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The "Amphi"-Brains of Amphipods: New Insights From the Neuroanatomy of Parhyale hawaiensis (Dana, 1853)

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The "Amphi"-Brains of Amphipods: New Insights From the Neuroanatomy of Parhyale hawaiensis (Dana, 1853)

Christin Wittfoth et al. Front Zool.

Abstract

Background: Over the last years, the amphipod crustacean Parhyale hawaiensis has developed into an attractive marine animal model for evolutionary developmental studies that offers several advantages over existing experimental organisms. It is easy to rear in laboratory conditions with embryos available year-round and amenable to numerous kinds of embryological and functional genetic manipulations. However, beyond these developmental and genetic analyses, research on the architecture of its nervous system is fragmentary. In order to provide a first neuroanatomical atlas of the brain, we investigated P. hawaiensis using immunohistochemical labelings combined with laser-scanning microscopy, X-ray microcomputed tomography, histological sectioning and 3D reconstructions.

Results: As in most amphipod crustaceans, the brain is dorsally bent out of the body axis with downward oriented lateral hemispheres of the protocerebrum. It comprises almost all prominent neuropils that are part of the suggested ground pattern of malacostracan crustaceans (except the lobula plate and projection neuron tract neuropil). Beyond a general uniformity of these neuropils, the brain of P. hawaiensis is characterized by an elaborated central complex and a modified lamina (first order visual neuropil), which displays a chambered appearance. In the light of a recent analysis on photoreceptor projections in P. hawaiensis, the observed architecture of the lamina corresponds to specialized photoreceptor terminals. Furthermore, in contrast to previous descriptions of amphipod brains, we suggest the presence of a poorly differentiated hemiellipsoid body and an inner chiasm and critically discuss these aspects.

Conclusions: Despite a general uniformity of amphipod brains, there is also a certain degree of variability in architecture and size of different neuropils, reflecting various ecologies and life styles of different species. In contrast to other amphipods, the brain of P. hawaiensis does not display any striking modifications or bias towards processing one particular sensory modality. Thus, we conclude that this brain represents a common type of an amphipod brain. Considering various established protocols for analyzing and manipulating P. hawaiensis, this organism is a suitable model to gain deeper understanding of brain anatomy e.g. by using connectome approaches, and this study can serve as first solid basis for following studies.

Keywords: Crustaceans; Hemiellipsoid body; Histamine; Lamina; Olfactory system; Peracarida; RFamide; Visual system.

Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The amphipod Parhyale hawaiensis and gross morphology of its nervous system. a Male and female of P. hawaiensis in precopula. b Orthoslice at the mid-level of the brain based on microCT analysis. The brain (yellow) is situated right between the compound eyes in the dorsal part of the head. c 3D reconstruction of the brain based on microCT analysis (light grey: optical system, yellow: neuropil, purple: organ of Bellonci, dark grey: somata) in anterolateral view (C1), frontal view (C2) and lateral view (C3). The red line displays the neuraxis of the brain with red dots indicating the roots of associated nerves to sensory organs and appendages to signify the arrangement of proto-, deuto-, and tritocerebrum. An interactive 3D PDF is available as supplement (Additional file 4). d 3D reconstruction of the central nervous system (yellow) in anatomical context based on microCT analysis in lateral view. The central nervous system is located ventrally and bent dorsally, anterior to the subesophageal ganglion. Inset (bottom right corner in B) shows position and orientation of depicted section in the brain. Abbreviations: I-III segmental ganglia of the pleosome, IV-VI fused ganglion of the urosome, a1 antenna 1, a1nv antenna 1 nerve, a2 antenna 2, a2nv antenna 2 nerve, br brain, cb central body, dcc deutocerebral commissure, dcl deutocerebral chemosensory lobe, ec esophageal connectives, es esophagus, g1–7 segmental ganglia of the pereon, gn2 second gnathopod, hn/tm hemiellipsoid body/ terminal medulla complex, la lamina, lan lateral antenna 1 neuropil, lo lobula, me medulla, obe organ of Bellonci, om ommatidia, p1–7 pereopods 1–7, pl1–3 pleopods 1–3, seg subesophageal ganglion, tc tritocerebrum. Scale bars: (a, d) 1 mm, (b) 200 μm, (c) 150 μm
Fig. 2
Fig. 2
Histological sections of the head. a Frontal section of the head with posterior part of the brain. The brain is surrounded by numerous lipid droplets (brownish spherules). The dorsal-most part of the brain is innervated by paired small nerves (double-arrow). The optical nerve forms a chiasm (red arrow). b Magnification of the chiasm (red arrow) of the optical nerve of the left brain hemisphere, frontal view. c Magnification of the first and second visual neuropil of the right brain hemisphere displaying a chambered appearance of the lamina, frontal view. Numerous terminations within each chamber are visible. Somata of cluster 1 appear darker and smaller. d Frontal section of the anterior part of the brain. A small section of the antenna 1 nerve projects to the deutocerebral chemosensory lobe (red arrows). The medial foramen is discernable (double-arrow). Insets (bottom left corner in A and D) show position and orientation of depicted sections in the brain. Abbreviations: a1nv antenna 1 nerve, ann antenna 2 neuropil, ao anterior aorta, ba brain artery, dcl deutocerebral chemosensory lobe, ec esophageal connective, es esophagus, hn/tm hemiellipsoid body/ terminal medulla complex, la lamina, lo lobula, maf a myoarterial formation a, me medulla, om ommatidia, onv optical nerve, pc protocerebrum, re retina, [numbers] somata cluster. Scale bars: (a) 100 μm, (b-d) 50 μm
Fig. 3
Fig. 3
Overview of the neuroanatomy of the brain. a Frontal vibratome section at the mid-level of the brain, double-labeled against acetylated tubulin (yellow-red) and RFamide (green). b Sagittal vibratome section of the brain, triple-labeled against acetylated tubulin (yellow-red), RFamide (green) and nuclei (blue, B1) as well as against acetylated tubulin, separately (black-white inverted, B2). Insets (bottom left corner in A and right upper corner in B1) show position and orientation of depicted sections in the brain. Abbreviations: ampn anterior medial protocerebral neuropil, ann antenna 2 neuropil, ao anterior aorta, cb central body, dcl deutocerebral chemosensory lobe, ec esophageal connective, hn/tm hemiellipsoid body/ terminal medulla complex, la lamina, lal lateral accessory lobe, lan lateral antenna 1 neuropil, lo lobula, maf a myoarterial formation a, onv optical nerve, pmpn posterior medial protocerebral neuropil, pnt projection neuron tract, tn tegumentary neuropil, [numbers] somata cluster. Scale bars: (a) 50 μm, (b) 100 μm
Fig. 4
Fig. 4
The protocerebrum. a Frontal vibratome section at the mid-level of the protocerebrum, double-labeled against acetylated tubulin (yellow-red on the left, black-white inverted on the right) and RFamide (green on the left). A bundle of neurites connects the lobula with the lateral part of the central body (double-arrows). b-c Horizontal vibratome section of the first and second visual neuropils of the left brain hemisphere. b Maximal projection of acetylated tubulin-like immunoreactivity (black-white inverted) showing the outer chiasm between lamina and medulla (yellow arrow). c Single optical slice, triple-labeled against acetylated tubulin (yellow-red), histamine (green) and nuclei (blue, C1) as well as against histamine, separately (black-white inverted, C2). Histamine-like immunoreactivity is concentrated peripherally within the spherical subunits of the lamina (asterisks) and reveals a two-layered organization of the lamina (brackets in C2). d Sagittal vibratome section of the visual neuropils showing crossing