1 The contribution of invertebrates to the understanding of the vertebrate 2 nervous system, its mechanisms, functions and pathological dysfunctions.

3 Martin Giurfa 1) and Hans-Joachim Pflüger2)

4 Research Center on Animal Cognition, Center of Integrative Biology, CNRS - University Paul Sabatier - 5 Toulouse III, 118 Route de Narbonne, 31062 Toulouse cedex 9, FRANCE 1), and Institute of Biology, 6 Neurobiology; Freie Universitaet Berlin, Koenigin-Luise-Strasse 1-3, 14195 Berlin, GERMANY 2)

7 The human brain and the nervous system are the immediate result of several (6-7) million years of 8 hominid evolution, and ultimately of organismic evolution as they bear many similarities to the brain 9 and nervous system of vertebrates. We readily accept that all vertebrate organisms such as fish, 10 amphibians, reptiles, birds and mammals, share many anatomical features and major organs 11 corresponding to our human organism: they have guts and intestines; they possess a circulatory system 12 with a heart, kidneys and an excretory system. Less well accepted, perhaps, is the fact that these 13 animals have brains and nervous systems which are also related to us. Novel analyses of the nervous 14 systems of vertebrates allow retracing the occurring evolutionary changes with unprecedented 15 precision using new technical tools from molecular biology, immunocytochemistry or molecular 16 genetics. These analyses reveal the evolution of particular brain structures in new environments and 17 their relation to different life styles, something that biologists call an “ecological niche”. We readily 18 accept that primates like chimpanzees have many similarities in their body shape and behaviour to us, 19 namely because they resemble to ourselves in many aspects. We even extend this judgement to our 20 pet animals, dogs and cats, which exhibit some similarities to certain aspects of our own behaviour. 21 Yet, we actually may be reluctant to grant such similarities to the brains of fish and frogs, despite the 22 fact that these animals belong to vertebrates and exhibit the so-called “common vertebrate Bauplan”.

23 If similarities appear difficultly to most people when thinking about the nervous system of humans and 24 that of amphibians or fishes, what could be said about the nervous system of invertebrates? Animals 25 without backbones, the “squishes and crunchies” so to speak, appear as so utterly distant from us, that 26 the question of similarity may even not be raised. Many people may even ask if creatures like 27 flatworms, earthworms, snails and mussels, and all insects and crustaceans, have brains or nervous 28 systems at all. Yet, all these animals feed, move and reproduce, thus revealing the presence of a 29 nervous system responsible of their different behaviors. This simple consideration indicates that 30 preconceptions in the case of invertebrates are not justified. As this argument may appear simplistic 31 to detractors of invertebrates, we discuss here a series of arguments and evolutionary facts that may 32 serve as an agenda to appreciate the enormous potential of invertebrates for research and 33 understanding of the nervous system, its function and pathological dysfunctions.

34 The origin of the nervous system has been a question that haunted (and still haunts!) zoologists since 35 a long time. With the advent of new molecular techniques, some answers to this particular question 36 have been obtained. One feature that appears obvious is that all vertebrates, including humans, and 37 most invertebrates, are in general bilaterally symmetrical and belong, therefore, to the Bilateria. 38 Although many asymmetries have occurred during evolution so that some organs are only found on 39 one side of the body (e.g. the liver), or in the nervous system the left and right hemispheres have 40 specialized to serve different functions, the common organization plan is bilaterally symmetric. In 41 addition, the whole body is separated into different portions or segments: a head is followed by a 42 thorax and an abdomen. At first glance, a big difference between the nervous systems of vertebrates 43 and invertebrates may be the position of the nervous system: a dorsal brain and dorsal spinal cord in 44 vertebrates contrast with a dorsal brain and a ventral nerve cord in invertebrates. However, the 45 discovery of developmental genes and their similarities (“homologies”) between, man, zebra fish, 46 mouse and fruit fly, to name only a few of the most studied organisms, has shown that developmental 47 processes mediated by these genes follow similar principles.

48 Neurobiologists, therefore, had a new look onto brains and nervous systems, a look that confirmed 49 that the nervous systems of all bilaterian animals, including humans and invertebrates, share many 50 common features and may be related. Based on this essential fact, we argue that research on 51 invertebrate animals, which are in most cases more accessible for studies at the neurobiological and 52 molecular level than vertebrate animals, can yield substantial advances and benefits for our 53 understanding of the nervous systems, its architecture and functioning. We maintain, furthermore, 54 that this research can contribute important information for characterizing pathological dysfunctions 55 of the vertebrate nervous system and for conceiving efficient strategies to fight against recurrent 56 diseases. (See Figure 1 and Footnote 1).

57 Below, we provide some examples illustrating our statements and underlining why research on 58 invertebrate neurobiology may be essential for Humans themselves beyond the acquisition of pure 59 biological knowledge.

60

61 1) Cephalopods like squids and octopods played an essential role for understanding how the 62 nervous system functions.

