report on the 12th symposium on invertebrate neurobiology held 31 august–4 september 2011 at the...

MEETING REPORT

Report on the 12th symposium on invertebrate neurobiologyheld 31 August–4 September 2011 at the Balaton LimnologicalResearch Institute of the Hungarian Academy of Sciences,Tihany, Hungary

Lindy Holden-Dye • Robert J. Walker

Received: 16 March 2012 / Accepted: 22 March 2012 / Published online: 6 April 2012

� Springer-Verlag 2012

Abstract In August 2011, the 12th international sympo-

sium of ISIN was held by Lake Balaton in Tihany, Hun-

gary. This convivial and stimulating meeting provided a

forum for discussion of a range of invertebrate organisms

in neuroscience research. Here the main topics covered at

the meeting are reviewed.

Keywords Invertebrate � Neuroscience � International �Meeting

Over 60 neurobiologists attended the 12th International

Society for Invertebrate Neurobiology held in Tihany, 31

August–4 September 2011. The participants came from 12

countries with the largest contingents coming from Russia,

Japan, Hungary and Germany. The programme consisted of

6 Plenary Lectures, including the Ernst Florey and Janos

Salanki memorial lectures, one special lecture, 25 oral

communications and 35 poster communications. As with

previous symposia, the animals of choice for study were

predominantly molluscs, particularly gastropods, and

arthropods, particularly insects. In addition, there were

presentations describing work on platyhelminthes, nema-

todes, annelids and ctenophores. Where possible, a refer-

ence has been included so the reader can access related

literature. For the posters, in general only one author who

attended the symposium is listed.

In the Ernst Florey memorial lecture, Professor Nicholas

Strausfeld (University of Arizona, Tucson, USA) provided

an in-depth historical background concerning the

relationship between the vertebrate and invertebrate brain

leading to their common evolution or possible conver-

gence. This included a review of the works of Dohrn, St.

Hilaire, Owen, Bellonci, Retzius, Nansen, Cajal and

Zavarzin. He presented molecular evidence for the basis of

their common origin. For example, the injection of sog

mRNA into Drosophila embryos resulted in the formation

of ventral denticle belts and ectopic patches of central

nervous system, while in Xenopus, sog mRNA caused

dorsal development of notochord and central nervous sys-

tem. The vertebrate homologue of sog, chd, behaved in a

similar manner, viz., chd mRNA injected into Xenopus

promoted dorsalization but when injected into Drosophila

embryos activated ventralization of cell fates (De Robertis

and Sasai 1996). Further evidence for a common molecular

plan for cephalization came from the expression of the otx

gene, the human homologue of the Drosophila homeobox

gene, otd, in otd mutant Drosophila (Nagao et al. 1998).

These authors found that otx gene expressed in an otd

mutant restored the cephalic defects of the mutant, pro-

viding further evidence that the cephalic region, including

the brain, of vertebrates and invertebrates could be

homologous. Evidence for a common origin of vertebrate

and invertebrate brains came from molecular biology

studies using a primitive annelid, Platynereis dumerilii, the

work of Tomer et al. (2010). Professor Strausfeld reported

that these authors demonstrated that regulating genes

expressed in mushroom bodies of the developing brain of

P. dumerilii corresponded to an identical group of genes

expressed in the developing mouse pallium, providing

evidence for homology between the vertebrate pallium and

mushroom bodies of annelids, insects, myriapods, cheli-

cerates and onychophorans, with the origin of the brain

arising from a common protostome/deuterostome ancestor.

Readers interested in further information on the origin of

L. Holden-Dye (&) � R. J. Walker

Centre for Biological Sciences, University of Southampton,

Southampton SO17 1BJ, UK

e-mail: [email protected]

123

Invert Neurosci (2012) 12:69–79

DOI 10.1007/s10158-012-0131-z

brains should consult Professor Strausfeld’s new book:

Arthropod Brains: Evolution, Functional Elegance, and

Historical Significance; Belknap Press, Harvard University

Press (2012).

In his Janos Salanki memorial lecture, Professor Leonid

Moroz (University of Florida, Gainesville and St. Augus-

tine, USA) reviewed his recent research and ideas on the

genomic bases of memory and neuronal evolution and

stated there is a genomics revolution in neuroscience. He

described the nervous system of ctenophores which have

two nerve nets, viz., a mesogleal nerve net throughout the

mesoglea and a more compact nerve net in the ectodermal

epithelium. Genes associated with the synthesis of classical

transmitters are absent with the exception of glutamate, but

there is evidence for over 50 neuropeptides. One of his

interests is in the genetic basis of neuronal identity and in

neuronal plasticity. In this research Professor Moroz has

developed techniques to analyse the genome of single

Aplysia neurons and is involved in sequencing the Aplysia

genome. Around 60 % of the Aplysia genome may be

expressed in a single giant neuron. He also discussed evi-

dence for the polyphyletic origin of the centralized nervous

system as opposed to the monophyletic hypothesis (Moroz

2009). He considers that from molecular data and animal

phylogeny that complex brains evolved up to nine times

during evolution. Taking molluscs as an example, complex

nervous systems might have arisen at least four times.

Secretory epithelial cells may represent the precursors of

neurons with peptides, ATP, glutamate and nitric oxide

(NO) evolving as the first interneuronal messengers. Pro-

fessor Moroz also discussed the possible role of injury and

regeneration resulting in massive gene expression leading

to neuronal evolution, viz., that injury might be a major

neurogenic factor in evolution. Injury might cause the

release of chemical messengers that could act as signal

molecules associated with injury and that memory of injury

could be a predecessor of all memory forms (Walters and

Moroz 2009). Peptides could also be released to act as

growth factors. Professor Moroz also reported studies on

the role of synaptic kinesin transport complex in memory

circuits. Transcriptome analysis of single synapses

revealed the presence of 256 RNAs per synapse. He is

investigating the genes that regulate memory and is also

interested in the epigenetics of memory with the proposal

that learning and memory are epigenetic processes

involving the interaction between 5-methylcytosine and

5-hydroxymethylcytosine. Professor Moroz’s group also

presented two posters, one on genome analysis of single

identified neurons from Aplysia californica and one on

genome analysis of neurotransmitter signalling in the

ctenophore, Pleurobrachia bachei.

