Reorganization of the nervous system during metamorphosis of a hydrozoan planula

Abstract. Laser scanning confocal microscopy is used to reveal the changes that occur in the RFamide-positive nerve net as a free-swimming, solid hydrozoan planula larva is transformed into a sessile, hollow, young polyp. Seven stages of development in Pennaria tiarella are described: planula competent to metamorphose, attaching planula, disc, pawn, crown, developing polyp, and developed primary polyp. The RFamide-positive nervous system undergoes dramatic reorganization during metamorphosis: (1) larval neurons degenerate; (2) new neurons differentiate and reform a nerve net; and (3) the overall distribution pattern of the nervous system changes. This study confirms earlier observations on RFamide-positive neurons of Hydractinia which also show the loss of these cells after the onset of metamorphosis.     

Nerve cells in hydra: monoclonal antibodies identify two lineages with distinct mechanisms for their incorporation into head tissue

The relationship between populations of nerve cells defined by two monoclonal antibodies was investigated in Hydra oligactis. A population of sensory nerve cells localized in the head (hypostome and tentacles) is identified by the binding of antibody JD1. A second antibody, RC9, binds ganglion cells throughout the animal. When the nerve cell precursors, the interstitial cells, are depleted by treatment with hydroxyurea or nitrogen mustard, the JD1+ nerve cells are lost as epithelial tissue is sloughed at the extremities. In contrast, RC9+ nerve cells remain present in all regions of the animal following treatment with either drug. When such hydra are decapitated to initiate head regeneration, the new head tissue formed is again free of JD1+ sensory cells but does contain RC9+ ganglion cells. Our studies indicate that (1) nerve cells are passively displaced with the epithelial tissue in hydra, (2) JD1+ sensory cells do not arise by the conversion of body column nerve cells that are displaced into the head, whereas RC9+ head nerve cells can originate in the body column, (3) formation of new JD1+ sensory cells requires interstitial cell differentiation. We conclude from these results that the two populations defined by these antibodies are incorporated into the h ad via different developmental pathways and, therefore, constitute distinct nerve cell lineages.     

Initial sex differences in neuron growth and survival within an avian song nucleus develop in the absence of afferent input

Only male zebra finches (Poephila guttata) sing, and nuclei implicated in song behavior exhibit marked sex differences in neuron number. In the robust nucleus of the anterior neostriatum (RA), these sex differences develop because more neurons die in young females than in males. However, it is not known whether the sexually dimorphic survival of RA neurons is a primary event in sexual differentiation or a secondary response to sex differences in the number of cells interacting trophically with RA neurons. In particular, since sexual differentiation of the RA parallels the development of dimorphisms in the numbers of neurons providing afferent input from the lateral magnocellular nucleus of the anterior neostriatum (lMAN) and the high vocal center (HVC), it has been hypothesized that sex differences in the size of these afferent populations trigger differential RA neuron survival and growth. To test this hypothesis, we lesioned either the lMAN or both the lMAN and HVC unilaterally in 12-day-old male and female zebra finches. Subsequently, RA cell death and RA neuron number and size were measured. Unilateral lMAN lesions increased cell death and decreased neuron number and size within the ipsilateral RA of both sexes. However, even in the lMAN-lesioned hemisphere, these effects were less pronounced in males than in females, so that by day 25 the volume, number, and size of neurons were sexually dimorphic in both the contralateral and ipsilateral RA. Similarly, the absence of both lMAN and HVC afferents did not prevent the emergence of sex differences in the number and size of RA neurons by 25 day posthatching. We conclude that these sex differences within the RA are not a secondary response to dimorphisms in the numbers of lMAN or HVC neurons providing afferent input. © 1995 John Wiley & Sons, Inc.     

