The Biological and Structural Organization of the Squid Brain

Abstract

Marine invertebrate models (squid, sepia, and octopus) made important contributions to description mammals’ nervous system. Being a very simple nervous system relatively easy to be manipulated experimentally and visualized by simple microscope or magnifying glass, the giant synapses at stellate ganglion and the large synaptosomes prepared from the squid photoreceptor neurons served as an attractive model to Histology and Anatomy studies. This sophisticated nervous system has elucidated synaptic transmission in detail with their numerous proteins at presynaptic terminal, synaptic vesicle biogenesis, neurotransmitter secretion, vesicle recycling and, allowed the study of postsynaptic complex with their membranes receptors. However, there are few studies with biochemical and molecular approaches, which lead to a better understanding of their physiological functions and verify operation of such nervous system.

1. Introduction

The brain is the control center that transmits the information of neurons to other groups of neurons. A single neuron can be affected simultaneously by excitatory and inhibitory stimuli from one axon or many other axons. Billions of neurons have different functions and constantly check the internal environment and external universe: light, touch, pressure, sound, balance, images, pain, emotion, consciousness. The nervous system from different external stimuli produces a continuous flow of information, memory, learning, and this conscious state throughout life seems infinite.

The nervous system of vertebrate is complex to be studied in individual or small groups of neurons. The cephalopods Coleoidea group (squid, sepia, and octopus), in addition to sea snails and nematodes, which contain few neurons and large structures, are relatively easy to manipulate experimentally and contributed to most of the current knowledge about the nervous system.

In general, these studies can be applicable to nervous system complexes, such as the human system. For example, mammals have a complexity of the Neuropil, an area composed of mostly unmyelinated axons, dendrites, and glial cell processes. For few (μ²) at Neuropil mammals, we find hundreds of bud’s terminals with their corresponding dendritic spines and pre-postsynaptic terminals, where pointing them out individually would be a difficult task.

It was noticed that the Neuropil from squid nervous system also has many pre-postsynaptic terminals such as a mammal. However, these are much larger in the squid nervous system and allow different types of experimental manipulations. In fact, light microscopy cannot resolve synaptic densities and synaptic vesicles, but the active zones in synapses can be observed easily from squid nervous system using low-power electron microscopy.

Thus, the squid nervous system with giant synapses and giant axon system have contributed to the current knowledge of Electrophysiology, Molecular Biology, and Biochemistry and allowed to verify the synaptic transmission and synaptic plasticity.

2. Nature history of Doryteuthis SSP: in the South and North Atlantic

According to the classification by Young [1], cephalopod can be grouped in the subclasses Nautiloidea (nautiluses) and Coleoidea (all the others) or in the general morphology to include in taxonomic descriptions [2].

During the summer in the South and North Atlantic, the mature specimens of Doryteuthis plei or Doryteuthis pealeii (or Doryteuthis ssp) were collected with supports of Marine Biology Center at University of São Paulo (CEBIMar-USP) and Marine Biological Laboratory (MBL) at Woods Hole, US, respectively.

The squid survives chasing food by capturing prey and escaping predators. An ability to accelerate quickly and make sudden changes in the direction of swimming help them to avoid danger. This agility is ensured by the sophisticated nervous system [3, 4] specialized for propellant jets: that pull the water into mantle and then, with the aid of a muscular body wall, which is rapidly contracted, the water is expelled, thus propelling it through the water.

To obtain this muscle contraction result, the squid requires a nervous system that can conduct signals with great speed throughout the body. The optic lobe or “brain” located on each side of the squid head is the control center that transmits the information to the chain of giant nerve cells in the mantle (Figure 1a).

Figure 1.

The optic lobes and stellate ganglion with giant nerve fibers. a) Illustration shows a squid animal model with optic lobes on each side of head and stellate ganglion with chain of giant nerve cells on each side of the midline in the mantle. b) The stellate ganglion in mantle (seen in pin). c) The stellate ganglion visualized in seawater with glass vision 10x magnification.

