Pigment-Dispersing Factor and GABA Synchronize Cells of the Isolated Circadian Clock of the Cockroach Leucophaea maderae

Introduction

The presence of an endogenous circadian clock in the brain of an animal was demonstrated first in the cockroach Leucophaea maderae (Nishiitsutsuji-Uwo and Pittendrigh, 1968; Sokolove, 1975; Page, 1982). However, the cellular basis of the clock remained elusive until pigment-dispersing factor (PDF)-immunoreactive (IR) neurons were proposed as circadian pacemaker candidates in the fruitfly and the cockroach (Homberg et al., 1991, 2003; Helfrich-Förster and Homberg, 1993; Stengl and Homberg, 1994; Helfrich-Förster, 1995). In Drosophila, these neurons express clock genes essential for circadian rhythmicity such as period and timeless (Helfrich-Förster, 1995; Kaneko and Hall, 2000) (for review, see Helfrich-Förster, 2004). The PDF-IR neurons are closely associated with the accessory medulla (AMe), a small neuropil in the optic lobe (Reischig and Stengl, 1996; Sato et al., 2002; Bloch et al., 2003; Sehadová et al., 2003; Závodská et al., 2003), which was identified as the circadian clock of the cockroach (Reischig and Stengl, 2003a). The PDF-IR cells are essential elements of the circadian clock controlling locomotor activity rhythms (Stengl and Homberg, 1994; Reischig and Stengl, 2003a). Associated with the AMe are ∼250 cells. More than 60% of these express nuclear PERIOD immunoreactivity (Fischer, 2002).

In the cockroach AMe, as in the mammalian suprachiasmatic nucleus (SCN), GABA-IR processes are abundant (Van den Pol and Tsujimoto, 1985; Moore and Speth, 1993; Petri et al., 1995, 2002). In addition, many different neuropeptide-IR neurons form clock subcompartments, which are dense knots of arborizations (noduli) with looser internodular neuropil and a shell of tracts embracing the AMe (Petri et al., 1995; Reischig and Stengl, 1996, 2003b). Approximately 25 GABA-IR neurons and the GABA-IR distal tract, the presumptive light entrainment pathway from the compound eye, arborize extensively in the noduli of the AMe (Petri et al., 2002; Reischig and Stengl, 2003b). The PDF-IR neurons, which branch in the internodular neuropil, control locomotor rhythms via projections to locomotor control centers in the superior lateral protocerebrum (Stengl and Homberg, 1994; Reischig and Stengl, 2003a). In addition, three of the 12 PDF-IR neurons connect both accessory medullas (AMae) and apparently serve to synchronize both pacemakers (Reischig and Stengl, 2004). Consistent with this hypothesis, PDF acts as a nonphotic input signal into the clock, delaying circadian locomotor activity rhythms (Petri and Stengl, 1997). In Drosophila, PDF is also assumed to be a circadian coupling signal that synchronizes clock cells in the midbrain to control locomotor activity rhythms (Peng et al., 2003; Lin et al., 2004).

Although considerable information about the molecular machinery of the core circadian pacemaker is available (Honma and Honma, 2003), little is known about the physiological properties of the clock. It is mostly unresolved how circadian coupling is accomplished and how the neuropeptides act within the clock. Here, we tested in extracellular recordings from excised AMae of the cockroach L. maderae whether GABA and PDF affect AMe neurons via modulation of neuronal activity. We discovered that AMe neurons express ultradian action potential oscillations (period-interspike interval) and are grouped into phase-locked assemblies via GABA-dependent synaptic interactions. We hypothesize that PDF-dependent phase locking of assemblies gates clock output to locomotor control centers.

Materials and Methods

All experiments were performed on AMae of adult male cockroaches during the day. Breeding colonies of the cockroach (L. maderae) were kept at the University of Marburg at 30°C and 30% humidity in 12 h light/dark cycles, with lights on from 7:00 A.M. to 7:00 P.M. Animals were provided with dry dog food, potatoes, and water ad libitum.

The experimental animals were decapitated, and the head capsule was opened to expose the optic lobes. After removal of fat body and the perineurial sheath around the optic lobes, the AMe was excised with a glass pipette (diameter, 150 μm; Flaming/Brown Micropipette Puller, model P-97; Sutter Instruments, Novato, CA). The location of the AMe was easily discernible beneath the bifurcation of a characteristic trachea. All experiments were performed at constant light. Altogether, 32 AMae were used in experiments involving application of GABA and PDF (see below) or of pharmacological agents; additional recordings involved controls (application of vehicle only) and changes of the ionic conditions. In summary, 623 experiments in 32 separate recordings were performed.