In her plenary lecture Professor Sakiko Shiga (Osaka

City University, Japan) reviewed her research to identify

the neurons and neuronal circuit involved in photoperiod-

ism in insects. While the neuronal circuit involved in

photoperiodism has been documented for Drosophila, the

photoperiodic response is difficult to measure in this insect

and so she used the blowfly, Protophormia terraenovae.

Interestingly, circadian clock genes are expressed in a wide

range of cells. Photoperiodic information is reflected in the

expression patterns of circadian clock genes per and tim

and in the subcellular distribution of PER (PERIOD)

(Muguruma et al. 2010). Ablation experiments have dem-

onstrated that the dorsal protocerebrum is an important

region for photoperiodism in insects and neurons in the

pars lateralis are key for controlling the hormones that

regulate diapause (Shiga and Numata 2009). Five types of

PER-immunoreactive neurons were identified, and Profes-

sor Shiga reviewed their roles in photoperiodism. Evidence

suggests that circadian clock neurons, s-LNVs (small lateral

ventral neurons), which drive behavioural rhythms, may

play a role in photoperiodism and that circadian behav-

ioural rhythms and photoperiodism share neuronal com-

ponents in their mechanisms. These s-LNVs, located on the

boundary between the optic lobe and mid-brain, are

immunoreactive to PERIOD, and pigment-dispersing fac-

tor and their ablation in P. terraenovae results in loss of

photoperiodic discrimination (Shiga and Numata 2009).

The photoperiodic clock provides neurons in the protoce-

rebrum with information on short and long days, on the

total number of days, and photoperiodic photoreceptors

have been identified in P. terraenovae (Shiga and Numata

2007). However, the process whereby the number of short

and long days is counted by the brain is unknown.

Professor Pavel Balaban (Institute of Higher Nervous

Activity, Russian Academy Sciences, Moscow, Russia)

presented his recent research on the regulation of synaptic

plasticity, using Helix lucorum as his experimental animal.

His simple experimental model consisted of a mechano-

sensory glutamatergic neuron which synapsed directly onto

the withdrawal behaviour interneuron (plasticity locus).

This synapse can be modulated by a reinforcing seroto-

nergic neuron that signals both presynaptically onto the

mechanosensory neuron and postsynaptically onto the

interneuron. He outlined several intracellular mediators,

including IP3, cAMP and calcium, which can impair

memory, but these are not selective for memory while

PKMzeta is selective for memory loss. NO also plays a role

in synaptic plasticity through its ability to S-nitrosylate

proteins and affect memory. Professor Balaban presented

evidence to support his proposal that during the relearning

process NO is involved in erasing old memory and in new

memory formation. In the presence of a NOS blocker

(L-NNA), habituation occurs faster. He also described

experiments involving contextual memory using the pro-

tein synthesis blocker, anisomycin, and L-NNA which

70 Invert Neurosci (2012) 12:69–79

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provided further evidence that in the absence of NO,

memory is not erased. This work was also described in a

poster presented by Dr. Tatiana Korshunova. Five further

posters presented work from Professor Balaban’s group,

viz., the role of dopamine and 5-HT in odour perception

and behaviour in Helix (Roshchin and Balaban 2012);

control of locomotion in Helix by peptides which can

increase or decrease locomotion (Dr. Igor Zakharov); evi-

dence that the hooks in the buccal apparatus of Clione

limacine originated from the radula (Dr. Aleksey Maly-

shev); early gene expression in the central nervous system

of Helix (Dr. Victor Ierusalimsky); and the command

neuron peptide 2 (DYPRLamide) plays a role in the

modulation of the Helix cardiovascular system (Dr. Niko-

lay Aseyev). Dr. Evgeny Nikitin, a member of Professor

Balaban’s group and a collaborator of Professor Gy}orgy

Kemenes, described experiments using Lymnaea stagnalis

where, following single-trial food-reward classical condi-

tioning fictive feeding behaviour, the membrane potential

of the giant serotonin cerebral cell (CGC) becomes long

term depolarized, 63–57 mV (Nikitin et al. 2008). In

untrained snails, direct current injection to depolarize the

CGC membrane potential results in the conditioned stim-

ulus triggering feeding. In cell culture the B1 buccal

motoneuron EPSP is potentiated when the co-cultured

CGC is depolarized, indicating that enhanced output from

the presynaptic terminals of the CGC. Optical recordings

employing calcium-sensitive and voltage-sensitive probes

showed that CGC soma depolarization spread to the distal

end of the axon side branch, increasing synaptic output

from the cell. Using 4-aminopyridine, evidence suggests

that an A-type potassium current controls propagation of

action potentials in the side branch. Calcium transients

evoked by action potentials at the distal end of the side

branch were increased following training, indicating that

training reduces attenuation along the side branch of the

CGC. This is linked with increased inactivation of a fast

transient potassium current at depolarized membrane

potentials in trained animals.

Professor Gy}orgy Kemenes (Sussex, UK) reviewed his

laboratory’s recent research on the molecular mechanisms

associated with learning and memory in Lymnaea stag-

nalis. He stressed the importance of choosing the appro-

priate animal model and stated that the feeding system in

terms of anatomy, physiology and behaviour is well

defined in L. stagnalis. The experiments are based on

single-trial food-reward classical conditioning using amyl

acetate (pear drops). On its own, amyl acetate does not

induce feeding, but in combination with sucrose, feeding is

induced and now amyl acetate alone triggers feeding, and

this effect can persist for 2–3 weeks. Following acquisition

of the memory, consolidation stabilizes a memory ‘trace’.

Consolidation consists of two phases, synaptic

consolidation occurring within a few hours after learning

followed by system consolidation that requires early post-

training gene transcription and protein synthesis, and

occurs over a period of weeks. A third process, reconsol-

idation, is where previously consolidated memories can

become labile through reactivation of the memory trace by

recall. Professor Kemenes discussed the roles of PKA and

Ca-calmodulin-dependent kinase II (CaMKII) in the

acquisition, consolidation and reconsolidation of long-term

memory (LTM). PKA is required for all three processes. If

PKA is blocked shortly after memory recall at six hours,

then reconsolidation is blocked, while if PKA is blocked

after memory recall at 24 h post-training, then there is no

effect on reconsolidation; thus, time of block is critical.

