cucaracha circadian - unam

REVIEW

Neural Organization of the Circadian System of theCockroach Leucophaea maderae

Uwe Homberg,* Thomas Reischig, and

Monika Stengl

Fachbereich Biologie, Tierphysiologie, Universitat Marburg,

Marburg, Germany

ABSTRACT

The cockroach Leucophaea maderae was the first animal in which lesion experiments

localized an endogenous circadian clock to a particular brain area, the optic lobe. The

neural organization of the circadian system, however, including entrainment pathways,

coupling elements of the bilaterally distributed internal clock, and output pathways

controlling circadian locomotor rhythms are only recently beginning to be elucidated.

As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac-

tive neurons of the accessory medulla of the cockroach are crucial elements of the

circadian system. Lesions and transplantation experiments showed that the endoge-

neous circadian clock of the brain resides in neurons associated with the accessory

medulla. The accessory medulla is organized into a nodular core receiving photic input,

and into internodular and peripheral neuropil involved in efferent output and coupling

input. Photic entrainment of the clock through compound eye photoreceptors appears to

occur via parallel, indirect pathways through the medulla. Light-like phase shifts in

circadian locomotor activity after injections of g-aminobutyric acid (GABA)- or Mas-

allatotropin into the vicinity of the accessory medulla suggest that both substances are

involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors

appear to be present in the optic lobe, but their role in photic entrainment has not

*Correspondence: Uwe Homberg, Fachbereich Biologie=Tierphysiologie, Philipps-Universitat

Marburg, D-35032 Marburg, Germany; Fax: þ49-6421-2828941; E-mail: [email protected]

marburg.de.

CHRONOBIOLOGY INTERNATIONAL

Vol. 20, No. 4, pp. 577–591, 2003

DOI: 10.1081=CBI-120022412 0742-0528 (Print); 1525-6073 (Online)

Copyright # 2003 by Marcel Dekker, Inc. www.dekker.com

577

been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent

output from the accessory medulla to several brain areas and to the peripheral visual

system. Pigment-dispersing hormone-immunoreactive neurons, and additional hetero-

lateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers.

The neuronal organization, as well as the prominent involvement of GABA and

neuropeptides, shows striking similarities to the organization of the suprachiasmatic

nucleus, the circadian clock of the mammalian brain.

Key Words: Insect brain; Circadian clock; Pigment-dispersing hormone; Light

entrainment; Accessory medulla; Cockroach; Leucophaea maderae.

INTRODUCTION

Internal circadian clocks are universal adaptations of living organisms to the 24h

periodicity of the environment. Their molecular organization and localization, the

analysis of clock-controlled effectors, and mechanisms for entrainment of these clocks

have been the focus of intensive research in organisms of all main phyla. Work in insects

has focused heavily on the fruit fly Drosophila melanogaster, owing largely to the

multitude of available molecular genetic tools, and has greatly advanced our under-

standing of the molecular machinery underlying the circadian system (Allada et al., 2001;

Stanewsky, 2002; Young and Kay, 2001). These studies also revealed: (i) in addition to a

‘‘master clock’’ in the brain for the control of behavioral output, additional clocks exist in

the nervous system and in peripheral organs (Giebultowicz, 1999); (ii) parallel mechan-

isms and pathways operate for light entrainment of the clock (Helfrich-Forster et al.

2001); and (iii) distinct neural and molecular pathways are required for the control of

specific outputs of the clock (Helfrich-Forster, 1998; McNeil et al., 1998; Zhang et al.,

2000). Comparative studies, however, showed that other insects may considerably differ

from D. melanogaster with respect to clock localization, entrainment mechanisms, and

molecular basis of the clock (Helfrich-Forster et al., 1998). For a general understanding

of the circadian system in insects, studies on a variety of insect species are, therefore,

essential. Insect species with a larger brain size than that of D. melanogaster, such as the

cricket Gryllus bimaculatus, the cockroach Leucophaea maderae, or the moth Antheraea

polyphemus, furthermore, offer the advantage of a cellular and physiological approach to

the circadian system.

Research on the circadian system of the cockroach L. maderae is favored by the

prominent circadian rhythm of locomotor activity of this insect. Owing to its large size and

robustness, L. maderae was the first animal in which lesion experiments have localized the

circadian clock to a particular brain region (Nishiitsutsuji-Uwo and Pittendrigh, 1968b).

