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Nissl substance and cellular structures involved in the intraneuronal and
neuroglial transport in the crayfish stretch receptor
G.M.Fedorenko1,2 and A.B. Uzdensky1
1
Southern Federal University, Rostov-on-Don, 344090, Russia
Southern Scientific Center RAN, Rostov-on-Don, 344006, Russia
2
The present paper describes ultrastructural elements involved in intra-neuronal and neuroglial transport processes in the
crayfish stretch receptor neurons and surrounding glial cells. Specific “tigroid” morphology of the neuronal perikarion is a
consequence of the necessity to supply remote parts of neuronal processes with proteins produced in the perikarion. In
large neurons the delivery of proteins from cell body to microtubules, through which macromolecules are transported to
neurite periphery, is hindered. Therefore, microtubule bundles separate the neuron perikarion by Nissl bodies - 2-3 µm
regions abundant with ribosomes, polysomes and rough endoplasmic reticulum where proteins are synthesized, Golgi
dictyosomes where these proteins are processed and sorted, and mitochondria that supply energy for these processes.
Dictyosomes are generally faced with their output trans-Golgi-network to microtubular bundles that indicates their
involvement in microtubule-mediated intraneuronal transport rather than in vesicular transport to the plasma membrane
and outside to glial cells. Neuroglial transport is mediated by diffusion through the narrow intercellular space (10-15 nm
width), by glial protrusions and corresponding invaginations in the neuron cytoplasm, which shorten the diffusion path
between glia and neuronal perikarion, and by double-wall vesicles – the captured protrusion tips containing large
fragments of the glial cytoplasm. Specific structural triads: “submembrane cisterns – vesicles of smooth endoplasmic
reticulum – mitochondria” are involved in formation of glial protrusions and double-wall vesicles. The tubular lattice in
glial cytoplasm may transfer ions and metabolites between glial layers. Thus, intense neuroglial exchange with cellular
components in the crayfish stretch receptor is mediated by a variety of mechanisms: diffusion, capture of big glial masses
and formation of double-walled vesicles, and transport through tubular lattices.
Keywords electron microscopy; neuron; glia; neuroglial interactions; Nissl bodies; Golgi; microtubules; double-wall
vesicles, tubular lattice
1. Introduction
Neurons are very large cells, whose neurites, axon and dendrites, reach several centimeters and in some cases even
meters in length. The supply of their remote regions with metabolites, proteins, mRNA, ribosomes and organelles is a
complicated task. This function is performed by specific intra-neuronal transport systems and by surrounding glial cells.
Ultrastructural study reveals the specific morphological elements involved in synthesis, intra-neuronal and neuroglial
transport of proteins. On the other hand, looking at the neuron from the “transport and supply” point of view, one can
understand the meaning of its specific morphology.
The spotted, “tigroid” morphology of the perikarion is characteristic for diverse nerve cells [1]. Nissl bodies, seen in
an optical microscope as dark spots, are dispersed throughout the neuronal perikarion. At the ultrastructural level, they
correspond to 1-4 µm regions abundant with granular endoplasmic reticulum, ribosomes and polysomes. These are the
centers of intense protein synthesis. After synthesis proteins are transported along neurites. However, it is not clear how
synthesis of proteins in Nissl bodies is coupled with their transport along neurites and how the non-uniform organelle
distribution within nerve cells is maintained. These questions are the parts of the general problem – intracellular
organization and integration of synthetic and transport processes.
Another significant problem is the study of the mechanisms of neuroglial interactions: which ultrastructural elements
are involved in the transport between neurons and surrounding glial cells. Glial cells, much more numerous than
neurons, provide the integrity of the nervous system. They support and isolate neurons, supply them with metabolites,
regulate the composition of the extra-neuronal medium, organize development of neural networks, etc. [2-4]. Neurons
and glial cells maintain survival of each other by means of the mutual exchange with neurotrophins and neuregulins [57]. The mechanism of the neuroglial exchange with metabolites, proteins and other cellular components is not well
understood.
The nervous system of invertebrates is much simpler than the central brain of mammals. However, even in this case,
it is not easy to identify neurons, to determine their functional state and to define their relationships with other neurons.