CaMKII is required for acquisition and late consolidation

but not for early or intermediate consolidation or retrieval

of LTM (Wan et al. 2010). The CaMK inhibitor, KN-62,

when injected 30 min before training, blocks acquisition

but fails to block memory formation if injected 30 min

after acquisition. There is also a critical window, around

24 h, when KN-62 will block late consolidation. Using the

NMDA receptor antagonist, MK-801, Professor Kemenes

reported that acquisition was dependent on NMDA recep-

tor activation in addition to CaMKII activation. However,

late memory consolidation does not require activation of

NMDA receptors. Pituitary adenylate cyclase–activating

polypeptide (PACAP) and its receptors occur in L. stag-

nalis (Pirger et al. 2010), and both are necessary for

memory formation after appetitive conditioning. There is

also a role for AMPA receptor activation as when these

receptors are blocked LTM expression is reduced. Phos-

phorylation of CaMKII at T305 is also necessary for

increased levels of AMPA receptors. From the same group

Dr. Ildiko Kemenes introduced the following question:

How does the brain decide between two behaviours? In her

experiments the pond snail, L. stagnalis, had to decide

between feeding and withdrawal behaviours. While the

circuit involved with feeding has been studied in depth,

that for withdrawal is less well known. Stimulation of the

skin leads to motoneuron activation and shell lowering and

head and foot shortening through modulation by possibly a

pedal neuron, PeD11. The preparation consisted of lip

sensory area, the buccal mass and columellar muscle

together with the central nervous system. Feeding was

monitored via a transducer attached to the buccal mass and

withdrawal by recording from the columellar muscle.

Sucrose induces rhythmic contractions of the buccal mass,

while the columellar muscle relaxes. In contrast, strong

touch induces the columellar muscle to contract and the

buccal system to relax. Which areas of the brain are

responsible for this behavioural switch? By ablating dif-

ferent brain areas Dr. Kemenes demonstrated that when the

parietal and visceral ganglia were removed there was no

Invert Neurosci (2012) 12:69–79 71

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effect on either behaviour. Withdrawal behaviour required

the pleural, cerebral and pedal ganglia, and backfilling the

columella nerve identified neurons including PeD11 and

the pleural coordinating interneuron, PIB. Recordings were

made from these neurons and their effects on buccal and

columella muscles noted from which possible sites for

decision-making were identified. Dr. Zsolt Pirger, also

from Sussex, extended this work and observed that depo-

larization of PIB inhibited feeding, while hyperpolarization

of PIB activated feeding. Sucrose application, while acti-

vating feeding, inhibited PIB activity. Depending on the

strength of stimulation to the lip, PIB activity can be

enhanced or inhibited. While there is no direct connection

between PIB and PeD11, and no direct effect of either

neuron on muscle activity, both indirectly influence buccal

and columella muscle activity. From his studies, Dr. Pirger

concluded that both PIB and PeD11 play key roles in

decision-making between feeding and withdrawal in

L. stagnalis. The effect of traumatic stress on the shadow

withdrawal reflex in L. stagnalis was discussed by Pro-

fessor Manabu Sakakibara (Numazu, Japan). Interneuron

RPeD11 is a key neuron for withdrawal behaviour, and in

traumatized animals RPeD11’s membrane potential

remains depolarized for 24 h and feeding is depressed. An

augmented withdrawal response can last for a week.

Trauma also reduced locomotion and respiration. There

was also a poster by Professor Sakakibara’s group on

aversion conditioning in L. stagnalis (Dr. Hiroshi Sunada

and Dr. Satoshi Takigami).

Professor Lindy Holden-Dye discussed data from a

collaborative project (with Dr. Vincent O’Connor and

Professor Robert Walker) on behavioural plasticity in the

free-living nematode, Caenorhabditis elegans (Mitchell

et al. 2010), particularly in respect of responses to food

deprivation and possible roles for neuropeptides, viz., FLPs

(FMRFamide-like peptides) and NLPs (neuropeptide-like

peptides). In the presence of food (bacteria) the radial

muscles of the pharynx contract and suck in bacteria. Pha-

ryngeal pumping can be recorded electrically as an EPG

(electropharyngeogram) using a suction electrode. Three

classical transmitters, viz., acetylcholine, serotonin (5-HT)

and glutamate, have a key role in the regulation of pharyn-

geal pumping (Franks et al. 2006). The pharynx of N2 wild-

type C. elegans normally pumps at around 200 pumps s-1 in

the presence of food, while in the absence of food, they

pump at a low rate for up to 2 h. Pumping then becomes

erratic over the following few hours. However, when

replaced on food they again pump at around 200 pumps s-1.

Professor Holden-Dye described experiments in which a

range of mutants were tested for the reaction to the absence

of food and this reaction compared with that observed using

N2 wild-type C. elegans. Aspects of Professor Holden-

Dye’s lecture were presented as a poster.

In a special lecture, Professor Adrian Horridge (Aus-

tralian National University, Canberra) reviewed his

research on the visual system of the honeybee, Apis

mellifera. This lecture was introduced by Professor Jozsef

Hamori (Budapest, Hungary), who had worked with Pro-

fessor Horridge on crustacean vision in the 1960s. Pro-

fessor Horridge provided a detailed analysis of the visual

processes in A. mellifera based on his extensive research.

The process involves the photoreceptors and small feature

detectors, the responses of which for areas and edges in a

local region of the eye sum to form a cue (Horridge 2009a).

The cues are the units of visual memory, and each has its

own identity. Concurrence of cues in a specific area of the

eye allows a landmark to be identified and remembered.

Landmark labels are learned at wide angles to each

other and are used to identify places and locate rewards.

A. mellifera remembers cues and detects parameters (parts of

the image outside the eye which display areas and edges), not

patterns or shapes. The resolution of cues varies and can be as

low as 2–3�. Those wishing to read a full account of Professor

Horridge’s research should consult his book that is available

in open access form (Horridge 2009b).