63 The basic ionic mechanisms accounting for the electrical excitability of neurons were precisely 64 discovered and studied for the first time in these animals. Neurons generate impulses called action 65 potentials that travel all along nervous circuits to convey specific messages; these signals act as 66 communication signals in the nervous system so that understanding how they are generated allows 67 understanding how the nervous system works. The fact that this discovery was first made in the giant 68 axon of squids, and then extended to vertebrates, underlines the similarity between the invertebrate 69 and vertebrate neural mechanisms. The flow of ions in these nervous systems corresponds to the flow 70 of currents, and Ohm’s law of current flow is fully applicable. Due to the characteristic semipermeable 71 properties of the cellular membrane of neurons with their ion channels, an action potential is 72 generated by a very short (approx. 1-2 milliseconds) inward current carried by two positive ions (either 73 sodium or calcium), followed by closure of the respective ion channels and return to the so-called 74 resting potential. Individual neurons, depending on the ion channels they possess, can fire from 10 to 75 800 action potentials per second, and fire in bursts, beats, or in other more complex rhythms. This 76 historic example shows how research on cephalopod neurons allowed understanding how neurons, 77 including ours, function.

78 Cephalopods continue to be attractive animals for studying how the nervous system organizes 79 behaviour. Research on the extraordinary motor abilities of cephalopods, which allow them to adopt 80 various body shapes or move and grasp objects with incredible flexibility using their arms, has led to 81 the development of prototypes and “soft robotics” simulating these capacities. In addition, studying 82 their fast communication system, which generates rapidly moving patterns over their whole body, or 83 which produces the most exquisite camouflage patterns, may yield results of general interest for 84 applications in many human (industrial) domains. The discovery of their impressive cognitive abilities, 85 which includes not only learning but also insight, and social learning by observation, has also received 86 wide attention from scholars interested in understanding how cognitive processes may arise in 87 nervous systems that are distant from that of humans (see Figure 2 and Footnote 2) 88 89 2) The existence of electrical synapses or gap junctions was first discovered in the crayfish. 90 Neurons communicate with each other and with all other target cells by specific contact sites called 91 synapses. These synapses are of a chemical nature and upon the arrival of a nerve impulse (action 92 potential; see above and Figure 2), a chemical transmitter is released in portions, binds to the target 93 neuron or tissue where it causes either excitation or inhibition. The delay between the release of the 94 transmitter and the first noticeable response in the target cell is small and can range between 1 and 95 several milliseconds. Therefore, each synaptic contact in a pathway adds to the time required for 96 conduction and from a neurobiological point of view “slows” information transfer. Yet, some 97 behaviours need to be very fast, for example escaping a predator. The crayfish nervous system was 98 chosen to study the powerful escape behaviours of these animals, which respond to potentially 99 dangerous situations by producing a tail flip that either leads to very fast backward swimming if the 100 predator is coming from the front, or first to a somersault and then to a fast backward swimming if the 101 predator comes from behind. It was found that the very fast reaction time between first sensory 102 detection of the predator and the reaction of the crayfish could hardly be explained by the involvement 103 of several chemical synapses. Additionally to the presence of fast conducting, large neurons, contacts 104 made by electrical synapses, or gap junctions, were found, which conduct escape signals immediately, 105 as it is required during flight responses. It was later discovered that such gap junctions (or electrical 106 synapses) are widespread in the central nervous system including our brain and that they play a very 107 important role during development as they do not only conduct electrical impulses without any delay 108 but also allow the exchange of all kinds of molecules between cells (see Figure 3 and Footnote 3). 109 110 111 3) GABA (gamma aminobutyric acid), a fundamental and widespread inhibitory 112 neurotransmitter, was first discovered in the crayfish.

113 Inhibitory cells, pathways and synapses are commonly found in all nervous systems. They are 114 extremely important for the functional organization of the brain as neurons need not only to get 115 excited, but also to be inhibited to enhance signal to noise ratio or to suppress reactivity to irrelevant 116 stimulations. -aminobutyric acid (GABA) is the neurotransmitter that is responsible for inhibition at 117 the great majority of inhibitory synapses. This discovery was made thanks to research performed in 118 Crustaceans. Work on their neuromuscular preparations showed that GABA was highly concentrated 119 in inhibitory axons dissected from crustacean nerve bundles. This finding was followed by the 120 demonstration that inhibitory neurons in the central nervous system of the lobster also contained high 121 levels of GABA. Furthermore, a specific sodium-dependent GABA uptake mechanism was identified in 122 crustacean neuro-muscular preparations that could serve to inactivate released GABA and stop 123 inhibition.

124 The function of GABA as an inhibitory neurotransmitter is now well known in the vertebrate nervous 125 system and in that of many other invertebrate animals, where it follows similar principles and mediates 126 similar functions. This example shows how outstanding contributions to the understanding of 127 important questions on our own nervous system such as the principle of inhibitory neurotransmission 128 are being made by using invertebrates rather than primates and rodents (see Figure 4 and Footnote 129 4).

