 min bin representing the same circadian time
were calculated. The circadian day was normalized to 24 h. The activity onset
of the first day of the examined activity episode was used as phase reference
point and set to CT 12.
Analysis of `arrhythmic' periodogram peaks
To analyse height and width of periodogram peaks in actual arrhythmic data
records, we selected 30 bilobectomised, arrhythmic animals (as judged by eye
on activity histograms and selective 2-periodogram analysis).
Using the scan periodogram analysis, we calculated consecutive 10-day
2-periodograms (day 1 to 10, day 2 to 11, etc.;
N=3445 for all animals) over the whole postoperative data record. The
means ± S.D. of the heights and widths of all periodogram peaks that
exceeded the SSL were calculated for every animal. Then, the total mean, as
well as the mean of the standard deviations, for all 30 animals was
calculated.
Moreover, we performed the periodogram peak analysis described above on randomly permutated activity data records (with Monte Carlo simulations) of 10 untreated, free-running cockroaches with prominent circadian rhythmic locomotor activity. The data records were about 10 weeks long, and every record was randomised and subsequently analysed 10 times. This resulted in a total of 7020 single periodograms, of which 88.1% showed peaks exceeding the SSL. The median height of these peaks was 10.2±9.8%, and the median width was 0.2±0.1 h; these values are within the range obtained by the respective evaluation of the generically arrhythmic animals and, therefore, further support the rhythmicity threshold selected for the Rhythm-Detector. Interestingly, the number of periodogram peaks exceeding the SCL obtained with the randomly permutated data was much higher than in the arrhythmic animals. This is apparently due to a more even distribution of activity over the whole data record after the randomisation compared with generically arrhythmic animals. This results in a more even distribution of the Qp values just below the SCLs in the periodograms and, therefore, leads to a higher probability of single Qp values slightly exceeding the SCL.
We further analysed permutated activity records of the mentioned rhythmic animals with our Rhythm-Detector analysis. Automated evaluation allowed us to perform 1000 permutations and subsequent analyses for every data record, with single periodogram lengths of 10 days. This resulted in a total number of 10 000 Rhythm-Detector analyses, of which nine (0.09%) indicated rhythmicity applying to the rhythmicity criteria stated above. Thus, the Rhythm-Detector judges 99.91% of randomly permutated data records as arrhythmic and, thus, has a negligible error rate.
Immunocytochemistry
Following activity recordings, brains of operated animals (together with
those of untreated animals to act as a control for staining) were dissected,
fixed in a formaldehyde solution and either embedded in gelatine/albumin or in
paraffin. Serial sections (gelatine, 30 µm; paraffin, 10 µm) were cut
and stained using anti-ß-PDH antiserum
(Dircksen et al., 1987) with
the three-step peroxidaseanti-peroxidase method according to
Sternberger (Sternberger,
1979
; see also Reischig and
Stengl, 1996
); detection of peroxidase was carried out with
3,3'-diaminobenzidine/H2O2. The paraffin sections
were counterstained in 1% methylene blue.
To determine whether control or test animals regain rhythmicity in the locomotor assays, cockroaches were left in the running-wheels for as long as possible. Once the cockroach appeared to approach its natural death (when it became weak and showed either decreased or strongly increased activity), it was sacrificed and its brain was removed for immunocytochemistry. This focus on the long-term analysis of locomotor activity records of transplanted and control animals necessarily takes into account that several of the operated animals will die unexpectedly before they can be examined immunocytochemically.
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Results |
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In a first set of transplantation experiments, we exchanged the remaining AMe between cockroaches of different endogenous periods (N=7). Animals were donors and hosts at the same time; thus, the graft was implanted into the space of the host's removed AMe (Fig. 1A). Two of the seven animals regained circadian locomotor activity (Tables 1, 2) four weeks after the transplantations. Immunocytochemistry in one of these specimens revealed PDH-ir somata (n=2) at the transplantation site as well as regenerated PDH-ir fibres in the midbrain (data not shown). Because it was difficult to unequivocally distinguish implanted PDH-ir neurons from remaining host PDH-ir cells, we continued with ectopic transplantations. In 45 experiments, the AMe-graft was inserted into the right antennal lobe of arrhythmic cockroaches, which had both optic lobes removed (Fig. 1B). In one control group (N=22), the remaining optic lobe was removed without further transplantations. In another control group (N=20), the remaining optic lobe was removed and grafts of medulla tissue next to the AMe were transplanted into the host's antennal lobe.