There were a number of talks and posters on sensory

systems in insects, particularly associated with olfaction

and vision. Professor John Hildebrand (University of Ari-

zona, Tucson, USA) described his research on the coding

of olfactory stimuli in the antennal lobe of his model insect,

the sphinx moth, Manduca sexta. The antennal lobes are

complex, composed of 63 glomeruli to which the olfactory

sensory cells project and synapse with interneurons that

project to other brain areas, including adjacent glomeruli.

Professor Hildebrand described two of the three glomeruli

of the male-specific macroglomerular complex, toroid 1

and the cumulus, and their specific projection neurons (Lei

et al. 2002). There are multiple coding mechanisms in the

macroglomerular complex. To analyse the population

coding for floral mixtures and component chemicals, a

combination of gas chromatography and multi-electrode

extracellular recordings in the antennal lobe of M. sexta

(Riffell et al. 2009) was used. Around 75 volatile com-

pounds are released from the flowers of the sacred thorn-

apple, Datura wrightii, and of these 9 were required to

produce the full response from M. sexta. These 9 were later

refined down to 3 key compounds, viz., benzaldehyde,

linalool and benzyl alcohol, since the same pattern of firing

in the antennal lobes is obtained when the 3 compounds are

used as when 9 are applied. This activity is in turn pro-

jected to neurons in the lateral horn of the protocerebrum.

This research has recently been extended through the

analysis of the antennal transcriptome of M. sexta where

the number of glomeruli for males was determined as 68

and 70 for females (Grosse-Wilde et al. 2011). These

authors also identified gene families associated with


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olfaction. Professor Friedrich-Wilhelm Sch}urmann (Got-

tingen, Germany) reviewed his research on filamentous

F-actin in the mushroom bodies’ Kenyon cells in the

cricket, Gryllus bimaculatus (Frambach et al. 2004). Ken-

yon cells are intrinsic to the mushroom bodies and receive

their input from sensory neurons. F-actin was identified in

Kenyon cell dendritic spines which make contact with

deuterocerebral chemosensory projection neurons. F-actin

did not appear to occur in GABA-containing sites. Intense

staining for b-actin occurred in Kenyon type III cells, and

there was evidence for colocalization between F-actin and

b-actin in the dendritic tips of Kenyon cells.

Professor Karoly Elekes (Tihany, Hungary) reviewed the

synaptology and 5-HT-immunoreactive innervation of the

cell body layer of the procerebrum, the olfactory centre of

Helix pomatia and Limax valentianus. The procerebrum is

the centre for olfactory learning and memory in these gas-

tropods, receiving projections from both the olfactory nerve

and other regions of the central nervous system. This area can

be compared to the mushroom bodies of insects. There are a

number of inputs to the globulus cells, indicating the regu-

latory role of local circuits in this region of the procerebrum.

5-HT is likely to be one of a number of transmitters/modu-

lators involved in these circuits; others may include NO, CO

and H2O2. The globulus cell bodies of H. pomatia are

strongly innervated by 5-HT-positive nerve terminals, but

there is no evidence that these cell bodies contain 5-HT. In

contrast, in L. valentianus, there is relatively little innerva-

tion of the procerebrum cell body layer though 1 mM 5-HT

evoked an excitatory effect on olfactory integration in L.

valentianus, inducing the globulus cells to exhibit bursting

activity. This provides evidence for 5-HT having a key role

in coordinating local field potential activity in the procere-

brum. In addition to this lecture there were two posters: one

described 5-HT innervation of the procerebrum in H.

pomatia, providing further evidence for a role for 5-HT in the

procerebrum of snails (Dr. Izabella Battonyai), and the other

poster summarized evidence for NO signalling in the pro-

cerebrum of H. pomatia (Dr. KalmanNacsa). There was a

poster describing immunohistochemical studies to identify

the structure of the dermal photoreceptor cells of L. stagnalis

(Dr. Satoshi Takigami, Dr. Hiroshi Sunada and Dr. Manabu

Sakakibara, Numazu, Japan).

Professor Ian Meinertzhagen (Halifax, Canada) described

serial section EM studies of the optic lobe of Drosophila

melanogaster which is subdivided into four neuropiles, viz.,

lamina, medulla, lobula and lobular plate (Fischbach and

Dittrich 1989). The cellular organization of the optic lobe is

complex with up to 70 different cell types although in any

one medulla column there are only about 35 such cells.

Analysis of a single column has shown the complexity of its

microcircuits and synaptic connections. The synapses are

divergent polyads with an average of four postsynaptic

elements abutting a T-bar ribbon at each release site. Each

medulla column has 10 inputs, viz., two receptor cells, five

lamina cells, two centrifugal cells and one enigmatic medulla

cell, T1. He showed reconstructions of 12 of the*35 mostly

transmedullary (Tm) cells so far constructed; Tm3 and Tm4

spread between columns. Reconstruction of a single column

through the medulla’s entire depth shows that it is composed

of around 10,000 presynaptic T-bar ribbons with around

40,000 postsynaptic dendrites. About 30 % of these synapses

are part of one column, with the column borders poorly

defined. Professor Meinertzhagen described some of the

connections between the different cell types. So far one

column has been analysed, with the aim of analysing a total

of seven columns, one plus its six neighbours. The findings

he reported clearly indicate that the circuits in the optic lobe

are far more complex than the textbook account of relay

circuits with single sites between single pre- and postsyn-

aptic components. Following this talk, Professor Reinhard

Wolf (Wurzburg, Germany) presented work on selective

visual attention in D. melanogaster where the response is to

only one of two or more competing visual stimuli. D. mel-

anogaster were attached to a torque metre and their flight

observed. Visual cues were used to direct the fly’s attention

to one or other of two visual half fields (Sareen et al. 2011).

Temporal and spatial separation of the cues was investigated

and described. It was concluded that a fly can temporarily

restrict some of its behaviour to a specific location in its

visual field.

Dr. Tibor Kiss (Tihany, Hungary) reported that 5-HT

inhibited dopamine (DA)-induced contractions of the sali-

vary duct muscle of the snail, H. pomatia. Previous studies

had shown the presence of both DA and 5-HT receptors in

this tissue and that DA contracts the muscle through acti-

vation of a D-1-like receptor while 5-HT has a biphasic

action, relaxation through a 5-HT2-like receptor and con-

traction through a 5-HT3-like receptor (Kiss et al. 2003).