130

131 4) Basic mechanisms of motor control are surprisingly similar in vertebrates and invertebrates

132 A fundamental characteristic of animals is that they can actively move, and for doing that, most animals 133 use contractile tissues such as muscles. For example, insects possess striated muscle with the same 134 molecular mechanism of contraction as that of vertebrates. Thanks to such contractions, appendages 135 like limbs or fins or wings are moved. Muscles contract by the action of motor neurons; usually these 136 contractions are rhythmical such as in walking, where a leg alternates between stance and swing 137 phases, or in flying where a wing alternates between up- and down-strokes. Alternating rhythmical 138 activity is generated within the central nervous system, for example, in the spinal cord of vertebrates 139 or the ventral cord of invertebrates. Circuits or networks of neurons called central-pattern generators 140 by neuroscientists, are responsible for this rhythmicity. Central pattern generators function in a 141 completely isolated nervous system without any sensory feedback; they pre-structure the activity 142 which is required for any locomotion behaviour. However, for the correct execution of limb 143 movements during walking, numerous sensory feedbacks are required, not only from external sense 144 organs (exteroceptors) to avoid stumbling or any disturbance of the movement trajectory, but also 145 from internal sense organs (proprioceptors) which monitor tensions, strain or any load forces acting 146 on joints. In general, very similar physical parameters have to be measured during movements 147 regardless of whether an insect or a cat is walking. The more uneven and rough the terrain is, the more 148 important sensory feedback becomes. How this wealth of sensory information is processed within the 149 central nervous system is only partly understood and, in principle, very similar mechanisms occur in all 150 animals. Therefore, knowledge from how invertebrates move are of immediate importance for 151 understanding how we move. One of the most advanced genetic experimental organism is the fruit fly 152 Drosophila, an insect for which transgenic fly lines have been developed. This allowed silencing or 153 activating particular neurons in mutant flies, a strategy that is of great importance for understanding, 154 which parts of the central nervous system are involved in motor control. Again, the wealth of 155 information gathered in invertebrate and vertebrate animals leads to a much deeper understanding 156 of particular mechanisms including severe human pathologies of motor systems (see Figure 5 and 157 Footnote 5).

158

159 5) A large marine snail, the Californian sea hare (Aplysia), was pivotal for understanding 160 mechanisms of various forms of learning.

161 Learning and forming a memory are capacities that can be found not only in humans but basically in 162 all animals. Learning is a prerequisite for survival, in particular, as it allows making predictions about 163 an environment that animals need to master to avoid enemies or noxious stimuli, or to find food , 164 partners, and other valuable resources. Just like us, animals have to learn to avoid aversive stimuli, 165 which could be harmful and life threatening, and to find appetitive stimuli, which may act as rewards 166 such as food, a partner or the way back to the nest. But what happens in the brain and nervous system 167 of a learning individual? It was the Nobel Prize winner Eric Kandel who discovered the great value of 168 using an invertebrate animal, a mollusk, with very big neurons to answer this question and unravel the 169 cellular mechanisms that underlie learning and memory formation. The large marine sea hare, Aplysia, 170 a mollusk with big-sized and limited number of neurons in the central nervous system (ca. 10 000), 171 offered an opportunity to study the changes in particular neurons after the animal had learned to 172 habituate (i.e. to diminish its response as a consequence of experience) to a repeated tactile stimulus 173 with no life-threatening consequences. Aplysia possesses a mantle which protects a gill and which is 174 contracted by the animal as a defensive mechanism to protect the gill (Figure 6A). This defensive 175 response can habituate upon repeated and regular stimulations of the mantle (or the siphon, a soft, 176 water-expelling structure) with a weak tactile stimulus (a mechanic contact without life-threatening 177 consequences). The mechanism by which this habituation is achieved takes places at the synaptic level, 178 i.e. at the contact place between two neurons. Upon every tactile stimulation, the sensory neurons 179 decrease the release of chemical transmitters onto motor neurons and interneurons so that excitation 180 becomes progressively weaker and no response is elicited anymore. This form of learning is considered 181 very simple and is termed non-associative as the animal learns to respond appropriately to a unique 182 stimulus, the tactile stimulation. Another form of non-associative learning also studied in Aplysia is 183 sensitization, the opposite of habituation. In this case, a repeated noxious stimulus such an electric 184 shock at the level of the tail leads to an increase of responses to a tactile stimulation of the mantle or 185 siphon. Again, this could be explained at the synaptic level (Fig. 6B).

186 In addition, associative learning could also be dissected in the sea hare. In this case, animals learn to 187 establish predictive links between two or more events in their environment. A typical case is Pavlovian 188 learning in which the association between a conditioned stimulus and an unconditioned stimulus is 189 learned. In Pavlov’s experiments, the former could be a bell that anticipated the arrival of the latter, 190 the food that the dog was expecting. Dogs thus learned to salivate to the bell sound, which anticipated 191 food delivery. In Aplysia, a tactile stimulation to the mantle/siphon was followed by and electric shock 192 to the tail. As the tactile stimulus anticipated the shock, mantle contractions became progressively 193 more important (Fig. 6C). The molecular mechanisms underlying this associative learning were also 194 unraveled in Aplysia, and highlighted the role of adenylyl cyclase as a coincidence detector of the 195 signals triggered by the tactile stimulus (calcium influx into the neuron) and by the shock (influx of 196 serotonin, also termed 5-Hydroxytrypthophane or 5-HT) and the concomitant conversion of ATP into 197 cAMP, leading to protein synthesis upon repeated coincidence detection. Similar cascades have been 198 found in several animals, from invertebrates to vertebrates.