The giant axon can reach 10 cm in length and is about one hundred times than the axon diameter of mammal [5, 6]. The giant axon model made important contributions to the description of axoplasmatic flow mechanisms [7], ion transport across the plasma membrane [8], and neurotransmission [9], and also allowed micro-injections using specific antibodies such a tool for the study of molecules involved in the synaptic function [10, 11]. In addition, the neuronal system contributed to discovery that mRNAs are present in the presynaptic region [12, 13] and new proteins synthesis occurs locally in this region [14].

Here, the stellate ganglion and the large synaptosome from photoreceptor neurons were isolated from squid nervous system and showed a biological and structural organization of brain by biochemical and immunohistochemistry studies.

3. Ultrastructure: giant synapse

Intact stellate ganglion in the mantle was removed (Figure 1b see in pin). The stellate ganglion in seawater can be observed by glass magnification (Figure 1c). In more detail, the presynaptic axon (Pre), postsynaptic axon (Post) and giant nerve fibers (Ax) are seen inFigure 2a.

Figure 2.

The giant synapse. a) Stellate ganglion visualized in seawater show presynaptic axon (pre); postsynaptic axon (post) and giant nerve fibers (Ax). The horizontal (line red) illustrates the cross-sectional region from stellate ganglion at giant synapse. b) Light microscopy of cross-sectional region from stellate ganglion shows synaptic contact region between the presynaptic and postsynaptic terminal at giant synapse visualized by H.E. with 40x magnification.

In light microscopy, cross-sectional region from stellate ganglion shows synaptic contact region between the pre- and postsynaptic terminal at giant synapse when visualized by H&E staining (Figure 2b).

Low-power electron micrograph of same region from stellate ganglion shows synaptic densities and clustering of synaptic vesicles that can be observed in the active zones (Figure 3). The presynaptic (Pre) terminal is lighter than the postsynaptic (Post) terminal and can be characterized by the presence of synaptic vesicles. In the contact areas, the postsynaptic sends digitiform processes and forms the active zones in the limits of interaction between presynaptic terminals. The electron micrograph shows two synaptic densities with clustering of synaptic vesicles in correspondence active zones at the giant synapse (arrows Figure 3).

Figure 3.

Ultrastructure of the giant synapse. Electron micrographs of the giant synapse showing clustering of synaptic vesicles (SV) and active zones (AZ). The insert left shows giant synapse with presynaptic and postsynaptic terminal visualized by H.E with 40x magnification. Scale bar represent 0.5 microns (courtesy of Dr. Jorge Moreira-FMRP).

In general, synapses are local of communication where the neurons pass signals through their axons to postsynaptic target (dendrites, axon or cell body of another neuron, muscle cells, or glandular cells). There are two types of synapses (electrical and chemical) that differ in structure and function. The neurons that communicate through electrical synapses outlets are connected by gap junctions, through which the electrical impulse signals are passed directly from pre- to postsynaptic terminals with high speed. On the other hand, chemical synapses contain the synaptic vesicles at the presynaptic terminal, which carry specific neurotransmitters and have ion channels in the plasma membrane. What differs between chemical and electrical synapses by electrophysiology approach is their impulse speed with a delay feature around 0.5 ms between them, respectively.

Each synaptic vesicles (SV) consists of an apparatus with hundreds of specific proteins to produce fusion of their membranes with the presynaptic membrane and secrete the neurotransmitters at the synapses. It is integrated by corresponding area of neuron, which contains a part of the postsynaptic density (PSD) with ion channel receptors at the postsynaptic membrane for neurotransmitters [10, 15].

The size of synaptic vesicles is variable and dependent of neurotransmitter type [16]. In general, there are two types of vesicles: electron-dense center vesicle and electron-lucent center vesicle. Electron-dense vesicles are subdivided into two types: containing catecholamines (80 nm) and, large synaptic vesicles that contain neuropeptides (200 nm). On the other hand, electron-lucent vesicles have 50 nm of diameter and been carried out with acetylcholine, glycine, GABA, or glutamate. These vesicles are accumulated close at the active zone with 0.5 mm of distance from the plasma membrane.

The membrane proteins (SV) are synthesized in the endoplasmic reticulum granular (REG) and carried through vesicles of the Golgi complex to the presynaptic terminal. The motor proteins (kinesin and dynein) make the transport through microtubules [7] and myosin proteins [17, 18, 19] through actin filaments [20]. The membrane proteins (SV) probably are selected in the primary endosome, which give rise to the synaptic vesicle that to be filled with neurotransmitters.