5-HT both reduced DA-induced contractions and electri-

cally stimulated contractions of salivary duct muscle, while

immunocytochemical evidence showed that both amines

occur in axons innervating the muscle. Both amines also

activated cAMP levels in salivary duct muscle with met-

oclopramide blocking this action of DA but not 5-HT.

Ergometrine blocked both DA and 5-HT receptors on the

muscle. Dr. Kiss discussed the possibility that both amines

might interact with a common receptor, a possibility that

has been proposed before on leech neurons (Sunderland

et al. 1980). The possibility that several ligands might

interact with a single receptor has been reviewed recently

(Mandrioli et al. 2007).

A number of papers and posters dealt with neuropep-

tides. Dr. Laszlo Mark (Pecs, Hungary) described the

technique of MALDI-TOF imaging, which enables the

localization and identification of neuropeptides in 8–20 lm


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cryosections from cerebral ganglia and whole embryos of

L. stagnalis. There was a joint poster presentation from

Tihany and Sussex on the pituitary adenylate cyclase–

activating polypeptide (PACAP) and its receptor from H.

pomatia and L. stagnalis (Pirger et al. 2010). This work

also used MALDI-TOF and immunohistochemistry to

show the occurrence of PACAP in the cerebral ganglia and

lip region of L. stagnalis and that it activated cAMP in the

cerebral ganglion. This action of PACAP was partially

blocked by the PACAP antagonist, PACAP6-38. PACAP

also has several physiological actions on snails including

reducing the number of TUNEL-positive apoptotic cells in

the salivary gland induced by colchicine, the levels of

PACAP are four times higher in the brain of active com-

pared to hibernating snails, plays a role in LTM and has an

effect on the heart. Professor Etsuro Ito (Sanuki, Japan)

presented evidence for a role of cerebral ganglion insulin in

long-term potentiation (LTP) and conditioned taste aver-

sion (CTA) and its consolidation into LTM (Azami et al.

2006). The molluscan insulin-related peptide II (MIP-II) is

only found in the cerebral ganglion in, for example, the

light green cells, but its receptor occurs throughout the

central nervous system of L. stagnalis, including around B1

buccal motoneuron. MIP-II is upregulated in L. stagnalis

showing CTA. Application of either mammalian insulin,

5 lM, or MIP-II activated the EPSP between CGC and B1

but trypsin blocked this potentiation by insulin. In the

absence of light green cells this increased EPSP is absent.

Application of an insulin receptor antibody also reduced

the EPSP recorded from B1. Injection of insulin receptor

antibody did not inhibit CTA but did block LTP and LTM

consolidation. Taken together, the results from Professor

Ito’s group clearly indicate a role for MIP-II LTM for-

mation in L. stagnalis and by analogy in other animals.

Professor Dick Nassel (Stockholm, Sweden) discussed the

control of insulin-secreting cells in D. melanogaster. There

are seven insulin-like peptides in D. melanogaster, with

DILP-2, 3 and 5 being most insulin-like, and are produced

by 12-16 protocerebral median secretory cells (insulin-

secreting cells) in the brain. DILP-2, 3 and 5 are released

into the haemolymph from the corpora cardiac and the

aorta. DILPs are released in response to nutritional changes

in D. melanogaster. It is not possible to measure insulin

release in flies, but it is possible to interfere with insulin

signalling and investigate the effects on metabolism and

behaviour. Receptors for short neuropeptide F (sNPF),

tachykinin (DTK), octopamine, GABA and 5-HT have

been located on the insulin-secreting cells. Octopamine and

sNPF activate these cells, while DTK, GABA and 5-HT

inactivate them. One insulin-like peptide, DILP-6, which is

more like an insulin-like growth factor, is produced by fat

cells, and insulin is very important in D. melanogaster for

its development (Nassel and Winther 2010).

Professor Joachim Schachner (Marburg, Germany) pre-

sented his research on the analysis of neuropeptides in the

olfactory system of the red flour beetle, Tribolium castane-

um, using MALDI-TOF mass spectrometry. T. castaneum

is a useful model insect, with a 4-week life cycle, but

which can live for up to 2 years. The genome has been

analysed (Li et al. 2008; Tribolium Sequencing Consortium

2008).

Professor Schachner described the different types of

sensilla which project to around 70 glomeruli and are the

same in both sexes of T. castaneum. A number of peptides

have been identified in the brain, including FMRFamide-

like peptides, allatostatin-B, tachykinin-related peptide,

adipokinetic hormone, myosuppressin, short neuropeptide

F, sulfakinin and SIFamide. No significant differences were

detected between the brain volumes of male and female T.

castaneum (Dreyer et al. 2010). Professor Schachtner’s

group also presented two posters on the distribution of

neuropeptides in the mushroom bodies and antennal lobes

of T. castaneum using conofocal laser scanning microscopy

and MALDI-TOF mass spectrometry. Interestingly the

levels of tachykinin immunoreactivity increases with the

age of both sexes. The brain of T. castaneum shows post-

metamorphic plasticity with the antennal lobe increasing

with age. Using a combination of immunocytochemistry

and mass spectrometry Professor Reinhard Predel

(Cologne, Germany) investigated the roles of neuropep-

tides in the antennal lobes of Periplaneta americana

(Neupert et al. 2012). He described the neurons involved in

the antennal lobes, viz., olfactory receptor neurons, local

interneurons and neurons projecting onto the protocere-

brum and tried to match different areas of the antennal

lobes with peptide expression. MALDI-TOF mass spec-

trometric analysis demonstrated up to 50 neuropeptides,

representing products from 10 neuropeptide genes,

including allatostatins, SIFamide, allatotropin, FMRFa-

mide-related peptides, crustacean cardioactive peptide and

tachykinin-related peptides from one glomerulus. Professor

Predel’s aim is to match the different types of antennal lobe

neurons with expression of selected neuropeptides and then

relate them to function. Mass spectrometric analysis of

single neurons demonstrated the presence of identified

peptides in single neurons; some neurons clearly show co-

localization of peptides, for example, co-localization

between tachykinin-related peptide and short neuropeptide

F in type I local interneurons. Professor Predel concluded

by saying that the feasibility of MALDI-TOF mass spec-

trometric profiling of single antennal lobe neurons is an

important precondition for combining electrophysiology

with peptide profiling on the single-cell level.

