Mutual coupling of neurons in the circadian master clock: What we can learn from fruit flies

Abstract

Circadian master clocks in the brain consist of multiple neurons that are organized into populations with different morphology, physiology, and neuromessenger content and presumably different functions. In most animals, these master clocks are distributed bilaterally, located in close proximity to the visual system, and synchronized by the eyes with the light-dark cycles of the environment. In mammals and cockroaches, each of the two master clocks consists of a core region that receives information from the eyes and a shell region from which most of the output projections originate, whereas in flies and several other insects, the master clocks are distributed in lateral and dorsal brain regions. In all cases, morning and evening clock neurons seem to exist, and the communication between them and other populations of clock neurons, as well as the connection across the two brain hemispheres, is a prerequisite for normal rhythmic function. Phenomena such as rhythm splitting, and internal desynchronization are caused by the "decoupling" of the master clocks in the two brain hemispheres or by the decoupling of certain clock neurons within the master clock of one brain hemisphere. Since the master clocks in flies contain relatively few neurons that are well characterized at the individual level, the fly is particularly well suited to study the communication between individual clock neurons. Here, we review the organization of the bilateral master clocks in the fly brain, with a focus on synaptic and paracrine connections between the multiple clock neurons, in comparison with other insects and mammals.

Keywords: Clock neurons; Drosophila melanogaster; Dual oscillator model; Flywire connectome; Multi-oscillator system; Neuropeptides.

Fig. 1

Clock neurons in the brain of Drosophila melanogaster A. Somata of the clock neurons in the lateral and dorsal brain. The Lateral Neurons (LN) can be divided into the anteriorly located dorsal LN (LN_d), ventral LN (LN_v), and 5th LN, as well as the posteriorly located Lateral Posterior Neurons (LPN). Of the six LN_d, three are Cryptochrome (CRY)-negative (yellow) and three are CRY-positive (orange and red). The LN_d in red does express the neuropeptide Ion Transport Peptide (ITP) and is therefore grouped with the ITP-positive 5th LN, in the LN^ITP. The LN^ITP have very similar arborization patterns (see Fig. 2). The LN_v can be further subdivided into the large LN_v (l-LN_v) and small LN_v (l-LN_v). Both express the neuropeptide Pigment-Dispersing Factor (PDF). The Dorsal Neurons (DN) can be divided into the anterior DN₁ (DN_1a), the posterior DN₁ (DN_1p), the DN₂, and the DN₃, which include the largest number of clock neurons, of which most have small somata. The DN₃ are the only clock cluster of which not all somata are drawn. The real number of DN₃ somata per brain hemisphere can be found in parenthesis behind the different subgroups. The DN_1p and the DN₃ are very heterogeneous in respect of neuropeptide expression and branching patterns. The DN_1p can be subdivided into five subgroups (A–E), of which only the first two subgroups (DN_1pA and DN_1pB) are CRY-positive. The DN₃ can even be subdivided into seven subgroups, of which the anterior projecting DN₃ (APDN₃) and the large centrally projecting DN₃ (l-CPDN₃) have larger somata and are CRY-positive. The other five subgroups project all centrally and have small somata (s-CPDN₃ A-E). B. Reconstruction of all clock neuron arborizations in one female brain stemming from the FlyWire connectome (same color code as in A). The neurites of the clock neurons overlap in four major fiber hubs: the accessory medulla (aMe), a hub in the Posterior Lateral Protocerebrum (PLP), a hub in the Superior Medial Protocerebrum (SMP), and one in the Superior Lateral Protocerebrum (SLP). The l-LN_v are the only clock neurons arborizing in the medulla (Me) of the optic lobes. Modified from Reinhard et al. (2024).