It is also difficult to reveal glial cells whose processes contact to the given neuron.
In this paper we consider the neuronal and glial structures involved in protein synthesis, sorting and intra-neuronal
transport, as well as in the transport of cellular components between neurons and satellite glial cells in the crayfish
abdominal stretch receptor taken as a simple but informative object. The part of these data has been recently published
[8-10].
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2. Materials and methods
The stretch receptor of the crayfish Astacus leptodactylus was isolated according to Florey and Florey (1955). It was
placed into a plexiglass chamber equipped with a device for receptor muscle extension and filled with van Harreveld’s
saline for invertebrates (mM: NaCl - 205; KCl-5.4; NaHCO3 - 0.24; MgCl2 - 5.4; CaCl2 - 13.5; pH 7.2-7.4). Neuronal
spikes were derived extracellularly from axons, amplified and processed by a personal computer. After isolation and 1
hour regular firing with a frequency of 6-10 Hz, the stretch receptor was fixed 1 hour with 2.5 % glutaraldehyde in 0.1
M phosphate buffer (pH 7.2). Then it was cut out together with the 2-3 mm piece of the receptor muscle so that the
preparation was T-shaped. It was washed in phosphate buffer, incubated 1 hour in 1% OsO4, contrasted by uranyl
acetate, dehydrated in a graded series of ethanol and acetone, and embedded into an epoxy resin. Ultra-thin sections
were obtained on the ultramicrotome Leica EM UC6 (Leica, FRG) and then studied on the electron microscope Tecnai
Spirit 12 (Phillips, Netherlands). For fluorescent visualization of the cell nuclei, the preparations were fluorochromed
with Hoechst 33342 [8].
3. Crayfish stretch receptor neurons
Each abdominal segment of a crayfish contains two bilateral stretch receptors that consist of a couple of
mechanoreceptor neurons, slowly and rapidly adapting, mounted on the corresponding receptor muscles (Fig.1) [11].
Their dendrites branch between muscle fibers and tightly contact to them [12]. Muscle extension stretches the dendrite
membrane at contact regions. The following depolarization induces receptor potential that triggers spikes propagating
along the axon. This supplies ventral ganglia with the information on position and movements of abdominal segments
that is necessary for control of animal locomotion. The rapidly adapting neuron responds only to the muscle extension
by a transient spike burst. At a constant length of the receptor muscle it is silent but the slowly adapting neuron
regularly fires with a steady frequency. Although their fine structure is similar, we studied the ultrastructure of the
slowly adapting mechanoreceptor neurons (MRN). These are large neurons with 50-100 nm body and several
centimeters long axon (Fig.1,2).
The advantages of this classical neurophysiologic object include its simplicity (only two identified neurons); well
known functional state, which is easily and precisely registered; simultaneous study of the neuronal activity and the
structure of MRN and surrounding glial cells. It has been used in studies of basic electrophysiological processes [1315]; neuroglial interactions [8,9]; neuron and glia responses to metabolism inhibitors [16-19], laser irradiation [20],
photodynamic effect [10,17-19]. The ultrastructure of its soma [21-23], axon [24], dendrite endings [12], and synapses
[23,25] has been carefully studied. Ultrastructural changes induced by adequate stimulation [21-23] or external impacts
[10, 26] have been studied as well.
Fig.1 Slowly adapting crayfish stretch receptor. (A) Brightfield microphotograph. (B) Fluorescence-microscopic microphotograph of
the stretch receptor fluorochromed with Hoechst 33342. (C) Scheme of morphology of the crayfish stretch receptor.
Mechanoreceptor neuron surrounded by glial cells is mounted on the receptor muscle (RM). Its axon and dendrites are filled with
numerous microtubules (MT). Dendrite endings (DE) are ramified between muscle fibers and contact to their membranes. N nucleus, Nl – nucleolus, Aff.Ax – afferent axons. Scale bar on B – 50 µm (Modified from [8]).
