Terry L. Page

Transplantation of the Cockroach Circadian Pacemaker

Surgical removal of the optic lobes of the cockroach Leucophaea maderae followed by transplantation of the optic lobes from another individual led to a restoration of the circadian activity rhythm in 4 to 8 weeks. The free-running period of the restored rhythm was determined by the period of the donor rhythm before surgery. The results suggest that the transplanted optic lobe contains a circadian clock that regenerates those neural connections with the host brain that are necessary to drive the circadian rhythm of activity.

Regeneration of the optic tracts and circadian pacemaker activity in the cockroachLeucophaea maderae

SummaryRecovery of the circadian rhythm of locomotor activity after bilateral section of the optic tracts (OT) of the cockroachLeucophaea maderae was investigated. After OT section rhythmicity consistently reappeared in 3–5 weeks (29±6.2 days,n=22) (Fig. 1), while removal of the optic lobes caused permanent (> 100 days) arrythmicity (n= 13) (Fig. 2A). Recovery of rhythmicity after OT section was likely due to regeneration since:(1) Histological examination showed structural regeneration had occurred (Fig. 3A). (2) Insertion of a glass barrier between the OL and midbrain prevented (> 75 days,n=6) or slowed (46±14.9 days,n=3) rhythm recovery (Fig. 2B). (3) Extracellular recording after optic tract section showed recovery of light evoked activity in the cervical connectives (Fig. 4) whose time course paralleled the recovery of behavioral rhythmicity (Fig. 5).The freerunning period (τ) of the rhythm after regeneration was strongly correlated with τ before surgery (r=0.87) but was slightly longer (Δτ=0.2±0.35 h) (Fig. 6). Also the phase of the rhythm, projected back to the day of surgery, was correlated with preoperative phase (r=0.61) (Fig. 7). Exposure to light cycles the first 10 days after OT section shifted the phase of the subsequent rhythm (Fig. 8). These results suggest that an entrainable circadian oscillation persists in the optic lobes after OT section.

Serotonin phase-shifts the circadian rhythm of locomotor activity in the cockroach.

Serotonin, a putative neurotransmitter in insects, was found to cause consistent phase shifts of the circadian rhythm of locomotor activity of the cockroach Leucophaea maderae when administered during the early subjective night as a series of 4-μl pulses (one every 15 min) for either 3 or 6 hr. Six-hour treatments with dopamine also caused significant phase shifts during the early subjective night, but 3-hr treatments with dopamine had no phase-shifting effect. Other substances tested in early subjective night (norepinephrine, octopamine, γ-aminobutyric acid, glutamate, carbachol, histamine, tryptophan, tryptamine, N-acetyl serotonin, or 5-hydroxy indole-3-acetic acid) did not consistently cause phase shifts. The phase-shifting effect of serotonin was found to be phase-dependent. The phase response curve (PRC) for serotonin treatments was different from the PRC for light. Like light, serotonin caused phase delays in the late subjective day and early subjective night, but serotonin did not phase-shift rhythms when tested at phases where light causes phase advances.

Effects of optic-tract regeneration on internal coupling in the circadian system of the cockroach

SummaryPrevious results have shown that following bilateral severence of the optic tracts or transplantation of the optic lobes in the cockroachLeucophaea maderae the circadian pacemakers in the optic lobes are able to regenerate those neural connections with the midbrain that are necessary to drive the activity rhythm (Page 1982, 1983 a). In the present experiments the possibility that the entrainment pathway that mutually couples the two optic-lobe pacemakers is also capable of regeneration was investigated. The results showed that (a) following unilateral optic-lobe transplantation or optic-tract section, regeneration of the coupling pathway did not occur, (b) an optic lobe that remained intact was able to completely suppress the expression of a contralateral pacemaker that had been forced to regenerate its output connections, and (c) if the host optic lobe had also been forced to regenerate its connections with the midbrain after unilateral optic-lobe transplantation, there was frequently evidence for two apparently independent periodicities in the activity pattern. The results indicate that the output pathway by which each oscillator controls activity is functionally independent of the output pathway that couples each oscillator to the contralateral optic lobe. The data also suggest that the regulation of activity by each optic-lobe oscillator may involve a pathway by which activity driven by the contralateral pacemaker can be suppressed.

