Planaria nervous system
Planarians (suborder Tricladida, phylum Platyhelminthes) are considered to be among the most primitive animals to have developed a central nervous system (CNS). (Taxonomy ID: 6157 in NCBI Taxonomy Browser)
According to morphological criteria, planarians were once positioned on a branch from the base of bilaterians before the protostome-deuterostome divergence, since the mouth and anus are not separate in planarians. However, on the basis of later molecular phylogenetic analysis using mitochondrial DNA sequences, the protostomes were separated into two groups, lophotrochozoa and ecdysozoa, and planarians were classified into lophotrochozoa (Aguinaldo et al., 1997). Later molecular phylogenetic analysis using genomic/EST database also confirmed this phylogenetic position (Dunn C. W. et al., 2008). Detailed molecular phylogenetic analysis using housekeeping genes categorized planarians into a basal position among CNS-possessing animals (Mineta et al., 2003), therefore molecular and cellular dissection of the planarian brain may be useful for understand the evolution of the brain in more complex animals.
Below are summarized the function and structure of the brain of a planarian, Dugesia japonica.
The body size of planarians along the anterior-posterior axis ranges from 3 to 20 millimeters. The planarian CNS is composed of a brain in the head region and a pair of ventral nerve cords (VNCs) that extend the length of the body (Fig.1A) (Agata et al., 1998; Tazaki et al., 1999). The brain consists of two lobes, forming an inverted U-shaped structure, and nine branches on the outer side of each lobe that project to the surface of the head region, forming sensory organs (Fig.1B)(Okamoto et al., 2005). A pair of eyes is located on the dorsal side of the brain. The size and number of brain neurons vary depending on the size of the planarian’s body. The minimum size of the brain (in a planarian with body size about 0.7mm) consists of about 8,000 neurons (Okamoto et al., unpublished).
Molecular studies showed that the brain is composed of structurally distinct and functionally diverse domains, which have been defined by morphological observations using a variety of molecular markers. In the first study on the organization of the planarian brain, a homolog of the homeobox-containing gene Orthopedia (Otp) from planaria, Djotp, was isolated. Djotp is specifically expressed in the branch structures of the brain (Umesono et al., 1997). In a subsequent study, two otd/Otx-related homeobox genes, DjotxA and DjotxB, were isolated from the planarian Dugesia japonica. Within the CNS, expression of both these genes appear to be restricted to the brain. DjotxA is expressed in the medial region of the brain and in visual cells. DjotxB is expressed in the major region of the brain (called the sponge region or main lobes) just lateral to the DjotxA-positive domain, but not in the more lateral branch structures, where Djotp is expressed. These expression data for DjotxA, DjotxB and Djotp suggest a molecular basis for subdivision of the mature planarian brain into at least four regions: a DjotxA-positive region, a DjotxB-positive region, a Djotp-positive region (branch regions), and a region lateral to the branches and negative for Djotp (Fig.2) (Umesono et al., 1999).interneurons, suggesting that the DjotxB-positive region may work as a signal processing center of the brain. The domain structure of the planarian brain is summarized in Fig.2.
Other studies showed more complex subdivision structures in the main lobes (Cebrià 2002b), but these small subdomains are not well characterized. Among these studies, the finding on the expression of one planarian brain factor homolog, DjfoxD, should be mentioned (Koinuma et al., 2003). The expression of DjfoxD is highly restricted to the mid-apex of the head, between the two lobes of the brain, where brain neurons are not present. The DjfoxD-expression domain clearly separates the right and left lobes (Saito et al., 2003).
Projection and Connection
To examine how individual neurons are connected to each other, antibodies were raised against visual neurons and chemosensory neurons and used to stain their axons (Sakai et al., 2000; Inoue et al., 2007). Individual neurons were also stained also by fluorescent dye tracing (Okamoto et al., 2005).
Results of immunostaining
Planarian eyes are composed of two cell types: pigment cells and visual neurons (photoreceptor cells). The pigment cells are arranged into a crescent-shaped eyecup, while the visual neurons are located outside of the eyecup. Studies using a monoclonal antibody specific to planarian photoreceptors (called VC-1) showed that dendrites from the visual neurons are distributed inside of the pigment eyecup to form a rhabdomeric structure containing opsins, and that the axons of the visual neurons project to the medial region of the main lobes (Agata et al., 1998; Sakai et al., 2000). They form an optic chiasma in the dorso-medial region of the brain. Each lateral branch neuron (chemosensory neuron) projects onto the lateral region of the brain (Inoue et al., 2007).