199 Sea hares, like humans, form memories, which result from what they learned. Aplysia memory, like 200 human memory, has different phases of different stability and durability. Like in vertebrates, memory 201 can be classified according to these properties into a short term and long-term memory and their 202 molecular underpinnings can be studied, leading to the discovery of basic principles such as the 203 requirement of protein synthesis to stabilize long-term memories. In vertebrates and humans, it was 204 later shown that the basic cellular and molecular mechanisms of learning and memory formation 205 largely correspond to those available in mollusks and insects, and can thus be regarded as evolutionary 206 conserved mechanisms. The fact that invertebrates have a much smaller number of neurons, which 207 are more accessible to network and modeling studies, shows the advantage of using these animals for 208 uncovering the cellular and molecular underpinnings of learning and memory. In addition, a genetically 209 advanced experimental organism like the fruit fly allows studying these phenomena at the level of 210 genes and gene cascades and thus allows characterizing their role and importance for learning and 211 memory (see Figure 6 and Footnote 6).

212 213 6) Aggression as an exemplary study case

214 Animals and humans alike are required to make decisions in agonistic situations such as when is it best 215 to flee rather than engage in a fight. Aggression is a key component of behavior as it allows the 216 defending or conquering important resources such as mates, food and/or territory. Not surprisingly, 217 aggression is widespread across species, from salmons to chimpanzees and humans. Yet, it can also be 218 found in insects where, for instance, ant colonies may execute lethal raids and fight against each other 219 for the possession of food resources. Precisely, the neural underpinnings of aggression have been 220 unraveled in the case of crickets, and have proved to be common to a large spectrum of species. Male 221 crickets engage in fights over a scarce resource, for example a burrows or a female. These fights occur 222 in bouts of increasing severity and usually end with a winner and a loser. These roles have been 223 associated with the presence and action of certain neuromodulators, i.e. substances that modulate 224 synaptic transmission between neurons and their electrical and biochemical properties. Research on 225 crickets has identified biogenic amines as well as Nitric oxide (NO) as key components of this 226 neuromodulation in an agonistic context. One of these amines termed octopamine (OA), the 227 invertebrate homologue of the vertebrate Noradrenaline, increases the motivation to fight, and 228 underlies stress and energy demanding behaviors. Winning seems to increase aggression and OA 229 levels, but the decision when to flee is independent from OA levels. In this case, the crucial substance 230 is NO, a gaseous modulator that neurons may also release, which dampens aggression and lowers the 231 threshold for when to flee. Crickets can also recover from loser effects and the important substance 232 here is the biogenic amine Dopamine (DA). There is also a role for serotonin (5-HT; see above), which 233 is released specifically after defeat to maintain a low threshold to flee, which is typical for crickets that 234 lose fights. All these results exhibit remarkable similarities to what is known from aggression in 235 rodents. Recent results even indicated that carefully monitoring of male cricket behavior allows 236 predicting its chances in a fight. “Aggressive individuality”, a term common in the analysis of human 237 social interactions, can thus be biologically assessed in the case of an insect. The advantages to study 238 aggression in crickets or fruit flies are manifold because their simple nervous system offers the 239 opportunity to study in detail cellular and synaptic mechanisms underlying aggressive encounters with 240 much higher precision than in vertebrates. Moreover, in the case of fruit flies, which also fight for 241 resources, the whole toolbox of genetic manipulations is also available for such studies, thus opening 242 fascinating perspectives for the characterization of the genetic bases of aggression (see Figure 7 and 243 Footnote 7).

244 245 246 7) Communication via pheromones: a widespread mechanism for animal communication

247 Pheromones are one of the most widespread and ancestral methods used by animals for intraspecific 248 communication. They are chemical substances that are released to the environment by exogenous 249 glands (glands that communicate with the environment through orifices) and that convey specific 250 messages such as sexual attraction, territorial defense, presence of food, nest entrances, among many 251 others. Given their role in animal communication, pheromones are termed ‘chemical messengers’ as 252 their role is to modify the behavior of receivers in an adaptive manner, i.e. in accordance with the 253 message received. In the absence of language, they play an essential role in animal communication 254 and in the regulation of social interactions between members of a species. The presence of 255 pheromones in humans has been the subject of vivid debates and no clear evidence exists for the 256 existence of this chemical communication in our species. Yet, it clearly exists in a broad spectrum of 257 species from invertebrates to vertebrates, in which hundreds of molecules have been characterized 258 for their role in communication. This research is of fundamental importance, for instance, in an 259 economic context: it has allowed conceiving lures and traps as a biological controlling method against 260 pests and parasites that may affect in a harmful way crops and human health. Interestingly, the 261 existence of pheromones was first discovered in a moth in the context of sexual attraction. Female silk 262 moths release a sexual pheromone made of two components, an alcohol and an aldehyde, which 263 attracts males from long distances given the extreme sensitivity of olfactory receptors on the male 264 antenna to detect these molecules, in particular the alcohol component, which is a long-distance 265 attractant (Figure 8 A-C). The two molecules building the sex pheromone of the silk moth were the first 266 pheromone components to be characterized and gave origin to the term pheromone. Beyond 267 discovering the existence of chemical attractants, this research had important consequences for our 268 understanding of olfactory processes. It characterized the nature and functioning of olfactory 269 receptors and the pathways, which convey and reshape the olfactory messages from the olfactory 270 receptors located in specialized hairs on the antenna to the brain (Figure 8 D,E). Similar architectures 271 exist in many olfactory systems so that the research on pheromones, started with the humble silk 272 moth, has expanded in a dramatic way our understanding of the neural bases of olfaction, besides its 273 fundamental economic importance (Figure 8 and Footnote 8).