Effects of light on circadian pacemaker development

Summary1.The effects of raising cockroaches,Leucophaea maderae, in non-24 h light cycles on circadian rhythms in adults were examined. The average period (Τ) of freerunning rhythms of locomotor activity of animals exposed to LD 11∶11 (T22) during post-embryonic development was significantly shorter (Τ=22.8±0.47 SD,n=85) than that of animals raised in LD 12∶12 (T24) (Τ=23.7±0.20 h,n=142), while animals raised in LD 13∶13 (T26) had significantly longer periods (Τ=24.3±0.21 h,n=65). Animals raised in constant darkness (DD) had a significantly shorter period (Τ=23.5±0.21 h,n=13) than siblings raised in constant light (LL) (Τ=24.0±0.15 h,n=10).2.The differences inΤ between animals raised in T22 and T24 were found to be stable in DD for at least 7 months and could not be reversed by exposing animals to LD 12∶12 or LD 6∶18.3.Animals raised in either T24 or DD and then exposed as adults to T22 exhibited average freerunning periods that were not different from animals not exposed to T22.4.Measurement of freerunning periods at different temperatures of animals raised in T22, T24, or T26 showed that the temperature compensation of t was not affected by the developmental light cycle. These results indicate that the lighting conditions during post-embryonic development can permanently alter the freerunning period of the circadian system in the cockroach, but do not affect its temperature compensation.

A circadian rhythm in neural activity can be recorded from the central nervous system of the cockroach

SummaryEvidence presented in this paper indicates that a robust circadian rhythm in the frequency of neural activity can be recorded from the central nervous system of intact cockroaches, Leucophaea maderae. This rhythmicity was abolished by optic lobe removal. Spontaneous neural activity was then used as an assay to demonstrate that the optic lobe is able to generate circadian oscillations in vitro. These results provide direct evidence that the cockroach optic lobe is a self-sustained circadian oscillator capable of generating daily rhythms in the absence of neural or hormonal communications with the rest of the organism.

Masking in Invertebrates

Masking effects are a common feature of daily rhythmicity in invertebrates; and, particularly with respect to activity/rest cycles in arthropods and mollusks, there are numerous examples of masking in response to external environmental stimuli. Internal masking, in which endogenous processes modulate circadian patterns, has also been documented in a few species. In general, however, because of the absence of appropriate experimental investigations on masking, the functional significance (in an ecological sense) of masking effects is not understood.

Neural and Endocrine Control of Circadian Rhythmicity in Invertebrates

The importance of the nervous and neuroendocrine systems in the control of daily rhythms in invertebrates did not escape the notice of early workers in the field. As early as 1911, Demoll suggested that color changes in arthropods were controlled by a periodic phenomenon in the nervous system. Kalmus, in 1938, concluded that the eyestalk neurosecretory system was the source of control of the crayfish activity rhythm, and Welsh (1941) proposed that “a regular variation in the activity of nervous inhibitory centers” was the major factor in the hormonal control of the rhythmic migration of retinal shielding pigments in the crayfish. In the past two decades, a large body of evidence has been obtained that firmly establishes the proposition, implicit in much of this early work, that it is the central nervous and neuroendocrine systems that are responsible for the generation and coordination of the circadian rhythmicity of many behavioral and physiological functions.

Circadian organization in cockroaches: Effects of temperature cycles on locomotor activity

Abstract The effects of various temperature cycles on the locomotor activity of the cockroach Leucophaea maderae were investigated. While bilateral ablation of the optic lobes abolished the free-running activity rhythm in constant conditions, rhythmicity was exhibited by lobeless animals placed in temperature cycles. The rhythms were driven by cycles as short as 12 h and as long as 48 h without evidence of frequency division or frequency demultiplication. The phase relationship between activity onset and the temperature cycle was dependent on both the relative durations of the “warm” and “cold” phases of the cycles and on the period of the cycle. Computer simulations indicated that these properties of the rhythm can be explained by proposing that activity is controlled by a damped oscillator, located outside the optic lobes, that can be forced by cycles of temperature.

Properties of Mutual Coupling between the Two Circadian Pacemakers in the Eyes of the Mollusc Bulla gouldiana

We examined, in vitro, the effects of changing the free-running period (τ) of one oscillator on the phase relationship between the circadian rhythms of impulse activity in the optic nerves that are driven by the bilaterally paired ocular pacemakers in Bulla gouldiana. One eye of the coupled pair was treated either with lithium artificial seawater (to lengthen τ) or with low-chloride artificial seawater (to shorten τ). The results suggested that the coupling is relatively weak, since the majority (9 to 16) of eyes were unable to maintain a stable phase relationship when τ differences between the eyes were only about 1 hr. When stable phase differences were achieved, the τ of the coupled system was intermediate between the τs of the individual oscillators, and the eye with the shorter intrinsic τ would invariably phase-lead the pair. Interestingly, in a few instances, pairs of eyes that had desynchronized by 9.5-10.5 hr resynchronized within a single cycle via a massive phase advance in the rhythm from the phase-lagging eye. The result suggests the existence of a novel phase-shifting mechanism that is part of the mutual coupling pathway. We found evidence that connection of the eye with the cerebral ganglion increases the τ of the ocular pacemaker, suggesting that efferent signals from the central nervous system influence τ. These signals may also modulate the phase-shifting response.