Figure 1.
Neuropils of the grasshopper central complex.
A: Sagittal section through the upper (CBU) and lower (CBL) division of the central body and one nodulus (N). Left side of the drawing is anterior and top is dorsal with respect to the grasshopper's body axis. The central body includes fronto-horizontal layers, three layers (layer I–III) plus the central body anterior lip (CBAL) in the upper division and six in the lower division (layer 1–6). The layers I and II of the upper division can both be subdivided into compartments a and b (modified after [18], [21]. B: Frontal section through the protocerebral bridge (PB), the upper (CBU) and lower (CBL) division of the central body and the lateral accessory lobes (LAL). Top of the drawing is dorsal and bottom is ventral with respect to the grasshopper's body axis.
Figure 2.
Muscarinic ACh receptor-immunoreactivity (mAChR-ir) in the central complex.
A–D: Frontal sections of the central complex. MAChR-ir is restricted to columnar fibers whose somata are located in the pars intercerebralis (PI). These neurons send small neurites into the protocerebral bridge (PB) (arrowheads in A, B and D). The main fibers project as large diameter neurites via four pairs of fiber bundles, the w-, x-, y- and z-bundles through the posterior chiasm (indicated by white asterisk in A, B, D and E) between the protocerebral bridge and the central body and innervate single columns of the lower division. The fibers run to the contralateral lateral accessory lobe (LAL) via the isthmus tract (indicated by white asterisk in C) and seem to terminate in the lateral triangle (indicated by arrow in C). E: Sagittal section through the central body with two types of mAChR-ir columnar fibers. One type (indicated by arrowheads) runs through layer I of the CBU and passes along the anterior border of the CBL, while the other type projects through layer III as part of the posterior vertical bundle (indicated by arrow) and innervates the CBL (The inset describes the projection pattern of the second type; modified from [18], [21]. F: Sagittal section of the CBL. The arborization pattern in the lower division is not restricted to a certain layer but extends rather diffusely throughout the entire CBL. Smooth appearance of neurites indicates their post-synaptic character. Scale bars = 50 µm in A, B, C and D; 20 µm in E; 10 µm in F.
Figure 3.
GABA immunoreactivity in the central complex.
A and B: Frontal sections through the midbrain (A) and the central body (B). The entire lower division (CBL) is densely innervated by GABA positive neurites, while only few neurites in the upper division (CBU) contain GABA. The somata of these fibers are located in the inferior-median protocerebrum (arrows in A and D) and in the inferior lateral protocerebrum (arrowhead in A). C and E: Sagittal sections through the central body. Staining in the CBU is restricted to layer II, while the other layers are devoid of GABA. The CBL is densely innervated by GABA-containing neurites. GABA positive fibers enter the CB via the posterior groove. D: Frontal section through one lateral accessory lobe (LAL). Fibers originating from cells in the inferior median protocerebrum run through the isthmus tract (indicated by asterisk) before they enter the CB. Knob-like staining is observed in the lateral triangle (double arrowheads). Additional intensive immunoreactivity is present in the median olive (arrowhead). F: Frontal section through the protocerebral bridge (PB) reveals the absence of GABA from this neuropil. G: Schematic drawing of a sagittal section through the CB. Regions highlighted in gray contain GABA positive fibers (modified from [18], [21]. CBAL, anterior lip of the central body upper division; N, noduli. Scale bars = 100 µm in A; 50 µm in B; 40 µm in C; 20 µm in D, E and F.
Figure 4.
Citrulline immunoreactivity in the central complex.
A–D: Frontal sections through the midbrain and central complex. E: Sagittal section through the central complex. Groups of citrulline immunoreactive cell bodies are located in the pars intecerebralis (PI, white arrows in A, B, D and E) and in the inferior median protocerebrum (arrowheads in A). Citrulline immunoreactivity is restricted to the layers II and III of the upper division (CBU) of the central body, while layer I and the entire lower division (CBL) was completely free of immunostaining. Two types of immunoreactive neurons were distinguished. Pontine neurons contributing to intensive labeling in the posterior chiasm (small arrowheads in B, D and E) and additional faint labeling in tracts entering the CB through the dorsal and posterior face (large arrowhead in E). Immunostaining in the posterior groove (black arrow in E) resulted from tangential neurons. The lateral accessory lobes (LAL) contained weak staining. Citrulline-ir was detected in the median olive (MO in C) and the ventral shell (white arrowhead in C) of the LAL. F: Schematic drawing of a sagittal section through the CB (modified from [18], [21]. Regions highlighted in gray contain citrulline positive fibers. CBAL = anterior lip of the central body, N = noduli. Scale bars = 50 µm in A, B and C; 20 µm in D and E.
