Lophotrochozoan neuroanatomy: An analysis of the brain and nervous system of Lineus viridis(Nemertea) using different staining techniques

Abstract

Background: The now thriving field of neurophylogeny that links the morphology of the nervous system to early evolutionary events relies heavily on detailed descriptions of the neuronal architecture of taxa under scrutiny. While recent accounts on the nervous system of a number of animal clades such as arthropods, annelids, and molluscs are abundant, in depth studies of the neuroanatomy of nemerteans are still wanting. In this study, we used different staining techniques and confocal laser scanning microscopy to reveal the architecture of the nervous system of Lineus viridis with high anatomical resolution.

Results: In L. viridis, the peripheral nervous system comprises four distinct but interconnected nerve plexus. The central nervous system consists of a pair of medullary cords and a brain. The brain surrounds the proboscis and is subdivided into four voluminous lobes and a ring of commissural tracts. The brain is well developed and contains thousands of neurons. It does not reveal compartmentalized neuropils found in other animal groups with elaborate cerebral ganglia.

Conclusions: The detailed analysis of the nemertean nervous system presented in this study does not support any hypothesis on the phylogenetic position of Nemertea within Lophotrochozoa. Neuroanatomical characters that are described here are either common in other lophotrochozoan taxa or are seemingly restricted to nemerteans. Since detailed descriptions of the nervous system of adults in other nemertean species have not been available so far, this study may serve as a basis for future studies that might add data to the unsettled question of the nemertean ground pattern and the position of this taxon within the phylogenetic tree.

Figure 1

Overview of the central nervous system of Lineus viridis. a: Head of a living specimen of Lineus viridis (Light microscopy, dorsal view) slightly squeezed, showing the location of the reddish brain (br) and the eyes (e). b: Schematic drawing (dorsal view) of the anterior nervous system of L. viridis (the neuronal somata and the cephalic nerves of the ventral part of the brain were omitted). The ventral commissural tract (vct) connects the two ventral lobes (vl) of the brain, while the dorsal commissural tract (dct) connects the two dorsal lobes (dl). Posteriorly, the ventral lobes of the brain merge with the medullary cords (mc). Medially, the esophageal nerves (en) arise from the ventral lobes. A dorsal nerve cord (dnc) arises from the dorsal commissural tract. Cephalic nerves (cn) extend from the dorsal lobes towards the anterior tip of the animal. The paired proboscidial nerves (pn) originate from the ventral commissural tract. c-f: Cross sections (Azan staining) of the head of L. viridis showing the structures of the central nervous system as shown in b. c: The brain (br) is composed of neuropil (n) which is surrounded by neuronal somata (s). The brain encloses the rhynchocoel (ry). d: Paired proboscidal nerves (pn) run the full length of the proboscis inside the proboscis musculature. c-f: The cephalic slits (cs) narrow each into a canal (ca) which leads to the cerebral organ (co). The cerebral organ (co) is attached posteriorly to the inferior branch of the dorsal lobes of the brain (idl). a anterior; bv blood vessel; d dorsal; ec esophageal commissure; in inner neurilemma; mo mouth opening; on outer neurilemma; p posterior; sdl superior branch of the dorsal lobe; v ventral. Scale bars: a = 500 μm; c-f = 200 μm.

Figure 2

Comparison of the neuroarchitecture of the brain. a-c: Horizontal sections through the ventral lobes of the brain. The brain is composed of neuropil which is surrounded by densely packed somata. The ventral lobes are connected by the ventral commissural tract (vct). Posteriorly, the ventral part of the brain merges with the paired medullary cords (mc). Medially, the esophageal nerves (arrowheads in a) arise from the ventral lobes. Cephalic nerves (arrow in a) extend from the ventral lobes of the brain towards the anterior tip of the animal. The cerebral organs (co) are attached to the brain posteriorly (c-e). d-f: Horizontal sections through the dorsal lobes of the brain. The dorsal lobes are connected by a dorsal commissural tract (dct), as well. The brain is penetrated by the proboscis (p) which exhibits a cylindrical plexus in the immunostainings (d, g). g-h: Cross sections demonstrate that the brain and its commissural tracts form a ring surrounding the proboscis (p). cs cephalic slits. Scale bars: a,d,g,h = 200 μm; b,c,e,f = 500 μm.

Figure 3

Cellular architecture of the brain of Lineus viridis. a: Azan stained cross section of the brain of L. viridis showing the location of the neuronal somata surrounding the neuropil of the brain. The neuropil within the ventral lobe (vl) and the dorsal lobe (dl) is interspersed with glia sheath (gs). b,c: Higher magnification images demonstrate that the neuronal somata differ in size and can be classified at least in three different types (s1-s3). dct dorsal commissural tract; in inner neurilemma; on outer neurilemma; pn proboscis nerve; pr proboscis; ry rhynchocoel. Scale bars: a = 100 μm; b,c = 20 μm.

Figure 4

Morphology of the cerebral organ. a: Overlay image of DAPI nuclear labeling in blue and FMRFamide-like immunostaining in red. a,b: The cerebral organ is attached to the dorsal lobe (dl) of the brain. Its anterior part is innervated by neurites (n) originating from the brain. The remainder of the cerebral organ is filled with densely packed small diameter somata (asterisk) and large diameter cells that contain voluminous cell nuclei (arrowheads). c,d: A canal (arrows) runs along the lateral aspect of the cerebral organ. e,f: In the most ventral part of the cerebral organ this canal opens into a ciliated epidermal canal (arrow) and a sac-like compartment (sc). Here the cells are more slender and densely packed (open arrowheads) than in the wider section of the canal. The duct connects the cerebral organ with the environment (c). bv blood vessel; ci cilia; ecm extracellular matrix; epi epidermis. Scale bars: a,c,e = 40 μm; b,d,f = 100 μm.

Figure 5

General anatomy of the peripheral nervous system. a: Horizontal section of the post-cerebral nervous system showing serotonin-like immunoreactive neurites and somata of the medullary cord (mc) as well as the subepidermal plexus (arrowheads) and the stomatogastric plexus (stp). b: Transverse section showing FMRFamide-like immunoreactivity in the medullary cord (mc), the subepidermal plexus (arrowhead), the commissural plexus (cp), the stomatogastric plexus (stp), and the plexus of the proboscis wall (pp). c: Serotonin-like immunoreactivity in a horizontal section reveals a diffuse arrangement of neurites in the subepidermal plexus (corresponding to arrowheads in a and b). d: FMRFamide-like immunoreactivity in a horizontal section demonstrates that neurites of the commissural plexus are preferentially oriented in circular manner. epi epidermis. Scale bars: a,c,d = 80 μm; b = 200 μm.