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Fig.2
The soma and initial parts of axon and dendrites of the crayfish
mechanoreceptor neuron. Large oval nucleuses with round nucleolus are in the
center of the neuron body. The perikarion has the spotted “tigroid” morphology. Ax
– axon; D – dendrite; GC – glial cells; FE – fibrillar envelope; N – nucleus; Nl –
nucleolus; PK – perikarion. Ob. 40x. The scale bar 10 µm. [9]
4. Glial envelope
The glial envelope of MRN consists of 10-30 layers formed by flattened sheet-like processes separated by collagen
layers (Fig.3). Glial nuclei are well visualized with DNA-specific fluorochromes such as Hoechst 33342 or DAPI
(Fig.1B). There are about 30-40 glial nuclei per a square of 100x100 µm2 around the MRN soma [18]. The cytoplasm of
separate glial cells is not well observed in an optical microscope because of the multilayer, roulette-like morphology. At
the electron-microscopic level, glial cytoplasm is less abundant with mitochondria, dictyosomes, ER cisterns and
ribosomes comparing to MRN perikarion, and glial processes are not as dense as the neuron soma (Fig.4-6). In some
places groups of relatively narrow finger-like glial protrusions inserted into each other contact with the neuron surface
(Fig.3B,4).
Such multilayer glial envelope in a crayfish stretch receptor plays a role of the blood-brain barrier. The majority of
metabolites should cross glial and collagen sheets before transportation into the neuron from the surrounding medium.
For example, staining of the crayfish stretch receptor with different photosensitizers (aluminum phthalocyanine,
hypericin) has demonstrated that they are accumulated in glial envelope rather than in neurons [18,27].
Fig.3 The transversal section of the axon and its glial envelope. A. Light layers – the cytoplasm of glial cells, darker sheets –
collagen. B. Dark dots inside the axon (rings at a higher magnification) are microtubules. The axon border is tortuous. Peripheral
mitochondria communicate with the axonal membrane through membrane cisterns (arrowheads). Gl - glial layers [8].
5.Transport pathways and Nissl bodies in the neuron perikarion
MRN neurites, axon and dendrites, are filled with hundreds of microtubules. These are the transport pathways, which
supply remote parts of neuronal processes with proteins and metabolites. Mitochondria that provide energy for transport
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processes are concentrated at the neurite periphery (Fig.3). One can suggest that in MRN microtubules pass not only
from the cell center to neurite periphery as in the most of cells but also along the proximal/distal cell axis from dendrites
to the axon through the cell body. Actually, the presence of non-centrosomal microtubules in neurons has been earlier
demonstrated [28]. It has been also shown that unlike “right” orientation of axonal microtubules with plus-end at the
periphery, the majority of microtubules in dendrites of invertebrate neurons (Drosophila) are oriented oppositely: with
minus-end at the periphery and plus-end at the neuron body [29]. Such orientation is, therefore, the same as in axons.
The similar trans-neuronal microtubular pathways may exist in the crayfish mechanoreceptor neurons.
Fig.4. The peripheral region of the crayfish stretch receptor neuron and surrounding glial cells. Bundles of microtubules (MT)
separate neuron cytoplasm by large parts – Nissl bodies containing ER, Golgi dictyosomes (AG), ribosomes and mitochondria. DW –
double-wall vesicle, Gl – glial cell, N – neuron, SC – subsurface cistern, TL – fragment of a tubular lattice [8].
What is the reason of the tigroid morphology of nerve cells? In order to provide long neuronal processes with
proteins that are synthesized mainly in the perikarion, such big cell as MRN has a potent protein-producing machinery
consisting of numerous ribosomes, polysomes and granular endoplasmic reticulum. These proteins are to be effectively
transported along microtubules with the help of anterograde or retrograde motors, kinesin or dynein. If a large
perikarion (up to 50-100 µm in the case of MRN) is not separated, proteins synthesized inside the perikarion and packed
in Golgi vesicles should diffuse tens micrometers to microtubular rails. Perikarion fragmentation by 2-3 µm Nissl
bodies significantly shortens the distance from Golgi dictyosomes to microtubules.