Professor Fred Libersat (Beer Sheva, Israel) described

his research involving the interactions between a cock-

roach, Periplaneta americana, and parasitoid wasp, the


123

jewel wasp, Ampulex compressa, in which the latter injects

a venom into the former to induce a lethargic, zombie-like,

state. If the wasp injects radioactive venom into the

cockroach the label is located in the cerebral ganglia,

specifically in the sub- and supraoesophageal ganglia,

which then induces a lethargic state. Following injection,

the cockroach grooms itself for 30 min and then enters a

hypokinetic state which can last 3–7 days. These animals

show less spontaneous and induced walking activity and an

increased threshold for walking initiated by foot shocks

though they can still swim and fly. Procaine injection into

the suboesophageal ganglion can also decrease neuronal

activity and walking in the cockroach. Professor Libersat

also reported that spontaneous and induced activity is

reduced in the suboesophageal ganglia of stung cock-

roaches, suggesting that this is the prime site for action by

the venom. A decrease in suboesophageal activity

decreased the drive for walking. The neurons involved in

this effect are not known though likely candidates are the

octopamine DUM cell (Gal and Libersat 2010).

Professor Joachim Pfl}uger (Berlin, Germany) presented

evidence for the roles of tyramine and octopamine during

fictive flight in M. sexta. In some insects the ratio between

cells containing the two amines varies between the brain

and the thoracic ganglia, viz., the brain has more tyramine

cells while the thoracic ganglia have more tyramine/octo-

pamine cells. Under stress some tyramine cells now

become tyramine/octopamine cells. Fictive flight in

M. sexta was induced using the octopamine agonist, chlo-

ridimeform. If tyramine is also applied, then more

depressor neurons are recruited, which can be reversed in

the presence of the tyramine blocker, yohimbine, without

affecting the elevators. D. melanogaster, which lacks

tyramine-b-hydroxylase, exhibits altered flight patterns and

cannot sustain long flight periods, confirming that octopa-

mine is needed for high energy–requiring processes. It is

likely that rather than being antagonistic, tyramine and

octopamine act on different components of the behaviour

(Brembs et al. 2007). For a recent review on neural control

of muscle metabolism and motor behaviour in insects,

consult Pfluger and Duch 2011.

Professor Chun-Fang Wu (Iowa City, USA) described

his work on the genomic analysis of behaviours, such as

social isolation associated with aggression and the inter-

action between ROS (reactive oxygen species) regulation,

social interaction and lifespan in D. melanogaster. When

flies are reared in isolation they show increased neuro-

muscular transmission. Two D. melanogaster mutants, Hk

(Hyperkinetic) and gsts1 (glutathionine-S-transferase-S1),

showed increased female aggression and muscle excit-

ability though they were group reared (Ueda and Wu

2009). When mutants were reared in isolation, there was no

increase in aggression. Products from both Hk and gsts1

genes are involved in ROS metabolism, and these mutant

flies also show increased signals from a ROS probe. It is

suggested that ROS regulation is involved in social isola-

tion stress. Professor Wu also described experiments using

mutants deficient in superoxide dismutase gene, Sod-1,

which are normally short-lived. Sod-1 mutants, when

housed with longer lifespan flies, increased their own

lifespan and stress resistance (Ruan and Wu 2008), indi-

cating a positive role for social interaction in lifespan.

Professor Elena Voronezhskaya (Moscow, Russia)

described her model for 5-HT-induced exogastrulation in

Lymnaea stagnalis in which early embryos were incubated

in 1 mM 5-hydroxytryptophan (5-HTP) to raise 5-HT

levels. Using this model she has investigated the intracel-

lular mechanisms involved in the delayed action of 5-HT

on the development of the embryos. Raised levels of both

intra- and extracellular 5-HT during early cleavage are

necessary for the induction of exogastrulation. Inhibition of

5-HT synthesis from 5-HTP prevented exogastrulation.

Evidence suggests the involvement of both adenylate

cyclase and protein kinase A and activation of a 5-HT2-like

receptor for exogastrulation. Following incubation in

5-HTP, there is also long-term activation of MAPKinase.

Professor Voronezhskaya also presented a poster with Dr.

Evgeny Ivashkin and Dr. Maria Khabarova showing that

5-HT occurs in L. stagnalis embryos at the early cleavage

and blastula stages, while its re-uptake by specific cells

only occurs at the late blastula stage. In a second poster

Professor Voronezhskaya and Dr. Khabarova presented

evidence that several 5-HT receptors are involved in the

regulation of locomotion and heart contraction during

Heliosoma development. Dr. Fofanova and Professor

Voronezhskaya also presented a poster on the roles of

5-HT, catecholamines and FMRFamide in the development

of a small marine annelid, Dinophius gyrociliatus, using

histo- and immunocytochemistry and laser scanning con-

focal microscopy. 5-HT neurons were identified in the

ventral nerve cords, FMRFamide-like immunoreactivity

around the cerebral ganglia, and evidence for catechola-

mines in the periphery. Dr. Oleg Tolstenkov (Moscow,

Russia) compared the localization of 5-HT and FMRFa-

mide in the nervous systems of 26 species of Digenea

trematodes using immunocytochemistry and confocal

scanning laser microscopy. He observed a successive

increase in complexity of the nervous system during the

life cycle of the different species of the Digenea (Tol-

stenkov et al. 2010). In redia and sporocysts the nervous

system is simple and extends in only two dimensions and

shows minor differences between species. During the

development from cercaria to metacercaria and adult, the

number of transverse commisures increases significantly,

however the number of marker 5-HT neurons remains

almost the same. It was concluded that the evolution of the


123

nervous system in Digenea has developed independently in

the major groups though with similar trends. Dr. Tolsten-

kov also presented a poster on the immunolocalization of

FMRFamide in the nervous system of two turbellarians,

Girardia tigrina and Polycelis tenuis. Dr. Vyacheslav

Dyachuk (Vladivostok, Russia) presented data on myo-

genesis and neurogenesis during development in two

bivalves, viz., Mytilus trossulus and Crassostrea gigas.