Results of DiI injection
Planarian neurons have some types of evolutionarily conserved molecules involved in guidance, adhesion and signaling. For example, a netrin homolog gene is isolated from Dugesia japonica and named Djnet1 (Cebrià et al., 2002b). This gene was expressed in the brain and VNCs. Also, homologs of vertebrate neural cell adhesion molecule (NCAM), Down Syndrome cell adhesion molecule (DSCAM), L1CAM and contactin were detected in the planarian Dugesia japonica, and designated DjCAM, DjDSCAM, DjLCAM and DjCTCAM, respectively (Fusaoka et al., 2006). DjCAM and DjDSCAM are differentially expressed in the CNS. In DjCAM-RNAi animals, abnormalities of axonal fasciculation in the lateral branches were observed using an lateral branch marker, anti-G protein beta antibidy. In DjDSCAM-RNAi animals, not only the number of lateral branches but anlo that of cell body clusters decreased. Furthermore, the a synaptotagmin homologue (Djsyt), a clathrin heavy chain gene (DjCHC) and the gene for synaptosome-associated protein 25 kDa (Djsnap-25) of Dugesia japonica were isolated (Tazaki et al., 1999; Inoue et al., 2007; Takano et al., 2007). RNAi of DjCHC prevented CNS regeneration by blocking the intermediate stage of regeneration prior to neural circuit formation. There are no obvious morphological defects in Djnsap25 knockdown head regenerated animals, but these animals have clear defect of movement related to negative phototaxis. Some kind of genes related to guidance and adhesion were also isolated from another planarian, Schmidtea mediterranea. For example, two netrin homolog genes and a netrin receptor gene were isolated and named Smad-netrin1, 2 and Smad-netR respectively (Cebrià et al., 2005). Following Smad-netrin1 RNAi, planarians could regenerate normally and no defects were observed either in the brain or VNCs. By contrast, Smad-netrin2 RNAi animals and Smad-netR RNAi animals couldn't regenerate a proper CNS. Also, homologs of the roundabout (robo) and slit family were detected in the Schmidtea mediterranea, and named Smad-roboA and Smad-slit respectively (Cebrià et al., 2007a.b). Smad-roboA RNAi disrupted the nervous system structure during cephalic regeneration: the newly regenerated brain and VNCs did not re-established proper connections. In Smad-slit RNAi planarians, many newly regenerated tissues at the midline, including the cephalic ganglia, VNCs, photoreceptores and the posterior digestive system, were collapsed.
Composition of neurons
Classical morphological studies revealed that planarian neurons more closely resemble the neurons of vertebrates than those of higher invertebrates (Sarnat and Netsky, 1985). Planarian neurons have a large egg-shaped nucleus, scattered agglomerated chromatin and large nuclear bodies. Synaptic vesicles are observed in the neuropile. The synaptic structure observed in the brain and peripheral ganglia is basically similar to the chemo-synapse of vertebrates. In both synaptic structures, thickened pre- and post-synaptic membranes and accumulation of clear vesicles in the pre-synaptic region are observed (Oosaki et al., 1965). Extensive molecular studies showed that the planarian brain is composed of functionally diverse neurons which express homologs of specific genes seen in mammalian neurons.
The planarian brain contains GABA (γ-aminobutyric acid) synthetic neurons (GABAergic neurons). GAD (glutatamic acid decarbocylase) is the rate-limiting enzyme for GABA biosynthesis, and converts glutamic acid into GABA. Recently, the planarian GAD gene (DjGAD) was isolated (Nishimura et al., 2008), and it was found that in the head region, DjGAD-expressing cells are distributed in a dotted pattern in the brain. Some of them are aligned in an inverted U-shape, while others are distributed in the medial part of the brain. The distribution pattern of DjGAD-immunopositive cells is very similar to that of DjGAD-mRNA stained by in situ hybridization (Fig.5B).
Several kinds of neurons synthesizing 5-HT, catecholamine and some neuropeptides have been also detected in the brains of other planarians, Girardia tigrina, Polycelis tenuis, Dendrocoelum lacteum and Schmidtea mediterranea (Reuter et al., 1995, 1996, Cebrià, 2008).
Planarians are known as not only one of the most primitive animals which have a brain, but also as animals that have high regenerative ability. They can regenerate an entire body, including a brain, within 5 days after amputation from a small piece of the body in which no brain tissues remain. The process of regeneration of the brain has been extensively analysed by whole-mount staining. The process of brain regeneration can be divided into five steps (Cebrià et al., 2002, Agata et al., 2008):
- anterior blastema formation
- brain rudiment formation
- pattern formation
- neural network formation
- functional recovery
The anterior blastema is formed after wound closure (step 1; Hwang et al., 2004) and then a brain rudiment is formed in the anterior blastema (step 2). These first two steps occur within 24 hours. The earliest gene known to be activated after amputation is a noggin-like gene, DjnlgA. This gene is activated by dosal-ventral interaction after wound closure (Ogawa et al., 2002). After formation of the brain rudiment, the regenerating brain starts to undergo pattern formation (step 3), in which the expression of three different otd/Otx-related and Wnt-related genes is detected 36 hours after amputation (Umesono et al., 1997, 1999, Kobayashi et al., 2007). In step 4, netrin homologues begin to be expressed, and then the eyes and brain, and the brain and VNCs become connected to each other (Cebria 2002b). N-CAM and cadherin family genes are also activated during this period (Fusaoka et al., 2006). The network structure of the CNS is completely reformed within 4 days. Although morphological recovery is complete, an additional day is needed for functional recovery (Inoue et al., 2004).
Gene profiling of single-neurons
To investigate the complexity of neural cells in planarians, a new method was developed for gene profiling of single neurons obtained using cell sorting. In this method, brain neurons are collected as single cells using FACS (Asami et al., 2002). After the collection of single neurons, RNA is extracted, and cDNA is synthesized and amplified by PCR. Each cDNA sample is then used as a template for semi-quantitative PCR analysis using specific primers. The results of such analyses were overlaid on a 3D planarian brain model and a 3D model based on the single-cell PCR database of the expression of about 1,000 genes in the planarian brain was created on a scale of about 1 to 10 and is available on the web (http://brain.brh.co.jp).
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