274

275 8) Understanding pathological dysfunction of the nervous system: fruit flies as a model for 276 Parkinson disease studies. 277 Parkinson's disease (PD) is a progressive nervous system disorder that affects a considerable 278 proportion of persons in our societies. Symptoms start with a light tremors but the disorder may evolve 279 to produce stiffness, slowing of movement and speech problems, rendering affected individuals unable 280 to conduct a normal life. Together with Alzheimer disease, it constitutes one of the major neural 281 dysfunctions associated to neural degeneration problems. The discovery of a genetic, heritable basis 282 of Parkinson's disease and the identification of genes that are recurrently associated with the 283 emergence of the disease constituted a critical advance for our understanding of the disease. This 284 allowed developing genetic models for the study of the mechanisms of the disease as an important 285 strategy to fight against it. Fruit flies, with their extended genetic tool kit have played an important 286 role in this research. It was possible to create transgenic individuals with tissue or neuron specific 287 expression of dominant mutations, and thus study PD in flies. These mutants do indeed show some of 288 the symptoms that human patients develop. For instance, they exhibit reduced locomotion, loss of 289 dopaminergic neurons, problems with reactive oxygen species, mitochondrial dysfunction, and protein 290 aggregation; these are all symptoms that humans with PD also exhibit.

291 Studies on fruit flies allowed identifying unknown aspects and genetic interactions underlying PD. For 292 example, perturbation of mitophagy, the selective elimination of dysfunctional mitochondria, is 293 associated with the development of PD. Studies in Drosophila showed that two PD genes, Parkin and 294 Pink1, interact at the level of the mitochondria and play a crucial role in the dysregulation of mitophagy 295 at the onset of PD (Figure 9). Other genes and genetic interactions characteristic of the disease have 296 been since then identified in PD flies, thus enhancing our understanding of the disease affecting 297 humans. Fly neurophysiology thus offers a unique opportunity to progress in the search of medical 298 solutions for PD (Figure 9).

299

300 Conclusion

301 Invertebrates have played a pivotal role for understanding functional principles of the vertebrate 302 nervous system, including that of Humans. Thanks to their large and accessible neurons, which allow 303 recurrent identification and characterization from one individual to the next, invertebrates have 304 pioneered the discovery of basic mechanisms of neurotransmission and neuromodulation. They have 305 been, therefore, fundamental models to increase our basic knowledge about how the nervous system 306 works and is organized. Although this role remains and promises more discoveries for the future, the 307 advent of the genomic era has added a new perspective in the case of invertebrate models regularly 308 used in neuroscience studies. Many of them had their genome entirely sequenced (Aplysia, fruit flies, 309 bees, ants, silk moths among many others), thus opening fascinating perspectives in terms of studies 310 identifying the role of specific genes and their interactions for specific behaviors and pathologies. 311 Identifying the molecular key players of behavioral components, as well as of neural diseases, will be 312 possible using transgenics and mutants as available in fruit flies and other invertebrates. Thus, paying 313 attention to invertebrates and appreciating the potential of their reduced nervous systems constitutes 314 a strategic relevant decision for understanding the human nervous system. As Nobel Prize winner 315 Santiago Ramon y Cajal used to say when comparing the nervous system of insects with that of 316 vertebrates: “As usually, the genius of life shines more in the construction of smaller than larger master 317 pieces”.

318

319

Figure 1. Evolution

A simplified scheme of animal phylogeny demonstrating that the nervous systems of all bilaterian animals and, thus, of both invertebrates and vertebrates, have a common origin going back to a hypothetical organism, an „urbilaterian“ that was already equipped with a nervous system. In the case of invertebrates, such a nervous system developed into a dorsal brain and ventral nerve cord while in the case of vertebrates, it developed into a dorsal brain and dorsal nerve, spinal, cord. Evidence for this comes from the great homology of developmental genes that are responsible for the development and segmentation of the nervous system (modified after Arendt D et al., From nerve net to nerve ring, nerve cord and brain—evolution of the nervous system, Nature, 17:61-72, 2016, and Arendt D and Nübler-Jung K, Inversion of dorsoventral axis?, Nature, 371:26, 1994). There is still a debate among zoologists whether concerning the possibility that “evolutionary attempts” were made to generate animals with a nervous system based upon slightly different principles. Comb jellies (Ctenophora) may be seen as evidence for such evolutionary attempts (Moroz LL et al., The ctenophore genome and the evolutionary origins of neural systems, Nature 510(7503):109-114, 2014)