In Nissl bodies of the crayfish mechanoreceptor neuron, dictyosomes are generally oriented so that their output
trans-side is faced to microtubular bundles that transfer proteins along neurites (Fig. 4,5) [8,9]. In this case the distance
between Golgi vesicle and microtubules is significantly shorter: tens and hundreds nanometers. Thus, the fragmentation
of the perikarion of big neurons by Nissl bodies is necessary for shortening the distance between protein-producing
structures and structures involved in the transport of cellular constituents.
Except intra-neuronal transport along microtubules, proteins may be transported by vesicles from dictyosomes to the
cell surface and further to the neighboring glial cells. In MRN the dictyosomes are very seldom faced to the plasma
membrane. Even when these are located near the plasma membrane, their trans-side may be exposed not to the neuronal
membrane but to the nearest microtubule bundle inside the perikarion (Fig.4,5). As a rule, Golgi vesicles don’t bypass
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the fibrillar layer and approach the plasma membrane (Fig. 3,4). Therefore, Golgi activity is associated with the
transport of proteins along microtubules rather than with the vesicular transport to the plasma membrane. The latter is
limited by the fibrillar envelope surrounding the perikarion [8]. However, this envelope is not continuous. In some
MRN regions organelles and vesicles get in touch with the neuronal membrane (Fig.3,6) and exocytosis or endocytosis
may occur in these places.
Fig. 5. The Nissl body at the perikarion periphery is separated
from the outer membrane by the microtubule layer (Mt). The
trans–side of the Golgi dictyosome (GA) is faced to the inner
microtubule bundle (Mt). ER – endoplasmic reticulum; Gl –
glial cell, GN – glial nucleus; Mit – mitochondria; TL – tubular
lattice [9].
6. Structural basis of the neuroglial exchange
The neuronal and glial membranes are separated by a 10-15 nm gap [8]. Small molecules and ions easily diffuse
through this space. Macromolecules like proteins and mRNA cannot cross cellular membranes. After vesicular transport
from the Golgi complex to the plasma membrane, proteins are released from the cell by means of exocytosis. Likewise,
cells capture an extracellular material by means of endocytosis. In MRN, however, the vesicular transport between
perikarion and the neuronal membrane is restricted by the fibrillar envelope surrounding the cell body. Rather few
vesicles are observed within this fibrillar layer. Vesicles approach the cell surface only in places where the fibrillar
envelope is interrupted (Fig.4,5). Glial cytoplasm contains much less dictyosomes than MRN and vesicles seldom
contact to glial membranes exposed to the neuroglial cleft. Hence, the level of exocytosis from glial cells is low (Fig.47). Although the vesicular transport of macromolecules between MRN and adjacent glial cells is limited, another
specific mechanism for delivery of big masses of glial material into the neuron exists.
Numerous glial protrusions 0.2-0.3 µm in diameter penetrate across the fibrillar layer up to 1 µm into the neuronal
cytoplasm (Fig.6). Corresponding invaginations are formed in the neuronal membrane. This shortens the diffusion path
between the glial cell and the neuron. As a result, the glial material is transferred into the neuron bypassing the fibrillar
envelope. Similar glial protrusions and neuronal invaginations called trophospongia have been earlier observed in
nerves and ganglia of crayfish [30,31], mollusks [32] and insects [33]. Moreover, the capture of tips of these protrusions
leads to formation of double-wall vesicles (DWV) at the MRN periphery. Their electron-light cytoplasm is typical for
the glial but not for darker neuronal cytoplasm (Fig.4,6). This confirms the glial origin of DWV and the glia-to-neuron
direction of such intercellular transport. With this mechanism, big masses of glial material including vacuoles and
fragments of mitochondria, microtubules and electron-dense inclusions consisting possibly of fat, a rich energy source,
are transferred into the neuron [8]. Similar DMV have been observed in vertebrate neurons [34-36]. Thus, glial
protrusions and double-wall vesicles facilitate large-scale delivery of a glial material to MRN.
Formation of glial protrusions and double-wall vesicles is possibly mediated by flattened submembrane cisterns
(SC), a specific kind of smooth ER cisterns associated with the inner side of the neuronal membrane (Fig.4-6) [8,9].