More recently, the organization of the putative circadian timekeeping center in the brain,

the accessory medulla, has been analyzed at the cellular level. The present paper reviews

the neuronal organization of the circadian system of L. maderae with special emphasis on

its internal organization, distribution of putative neurotransmitters and neuropeptides

operating in the circadian system, light entrainment pathways, clock outputs, and bilateral

coupling mechanisms.

578 Homberg, Reischig, and Stengl

LOCALIZING THE INTERNAL CLOCK THROUGH

LESIONS AND TRANSPLANTATIONS

Cockroaches show robust circadian rhythms in wheel running activity (Roberts, 1960)

and in electroretinogram recordings (Wills et al., 1985). Both assays have been used to

monitor circadian clock function after various lesions to the cockroach brain. Transections

of the optic lobe at various levels showed that the circadian pacemaker controlling

locomotor activity (Nishiitsutsuji-Uwo and Pittendrigh, 1968b; Roberts, 1974), neural

activity in the ventral nerve cord (Colwell and Page, 1990), and electroretinogram rhythms

(Wills et al., 1985) resides in the optic lobe, and is associated with the medulla=lobula

but not with the lamina. A sustained electroretinogram rhythm could be recorded from the

isolated optic lobe for several days (Colwell and Page, 1990), and transplantation of

the optic lobe into an animal, whose own optic lobes had been removed, restored circadian

rhythmicity of locomotor activity after several weeks (Page, 1982). These experiments

showed that the optic lobe contains a self-sustained circadian clock controlling locomotor

behavior and visual sensitivity.

Microlesions using tungsten microelectrodes further specified the site of the clock

(Page, 1978; Sokolove, 1975) and showed that lesions in or near the lobula, particularly in

the ventral cell body cortex between the medulla and lobula, produced a high percentage of

arrhythmic cockroaches. Immunocytochemical studies revealed that this area contains the

cell bodies of neurons with arborizations in the accessory medulla, a small neuropil at the

anterior ventromedian edge of the medulla (Homberg et al., 1991; Petri et al., 1995).

Distinct and partly overlapping subpopulations of these neurons are immunoreactive with

antisera against serotonin, g-aminobutyric acid (GABA), and a variety of neuropeptides,

including Mas-allatotropin, Dip-allatostatin, FMRFamide, pigment-dispersing hormone

(PDH), and leucokinin (Petri et al., 1995; 2002). These neurons send processes into

the accessory medulla, and most of them have additional projections into other areas of the

brain. These studies suggest that neuropeptides play a key role in the circadian system of

the cockroach and draw attention to the accessory medulla as the possible synaptic

integration center of the clock.

Immunostaining with an antiserum against the crustacean peptide PDH (Rao, 2001) is

particularly selective for neurons of the accessory medulla, and throughout the brain of the

cockroach, only three groups of neurons are immunoreactive. Peptides closely related to

crustacean PDH, termed pigment-dispersing factors (PDFs), have been identified in several

insect species, including the cockroach Periplaneta americana (Rao, 2001). Two groups of

PDH-immunoreactive neurons with cell bodies at the posterior dorsal and posterior ventral

edge of the lamina were termed PDFLa cells and, a third group of 16 neurons near the

accessory medulla, PDFMe neurons [Homberg et al., 1991; Reischig and Stengl, 1996;

Fig. 1(A)]. The PDFLa neurons have arborizations throughout the lamina and probably

contribute processes to a fan of immunostained fibers along the distal face of the medulla

[Fig. 1(A)]. Their possible involvement in the circadian system is unknown. The role of the

accessory medulla and the contribution of PDFMe neurons to the circadian system was

addressed in lesion and transplantation experiments (Reischig and Stengl, 2002; Stengl

and Homberg, 1994). Bilateral transections of the optic stalk in the cockroach abolished

circadian wheel-running activity. In many individuals, however, the circadian rhythm

reappears after several weeks corresponding with mass regeneration of fibers from the

optic lobe to the median protocerebrum (Page, 1983a).