Figure 2. The squid giant fiber

A) The squid giant-axon system. The giant fiber, a merger of axons from neurons of the stellate ganglion, mediates fast action potentials to the mantle to allow escape responses of the squid. It includes a giant axon (800 µm in diameter) surrounded by smaller axons. B) A cross section of the stellate nerve showing the size of the giant axon in comparison to the cross sections of other smaller axons. C) The squid giant fiber axon was pivotal in revealing the ionic mechanisms of the action potential (in blue) with the peak of the action potential corresponding to an increase in sodium conductance (influx of sodium ions into the neuron; in red, full line) and the repolarization corresponding to a delayed increase of potassium conductance (outflow of potassium ions from the neuron; in red, dashed line). For their research on the squid giant axons and on electrical conductance a Nobel Prize in Physiology or Medicine was awarded to Sir John Carew Eccles, Alan Lloyd Hodgkin and Andrew Fielding Huxley in 1963. The fact that action potentials ares based on the flow of ions across the cellular membrane through specific ion channels was later proven by an elegant electrophysiological technique, the patch clamp technique, which allowed to measure the currents through one ion channel. This also lead to the award of a Nobel Prize in Physiology or Medicine in 1991 to Erwin Neher and Bert Sakmann. Pioneering work on cephalopods, and in particular, squids, was carried out by J.Z. Young (Some comparisons between the nervous systems of cephalopods and mammals. In: C.A.G. Wiersma (ed.): Invertebrate Nervous Systems. Their Significance for Mammalian Neurophysiology, University of Chicago Press, Chicago and London, pp. 353–362, 1967; see also a recent publication: Shigeno S et al., Cephalopod Brains: An Overview of Current Knowledge to Facilitate Comparison With Vertebrates. Front. Physiol. 9:952, 2018)

Figure 3. The electrical synapse A) Circuitry for crayfish escape behaviour. The colours of the crayfish (top) indicate the areas of sensory stimulations (red for the rear end and blue for the frontal part). The sensory neurons and their connections to the so-called giant fibers [axons of respective interneurons; the lateral giant (LG, red) and the medial giant fiber (MG, blue)] are also colour-coded. These giant fibers make electrical synapses with giant motor neurons (MoG) in particular abdominal segments, which lead to different bending movements indicated at the bottom. Mechanical stimulation of the frontal part leads to a fast retraction movement (blue crayfish at bottom) and mechanical stimulation of the rear end leads to a somersault (red crayfish at bottom) and then retraction. The multisegmental nature of the LG, which is an electrically well-coupled chain of segmental neurons, each with its own dendrites, is indicated. Colored asterisks mark phasic flexor muscles of segments 2–5 that are used in each type of GF reaction. In addition to this very special circuitry of the fast escape response, only used in life-threatening circumstances, a second system allows fast swimming movements. This circuitry, indicated by the label ‘non-G’ uses so-called fast flexor motor neurons (FF), which are activated by interneurons (white circles) and also the two giant fiber systems (LG and MG) via so-called segmental giant interneurons (SG, green). This system is used for swimming movements exclusively. Inhibitory connections from the LG to this system ensures that the non-giant swimming system is not active during an escape tail flip (from Edwards DH, Heitler WJ and Krasne FB, Fifty years of a command neuron: the neurobiology of escape behavior in the crayfish. Trends in Neuroscience 22:153-61, 1999). B) Transmission of a nerve impulse across an electrical synapse. The presynaptic fibers was electrically stimulated by an electrode and the action potential of the pre- and postsynaptic fiber (axon) recorded intracellularly. The measured amplitudes of the pre- and postsynaptic action potential were 92 and 70 mV respectively. Note the very short delay (fraction of a msec) between the pre- and postsynaptic action potentials. The delay time of a chemical synapse is usually 1 msec or more. (From Furshpan EJ and Potter DD. Mechanism of Nerve-Impulse Transmission at a Crayfish Synapse. Nature 180:342-43, 1957).

B) A) i)

-aminobutyric acid (GABA)

ii)

iii)

Figure 4. GABA as an inhibitory transmitter

A) A simple experimental system consisting of nerve and muscle fibers from the crayfish provided the basis for proving that -aminobutyric acid (GABA) is an inhibitory transmitter. The excitatory axon, coming from the main nerve bundles near the midline of the body, causes the muscle fiber to contract. The stretch receptor conveys information about the contractional state of the muscle. The inhibitory axon blocks muscular contraction and the firing of the stretch receptor. In the 1960's several lines of evidence (including the fact that blockers of GABA readily and reversibly cancel the inhibitory effect) showed that GABA is the inhibitory transmitter in this system (From Gottlieb DI, 1988, GABAergic neurons, Scientific American 258, No. 2: 82-89). B) Inhibition induced by GABA application on lateral giant interneuron (LG), an interneuron in the abdominal nerve cord of crayfish (see Figure 3). i)

Normalized size of Vprox, the voltage produced by current injected into the initial segment of the axon of the neuron, over the session. GABA, period during which 5 x 1O-4 M GABA was applied resulting in a significant reduction of Vprox. Inset shows two superimposed sample traces, one before and one during GABA application. ii) Normalized LG membrane potential from same preparation as in i). iii) In a different preparation than in i), 7 x 1O-6 M picrotoxin (PTX), an antagonist of GABA receptors, was continuously perfused. Vprox was repeatedly measured and normalized to the mean value before GABA -4 application. Application of 5 x 10 M GABA no longer caused a reduction in Vprox, showing the antagonistic effect of PTX on GABA receptors (from VU ET, Krasne FB, 1993, Crayfish tonic inhibition: prolonged modulation of behavioral excitability by classical GABAergic inhibition. Journal of Neuroscience 13(10): 4394-4402)