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After transplantation or control surgery, in most cases the locomotor activity was disrupted for several days and then became arrhythmic. The amounts and patterns of arrhythmic locomotor activity largely varied between individuals as well as within the postoperative life span of a single individual. Rhythmic locomotor activity returned in several animals but often became arrhythmic again. Based on these observations, we developed a new method for an automated search of shorter rhythmic episodes in long data records with rather strict standards for rhythmicity, to avoid biased judgement of periodicity (see Materials and methods).
Applying this analysis, a total of 18 (21%) of the 87 animals examined
regained rhythmic locomotor behaviour (Tables
1,
2). Among the rhythmic animals
were 13 cockroaches with AMe transplants and five controls (Tables
1,
2; Figs
2,
3). Significantly more animals
regained rhythmicity in the transplantation group (29%) versus the
control group (12%), as tested with a single-tailed two-by-two frequency
table, which involves a 2 test with one degree of freedom
(
2=3.82, P<0.05, d.f.=1). As a second test, we
applied a G-test of association, which compared the distributions of
values between the AMe-transplanted and control groups. We assumed the results
of the control operations as the predicted values for the AMe
transplantations, if the transplantations would have no effect. Therefore, we
would expect six rhythmic animals in the 45 AMe transplantations. However, the
frequency of rhythmic animals differed significantly from those predicted by
the control operations (G=7.44, P<0.05, d.f.=1).
The regenerated rhythmic behaviour of all these animals differed in at least one of the following criteria from rhythmic behaviour of normal animals: (1) rhythmicity was only transiently maintained (Table 2), (2) the onsets of locomotor activity were more variable, (3) phases of rhythmic activity were sometimes interrupted by bursts of continuing activity, (4) the amount of activity often fluctuated from one circadian day to the next, (5) unusual period lengths sometimes occurred (Table 2) and (6) rhythmic activity phases were often introduced by long bursts of activity (Fig. 3A). No correlation was detectable between the periods of donors and hosts (Table 2).
Of the 45 AMe transplantations, 22 (49%) animals could be examined histologically before they died. Of these, PDH-ir somata (n=15) in the antennal lobe and PDH-ir midbrain arborisations were observed in all of the cockroaches that regained rhythmicity after transplantation of the AMe (N=4; Tables 1, 2; Fig. 4BD). Among the transplanted PDH-ir neurons, mostly two of their three size classes the medium-sized (1216 µm) and large (>16 µm) but only one of the small (<12 µm) class PDH-ir somata were found (Table 3). Additionally, 10 (31%) of the 32 arrhythmic AMe-transplanted animals expressed PDH-ir terminals in the midbrain (Table 4). In all rhythmic animals examined (including one rhythmic control animal) regenerated PDH-ir fibres arborised in the superior medial and superior lateral protocerebra (SMP and SLP, respectively; N=5), but PDH-immunoreactivity in the ventro- or inferior lateral protocerebra or in the posterior optic tubercles was not found in all rhythmic animals (Table 4). In the AMe-implanted antennal lobes, we did not find any AMe-like neuropil structure retained from the implanted tissue, but regenerated PDH-ir fibres in the antennal lobe showed varicosities.
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Regained rhythmicity in the one control animal, which could be histologically examined, also correlated with the presence of regenerated PDH-ir arborisations in the midbrain (Tables 2, 3, 4). The PDH-ir somata were found in the antennal lobe, which was implanted with medulla tissue, as well as in the stump of one sectioned optic lobe. Moreover, two other arrhythmic control animals each had one PDH-ir soma in an optic lobe stump. Because three of the 22 histologically examined controls exhibited PDH-ir neurons, the expected error rate for these difficult control surgeries was 14%.