Neural Organization of Leucophaea maderae 579

Immunocytochemistry using PDH antiserum shows a strong correlation between

ingrowth and reinnervation of original targets in the lateral and superior protocerebrum by

fibers from PDFMe neurons in animals that regained circadian activity after lesions (Fig. 2;

Stengl and Homberg, 1994). This suggests that PDF acts as an output signal of the clock to

particular brain areas involved in motor control. Finally, ectopic transplantations of

Figure 1. Arborizations of PDH-immunoreactive neurons in the brain of L. maderae (A) and

anatomical organization of the accessory medulla (B). (A) Camera lucida reconstruction of PDH-

immunoreactive neurons embedded in a computer-aided three-dimensional reconstruction of the

cockroach brain (modified after Reischig and Stengl, 2002). PDFMe neurons have cell bodies near

the accessory medulla (AMe). The neurons have dense ramifications in the AMe and send processes

through the distalmost layer of the medulla (Me) to the lamina (La) and a second set of axonal fibers

to various areas in the median protocerebrum. Immunoreactive fibers in the anterior and posterior

optic commissures (AOC, POC) provide heterolateral connections between the two AMea. Two

additional groups of PDH-immunoreactive neurons with somata near the posterior dorsal (dPDFLa)

and posterior ventral edge of the medulla contribute to immunostained arborizations in the lamina

and first optic chiasma. (B) Computer-aided three-dimensional reconstruction corresponding to the

boxed area in (A). Somata of the AMe-neurons are organized in six groups: median neurons (MNe),

ventro-median neurons (VMNe), medial frontoventral neurons (MFVNe), ventral neurons (VNe),

distal frontoventral neurons (DFVNe), and ventroposterior neurons (VPNe). The core of the AMe,

composed of dense nodular neuropil (NN) and coarse internodular neuropil (IN) is surrounded by a

shell of coarse neuropil (SN). Two of the six cell groups (VNe, VMNe) are shown separately in the

right panel. Lo, lobula. Scale bars: 200 mm (A), 20mm (B).


accessory medullae and adjacent cell bodies into animals whose own optic lobes had been

removed restored circadian activity (Reischig and Stengl, 2003). In these experiments, a

small piece of tissue (about 200 mm diameter) including the accessory medulla and its

neurons was transplanted from a donor cockroach to the optic or antennal lobe of a host

cockroach whose accessory medullae or optic lobes had been removed. In nearly 30% of

Figure 2. Reappearance of circadian wheel-running activity after optic stalk severance correlates

with mass regeneration of PDH-immunoreactive fibers from the AMe to the midbrain (modified after

Stengl and Homberg, 1994). (A) Activity record of a cockroach kept in DD, which received

transection of the right optic stalk and crushing of the left optic stalk at day 22 (arrowhead). After the

operation, activity is reduced and the animal becomes arrhythmic. Five days after reappearance of

rhythmic activity at day 40 the cockroach was sacrificed and the brain was examined for PDH

immunostaining. (B) Pigment-dispersing hormone immunostaining in the brain of the cockroach in

(A) at day 44. Mass regeneration of PDH-immunoreactive fibers has occurred predominantly from

the right optic lobe to the median protocerebrum. Many fibers have reinvaded their original targets in

the superior median, superior lateral, inferior lateral, and ventro-lateral protocerebrum (SMP, SLP,

ILP, VLP) or have entered the posterior optic commissure (POC). Scale bar: 200 mm.


these animals, a circadian rhythm of locomotor activity reappeared and in all animals that

regained rhythmicity, transplanted PDFMe neurons had regenerated fibers to their original

targets in the protocerebrum (Reischig and Stengl, 2003). These data further confirm that

the accessory medulla is the site of the circadian pacemaker controlling locomotor activity

rhythms in the cockroach and suggest that neural connections by PDH-immunoreactive

fibers from the accessory medulla to their original targets in the brain may be essential for

clock control of locomotor activity.