Figure 5.
Nitric oxide-stimulated cGMP immunoreactivity in the central complex.
A: Frontal section, through the central complex and surrounding midbrain structures. Within the central complex cGMP-ir is restricted to a particular layer of the CBL. Labeled cell bodies are located in the inferior median protocerebrum (indicated by arrows). B and C: Frontal sections through the CBL. Strong cGMP-ir is associated with fibers of tangential neurons projecting close to the anterior border of the CBL. The neurites enter the CBL from posterior direction and innervate the CBL in a fan-shaped fashion (best seen in C). Immunostaining in the CBL appears to be beaded (best seen in B and D) suggesting that labeled neurites represent presynaptic structures. D: Sagittal section through the central body: Staining in the CBL is restricted to layer II, while the other layers of the CBL and the entire CBU are completely devoid of staining (compare with F). E: Frontal section through one lateral accessory lobe (LAL) containing faint labeling in the lateral triangle (LT) and the median olive (MO). F: Schematic drawing of a sagittal section through the CB (modified from [18], [21]. Regions labeled black contain cGMP immunopositive neurites. CBAL = anterior lip of the central body, N = noduli. Scale bars = 100 µm in A; 20 µm in B, D and E; 10 µm in C.
Figure 6.
GABA, mAChR and NO-stimulated cGMP immunoreactivity in the central complex.
A1–B3 Double labeling of GABA (green) and cGMP (magenta) in frontal sections through the midbrain. Colocalization (highlighted in white) was detected in tangential neurons of the central body lower division. All cGMP-immunoreactive neurites also contain GABA immunoreactivity, whereas only a subset of GABA-immunoreactive neurites accumulated detectable amounts of cGMP upon nitric oxide stimulation. Colocalisation of GABA and cGMP immunoreactivity was detected in five cell bodies per hemisphere in the infero-median protocerebrum (two of them are marked by arrows in B1–B3). C1–D3 Double labeling of mAChR (green) and cGMP (magenta) on frontal sections through the central complex. No colocalisation was detected, indicating that NO has no direct effect on mAChR-expressing columnar neurons of the central complex. Scale bars = 50 µm in B1–C3; 20 µm D1–D3; 10 µm in A1–A3.
Figure 7.
Pharmacological stimulation of sound production by pressure injection of neuroactive substances into the central complex of restrained male Ch. biguttulus.
A: The duration of stridulation stimulated by 10−4 M muscarine (black column) was reversibly reduced by a single injection of the NO donor SNP (10−3 M) to the same site in the central complex. Significant differences (**: p≤0.01; *: p≤0.05) were calculated with respect to the muscarine pulses before SNP application. B: After overnight storage in an unsealed vessel, SNP solution loses its potency to generate NO upon injection into the central complex. Inactivated SNP had no inhibitory effect on muscarine-stimulated stridulation. C: The duration of stridulation stimulated by 10−3 M picrotoxin was unaffected by co-application of SNP (10−3 M) to the same site in the central complex.
Figure 8.
Information flow in the central body with respect to the control of grasshopper sound production.
Each type of neuron included represents bilateral groups of several cells. A: Columnar neurons (green) are activated by both nicotinic and muscarinic input from unknown presynaptic neurons. Sufficient cumulative activity of columnar neurons initiates stridulation via direct or indirect activation of descending stridulatory command neurons. Tangential neurons release GABA in the lower division of the central body (CBL) that most likely directly inhibits columnar neurons through picrotoxin-sensitive ionotropic GABAA receptors. Part of the synaptic release of GABA in the CBL is potentiated by nitric oxide (NO) released in the upper division (CBL). B: Three transmitters known to promote (ACh) or suppress (GABA and NO) grasshopper sound production converge on GABAergic synapses in the CBL. NO released from neurites in the CBU activates soluble guanylate cyclase (sGC) in a subpopulation of GABAergic tangential neurons in the CBL. Accumulation of cGMP potentiates GABA release from these tangential cells. The balance of activating and inhibiting inputs to these columnar neurons determines whether sound production will be executed.