In summary, when an action potential reaches in the presynaptic terminal least vesicles fuse with specify region at the presynaptic membrane. The action potential in the presynaptic terminal is a very fast process, which have voltage-operated channels allowing rapid and transient entry of calcium (Ca²⁺) that interacts with several group of vesicular proteins and membrane protein into presynaptic terminal [7, 21, 22]. However, are many proteins involved with endocytosis and exocytosis process of synaptic vesicles.

The calcium triggers fusion of synaptic vesicle with the presynaptic membrane, whereas the central protein involved in fusion of synaptic vesicles is Synaptotagmin protein, which is defined such a calcium sensor due to its C2A domain. The C2A domain through its interaction with calcium undergoes a conformational change, causing it to fuse phospholipids from vesicle and presynaptic membranes. On the other hand, the C2B domain of Synaptotagmin is involved with recycling of synaptic vesicles [23, 24, 25]. Finally, electrophysiology and biochemical approaches of synaptic events have been obtained from the squid nervous system studies, in which specific antibodies and homologous proteins were used for knowledge of synaptic events [10, 11, 15, 26, 27, 28], and also more recently, genetic approach was showed a knockout of the squid pigmentation gene [29]. This method demonstrated efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9 and should be readily adopted by other research groups because this method does not require specialized equipment and squid are available worldwide.

4. Immunohistochemistry and immunofluorescence studies: squid optic lobe and synaptosome

The complexity of subcellular domains in neurons and their distances of nucleus demand a spatial and temporal control of protein synthesis. We have identified a novel member of hnRNP A/B type, of 65 kDa, in the presynaptic terminal of squid neuron [30, 31, 32]. Squid p65 was phylogenetically conserved hnRNP type A/B protein in mammals [33]. In this scenario, these hnRNPs have emerged as major components of mechanisms for the local protein synthesis and synaptic plasticity.

To assess their presynaptic terminal location in squid photoreceptors neurons, the immunohistochemistry of slices from optic lobe was incubated with the anti-synaptic vesicle glycoprotein 2A (ɑ-SV2) antibody and developed by peroxidase-DAB (see in method). The ɑ-SV2 antibody recognizes the corresponding bands in the outer plexiform layer (Figure 4a), which is a synaptic connection region [34]. This image was similar to those previously obtained with the Synaptotagmin antibody [31]. These data showed an anatomy is highly complex with outer cortical layers and a central medulla from squid optic lobe [35, 36, 37].

Figure 4.

Immunolocalization of presynaptic terminal in the optic lobe “chickpeas-like.” a) Immunohistochemistry of the 10 nm slice through the squid optic lobe labeled with anti-vesicle protein 2 antibody (ɑ-SV2) and secondary antibody by the peroxidase-DAB (method described by [31]). The arrows indicate the immunopositive bands for both antibodies in the outer plexiform layer (opx). The morphological layers of the optic lobe cortex are indicated to the right: Outer nuclear (on), outer plexiform (opx), inner nuclear (in), inner plexiform (ipx), and mononuclear (mn) layers with optic nerve terminals at the outer plexiform layer (opx) from optic lobe cortex. Scale bars 100 μm. b) Confocal images of two representative synaptosomes that were double immunolabeled with anti-sqRNP2 antibody (ɑ-sqRNP2, green) and anti-vesicle protein 2 antibody (ɑ-SV2, red). The merged images show yellow where overlap occurs. Scale bars 2 μm.

To further indicate the subcellular localization of hnRNPs is involved in presynaptic terminal localization in squid photoreceptors neurons, the synaptosomes isolated from optic lobes were double probed with ɑ-SV2 antibody and anti-squid ribonucleoprotein motif 2 (ɑ-sqRNP2) antibody raised in rabbits [30]. Immunofluorescence microscopy of synaptosomes showed intense granular staining with both antibodies frequently close to the plasma membrane, suggesting a spatial relationship between both proteins to clustering synaptic vesicle at the presynaptic terminal (Figure 4b).