Figure 5. Locomotion

A to C) Walking of a stick insect, a lobster and a cat involves rhythmical alterations between groups of antagonistic muscles such Remotors and Promotors (stick insect and lobster) or Flexor and Extensor (cat). The colored arrows refer to the muscle group and direction of movement. On the right side the electromyograms from the respective muscles are shown during walking. The calibration bar corresponds to 1 s. (courtesy of Prof. A. Büschges, University Cologne). D) The development of walking in an in-vitro preparation of the isolated spinal cord of a mouse is shown in this graph, and particular key stages are indicated by arrows and inserts. The time axis refers to the embryonic stages (E10 to E22), and the stages after birth (P0 to P16). The first rhythms of the Central Pattern Generator (CPG) appear between E16/17 followed by maturation of the pattern at E18 and E20/21. Between E15 and birth (E22/P0) many motor neurons die (apoptosis) and at birth flexor (F) and extensor (E) motor neurons are matured differently with the extensors lagging behind. The first motor neurons appear between E13/14 and are electrically coupled at first. At stage P7, the motor neurons are no longer electrically coupled. Other key points are the first growth of axons containing the transmitter serotonin at E17 and the formation of tracts that connect the spinal cord with the cortex (corticospinal tract) and the full maturation of the cerebellum at P16. In addition, first monosynaptic reflexes occur E18. At birth the pups show leg movements such as swimming movements and stepping movements (air stepping), followed by elevation of head and shoulders starting at P2/3. Finally an upright posture (elevation of pelvis) at P10 and the start of real walking at P12 before mature walking at P15. Simultaneously to first walking, senses such as hearing and vision get mature whilst olfaction develops earlier. This developmental scheme applies to any walking animal, including humans, with species- specific time scales. Even insects with a complete metamorphosis during the pupal stage exhibit similar key features. (For further reading see Büschges A et al., New moves in motor control, Curr. Biol. 21(13):R513-24, 2011; and Bidaye SS et al., Six-legged walking in insects: how CPGs, peripheral feedback, and descending signals generate coordinated and adaptive motor rhythms. J Neurophysiol 119: 459-475, 2018)

C D

Figure 6. Learning and memory

A) A dorsal view of Aplysia showing the gill, the animal’s respiratory organ. A light touch to the siphon (mechanical stimulus) with a fine probe causes the siphon to contract and the gill to withdraw. Here, the mantle shelf is retracted for a better view of the gill. Sensitization of the gill-withdrawal reflex, by applying a noxious stimulus (electric shock) to another part of the body, such as the tail, enhances the withdrawal reflex of both the siphon and the gill. B) Spaced repetition converts short-term memory into long-term memory in Aplysia. Before sensitization training, a weak touch to the siphon causes only a weak, brief siphon and gill withdrawal reflex. Following a single noxious, sensitizing, shock to the tail, that same weak touch produces a much larger siphon and gill reflex withdrawal response, an enhancement that lasts about 1 hour. More tail shocks increase the size and duration of the response. (from Kandel ER, 2001, The Molecular Biology of Memory Storage: A Dialogue Between Genes and Synapses, Science 294: 1030-1038). C) Cellular and molecular mechanisms underlying the switch from short-term to long-term sensitization memory in Aplysia. In short-term memory, an external stimulus, such as an electric shock to the tail of the animal, causes the sensory neuron to send information in the form of chemical signals (5-HT) across the synapse to a motor neuron. The structure of neither cell is changed. In long-term memory, repeated stimuli recruit the gene transcription factor cyclic AMP (cAMP) response-element–binding protein (CREB), which activates downstream genes and results in the growth of new synapses in the sensory cell, thus strengthening the connection between neurons (i.e., long-term potentiation). 5-HT denotes 5-hydroxytryptamine, AMPA α-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid, CRE cyclic adenosine monophosphate response element, G guanine nucleotide–binding, MAPK mitogen-activated protein kinase, NMDA anti–N-methyl-D-aspartate, and PKA protein kinase A (from Kandel ER & Kandel DB, 2014, A Molecular Basis for Nicotine as a Gateway Drug. N Engl J Med 2014; 371:932-943). D) Classical conditioning in Aplysia. The conditioned stimulus (CS) is a weak tactile stimulus (0.5 s) to the siphon delivered either by a nylon bristle or by implanted electrodes in the siphon skin. The unconditioned stimulus (US) is a strong electrical shock to the tail (1.5 s) delivered either by hand-held spanning electrodes or by implanted electrodes in the tail. The right traces show the experimental groups; the paired group received contingent CS and US presentations; the unpaired group received non-contingent CS and US presentations. The US alone group received the same amount of electric shocks as the paired and the unpaired groups. After training, all three groups were tested with the CS once every 24 hr. Within group comparisons showed that the paired group exhibited significant retention of the learned response up to and including day 3 (red curve). The US alone group, while significantly lower than the paired group, showed significant sensitization up to and including day 2 (black curve). Finally, the unpaired group showed no significant variation of responses (grey curve) (from Carew TJ, Walters ET, Kandel ER, 1981, Classical conditioning in a simple withdrawal reflex in Aplysia californica; J Neurosci 1:1426-1437).