INTERNAL ORGANIZATION AND CHEMOARCHITECTURE OF

THE ACCESSORY MEDULLA

The accessory medulla has a pear-shaped form with a maximum longitudinal size

of about 90 mm (Reischig and Stengl, 1996). It is composed of a core consisting of

dense knots of neuropil termed noduli. The nodular core is embedded in coarse

internodular neuropil and is surrounded by a shell of coarse neuropil, which is

anteriorly continuous with the adjacent neuropil of the medulla [Fig. 1(B)]. Somata

arranged in six groups in the cell cortex antero-ventrally to the accessory medulla

contribute processes to the accessory-medulla neuropil [Fig. 1(B)]. Eight of the 16

PDFMe neurons have somata in the VNe group, four small somata are in the DFVNe

group (see Fig. 1 for nomenclature), and the four remaining somata are in the cell

cortex posterior to the medulla=lobula neuropil not shown in Fig. 1. The other cell

groups contain accessory-medulla neurons immunostained with antisera against other

neuropeptides and transmitter candidates, e.g., GABA-immunstained neurons in group

MNe and Mas-allatotropin-immunostained neurons concentrated in the DFVNe and

MFVNe cell groups (Petri et al., 1995; see Fig. 1 for nomenclature). The three

compartments of the accessory medulla, noduli, internodular neuropil, and peripheral

shell, are differentially supplied by immunocytochemically characterized populations of

AMe neurons. Neurons immunostained with antisera against GABA, Mas-allatotropin,

and leucokinin have processes concentrated in the nodular core of the accessory

medulla (Petri et al., 1995; 2002). These neurons are either local interneurons of the

accessory medulla (Mas-allatotropin) or connect the distalmost layer of the medulla

(leucokinin) or a median layer of the medulla (GABA) to the nodular core of the

accessory medulla. In contrast, neurons immunostained with antisera against serotonin,

PDH, FMRFamide, Dip-allatostatin, and corazonin have processes concentrated in the

internodular neuropil and peripheral shell of the accessory medulla (Petri et al., 1995;

2002). These neurons connect the accessory medulla through a fan of fibers with the

distalmost layer of the medulla and with the lamina (PDH, FMRFamide, serotonin) and

have axonal projections to several areas in the median protocerebrum and to the

contralateral optic lobe (PDH, FMRFamide). The concentration of neuropeptides in the

accessory medulla correlates with an abundance of dense core vesicles in fiber profiles

in the accessory medulla revealed by ultrastructural examination. Based on differences

in size and electron density, at least four different types of dense core vesicles have

been distinguished, which are differentially distributed in the nodular core, in the

internodular and shell neuropil. Granular vesicles are almost completely confined to

the nodular core, while subpopulations of profiles with PDH-immunoreactive

large dense-core vesicles are concentrated in the internodular neuropil, and


PDH-immunoreactive medium size dense-core vesicles are most common in the shell

neuropil of the accessory medulla (Reischig and Stengl, 1996).

PARALLEL PATHWAYS FOR LIGHT ENTRAINMENT

Light entrainment of the circadian clock of L. maderae occurs through photoreceptors

in or near the compound eye. Bilateral transections of the optic nerves connecting the

compound eyes to the optic lobes result in free-running circadian locomotor activity, while

removal of the ocelli had no effect on light entrainment (Nishiitsutsuji-Uwo and

Pittendrigh, 1968a; Roberts, 1965; 1974). These data indicate that ocellar photoreceptors

are not essential for photic entrainment and provide no evidence for the involvement of

other nonretinal photoreceptors. A series of bilateral lesion experiments, moreover,

demonstrated that light entrainment of each of the two bilaterally distributed circadian

pacemakers occurs via photoreceptors in the ipsi- and contralateral compound eye (Page,

1978; Page et al., 1977).

Recent studies from our laboratories showed that neural connections from photo-

receptors to the accessory medulla involve intercalated interneurons and may be organized

in several parallel pathways. Histamine immunostaining used to label all compound-eye

photoreceptor cells showed that photoreceptors terminate in the lamina and in distal layers

of the medulla, but not in the accessory medulla (Loesel and Homberg, 1999). Intracellular

recordings showed that only certain cell types of the accessory medulla respond to photic

stimuli (Loesel and Homberg, 2001). Excitatory responses to light were found in neurons

with tangential dendritic processes in median layers of the medulla, projections to the

nodular core of the accessory medulla, and fan-shaped, apparently axonal, processes to the

lamina [Fig. 3(A); Loesel and Homberg, 2001]. These neurons are, therefore, likely

candidates for photic input elements to the accessory medulla, but whether they receive

direct input from compound eye photoreceptors remains to be demonstrated. In the same

study light-sensitive commissural neurons were identified, which interconnect median

layers of the medulla and nodular core of the accessory medulla of both hemispheres.