In fact, hnRNP protein shuttles mature mRNA from nucleus to the cytoplasm and are also involved in packaging mRNAs into cytoplasmic granule transport, which have been more clearly evidenced in dendrite cells. However, the exact function of hnRNPs in the presynaptic terminal has yet to be clarified [31, 38].

5. hnRNP proteins and degenerative diseases

Several cellular compartments not enclosed by membranes are called ribonucleoprotein granules, due to the high concentration of proteins and mRNAs (mRNPs). For example, mRNPs can be found in the nucleus in Cajal bodies, paraspeckles, speckles, etc., but they can also be found in the cytoplasm, such as stress granules and processing bodies (Pbodies).

Stress granules are cytoplasmic complexes made up of proteins and RNA, found in most cell types in culture (from yeast to humans), and are formed under specific conditions of cellular stress [39]. In vitro experiments with different conditions can induce the formation of stress granules such as lack of nutrients, heating, protein complex and protein degradation inhibitors (proteasome), genotoxic drugs (such as UV radiation), and drugs that cause oxidative stress (sodium arsenite) or osmotic agent (sorbitol) [39, 40]. Many studies showed an association of mRNPs to neurodegenerative diseases [41, 42, 43, 44, 45].

Of course, stress granules are generated as a cellular response to a physiologically unfavorable environment, and their prevalence in neurological disorders suggests that their formation in affected neurons is, at least initially, a cytoprotective response to disease-associated stress. However, the persistence of stress granules can contribute to the development of several degenerative diseases, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and some myopathies and neurodegenerative diseases [46].

The mutations in RNA-binding proteins appear to increase the propensity of these proteins to aggregate and form stress granules. For example, diseases linked to mutations promote aggregation in the proteins such as TDP-43 (TAR DNA-binding protein 43) and FUS (Fused in Sarcoma) [43, 44, 45, 47, 48, 49].

In previous studies [30, 50] showed molecular and biochemical evidence that p65 is a dimer resistant to SDS treatment, composed of two protein subunits of the hnRNP A/B type with molecular weight around 37 kDa, and this propensity of these proteins to aggregate and form stress granules involves endogenous modification factors. Nevertheless, it did not clear show how such endogenous factors could act to produce alterations in hnRNP A/B-like protein that induces dimerization. Immunohistochemical studies demonstrate the presence of p65 in presynaptic nerve terminals and its propensity to oligomerize led us to further investigate their cellular and molecular properties in presynaptic nerve terminals [50].

In conclusion, based on these points presented here, we suggest that hnRNP A/B protein could be a link between local RNA processing and synaptic function at the presynaptic terminal. Understanding this can bring insights into evolution of several neurodegenerative diseases and verify if they resemble the function performed in vertebrates.

This brief summary has shown that the cephalopods have structure’s power that served in comparative studies and useful alternative to invertebrate experimental research that, in general, is applicable to mammalian systems. However, are few studies with biochemical and molecular approach, which could lead to a better understanding of their physiological functions.

6. Materials and methods

6.1 Animals and tissue preparation

The optic lobes were dissected from freshly killed D. pealei obtained from the Marine Biological Laboratory in Woods Hole or from D. plei obtained from the Centro de Biologia Marinha-CEBIMar, University of São Paulo, São Sebastião, Brazil. For immunohistochemistry and immunofluorescence procedures freshly dissected optic lobes are from D. ssp [31]. The synaptosomes (isolated nerve terminals from photoreceptor cells) were prepared from tissue according to Pekkurnaz et al. [51] with slight modifications. Briefly, optic lobes were quickly dissected from squid onto ice-cold Petri dishes and weighed. Each g of tissue was homogenized in 5 ml of ice-cold homogenization buffer (HB) (1.0 M sucrose in 20 mM Tris-HCl, pH 7.4) in a Wheaton glass homogenizer with a loose-fitting pestle, by 10–15 gentle strokes. The homogenate was spun at 1000xg for 11 min at 4 °C and then spun at 13,000xg for 45 min. The floating synaptosome layer was carefully decanted into a small Petri dish, gently washed in HB and resuspended in 0.5 ml of HB.