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Discussion |
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Complete arrhythmicity after removal of both optic lobes was repeatedly
demonstrated in L. maderae, as in other cockroaches
(Roberts, 1974;
Sokolove, 1975
;
Lukat and Weber, 1978
;
Page, 1982
;
Stengl and Homberg, 1994
),
crickets (Loher, 1972
;
Tomioka and Chiba, 1984
;
Abe et al., 1997
) and wetas
(Waddell et al., 1990
). Thus,
re-established rhythmicity after AMe transplantation in cockroaches without
optic lobes strongly suggests that the transplanted AMe-grafts indeed
contained the circadian pacemaker. This is supported by the significant
difference in the number of rhythmic animals in the transplantation group
versus the control group. Furthermore, only the presence of circadian
rhythmicity argues for the presence of an intact circadian clock, but the
absence of circadian locomotor rhythms does not prove the lack of an intact
circadian clock (Stengl and Homberg,
1994
; Stengl,
1995
). This is also shown by the occurrence of 41% apparently
arrhythmic cockroaches with one intact circadian clock in the foreruns of the
locomotor activity assays of the current study. Thus, the presence of
successfully transplanted PDH-ir neurons in arrhythmic animals does not weaken
the conclusion that the transplanted tissue contains the circadian clock. In
addition, the selective transplantation of medulla control tissue next to the
AMe, but within the predicted pacemaker location according to Sokolove
(1975
), restored rhythmicity
in arrhythmic animals significantly less often than did AMe transplants. With
an error rate of 14%, we also transferred PDH-ir neurons during our control
transplantations, or single PDH-ir medulla neurons were accidentally left in
the remaining stump after optic lobe excision. This is not surprising because
PDH-ir somata (and possibly other neurons of the AMe) are sometimes not
directly beneath the AMe but slightly dislocated towards the medulla or
lobula, where we set our cut. Thus, it is likely that in all of the 12%
rhythmic controls, rhythmicity was generated by accidentally transferred or
leftover AMe neurons, as shown by immunocytochemistry in three control animals
(Table 4).
Thus, because cockroaches with intact circadian pacemakers sometimes show
only short or no periods of rhythmicity, any episode of clear rhythmicity
indicates that these animals contain circadian pacemakers, while arrhythmicity
allows no final conclusion about the presence of an intact clock. Since no
clear, objective measures for transient rhythmicity in long data sets had been
published before, we took great care to develop new software and standards to
distinguish rhythmic from arrhythmic episodes in long data sets. Because
different analysis methods such as MESA and 2-periodogram
analysis, as well as a subjective judgement by eye, confirmed our own software
we consider our analysis program to be very reliable. In addition, because
rather strict criteria were used for the distinction of rhythmicity
versus arrhythmicity, we very likely underestimate the number of
rhythmic animals in the transplantation group.
Because the lack of circadian rhythmicity in cockroaches without optic
lobes is well established by lesion experiments from different laboratories
(Roberts, 1974;
Sokolove, 1975
;
Lukat and Weber, 1978
;
Page, 1982
;
Stengl and Homberg, 1994
), the
return of rhythmicity in transplanted animals demonstrated in the present
study shows that the transplanted tissue contains circadian pacemaker neurons.
However, it does not distinguish which of the transplanted cells are circadian
pacemaker cells. The correlation between the presence of regenerated PDH-ir
processes in original target areas in all histologically examined rhythmic
animals suggests a role for PDH-ir neurons as circadian pacemaker candidates.
But, because we focused on long-term behavioural analysis at the expense of
histological examination, only five of the cockroaches with regained
rhythmicity could be examined histologically. Thus, we cannot draw a
statistically significant conclusion about the cellular nature of circadian
pacemaker neurons within the AMe transplants. But it is likely that at least a
subgroup of PDH-ir neurons relays the circadian information to the midbrain
because regeneration of PDH-ir neurons to original midbrain targets also
correlated with regained circadian activity rhythms after transection of the
optic stalk (Stengl and Homberg,
1994
).
That the circadian pacemaker is at least partly composed of the PDH-ir
neurons is further supported by findings in the fruitfly Drosophila
melanogaster. In the fruitfly, pigment-dispersing factor is colocalised
with the clock proteins PERIOD and TIMELESS in the same circadian pacemaker
candidates, the lateral neurons (Helfrich-Förster,
1995,
1998
). In addition, PDH is
thought to be the crucial circadian output and coupling neuropeptide in
insects (Renn et al., 1999
;
Blanchardon et al., 2001
;
Taghert, 2001
;
Reischig and Stengl, 2002
).
The importance of PDH-ir neurons for circadian activity is further supported
by our observation that regained rhythmic activity strictly correlated with
regeneration of transplanted PDH-ir neurons into the SMP and SLP, which are
the clock's presumed output regions to locomotor centre pathways in wild-type
cockroaches as well as in Drosophila
(Homberg et al., 1991
;
Renn et al., 1999
).