These neurons are candidates for providing light entrainment from the contralateral eye

[Fig. 3(C)]. Another line of evidence supporting the role of the nodular core of the

accessory medulla as the recipient for light entrainment signals comes from phase shifting

experiments. Injections of GABA or Mas-allatotropin into the medulla result in phase-

dependent phase shifts of circadian locomotor activity (Petri et al., 2002). The phase

response curves have striking resemblence to phase response curves obtained for 6h light

pulses [Fig. 4(A)], and suggest that both substances are involved in light entrainment. At

least one GABA-immunoreactive neuron is a member of the light-responsive tangential

inputs from the medulla, many others connect the medulla via the distal tract, a fiber

bundle along the distal face of the medulla (Reischig and Stengl, 1996), to the nodular core

of the accessory medulla. Mas-allatotropin-immunostained local neurons of the nodular

core likewise appear to be involved in the photic input pathway.

In addition to light signalling pathways from compound-eye photoreceptors via

medulla interneurons to the accessory medulla, two additional pathways might contribute

to light sensitivity in the accessory medulla. A class of apparently centrifugal neurons with

ramifications in the median protocerebrum and axonal projections to the accessory

medulla is strongly inhibited by light (Loesel and Homberg, 2001). One of the recorded


neurons was an ocellar interneuron. A prominent role of this pathway in photic entrain-

ment is unlikely, given the evidence from lesion studies cited above, but a modulatory role

in adjusting light sensitivity, as shown for an ocellar contribution in a cricket (Rence et al.,

1988), or a role in entrainment under particular lighting schedules as shown for the

geniculo-hypothalamic pathway in rats (Edelstein and Amir, 1999), appears possible.

Figure 3. Camera lucida reconstructions of single intracellularly recorded and stained neurons of the

accessory medulla (AMe) (modified after Loesel and Homberg, 2001). (A) Candidate neuron involved

in photic entrainment of the clock. The neuron has its cell body in cell group MNe (see Fig. 1). It has

tangential arborizations in a median layer of the medulla (Me), innervates the nodular core of the AMe

and projects via a fan of fibers to the lamina (La) and to small accessory neuropils of the lamina

(arrowhead). The neuron was strongly excited by light during daytime. (B) Candidate output neuron of

the AMe with cell body in cell group VNe. The neuron has sparse ramifications in the shell and

internodular neuropil of the AMe and sends axonal processes to the La and to the median

protocerebrum near the pedunculus (P) of the mushroom body. The neuron was unresponsive to

light stimuli at daytime. (C) Candidate neuron involved in light entrainment via the contralateral eye. Its

cell body lies in group VMNe (see Fig. 1). The neuron has bilateral arborizations in the Me and AMe.

The neuron was strongly excited by light and showed pronounced polarization sensitivity. aL, Ca,

a-lobe, and calyces of the mushroom body; Lo, lobula. Scale bars: 100mm in (A) and (B); 200mm in (C).


Finally, ultrastructural studies and immunoreactivity for the blue light photopigment

cryptochrome provided evidence for the presence of two extraocular photoreceptor organs

in the optic lobe of the cockroach (Fleissner et al., 2001). The lamina organ is an elongated

structure which runs parallel to the lamina in front of the first optic chiasm, and the second

organ, termed lobula organ, lies near the accessory medulla in front of the second optic

chiasm. So far, however, a possible involvement of these organs in photic entrainment of

the clock has not been investigated at any level.

OUTPUT PATHWAYS

Reappearance of circadian wheel-running activity after bilateral optic stalk severance

or transplantations of the accessory medulla correlates with regenerated PDH-immuno-

reactive fibers from PDFMe neurons to the median protocerebrum (Fig. 2; Reischig and

Stengl, 2003; Stengl and Homberg, 1994). Therefore, PDFMe neurons might provide

the connection between the pacemaker and motor control areas in the protocerebrum.