6.2 Antibodies

Anti-squid ribonucleoprotein motif 2 (ɑ-sqRNP2) antibody was raised in rabbits. The ɑ-sqRNP2 antibody raised in rabbits against the synthetic peptide CLFIGGLSYDTNEDTIK corresponds to an internal sequence determined by mass spectrometry from tissue-purified p65 [30]. The rabbit serum after inoculation was purified on a HiTrap Recombinant Protein A column (GE Health Science, Chalfont St.Giles, UK) by Fast protein liquid chromatography (ÄKTAFPLC system). The monoclonal anti-synaptic vesicle glycoprotein 2A (ɑ-SV2) antibody (64,051, Invitrogen, Carlsbad, CA) was raised in mouse and secondary antibodies conjugated to Alexa 488 for immunofluorescence from Molecular Probes (Invitrogen, Carlsbad, CA).

6.3 Ultrastructure

The stellate ganglion was removed from squid mantle on the seawater, fixed by immersion in 2% formaldehyde plus 2% glutaraldehyde in buffered calcium-free seawater, postfixed in osmium tetroxide, stained with uranium acetate, dehydrated, and embedded in Araldite plastic CY212 (EMS). Ultra-thin sections in carbon-coated single-slot grids were contrasted with uranyl acetate and lead citrate. Electron micrographs were taken at an initial magnification of x14,000 and photographically enlarged to a magnification of x35,000.

6.4 Immunohistochemistry

Fixed, 1-mm transversal slices of optic lobes were included in Paraplast (Oxford Labwase, St. Louis, MO, USA) following the manufacturer’s instructions. Microtome slices of 10 μm were cut, transferred to glass slides, de-parafinized, and rehydrated by standard procedures. Slices were incubated in PBS pH 7.4 containing 0.1 M glycine for 30 min at 4°C to block aldehyde groups and then washed three times for 10 min in PBS. They were then incubated in the dark at room temperature for 30 min in methanol containing 0.9% hydrogen peroxide solution to inhibit endogenous peroxidase activity, followed by washing in PBS. The samples were permeabilized and blocked by incubation in PBS containing 1% Triton X-100, 3% BSA, and 0.5% sheep serum and then incubated for 1 h with primary antibodies in PBS containing 0.1% Triton X-100, 3% BSA, and 0.5% sheep serum. After washing in PBS containing 0.1% Triton X-100, the slices were incubated for 1 h with secondary antibodies conjugated to horse radish peroxidase (KPL, Gaithersburg, MD, USA) diluted 1:400 in PBS containing 0.1% Triton X-100, 3% BSA, and 0.5% sheep serum and developed using 3,3-diaminobenzidina (DAB) as substrate.

6.5 Immunofluorescence

Synaptosomes in suspension were fixed in 1% paraformaldehyde for 6 hr. at room temperature, adhered to glass microscope slides by incubation for 1 hr. After gentle washing with PBS, slides were incubated for 30 min in 0.1 M glycine, 0.3% Triton X-100 in PBS, pH 7.4, and washed and blocked in 1 mg/ml BSA, 1% goat serum, and 1% Triton X-100 for 1 hr. at room temperature. The slides were washed with PBS containing 0.3% Triton X-100 and incubated with primary antibody in PBS containing 1 mg/ml BSA, 1% goat serum, and 0.3% Triton X-100 for 2 hrs. at room temperature. The slides were washed in PBS and incubated with appropriate secondary antibody for 1 hr. and washed again. The slides were mounted in Fluoromount G (EMS) diluted 2:1 in PBS and examined on a Zeiss 510 confocal microscope.

Acknowledgments

Dr. Gabriel Sarti Lopes and Dr. Jorge E. Moreira of University of São Paulo, FMRP/USP and Dra. Janaina Brusco from University of British Columbia, Can. Financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) are acknowledged. Special thanks to Dr. Roy E. Larson of University of São Paulo, FMRP/USP (past advisor). Infrastructure support from Marine Biology Center at University of São Paulo (CEBIMar-USP) and Marine Biological Laboratory, Woods Hole-US. Special thanks to Patricia Horta, PhD, IFSP, that revised this chapter.

Conflict of interest

The author declares that there is no conflict of interests regarding the publication of this chapter.