Arborisations in the ventrolateral and inferior lateral protocerebrum, or
posterior optic tubercle, which are also arborisation sites for PDH-ir
terminals in wild-type cockroaches
(Homberg et al., 1991
), were
not necessary for regained locomotor activity. Because rhythmicity resumed
within
10 days of the operation (Table
2), circadian outputs to locomotor centre pathways appear to rely
on regenerated neuronal connections but not on diffusible factors as shown in
vertebrates (see Silver et al.,
1996
).
In both the fruitfly and the cockroach, there are different size groups of
PDH-ir neurons next to the AMe. In Drosophila, small PDH-ir lateral
neurons project to the superior lateral protocerebrum, and large PDH-ir
lateral neurons appear to connect both optic lobes. In the cockroach, only
subgroups of the large- and medium-sized PDH-ir neurons project to the
protocerebrum and appear to connect both accessory medullae
(Reischig and Stengl, 2002).
Because in the current study large- and medium-sized transplanted PDH-ir
neurons (Table 3) were found to
regenerate to the superior lateral protocerebrum, it is likely that at least
these two subgroups of the PDH-ir neurons are circadian pacemaker neurons that
can drive circadian locomotor behaviour. This is in contrast to some findings
in Drosophila indicating that only the small lateral neurons have
pacemaker function (Park et al.,
2000
). However, since in disco mutants a single large
PDH-ir neuron with aberrant connections to the superior lateral protocerebrum
correlates with rhythmic locomotor behaviour
(Helfrich-Förster, 1998
),
in the fruitfly, as in the cockroach, all PDH-ir neurons might be circadian
pacemakers.
In contrast to the transplantation studies of Page
(1982), our experiments could
restore rhythmic behaviour but not period length. The regained period lengths
were dissimilar to the donors' periods and ranged from 20.4 h to 28.5 h,
closely reflecting the range of periods of non-coupled vertebrate SCN
pacemaker neurons in vitro (Honma
et al., 1998
). Our immunocytochemical results indicate that only a
few of the transplanted AMe neurons survived in the host's antennal lobe and
that the neuropil of the AMe is lost in the host. Apparently, coupling
interactions between transplanted AMe neurons that might generate the
characteristic period of the wild-type cockroach are strongly reduced or
missing. Thus, it is likely that an insect, as well as a vertebrate, circadian
pacemaker constitutes its period via coupling in an interconnected
neuronal network rather than via single independent pacemaker neurons
(Honma et al., 1998
). In
addition, we assume that, adjacent to the PDH-ir neurons, other neurons of the
AMe are also circadian pacemakers, because no correlation between the number
of surviving PDH-ir somata and overt period lengths was observed
(Table 2). The different period
lengths might possibly indicate varying amounts of coupling between different
pacemaker cells in the transplanted grafts
(Michel and Colwell, 2001
).
This further adds to the assumption that the period of the circadian system
depends on the period of single pacemaker cells as well as on the coupling
between the pacemakers. Furthermore, in hamsters (Mesocricetus
auratus), it was shown that quality and period lengths of rhythmicity
after SCN transplantation are influenced by the number of re-established
neuronal connections, the graft volume and the attachment site of grafts
(Davis and Viswanathan, 1996
;
LeSauter et al., 1997
).
Therefore, the lack of the normal AMe neuropil might explain why regenerated
rhythmicity occurred only transiently and why some animals did not regain
rhythmic locomotor activity, even in the presence of successfully transplanted
PDH-ir neurons. However, it cannot be determined whether these arrhythmic
animals lacked a functional clock, since about one-third of non-operated
cockroaches with intact circadian clocks did not express circadian locomotor
activity in running-wheel assays.
With the exception of the transplantations of whole optic lobes by Page
(1982), transplantation of
small, defined brain regions containing circadian oscillators succeeded only
in vertebrate species (Sawaki et al.,
1984
; Lehman et al.,
1987
; Ralph et al.,
1990
; Grosse and Davis,
1998
), thus identifying the suprachiasmatic nucleus as the
circadian pacemaker centre controlling locomotor activity in mammals. Only
transplantations of embryonic or developing tissue within a narrow time window
after birth of the donors succeeded
(Romero et al., 1993
;
Kaufman and Menaker, 1993
).
Reorganisation of identifiable, fully differentiated central nervous system
(CNS) neurons after ectopic transplantation from and into adult animals has
not been reported before. Thus, the cockroach is not only an excellent model
organism to study the neurophysiology of circadian timing but is also an
interesting system for studies of neuronal regeneration after CNS damage or
transplantation, because of its dramatic power in repairing severed neuronal
connections.
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Acknowledgments |
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