Fig. 2

Clock neurons with prominent contralateral projections A. The four CRY- and PDF-positive l-LN_v project via the posterior optic commissure (POC) into the ipsilateral and contralateral accessory medulla (aMe) and medulla (Me). B. The two CRY- and ITP-positive LN^ITP project to the ipsilateral and contralateral SMP. Ipsilaterally they arborize in the PLP and aMe. C. The two CRY-positive LN_d (LN_d^CRY+) arborize densely in the ipsilateral and contralateral SMP, and ipsilaterally they send few fibers to PLP and aMe. D. The CRY-positive large centrally projecting DN₃ (l-CPDN₃) arborizes in the ipsi- and contralateral SMP, SLP and PLP and sends fibers into the ipsilateral aMe. E. The four CRY-positive DN_1pA have the densest contralateral projections in the SMP and SLP, and ipsilaterally they extend to the PLP. F. The fibers of the two CRY-negative DN₂ remain in the dorsal protocerebrum and invade ipsi- and contralaterally the SMP and SLP. Ipsilaterally they project toward the anterior optic tubercle. On the right hemisphere, one DN₂ is missing within the FlyWire connectome.

Fig. 3

Typical actograms of a wildtype fly and two mutants (gl^60J and Pdf⁰) that putatively affect the coupling between the clock neurons All flies were first recorded under 12:12 h light-dark cycles (LD, see shaded areas for the light phase). After 8 days the light-dark cycle (LD1) was phase-delayed by 8 h (LD2) for the wild-type fly and glass mutant (gl^60J) to show that they can re-entrain quite fast to a new light-regime. In contrast to the wild-type fly, the gl^60J mutant showed a broad scattered activity of which some activity components re-entrained only slowly to the new light regime (open arrows). After transfer to constant darkness (DD), the broad and scattered activity of the gl^60J mutant persisted, but the fly did not become arrhythmic. In contrast, the Pdf⁰ mutant, which was transferred to DD directly after the first LD cycle, initially showed a narrow activity band band that continuously broadened leading finally to arrhythmicity. This mutant free-ran with a short period before it became arrhythmic. Modified from Helfrich-Förster et al. (2001).

Fig. 4

Ipsilateral synaptic connections between the clock neurons according to the hemibrain. The size of the colored bars at the margin of the half-circle, which represents the different clock neurons, is plotted proportionally to their synaptic connections, meaning that those clock neurons that have virtually no synaptic connections within the ipsilateral clock network, such as the four s-LN_v and four l-LN_v are represented by very slim black bars, whereas the ones that have plenty connections such as the two LN^ITP are represented by thick dark red bars. The direction of the arrow indicates the flow of information. Only connections with >9 synapses were taken into account. Please note that the l-LN_v synapse only onto each other, and that the s-LN_v have extremely few synapses with other clock neurons, but not with the E neurons (LN^ITP and LNd^CRY+). See text for further explanations. Modified after Reinhard et al. (2024).

Fig. 5

Morning (M) and evening (E) activity of wild-type and mutant flies, which are controlled by M and E neurons A. Double-plotted actogram of a wild-type fly, in which the M activity (blue line) persists in constant darkness (DD) and free-runs in parallel to the E activity (red line). B. Double-plotted actogram of a wild-type fly, in which the M activity free-ran with a shorter period than the E activity. C. Typical activity pattern of a sol¹;so¹ mutant that has extraordinarily high PDF levels in the central brain, which shortens the free-running period of the M oscillator and lengthens that of the E oscillator leading to an internal desynchronization of both oscillators. D. Typical activity pattern of a cry^b mutant in LD and in constant light (LL). Due to not functional CRY, the fly remains rhythmic in LL, and light from the compound eyes to the l-LN_v increases PDF secretion into the accessory medulla, shortening/lengthening the periods of M and E oscillators, respectively, and causing internal desynchronization. E. M (blue) and E (red) clock neurons in the Drosophila brain. The yellow arrows indicate the light input to the l-LN_v which activates them and leads to enhanced PDF secretion into the accessory medulla. Modified from Helfrich-Förster (2024).