For the extracellular recordings, the AMe was transferred to a Petri dish (diameter, 4 cm). All chemicals used were purchased from Sigma-Aldrich (Taufkirchen, Germany). The osmolarity of the extracellular saline (156 mm NaCl, 4 mm KCl, 6 mm CaCl₂, 10 mm HEPES, 5 mm glucose, 0.01 g/L phenol red, pH 7.1) was adjusted with mannitol to 380 mOsm. In the Ca²⁺-free extracellular solution, CaCl₂ was replaced by 1 mm EGTA. PDF (NSELINSLLSLPKNMNDA-NH₂), GABA, picrotoxin (PTX), and tetrodotoxin (TTX) were dissolved and diluted in saline. Bicuculline was dissolved in chloroform and diluted in saline. The tissue was continuously superfused with 10 ml of saline per hour at room temperature. Drugs were applied to the tissue either by pressure ejection via glass capillaries (Picospritzer II; General Valve Corporation, Fairfield, NJ) or via bath application to a chamber with a volume of 5 ml and a flow rate of 30-40 ml/h. The doses of applied materials varied according to the following two delivery modes: (1) pressure ejection (PDF, 24-800 fmol; GABA, 1-5 pmol; PTX, 100-200 pmol; bicuculline, 0.4-2 nmol), and (2) bath application (GABA, ranging from 1 to 1000 μm; PTX, ranging from 1 to 1000 μm; bicuculline, 10 μm; TTX, 0.1 μm).

Extracellular electrical activity of the excised AMe was recorded with glass electrodes (0.3-1.5 MΩ) connected to an extracellular amplifier (EXT-01C/DPA 2F; NPI Electronics, Tamm, Germany). Because of the low resistance of the recording electrodes, multiunit action potentials (events) were recorded as upward and/or downward deflections of the baseline. The output of the amplifier was high-pass filtered (3 Hz) to eliminate electrode offset and low-pass filtered (1.5 kHz) to avoid high-frequency noise and aliasing. The signal was digitized (DIGIDATA 1322A; Molecular Devices, Union City, CA) with a sampling rate of 5 kHz and stored on a disk for additional analysis. Event detection via threshold search was performed off-line with SPIKE II software (Cambridge Electronic Design, Cambridge, UK).

The mean frequency (number of events per second) was calculated to evaluate the effects of applied drugs on the firing rate of the cells. Interevent-interval distributions were generated using 1 ms bin width and different periods (depending on the experiment, with a minimum of 100 s) at different time points of the experiment to identify changes in the regularity of the electrical activity. Instantaneous frequency (1/interevent interval) plots were calculated over the whole time course of the experiments to visualize the regularity of electrical activity.

Electrical activity originating from irregularly spiking neurons or from neurons spiking with different rates or phases that are not integer multiples of each other result in a broad cloud of instantaneous frequencies. A defined narrow band of instantaneous frequencies indicates that all recorded cells fire action potentials very regularly at the same or integer multiples of the same interevent interval (harmonic frequencies). Parallel bands in the instantaneous frequency plot indicate that at least two cells fire with the same or integer multiples of the same interevent interval but with different phase relationships. If more than one cell fires with different noninteger multiples of interevent intervals and with different phase, the bands cross each other (Pikovsky et al., 2001). Fusion of parallel bands to one band indicates that the recorded cells now fire with the same phase and the same ultradian period (same interevent intervals). Autocorrelograms were generated using a bin width of 1 ms for an interval of 1 s over 5-30 min periods to identify rhythmic firing patterns (Groves et al., 1978). Regular interevent intervals cause multiple peaks in the autocorrelogram (Tepper et al., 1995).

Results

In extracellular recordings, we tested whether the neuropeptide PDF and the neurotransmitter GABA affect the electrical activity of interneurons of the AMe of the cockroach L. maderae. We show that neurons of the AMe are ultradian oscillators that are grouped into phase-locked assemblies via GABAergic synaptic interactions. PDF transiently phase locked assemblies of neurons apparently via inhibition of GABAergic interneurons, thereby disinhibiting their postsynaptic cells. We hypothesize that this disinhibition activates PDF-IR outputs to locomotor control centers via resonance (see Fig. 9). We assume that circadian clocks are indispensable for phase control of neuronal oscillators also at the time scale of milliseconds, which underlies temporal encoding of the brain.

