nissl substance and cellular structures involved in … · nissl substance and cellular structures...

Nissl substance and cellular structures involved in the intraneuronal and

neuroglial transport in the crayfish stretch receptor

G.M.Fedorenko1,2 and A.B. Uzdensky

1

1Southern Federal University, Rostov-on-Don, 344090, Russia 2Southern Scientific Center RAN, Rostov-on-Don, 344006, Russia

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 [5-

7]. 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].

Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.)

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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 [13-

15]; 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.4-

7). 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 – double-

membrane 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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