neurites between lamina and medulla (outer chiasm) as well as between medulla and lobula (putative inner chiasm, yellow arrow), labeled against acetylated tubulin (black-white inverted). e Frontal view on the central body and the tracts w, x, y, and z, double-labeled against acetylated tubulin (yellow-red), RFamide (green, E1), as well as against acetylated tubulin, separately (black-white inverted, E2). Some neurites of z-tracts form chiasmata within the central body (single arrow). The double-arrow points to the bundle of neurites that connects the central body with the lobula. Insets (bottom left corner) show position and orientation of depicted sections in the brain. Abbreviations: ampn anterior medial protocerebral neuropil, cb central body, hn/tm hemiellipsoid body/ terminal medulla complex, la lamina, lal lateral accessory lobe, lo lobula, me medulla, pcc protocerebral commissure, pmpn posterior medial protocerebral neuropil, pnt projection neuron tract, w/x/y/z tracts, [numbers] somata cluster. Scale bars: (a-c) 50 μm, (d-e) 25 μm
Fig. 5
Fig. 5
The deutocerebrum and central olfactory system. a Frontal vibratome section of the posterior part of the brain showing the projection neuron tract, labeled against acetylated tubulin. b Magnification of the chiasm of the projection neuron tract with neurites projecting contra- and ipsilaterally (red arrows), labeled against acetylated tubulin (black-white inverted). c Magnification of the deutocerebral chemosensory lobe of the right brain hemisphere showing the peripheral arrangement of spherical to wedge-shaped glomeruli, labeled against acetylated tubulin (black-white inverted). d Horizontal view on the deutocerebral chemosensory lobe of the right brain hemisphere showing three RFamide-like immunoreactive neurons located in cluster 9/11 (asterisks), triple-labeled against acetylated tubulin (yellow-red), RFamide (green) and nuclei (cyan, D1) as well as against RFamide, separately (black-white inverted, D2). Insets (bottom left corner) show position and orientation of depicted sections in the brain. Abbreviations: dc deutocerebrum, dcl deutocerebral chemosensory lobe, hn/tm hemiellipsoid body/ terminal medulla complex, la lamina, lan lateral antenna 1 neuropil, lo lobula, maf a myoarterial formation a, og olfactory glomeruli, pb protocerebral bridge, pmpn posterior medial protocerebral neuropil, pnt projection neuron tract. Scale bars: (a) 50 μm, (b-d) 25 μm
Fig. 6
Fig. 6
Synapsin labeling of the brain of Parhyale hawaiensis. a Frontal vibratome section of the anterior part of the lateral and medial protocerebrum of the brain hemisphere. (A1) Magnification of the lamina showing the organization in spherical subunits with peripherally concentrated immunoreactivity. (A2) Cross section through the dorsomedial part of the lateral protocerebrum showing a weak distinction of a cap-like structure that potentially represent the hemiellipsoid body. b Sagittal vibratome section of the lateral antenna 1 neuropil showing a weak horizontal division into two equally sized parts. c Frontal vibratome section of the deutocerebral chemosensory lobe of the left brain hemisphere. The medial and lateral foramina are discernable (double-arrows). (C1) Magnification of the olfactory glomeruli showing the spherical to wedge-shaped appearance as well as a weak division into cap and base (brackets). Insets (bottom left corner) show position and orientation of depicted sections in the brain. Abbreviations: ampn anteromedial protocerebral neuropil, dcl deutocerebral chemosensory lobe, hn/tm hemiellipsoid body/ terminal medulla complex, la lamina, lal lateral accessory lobe, lan lateral antenna 1 neuropil, lo lobula, me medulla, og olfactory glomeruli, pmpn posteromedial protocerebral neuropil, [numbers] somata cluster. Scale bars: 25 μm
Fig. 7
Fig. 7
Three-dimensional reconstructions of the brains of one female and one male based on histological sections. Neuropils of the protocerebrum (reddish: visual neuropils, blue: hemiellipsoid body/terminal medulla complex, purple: central body), deutocerebrum (yellowish) and tritocerebrum (green) as well as the projection neuron tract (grey) are shown. In the right hemispheres of both brains, all somata are depicted. Interactive 3D PDFs are available as supplements (see Additional files 5 and 6). Abbreviations: ann antenna 2 neuropil, cb central body, dcl deutocerebral chemosensory lobe, ec esophageal connective, hn/tm hemiellipsoid body terminal medulla complex, la lamina, lan lateral antenna 1 neuropil, lo lobula, me medulla, pnt projection neuron tract, vnc ventral nerve cord. Scale bars: 100 μm
Fig. 8
Fig. 8
Schematic representation of the brain of Parhyale hawaiensis in anterior view. Abbreviations: ampn anterior medial protocerebral neuropil, ann antenna 2 neuropil, ao anterior aorta, ba brain artery, cb central body, dcl deutocerebral chemosensory lobe, ec esophageal connective, hn/tm hemiellipsoid body/ terminal medulla complex, la lamina, lal lateral accessory lobe, lan lateral antenna 1 neuropil, lo lobula, maf a myoarterial formation a, man medial antenna 1 neuropil, me medulla, pb protocerebral bridge, pmpn posterior medial protocerebral neuropil, pnt projection neuron tract, tn tegumentary neuropil

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