Distribution and daily oscillation of GABA in the circadian system of the cockroach Rhyparobia maderae

Abstract

Gamma-aminobutyric acid (GABA) is the prevalent inhibitory neurotransmitter in nervous systems promoting sleep in both mammals and insects. In the Madeira cockroach, sleep-wake cycles are controlled by a circadian clock network in the brain's optic lobes, centered in the accessory medulla (AME) with its innervating pigment-dispersing factor (PDF) expressing clock neurons at the anterior-ventral rim of the medulla. GABA is present in cell clusters that innervate different circuits of the cockroach's AME clock, without colocalizing in PDF clock neurons. Physiological, immunohistochemical, and behavioral assays provided evidence for a role of GABA in light entrainment, possibly via the distal tract that connects the AME's glomeruli to the medulla. Furthermore, GABA was implemented in clock outputs to multiple effector systems in optic lobe and midbrain. Here, GABAergic brain circuits were analyzed further, focusing on the circadian system in search for sleep/wake controlling brain circuits. All GABAimmunoreactive neurons of the cockroach brain were also stained with an antiserum against the GABA-synthesizing enzyme glutamic acid decarboxylase. We found strong overlap of the distribution of GABA-immunoreactive networks with PDF clock networks in optic lobes and midbrain. Neurons in five of the six soma groups that innervate the clock exhibited GABA immunoreactivity. The intensity of GABA immunoreactivity in the distal tract showed daily fluctuations with maximum staining intensity in the middle of the day and weakest staining at the end of the day. Quantification via enzyme-linked immunosorbent assay and quantitative liquid chromatography coupled to electrospray ionization tandem mass spectrometry, likewise, showed higher GABA levels in the optic lobe during the inactivity phase of the cockroach during the day and lower levels during its activity phase at dusk. Our data further support the hypothesis that light-and PDF-dependently the circadian clock network of the cockroach controls GABA levels and thereby promotes sleep during the day.

3D‐atlas of the brain of the cockroach Rhyparobia maderae

“…The antiserum against GABA (Sigma–Aldrich cat# A2052, RRID: AB_477652; Table 1) was raised in rabbit against GABA conjugated to bovine serum albumin. On brain sections of R. maderae the pattern of immunolabeling was identical to that obtained with an antiserum against the GABA‐synthesizing enzyme glutamic acid decarboxylase (Massah et al., 2022). Liquid‐phase preadsorption of the diluted antiserum with 10 μmol L −1 GABA‐BSA conjugate abolished all immunostaining on brain sections of R. maderae (Giese et al., 2018).…”

“…It takes a posterior course ventrally along the LH, ends in the superior intermediate protocerebrum (SIP) and defines boundaries of the superior neuropils (SNP). The PLF contains GABA‐immunoreactive fibers with somata in the anterior soma rind below the lobula complex (LOX) (in the G7 cluster; Massah et al., 2022). These fibers run through the superior clamp (SCL) toward the crepine (CRE) and have no connection to the other reconstructed tracts.…”

“…All of these neuropils process visual signals from the compound eye. The three‐layered LA and 10‐layered ME are connected by fibers in the first optic chiasma and have been investigated in detail before (Arnold et al., 2020; Giese et al., 2018; Massah et al., 2022). Five ALAs are associated with the LA.…”

“…Five ALAs are associated with the LA. They are small oval and synapsin‐rich areas at the proximal posterior edge of the LA, three at the dorsal posterior edge (dALAs) and two at the ventral edge (vALAs), clearly separated by the first optic chiasma between the LA and ME (Figures 5e–f, 6a, and 6c) and innervated by PDH‐immunoreactive processes (Massah et al., 2022). The AME (Figures 5a–d and 6a–c), the master circadian pacemaker of the cockroach, is a small neuropil anteriormedial to the ME (Homberg et al., 2003b; Petri et al., 1995; Reischig & Stengl, 2003a,b).…”

“…The two CAs, the medial (MCA) and lateral CA (LCA), extend over almost the entire dorsal surface of the protocerebrum (Figures 1 and 6e). Each CA is further divided into an outer sheet (OCA) innervated by large GABA‐ and SIFamide‐labeled fibers (Arendt et al., 2016; Massah et al., 2022) and an inner sheet (ICA) that contains neurites of Kenyon cells (Strausfeld & Li, 1999). The ICA is free of GABA‐ and synapsin immunostaining (Figures 3–5) and can be described as a bifurcated extension of the PED (Figure 6d, yellow asterisks).…”