Extracellular recordings of electrical events of the excised AMe lasted for several hours (Fig. 1). The average peak-to-peak noise amplitude was ∼40 μV. Event amplitudes ranged from 50 to 150 μV and were observed as upward and downward deflections from the baseline, depending on the impedance ratio between the recorded neurons, the recording electrode, and the indifferent electrode. Application of the sodium channel blocker TTX (10^-8m) reversibly blocked all electrical events within several hours after application to the bath solution (data not shown; n = 3 experiments in three different preparations).

Extracellular recordings usually were multiunit recordings and were composed of activity from more than one cell with different event amplitudes (Fig. 1a). Less than five recordings appeared to result from single cells. These showed constant action potential amplitude and regular firing pattern, which resulted in a sharp peak of the interevent-interval histogram (data not shown for single cells) (Fig. 1b, multiunit recording). Likewise, >80% of the multiunit recordings (of 32 preparations) showed sharp multiple peaks in the autocorrelograms, indicating regular firing modes of recorded neurons (Tepper et al., 1995) (Figs. 1c, 2b,d,f).

In 72% of all recordings, spontaneous activity occurred in one to seven parallel bands in the instantaneous frequency plot (1/interevent interval; see Materials and Methods). Activity in one band originates from spontaneously active cells that fire at the same phase and the same or integer multiples of the same interspike interval (harmonic frequencies) (Figs. 1d, 2a). Parallel bands indicate that neurons with the same or with integer multiples of the same frequency fire with a stable phase difference (Figs. 2c, 3c) (see Figs. 5c, 7c, 8c). Thus, spontaneously active ultradian oscillators are grouped into assemblies. All cells within an assembly fire phase locked with the same or with harmonic frequencies, whereas cells between assemblies fire at a stable phase difference.

To examine whether the regular action potential activity of AMe neurons is a property of the single cells or whether it is caused by synaptic synchronization of irregularly spiking neurons, we superfused the AMe with Ca²⁺-free or high Mg²⁺ saline to inhibit neurotransmitter release (Fig. 3). Application of Ca²⁺-free saline resulted in an increase of the mean event frequency and amplitude, apparently as a result of release from synaptic inhibition. In 19 of 23 experiments (involving 13 preparations), the sharp band of instantaneous frequencies broadened and gradually increased to a higher frequency level. Simultaneously, several new bands of lower frequency appeared parallel to the high-frequency band. Thus, the previously single assembly split into different assemblies. Superfusion with saline containing 10 mm Mg⁺² mimicked the effects of low-divalent solutions (n = 3) (data not shown), suggesting that the observed changes in activity are caused by loss of synaptic connections and not only by changes in the extracellular Ca²⁺ concentration.

Several events appear to underlie these observed phenomena. Neurons of one assembly, firing in synchrony with the same phase, appear to gradually shift to a new constant phase while increasing their firing frequency. In addition, previously silent neurons appear to discharge action potentials but at the same interspike interval and with constant phase relationship to the firing neurons. Thus, without synaptic communication, cells remained coupled ultradian oscillators but split into different assemblies that fired with constant phase difference and with higher frequency. After returning to extracellular saline with 6 mm Ca²⁺ concentrations, the parallel bands smoothly declined and fused to one lower frequency band (n = 10). These findings indicate that all cells gradually returned to firing in synchrony. Apparently, via inhibitory synaptic interactions again, they formed an assembly and fired at the same phase and at the same lower frequency (Fig. 3c). Frequently, a sharp drop in instantaneous frequency occurred when we switched back to high-Ca²⁺-containing extracellular saline but before complete exchange of the low-divalent extracellular solution. It is possible that this sudden inhibition of activity is caused by sudden synchronous release of an inhibitory neurotransmitter either via Ca²⁺ release from intracellular stores or via Ca²⁺ influx.

Dense GABA immunostaining in the AMe (Petri et al., 2002) suggests that GABA essentially is responsible for the inhibitory synaptic interactions between AMe neurons. Application of GABA (1-5 pmol via picospritzer; 1 μm to 1 mm via bath) resulted in a dose-dependent strong inhibition of electrical activity (Fig. 4) in 98% of all experiments (n = 453; 15 preparations). Reduced spiking activity developed within 1 to 2 s after application. Activity returned within 10 to 100 s, depending on the applied dose. In approximately one-third of all experiments in instantaneous frequency plots, GABA-dependent phase locking of cells was observed before electrical activity ceased (data not shown). Application of the vertebrate GABA_A receptor antagonist bicuculline (0.4-2 nmol) showed no effect (n = 5; five preparations). Because bicuculline is also without effect on GABA_A receptors in other invertebrate preparations, whereas the chloride channel blocker picrotoxin was used successfully as a GABA channel antagonist (Lee et al., 2003; Sattelle et al., 2003), we used picrotoxin. Picrotoxin (1 μm to 1 mm) reproducibly opposed GABA-dependent inhibition. Picrotoxin application increased electrical activity, the instantaneous frequency band smoothly increased, and several parallel bands appeared. Thus, in the presence of picrotoxin, cells split into different assemblies and remained synchronized but resumed a stable phase difference and fired with higher frequencies (n = 6; five preparations) (Fig. 5). The density of the bands increased after picrotoxin application and smoothly shifted to higher instantaneous frequencies. This indicates that previously silent cells gradually started to fire. However, these neurons always remained phase locked at a stable phase difference, because several parallel bands appeared, not just noise, or only one band at a higher instantaneous frequency. Because picrotoxin is not specific for GABA receptors but blocks different chloride channels, we cannot exclude that it might also inhibit other ligand-gated chloride channels such as glutamate receptors.

