**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**).

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 *The Biological and Structural Organization of the Squid Brain DOI: http://dx.doi.org/10.5772/intechopen.107217*

#### **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.*

detail, the presynaptic axon (Pre), postsynaptic axon (Post) and giant nerve fibers (Ax) are seen in **Figure 2a**.

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**).

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

#### **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.*

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 Biological and Structural Organization of the Squid Brain DOI: http://dx.doi.org/10.5772/intechopen.107217*

#### **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).*

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 [17–19] 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 [23–25]. 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, 26–28], 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.