Both species have trochophore, veliger and pediveliger

stages with apical neurons and pioneer neurons being first

to differentiate in the trochophore (Voronezhskaya and

Ivashkin 2010). Neurogenesis begins in the apical organ

with the appearance of 5-HT- and FMRFamide-containing

neurons, but they are not co-localized. Dr. Dyachuk

described the appearance of the major ganglia and the

innervation of the developing muscles in the two species by

5-HT- and FMRFamide-containing axons. He concluded

that while the pioneer neurons are probably not homolo-

gous in these two species, the final pattern of muscle and

neuronal structures is similar.

Dr. Zoltan Serfoz}o (Tihany, Hungary) reported that NO

is required for regeneration of H. pomatia tentacular gan-

glion (Serfozo and Elekes 2010). The tentacular ganglion

contains six large and a lot of small neurons and is

responsible for sensory processing. Following ablation, this

ganglion takes around 15 weeks to regenerate and provides

an example of peripheral neurogenesis. NOsynthase (NOS)

is present in the intact ganglion and appears from week 10

during regeneration, and at the 13th week of regeneration,

both NOS and cGMP reached peak levels. High PKG

levels were found during weeks 1–5 and 11–15 of regen-

eration. Injection of L-NAME at week 13 of regeneration

delayed regeneration by about a month. During tentacular

injury there is no evidence for the inactivation of the

inflammatory NO signal generated by the inducible iso-

form of NOS (iNOS) in vertebrates. These results strongly

suggest that NO, cGMP and PKG are involved in tentacular

regeneration. Dr. Serfoz}o is currently investigating the

cells involved in regeneration. Dr. Ryota Matsuo (Kagawa,

Japan) presented results on DNA endoreplication, that is,

DNA synthesis in the absence of cell division, in giant

neurons during growth in the slug, L. valentianus (Ya-

magishi et al. 2011). Two groups of slugs were studied: one

group was well-fed, while the other group was starved for

44 days. Brain size, neuron size, DNA synthesis and gene

expression all increased during growth. Dr. Matsuo mea-

sured DNA synthesis in cells that synthesize the cardio-

excitatory neuropeptide, achatin-I, and found that the

expression level of achatin-I mRNA per neuron was higher

in well-fed compared to starved L. valentianus though the

number of achatin-I-positive cells did not change. The

number of achatin-I transcripts per neuron was around five

times greater in well-fed compared with starved snails.

In the final talk of the Saturday afternoon session Dr.

Annelise Garcon-Bocquet (Lille, France) introduced her

work on microglial recruitment during central nervous

system repair in the medicinal leech, Hirudo medicinalis

(Croq et al. 2010). Microglial cells, which are normally

dispersed through the central nervous system, migrate to

lesion sites, and this phenomenon is essential for repair

mechanisms. Following a control study using conditioned

medium, medium in which eight crushed nerve cords of

leech were incubated, HmIL-16 and HmC1q were identi-

fied. These molecules are homologous to mammalian

interleukin-16 (IL-16) and human C1q/TNF family. Che-

motaxis assays showed that both HmIL-16 and HmC1q can

recruit microglial cells. The recruitment is inhibited when

microglial cells are pre-incubated with either antiHmIL-16

antibodies or anti-HmC1q antibodies. HmIL-16 presents

many different forms including one active form. HmC1q

recruits the leech microglial cells via gC1qR (CqBP) rec-

ognition. For the first time, throughout vertebrate and

invertebrate models, this interaction (C1q–gC1qR) is

implicated in microglial recruitment. Interestingly, HmIL-

16, active form, can stimulate human CD4? T cell

migration, showing that HmIL-16, active form, is func-

tionally analogous to human IL-16. Incubation with anti-

human gC1qR antibodies reduced recruitment of microglia

by HmC1q (Tahtouh et al. 2012).

The final lecture of the symposium was given by Dr.

Laszlo Hernadi (Tihany, Hungary) who described the

structure of three flexor muscles and their innervation.

These muscles are responsible for space positioning of the

olfactory tentacles of H. pomatia. These muscles oppose

the haemolymph hydrostatic pressure and allow lateral

bending of the tentacle. Cerebral ganglion neurons that

innervate these muscles were identified using anterograde

neurobiotin tracing, and 5-HT immunocytochemistry was

used to identify 5-HT neurons innervating these flexor

muscles. This talk was complemented by a poster investi-

gating the transmitters involved in contracting and relaxing

these muscles (Dr. Nora Krajcs, Tihany, Hungary). Ace-

tylcholine and dopamine contracted the muscles, while low

concentrations of 5-HT also contracted the muscles but

high concentrations relaxed the muscles. Evidence was

presented for both D1A and D2 receptors, while 5-HT and

tyrosine hydroxylase immunoreactivities were found in the

nerve axons innervating the muscles.

In addition to the posters linked to talks that have

already been mentioned, there were a number of other

posters which will now be briefly reviewed. Dr. Suguru

Kokayashi (Kagawa, Japan) recorded slow oscillations,

0.5–1.0 Hz, from the procerebrum of L. valentianus and

applied GABA. GABA increased the frequency of the

oscillations, while metabotropic GABA antagonists

decreased their frequency. GABA immunoreactivity was


123

located in the cell body layer and neuropile of the proce-

rebrum. Fluorescence immunohistochemistry was used to

visualize the distribution of KV1.2, KV1.4, KV1.42 and

KV4.3 potassium channels in the central nervous system of

H. pomatia (Dr. Izabella Battonyai, Tihany, Hungary).

Using immunostaining for NMDA receptors and vesicular

glutamate transporters, evidence was obtained for NMDA

receptors on glutamate-containing neurons in L. stagnalis

(Hernadi and Kemenes). This suggests the presence of a

glutamate positive feedback system in the central nervous

system of L. stagnalis. A further poster by Dr. Laszlo

Hernadi (with Zoltan Serfoz}o and Agnes Vehovszky) used

immunostaining to provide evidence for an interaction

between dopamine- and 5-HT-containing neurons in

L. stagnalis central nervous system. Evidence suggested

that this system might be used during feeding arousal.