Application of PDF (24-800 fmol with picospritzer) during the day (Zeitgebertime ZT 4-11) changed the electrical activity of AMe neurons in 78% of all experiments. Peptide application either transiently decreased (n = 58; seven preparations) or increased (n = 76; four preparations) neuronal activity after delays of 1-100 s (Fig. 6). In low-divalent solutions, which disrupt synaptic transmission, PDF always led to neuronal inhibition (n = 5, involving three preparations) (Table 1), suggesting that the excitatory effects in normal saline were indirect effects through inhibitory interneurons. Indeed, after PDF-induced transient activations, removal of extracellular Ca²⁺ or elevation of Mg²⁺ abolished the PDF effect (n = 7; three preparations). In the presence of picrotoxin (Table 1), in two experiments, PDF-dependent activation was seen (n = 2; one preparation) whereas in 11 experiments, PDF-dependent inhibition was observed (n = 11; one preparation). Thus, PDF-dependent inhibition/disinhibition appears not to be mediated via chloride channel opening. PDF-dependent increases in the event amplitudes (Figs. 6a, 7a) were observed, which resulted from either disinhibition of previously inhibited cells or PDF-dependent synchronization of cells, which were not phase locked previously to the same phase. High-amplitude events were abruptly and transiently elicited superimposed on the background activity accompanied by an increase in the mean frequency (Fig. 6a); thus, PDF disinhibited new units. In other recordings, the event amplitudes increased gradually while the mean frequency decreased (Figs. 7a,b). These PDF-dependent increases in the event amplitudes resulted from synchronization of units, which previously belonged to different assemblies, as shown in instantaneous frequency plots (Fig. 7c).

In recordings with several instantaneous frequency bands, PDF caused a transient, smooth fusion of the bands to a higher synchronized instantaneous frequency compared with the lowest frequency band before application but to a lower instantaneous frequency compared with the highest band before application (Fig. 7). Because the previously parallel bands are fused to one dense band, PDF strictly synchronizes and phase locks the recorded units to the same phase (Fig. 7c). Because after PDF application the fused band maintains a higher instantaneous frequency than the lowest frequency band before application, PDF disinhibited AMe neurons (n = 16; five preparations) (Fig. 7). At the same time, PDF inhibited previously active cells, because the fused band maintains a lower synchronized instantaneous frequency compared with the highest frequency band before application. During the fusion of the bands, the event amplitudes in the original recordings transiently increased (by ∼20%) (Fig. 7a) while the mean frequency dropped. This effect of PDF application indicates superposition of action potentials of different cells, which now fire at the same phase and the same or integer multiples of the same interevent intervals. In addition, the mean frequency increased while the peptide-dependent fusion of the instantaneous frequency bands gradually disappeared. This particular result suggests that during washout (because of perfusion of the dish) of PDF, the phases of the neurons smoothly drifted apart again until their previous stable frequency and phase relationships were restored. The smoothness of transitions from parallel bands to one fused band and back to parallel bands indicates that the recorded AMe neurons always remained synchronized. Thus, PDF only synchronized the phase of perpetually coupled AMe neurons, which previously belonged to different assemblies. Control applications of vehicle resulted in none of the physiological modulations as described for the PDF effects (Fig. 8) (n = 9; five preparations).

Discussion

Extracellular recordings from the excised AMe, the circadian clock of the cockroach L. maderae, investigated the function of PDF and of GABA on neuronal spike discharges. Circadian pacemaker candidates fire action potentials with regular interspike intervals and are coupled via synaptic and nonsynaptic mechanisms. They are organized into phase-locked assemblies via GABAergic interneurons. Different assemblies distinguished by a stable phase difference were transiently synchronized to the same phase and period by PDF. We assume that PDF-dependent phase locking activates outputs to locomotor control centers (Fig. 9).