Figure 7. Aggression

A) Increasingly aggressive encounters between two male crickets from level 0 to 6, which ultimately will result in a winner and a loser (from: Stevenson PA and Rillich J. Neuromodulators and the Control of Aggression in Crickets. In Horch HW, Mito, T Popadic A, Ohuhi H, Noji, S (editors) The Cricket as a Model Organism, pp 169-196, Springer, New York Berlin, 2017). B) A number of different neuromodulators such as octopamine (OA), dopamine (DA), serotonin or 5-Hydroxytryptamine (5-HT or serotonin) and nitric oxide (NO) are involved in the decision to either fight on or flee, and their influences and actions are depicted in this scheme. (from: Stevenson PA and Rillich J (2019). Fight or flee? Lessons from insects on aggression. Neuroforum 25(1), https://doi.org/10.1515/nf-2017-0040). The same neuromodulators and their various receptor molecules are part of the human brain and at least some of them were also identified to play a role in human aggression. (For further reading: Stevenson PA and Schildberger K. Mechanisms of experience dependent control of aggression in crickets. Curr Opin Neurobiol 23(3):318-23, and Stevenson PA and Rillich J. Controlling the decision to fight or flee: the roles of biogenic amines and nitric oxide in crickets. Curr Zool 62(3):265-275, 2016)

A) B) C)

D) E)

Figure 8. Pheromones

A) Male of the silk moth Bombyx mori, with developed antennae fort the detection of the sex pheromone released by the female. B) Tip of the abdomen of a female exposing the pheromone- producing gland to the environment. C) The two components of the sex pheromone of Bombyx mori, bombykol (alcohol) and bombykal (aldehyde), which can be found in a ratio of 10:1 in the pheromone. D) Scheme of a sensillum trichodeum, a specialized hair on the antenna of the male of Bombyx mori, which hosts two olfactory receptor neurons (ORNs, colored red), one of which responds to bombykol and the other to bombykal. The ORNs are surrounded by three auxiliary cells (colored green), which build the hair and define the cavity containing a receptor hemolymph (colored blue) containing odorant binding protein (OBP) or pheromone binding protein (PBP). The dendrites of the ORNS bath within that hemolymph. Arrows show the position of electrodes for recording the activity of ORNs upon stimulation of the antenna with bombykol or bombykal. (from K.E. Kaissling, 2019, Responses of Insect Olfactory Neurons to Single Pheromone Molecules, In J.-F. Picimbon (ed.), Olfactory Concepts of Insect Control - Alternative to Insecticides, Springer, pp 1-27). E) Antennal lobe (AL) of a male of Bombyx mori. The AL is the primary olfactory center in the insect brain. It is constituted by globular subunits termed ‘glomeruli’. Axons from a ORN type arrive at the same glomerulus so that each glomerulus responds to a specific odor. In the left panel, a transversal section of a male AL allows appreciating ‘ordinary’ glomeruli (Gs), and hypertrophied glomeruli termed the macroglomerular complex (MGC), which are located at the entry region of the AL, where the antennal nerve (AN) arrives. Two of these macroglomeruli , the torus (T) and the cumulus (C), are shown. The MGC specializes in responding to the pheromone components of the sex pheromone. In consequence, the MGC is only found in males and is absent in females. In the middle and right panels, 3-D reconstruction images of the AL are shown from two different directions (anterior and posterior). In the anterior image, the toroid and the cumulus are visible. Another macroglomerulus, the horseshoe (H), is not visible because this compartment resides near the posterior surface. D, dorsal; L, lateral. Scale bar, 100 µm. (from Ai H, Kanzaki R, 2004, Modular organization of the silkmoth antennal lobe macroglomerular complex revealed by voltage-sensitive dye imaging, Journal of Experimental Biology 207: 633-644).

Figure 9. Genes responsible for autosomal recessive Parkinson's disease are involved in pathways that regulate mitochondrial morphology and integrity in Drosophila.

Loss-of-function mutations in the Drosophila orthologs of parkin or PINK1 lead to early mitochondrial pathology in flight muscles, associated with muscle degeneration, impaired flight and climbing abilities, and a peculiar wing phenotype (downturned wing). Similar phenotypes are observed in DJ-1 mutant flies. Overexpression of parkin rescues the phenotype of PINK1 mutant flies, whereas PINK1 does not prevent the defects observed in Parkin-deficient flies, suggesting that these genes are involved in a common pathway in which parkin acts downstream of PINK1. Genetic interaction studies demonstrated involvement of the gene encoding the intramitochondrial membrane protease Rhomboid 7 upstream of PINK1 in the PINK1/parkin pathway. The gene coding for the matrix serine protease Omi/Htra2 may act downstream of PINK1 in a pathway parallel to the PINK1/parkin pathway. Overproduction of DJ-1 rescues some aspects of the phenotype caused by loss of PINK1 function but does not affect the phenotype in parkin mutant flies, pointing to the existence of a pathway independent from the PINK1/parkin pathway, in which DJ-1 acts downstream of PINK1. The concerted effects of PINK1 and Parkin in the PINK1/parkin pathway may be mediated by direct protein-protein interaction (from Corti O, Lesage S, Brice A., 2011, What Genetics Tells Us About the Causes and Mechanisms of Parkinson’s Disease. Physiol Rev 91: 1161–1218)