After formation of an invagination of the neuronal membrane and even after the capture of its tip and DWV formation,
SC remains to be associated with their surfaces (Fig. 4,6). As a rule, submembrane cisterns are combined with
neighboring smooth ER cisterns, mitochondria, and sometimes with dictyosomes (Fig.4,6). Such triads “Submembrane
cistern - ER cistern - Mitochondria” are observed in almost all protrusions and DWV [8,9]. Similar SCs have been also
observed in the nervous system of different vertebrates [37-39]. These have been proposed to store Ca2+, which
regulates the assembly of actin filaments involved in the membrane bending and formation of protrusions [34,36,38,39].
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Fig.6. Glial protrusions, corresponding invaginations (In) in the neuron cytoplasm and double-wall vesicles (DW) within the neuronal
perikarion. AG – Golgi apparatus; Gl – glial cell; N – neuron; SC – subsurface cistern [8].
Neuron cytoplasm contains also large, 0.5-1 µm autophagosomes. Unlike DWV, these are surrounded by one
membrane and contain destructed organelles that have to be excreted from a neuron (Fig.7) [8].
The glial cytoplasm contains much less mitochondria and other organelles comparing to the neuron perikarion. Only
smooth ER cisterns and vesicles are usually observed in glial processes (Fig.3-7) [8]. It is of interest, how glial
processes, especially their distal parts are supplied with metabolites and biopolymers and how macromolecules
synthesized in the glial perikarion are transported along thin glial sheets and then reach the neuron? Microtubules,
which mediate the intracellular protein transport, are seldom observed in glial processes (Fig.4-7). Small metabolites
may diffuse through the extracellular space and possibly through the collagen sheets that separate glial layers. How
proteins, ribosomes and organelles are transported along the glial processes is unknown.
Fig.7. Tubular lattices (TL) in glial cells (GL). (A) Tubular lattice sometimes contacts to a collagen layer. (B) Tubular lattice may be
formed from small vesicles or may be disintegrated to a chain of numerous such vesicles. AP – autophagosome, DW – doublemembrane vesicle; N - neuron.
Tubular lattice is another specific element possibly involved in transport processes. It is found in the cytoplasm of
diverse invertebrate glial cells [40,41]. This is a polygonal cluster consisting of joint penta- or hexagons formed from
50-nm vesicles connected with 30-nm width tubules (Fig.7). The observation that these tubules are opened into the
perineuronal space and, at the opposite side, into the collagen layer has suggested that tubular lattices represent multiple
parallel pathways for transport of some cellular constituents. These structures have been first suggested to remove
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rapidly the excessive K+ ions, which are excreted by intensely firing nerves [40,41]. In the crayfish stretch receptor, the
tubular lattices were, however, found in remote rather than adaxonal glial layers (Fig.7). They are opened into other
glial layers or into collagen sheets (Fig.7A) but not into the perineuronal space. This suggests that tubular lattices may
be involved in the transport between different glial layers rather than from a neuron to glia. Fragments of tubular lattices
and chains of small vesicles are sometimes observed near the whole structures (Fig.7B) thus indicating that tubular
lattice is a dynamic, self-assembling structure capable of formation or decomposition.
Collagen layers that separate glial sheets have been suggested to be permeable for metabolites [42]. Therefore, some
substances may be transferred between glial layers directly through collagen layers.
7. Conclusion
Intercellular exchange with ions, metabolites, proteins and bigger particles is the important aspect of neuroglial
interactions. This transport is presumably bilateral – from a neuron to surrounding glial cells and from glial cells to a
neuron. Diverse structural elements are involved in the neuroglial exchange in the crayfish stretch receptor: simple and
facilitated diffusion across the intercellular cleft, vesicular transport including endo/exocytosis, and large-scale
fagocytosis. Glial protrusions and double-wall vesicles may transfer big cytoplasm fragments into the neuron. Neurons
may excrete autophagosomes containing the used or damaged cytoplasm fragments. Different substances may be
transported between glial layers through extracellular collagen layers and tubular lattices.
Acknowledgements. The support by RFBR (grants 05-04-48440 and 08-04-01322) and Minobrnauki RF (grant 2.1.1/6185) are
gratefully acknowledged.
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