While in animals with optic stalk severance, equal reinnervation of all original targets in

the protocerebrum was observed (Stengl and Homberg, 1994), regeneration in animals

with transplanted accessory medulla occurred predominantly to the superior median and

lateral protocerebrum, suggesting that these areas might be more important for clock

Figure 4. Phase response curves demonstrate different temporal sensitivities of the circadian clock

to injections of Mas-allatotropin, GABA (A modified after Petri et al., 2002) and PDF (B, after Petri

and Stengl, 1997). (A) Injections of Mas-allatotropin and GABA into the medulla lead to phase-

dependent phase shifts in circadian locomotor activity which show striking resemblance to the phase-

shifting effects of light. This suggests that GABA and a peptide related to Mas-allatotropin are

involved in light entrainment pathways. (B) Injections of PDF into the medulla lead to an all-delay

type phase-response curve with maximum phase shifting effects in the late subjective day. This

suggests that PDF is involved in non-photic inputs to the circadian clock. Data are merged into 2h

bins. Solid stars indicate phase delays that are significantly different from phase shifts following

control injections. Phase delays at CT 8:00–10:00h (double star) and at CT 10:00–12:00h (single

star) are significantly different ( p< 0.05) from PDF injections at other circadian times (open stars).


control of locomotion than others (Reischig and Stengl, 2003). PDFMe neurons also have

fan-like processes through distalmost layer 1 of the medulla toward the lamina [Fig.

1(A)], suggesting that they are also involved in circadian control of visual sensitivity at

the level of the lamina. In single-cell recordings, neurons of the accessory medulla that

were unresponsive to light stimuli during the day were regarded as candidate output

neurons of the clock, since light does not cause phase shifts during the day. Like PDFMe

neurons, these neurons had ramifications confined to the internodular and peripheral shell

neuropil of the accessory medulla. In all cases, their axonal fibers had two targets, fan-like

distal projections to the lamina or first optic chiasm, and proximal projections to certain

areas in the median protocerebrum [Loesel and Homberg, 2001; Fig. 3(B)].

COUPLING OF THE BILATERALLY SYMMETRIC CLOCKS

The two pacemakers in the right and left optic lobe of the cockroach are coupled

through neural pathways (Page, 1978; 1983b; Page et al., 1977). Lesions of one of the two

pacemakers invariably leads to an increase in the free-running period of the rhythm,

indicating that coupling of both pacemakers accelerates the system, possibly through

mutual inhibition (Page, 1983b). Several lines of evidence suggest that PDF may be

involved in the coupling pathway. Dextran tracing studies and double-labeling of

commissural neurons and PDH immunostaining showed that three of the 16 PDFMe

neurons are commissural neurons that interconnect the two accessory medullae (Petri,

1998; Reischig and Stengl, 2002). The neurons project via the anterior and posterior optic

commissures, innervate the internodular and shell neuropil of the accessory medulla, and

send a fan of fibers along the distal surface of the medulla into the lamina [Fig. 5(B)].

Neurons of this morphological type were intracellularly stained [Fig. 5(A)] and were found

to be unresponsive to light stimuli during the day (Loesel and Homberg, 2001). Injections

of PDF into the medulla caused phase shifts in circadian wheel-running activity which

provides additional support for a role of PDF as a coupling signal in the circadian system

(Petri and Stengl, 1997). The resulting phase-response curve is monophasic with phase

delays during the late subjective day and, therefore, differs considerably from the phase-

response curve obtained with light pulses (Fig. 4). Pigment-dispersing factor is, therefore,

not part of the light entrainment pathway but participates in non-photic input to the clock.

Taken together with the immunocytochemical data, PDH-immunoreactive commissural

neurons are therefore, strong candidates for the bilateral coupling pathway of the two optic

lobe pacemakers. Evidence supporting a dual role of PDH-immunostained neurons as

output and coupling pathways is provided by ultrastructural studies, which revealed input

as well as output synapses from PDH-immunoreactive neurons in the accessory medulla

(Reischig and Stengl, 2003). Computer simulations, finally, demonstrated that in addition

to all-delay-type interactions as found with PDF injections, all-advance-type interactions

between the two pacemakers are required to explain all experimental findings on mutual

pacemaker coupling in L. maderae (Petri and Stengl, 2001). It is, therefore, likely, that

additional non-PDH-immunostained commissural neurons participate in bilateral coupling.

Candidates are commissural FMRFamide-immunostained connections between the two

accessory medullae, but the effects of FMRFamide-related peptides on locomotor activity

have not been tested so far.