A poster presented by Dr. Ilya Chistopolsky (Moscow,

Russia) described the influence of distal gut activity on

buccal rhythm and its possible effect on fictive feeding in

L. stagnalis (Dyakonova and Dyakonova 2010). Stimula-

tion of the oesophagus was able to initiate or modify buccal

mass activity, and this effect was lost when the dorsal

buccal nerves were sectioned, demonstrating that signals

from the gut could modify feeding activity. In another

poster on the buccal mass feeding system in L. stagnalis,

the role of 5-HT was investigated during development

(Dr. Gabor Balog, Tihany, Hungary). Both 5-HT levels and

a 5-HT uptake system increased through development.

Evidence suggested the presence of three 5-HT receptors in

the regulation of muscle activity, viz., 5-HT1-like, 5-HT6-

like and 5-HT7-like receptors, suggesting a complex role

for 5-HT in feeding regulation in L. stagnalis. There were

two posters on the field cricket, G. bimaculatus. In the first

poster genes were identified which were involved in the

synthesis of 5-HT, octopamine and dopamine, together

with their expression patterns (Dr. Tohru Watanabe, Sap-

poro, Japan). In the second poster the role of visual cues in

the intensity of fighting in male white-eye mutants was

investigated (Dr. Midori Sakura, Kobe, Japan). The results

indicated that unlike normal crickets, white-eye mutants

were unable to use visual cues during fighting. Dr. Hitoshi

Aonuma (Sapporo, Japan) also assisted in the presentation

of both posters on G. bimaculatus and presented a poster on

the role of octopamine in aggression in the ant, Formica

japonica. Octopamine levels in the brains of forager and

guard ants which show increased aggression were greater

than in nest builders which show less aggression. Social

isolation altered the levels of aggression and octopamine. It

was concluded that central nervous system octopamine

regulates task-dependent aggression in F. japonica. Rela-

ted work by Dr. Aonuma’s group on the role of dopamine

and octopamine on starvation stress and social interaction

in F. japonica has recently been published (Wada-Katsu-

mata et al. 2011). Dr. Ryuichi Okado presented a poster

describing a model for bee foraging-related behaviour

(waggle dance) using video analysis to obtain biological

Fig. 1 Balaton Limnological Research Institute of the Hungarian Academy of Sciences. The Institute was opened on 5 September 1927 and has

provided the venue for ISIN meetings for the last 44 years, since 1967


123

parameters for the model. It was found that accurate

transfer of information in the waggle dance was important

for successful visits to a food source. Dr. Tohru Moriyama

(Nagano, Japan) presented evidence that when pill bugs,

Armadillidium vulgare, form into a ball (conglobate),

various internal factors, possibly including the central

nervous system, influence the duration of this behaviour. A

poster on the effect of varying salinity on larval growth and

levels of FMRFamide and 5-HT in the presence of anti-

biotics in the mussel, Mytilus trossilus, was presented by

Dr. Evgeny Ivashkin (Moscow, Russia). The presence of an

antibiotic enhanced larval survival while changing salinity

altered growth, survival and expression of FMRFamide and

5-HT. The bursting activity of enteric nervous system

neurons was found to be responsible for rhythmic action of

peristalsis in the gut of Aplysia and Lymnaea (Dr. Makoto

Kurokawa, Tokyo, Japan). The pacemaker region was

different in the two animals, being in the gizzard of Aplysia

and in the crop of Lymnaea. Both of these regions are distal

to the peristalsis region in both animals. In the final poster

Dr. Agnes Vehovszky (Tihany, Hungary) reported on her

work testing the actions of extracts of cyanobacteria on the

heart of H. pomatia and on central nicotinic acetylcholine

receptors from H. pomatia and L. stagnalis. Some extracts

blocked both excitatory and inhibitory acetylcholine

response on neurons, while other extracts potentiated the

responses to acetylcholine. In contrast to the inhibitory

effect of acetylcholine on the heart, the extracts excited the

heart, suggesting a non-cholinergic site of action. None of

the known cholinergic cyanotoxins were found when these

extracts were analysed.

The final session of the symposium was followed by a

general discussion on where to publish invertebrate neu-

robiology. Editors from several journals were present, and

each gave a short presentation about their journal, viz.,

Invertebrate Neuroscience, Lindy Holden-Dye; J. of

Comparative Neurology, Ian Meinertzhagen; J. of Com-

parative Physiology A, John Hildebrand and Hans-Joachim

Pfl}uger; Arthropod Structure and Development, Nicholas

Strausfeld; and J. of Neurogenetics, Chun-Fang Wu. There

was a general discussion as to whether it was better to

publish in a general neurobiology journal to reach a wide

audience or a specialized invertebrate journal where the

audience was relatively restricted. In general it was felt that

authors should decide which audience they wanted to reach

and publish accordingly. Thus, if their paper was of med-

ical interest, then they should try and publish in an

appropriate journal even if it might not be read by many

invertebrate neurobiologists. There was also a discussion

on the value of impact factors, and in general, they were

considered to be a negative influence on the publication of

scientific papers, and where possible the most appropriate

journal should be used irrespective of its impact factor.

This was followed by a general meeting of the ISIN at

which a new President, Professor Etsuro Ito, was elected

together with a new Executive Secretary, Professor Karoly

Elekes. The new executive committee consisted of the

President, the Executive Secretary, the past President, Dick

Nassel, and seven others, viz., Pavel Balaban, Lindy Hol-

den-Dye, Gy}orgy Kemenes, Fred Libersat, Ian Meinertha-

gen, Leonid Moroz and Hans-Joachim Pfl}uger.

Every 4 years for the past 44 years, the ISIN meeting has

brought together junior and experienced researchers who

work on an excitingly diverse range of invertebrate species.

This year again the quality and breadth of the talks and

posters served to emphasize the vibrant role that inverte-

brate neuroscience continues to play in the shared goal of all

neuroscience, to rise to the challenge of providing a fun-

damental understanding of the link between neural function

and behaviour. The role that invertebrates still have to play

in this long-term game plan should not be underestimated.

With this in mind, the intention is to reconvene the next

meeting of ISIN in 4 years in the summer of 2015. The

beautiful venue at the Limnological Institute at Lake Bal-

aton (Fig. 1) will once again provide the setting for stimu-

lating conversations on invertebrate nervous systems.

Acknowledgments The authors gratefully acknowledge the critical

reading and comments on the manuscript by the presenting authors at

this meeting.

Conflict of interest None.

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