Representation of the brain's superior protocerebrum of the flesh fly, Neobellieria bullata, in the central body

The central complex of the insect brain has been shown to be involved both in sensory integration and the control of behavioral actions (Strauss et al., 1992; Liu et al., 2006; Ridgel et al., 2007; Heinze and Homberg, 2007; Ritzmann et al., 2008). It comprises four interconnected midline neuropils. These are the protocerebral bridge, the fan-shaped body, the ellipsoid body, and the paired noduli. A number of satellite neuropils, such as the pair of lateral accessory lobes and their subdomains (Muller et al., 1997), receive connections from the central complex and are critical in the expression of motor actions (Iwano et al., 2010). The components of the central complex are notable for the precise organization of their constituent neurons (Williams, 1975). In many insect species, neuronal processes projecting within and among central complex neuropils reveal a repeat modular organization (Strausfeld, 1976, 1999; Hanesch, 1989; Heinze and Homberg, 2008). Other descriptions stress the presence of tangential processes that intersect these modules, and which derive from neuropils that are lateral to the central complex (Hanesch et al., 1989; Pereanu et al., 2011). Almost unrecognized, other than in a few accounts (Liu et al., 2006; Ito and Awasaki, 2008; Young and Armstrong, 2010; Li et al., 2010), are inputs to the fan-shaped body that originate from the brain’s superior protocerebrum, a neuropil that is essentially terra incognita, having received very little attention because of its tangled appearance and lack of connections to primary sensory neuropils. These neuropils, which constitute the larger volume of the midbrain, have been referred to as “unstructured” (Liu et al., 2006). However, the superior protocerebrum is indeed structured (Strausfeld, 1976, 2012; Ito et al., 2012), and its importance in determining behavioral actions is suggested by the observation that many output neurons from the mushroom body terminate in the medial and lateral protocerebrum (Li and Strausfeld, 1997, 1999). Together these pathways imply that a crucial system of protocerebral relays are likely involved in linking the mushroom body indirectly to the central complex (Strausfeld, 2012). Notably, the central complex does not receive inputs directly from primary sensory neuropils of the brain, nor from ascending sensory relays from thoracic and abdominal ganglia. Mass dye-fills into the ventral nerve cords never show processes in the central complex. Nevertheless, neurons responsive to sensory parameters project to the central complex (Ritzmann et al., 2008), not from primary sensory neuropils but from areas of the brain that receive relays from sensory processing neuropils. For example, in grasshoppers and monarch butterflies neurons sensitive to the e-vector of polarized light project into the protocerebral bridge in a highly organized manner (Heinze and Homburg, 2009; Heinze and Reppert, 2011). These neurons have their dendrites in neuropils lateral to the central complex, in a region called the posterior optic tubercle supplied by outputs from the accessory medulla of the ipsilateral optic lobe (Heinze and Homberg, 2007). Other visual inputs to the bridge are as indirect, such as those originating from the optic glomerular complex (Phillips-Portillo, 2012). The protocerebral bridge, in turn, sends modular relay neurons into the central body (Williams, 1975; Hanesch et al., 1989; Vitzthum and Homberg, 1998; Boyan et al., 2008). Experimental evidence supports the proposition, made by several authors, that the central complex plays a cardinal role in motor actions, particularly those that involve jointed appendages (Huber, 1960; Strausfeld, 1999; Heinrich et al., 2001; Poeck et al., 2008; Kahsai et al., 2010; Bender et al., 2010). Developmental studies of the mealworm Tenebrio molitor have shown that the elaboration and differentiation of central body architecture matches the development of the legs (Wegerhoff and Breidbach, 1992; Wegerhoff et al., 1996) and that taxa having the most finely controlled leg movements possess fan-shaped body neuropils with exaggerated modular organization (Strausfeld, 1999, 2012). Genetic manipulations of Drosophila have shown that an intact central complex is also necessary for proper coordination of the limbs during asymmetrical movements such as turning, as evidenced by the mutant nobridge (nob), in which the protocerebral bridge is incompletely formed, disrupts turning (Strauss et al., 1992; Strauss and Heisenberg, 1993; Strauss, 2002). Ridgel et al. (2007) found that lesions to the central complex in the cockroach Blaberus discoidalis similarly result in animals that either made continuous turning movements during walking, or made inappropriate turns when presented with an obstacle that necessitated a change of course. The central complex has also been suggested to be important in executing actions arising from visual discrimination and learning (Liu et al., 2006; Pan et al., 2009; Triphan et al., 2010). Liu et al. (2006) used a tethered flight preparation (see Wolf and Heisenberg, 1990) to test the ability of wildtype and mutant Drosophila to learn to distinguish between two similar visual patterns. Wildtype animals readily learn to avoid flying toward punished patterns, whereas animals that express a mutant form of the adenylyl cyclase rutabaga protein required for learning (Levin et al., 1992; Zars et al., 2000) perform no better than chance. These studies have drawn special attention to neurons, called “tangential neurons,” that connect the superior protocerebrum with the fan-shaped body where their terminals extend across its extent (Liu et al., 2006). It was proposed that different types of tangential neurons carry information about the discrimination of different pattern parameters — the height of the pattern’s center of mass (elevation), or pattern inclination (contour orientation). Driving expression of rutabaga in these neurons against a mutant rutabaga- deficient background rescues the learned ability to discriminate elevation or orientation (Liu et al., 2006; Wang et al., 2008). As a step to understand how the central complex relates to the neuronal organization of the superior protocerebrum, we here describe results using reduced silver and anti-synapsin-stained brains that identify distinct anatomical domains of the protocerebrum. Into these have been mapped the arborizations of dye-filled neurons that provide tangential endings across levels of the fanshaped body. We show that, whereas the processes of many of these neurons superficially appear identical in the fan-shaped body, on close examination they constitute distinctive cell types that invade overlapping strata. Their distal arborizations reveal very different morphologies, each relating to a characteristic domain of the superior protocerebrum. These domains, which are associated with systems of local interneurons, feedback pathways, and inputs from other brain area, are likely candidates for the location of integrative circuits between inputs to the dorsal protocerebrum and outputs from it to the central complex.