COMPARISON BETWEEN FLIES, COCKROACH, AND MAMMALS

The circadian system of L. maderae, particularly with respect to the involvement

of PDF-containing neurons shows striking similarity to the well-studied circadian system

of the fruitfly D. melanogaster. In D. melanogaster, the so-called ‘‘lateral neurons’’ (LNs)

near the medulla are circadian pacemaker cells (reviewed by Helfrich-Forster et al.,

1998; Kaneko, 1998; Stanewsky, 2002). The neurons express the clock genes period,

timeless, and doubletime and the presence of these neurons as well as their

cyclical expression of period is essential for behavioral circadian rhythmicity (Helfrich-

Forster, 1998; Kaneko et al., 1997). A subset of the LNs contain PDF and mutations of the

pdf-gene or ectopic expression of pdf leads to severe deficits in circadian rhythms

Figure 5. Coupling pathways between the bilaterally distributed pacemakers in the AMe. (A)

Hypothetical neuron involved in coupling of both pacemakers. The neuron interconnects the AMe,

the distalmost layer of the medulla and the lamina of both hemispheres. The original Lucifer

injection (modified from Loesel and Homberg, 2001) showed incomplete staining of the neuron with

ramifications in the right brain hemisphere and a commissural fiber in the posterior optic commissure

(POC) toward the left optic lobe. Mirror-symmetric arborizations in the left optic lobe were added

using Adobe Photoshop 5.0 software because evidence from dextran backfills suggests that this cell

type has bilaterally symmetric arborizations in both optic lobes. The neuron was unresponsive to

light stimuli. (B) Schematic diagram showing two presumptive PDH-immunoreactive circadian

coupling pathways through the anterior (AOC; dark gray) and posterior optic commissure (POC;

light gray) as revealed by tracer injections combined with PDH immunostaining (modified after

Reischig and Stengl, 2002). ILP, inferior lateral protocerebrum; La, lamina; Me, medulla; POTu,

posterior optic tubercle; SLP, SMP, superior lateral, resp. median protocerebrum. Filled circles

indicate possible outputs and=or interactions between the two systems. Scale bar: 200mm in (A).


(Helfrich-Forster et al., 2000; Renn et al., 1999). As in the cockroach, distinct subpopula-

tions of pdf-expressing neurons with different branching patterns can be distinguished in the

fly, consistent with a double role of the peptide as an output and bilateral coupling signal of

the clock. Differences between D. melanogaster and L. maderae appear to exist with respect

to light entrainment pathways; in the fly the pdf-expressing pacemaker cells possess their

own extraocular photopigment, cryptochrome, which is apparently not present in the

PDFMe neurons of the cockroach (Emery et al., 2000; Fleissner et al., 2001). Furthermore,

a direct synaptic input from an extraocular photoreceptor organ, termed the Hofbauer-

Buchner eyelet in the fly (Helfrich-Forster et al., 2001), has also not been found in the

cockroach. While in D. melanogaster strong evidence suggests that the pdf-expressing cells

are pacemaker cells, the identity of clock cells in the circadian system of L. maderae has not

been determined. Current experiments aimed at identifying the clock molecules PER and

TIM in the cockroach brain might help to solve this question.

While a distinct accessory medulla is hardly visible in D. melanogaster owing to fusion

with the adjacent medulla, the anatomical and neurochemical organization of the accessory

medulla of the cockroach bears considerable similarities to the organization of the

mammalian circadian pacemaker, the suprachiasmatic nucleus (reviewed by Moore et al.,

2002; Reghunandanan et al., 1993; van Esseveldt et al., 2000). Both areas are densely packed

with neuropeptides and have a clear internal substructure into a core receiving retinal input,

and a shell (internodular and shell neuropil in the cockroach) receiving nonphotic input. In

mammals outputs to effector systems exit the core as well as the shell, while in the cockroach,

outputs appear to exit primarily from the internodular and peripheral neuropil. These

similarities may be accidental, but more likely represent similarly optimized solutions for the

neural organizations of brain circadian pacemakers.

ACKNOWLEDGMENTS

Research in the authors’ laboratories on the insect circadian system has been

supported by DFG grant HO 950=9 to U.H. and by DFG grant STE 531=7 and a grant

from Human Science Frontier to M.S.

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