Neuronal evolution: analysis of regulatory genes in a first-evolved nervous system, the hydra nervous system

Cnidarians represent the first animal phylum with an organized nervous system and a complex active behavior. The hydra nervous system is formed of sensory-motoneurons, ganglia neurons and mechanoreceptor cells named nematocytes, which all differentiate from a common stem cell. The neurons are organized as a nerve net and a subset of neurons participate in a more complex structure, the nerve ring that was identified in most cnidarian species at the base of the tentacles. In order to better understand the genetic control of this neuronal network, we analysed the expression of evolutionarily conserved regulatory genes in the hydra nervous system. The Prd-class homeogene prdl-b and the nuclear orphan receptor hyCOUP-TF are expressed at strong levels in proliferating nematoblasts, a lineage where they were found repressed during patterning and morphogenesis, and at low levels in distinct subsets of neurons. Interestingly, Prd-class homeobox and COUP-TF genes are also expressed during neurogenesis in bilaterians, suggesting that mechanoreceptor and neuronal cells derive from a common ancestral cell. Moreover, the Prd-class homeobox gene prdl-a, the Antp-class homeobox gene msh, and the thrombospondin-related gene TSP1, which are expressed in distinct subset of neurons in the adult polyp, are also expressed during early budding and/or head regeneration. These data strengthen the fact that two distinct regulations, one for neurogenesis and another for patterning, already apply to these regulatory genes, a feature also identified in bilaterian related genes.     

Kisspeptins Modulate the Biology of Multiple Populations of Gonadotropin-Releasing Hormone Neurons during Embryogenesis and Adulthood in Zebrafish (Danio rerio)

Kisspeptin1 (product of the Kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in Kiss1 and the kisspeptin receptor (GPR54/Kiss1r) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with Kiss1 or Kiss2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only Kiss1, but not Kiss2, stimulated proliferation of terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that Kiss1, but not Kiss2, increased synaptic contacts on the cell body and along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of Kiss1 and Kiss2 on spontaneous action potential firing frequency and membrane potential. Kiss1 increased spike frequency and depolarized membrane potential, whereas Kiss2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, Kiss1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while Kiss2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide that inhibits electrical activity of hypophysiotropic GnRH3 neurons in the adult.     

Postembryonic neuronogenesis in the procerebrum of the terrestrial snail,Helix lucorum L.

Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70–80%) of the snail procerebrum are produced during the first 1–2 months of posthatching development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 271–276, 1998     

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.     

Egg incubation temperature differently affects female and male hatching dynamics and larval fitness in a leafhopper

Temperature effects on ectotherms are widely studied particularly in insects. However, the life-history effects of temperature experienced during a window of embryonic development, that is egg stage, have rarely been considered. We simulated fluctuating temperatures and examined how this affects the operational sex ratio (OSR) of hatching as well as nymph and adult fitness in a leafhopper, Scaphoideus titanus. Specifically, after a warm or cold incubation we compared males and females hatching dynamics with their consequences on the sex ratio in the course of time, body size, weight, and developmental rate of the two populations, all reared on the same posthatching temperature. Males and females eggs respond differently, with females more sensitive to variation in incubation temperature. The different responses of both sexes have consequences on the sex ratio dynamic of hatchings with a weaker protandry after warm incubation. Temperatures experienced by eggs have more complex consequences on posthatching development. Later nymphal instars that hatched from eggs exposed to warm temperature were larger and bigger but developmental rate of the two populations was not affected. Our study demonstrates how incubation temperature could affect operational sex ratio and posthatching development in an insect and how this may be critical for population growth.     

Orexin/hypocretin neurons: chemical phenotype and possible interactions with melanin-concentrating hormone neurons

We showed earlier that a specific neuron population of the rat lateral hypothalamus, differing from the codistributed melanin-concentrating hormone (MCH) neurons, express both dynorphin (DYN) and secretogranin II (SgII) genes. We demonstrated later that this population corresponds in fact to the newly identified orexin/hypocretin (OX/Hcrt) neurons. In the present study, by revisiting the chemical phenotype of these neurons, we confirm that all of them contain DYN B- and SgII-immunoreactive materials. The roles played by these peptide/protein in OX/Hcrt neurons are still unclear.Double immunocytochemical stainings highlight putative somasomatic, axosomatic and axodendritic contacts between OX/Hcrt and MCH neurons. Adding OX/Hcrt to the culture medium of hypothalamic slices from 8-day-old rats results either in a significant increase of MCH mRNA after 24 h survival or a strong fall after 10 days culture. These results taken together suggest that OX/Hcrt can directly and/or indirectly affect MCH expression, and that both OX/Hcrt and MCH neuron populations interact to respond in a coordinated manner to central and peripheral signals.     

Calbindin immunoreactivity in the enteric nervous system of larval and adult zebrafish (Danio rerio)

Calbindin is a calcium-binding protein, commonly found in certain subpopulations of the enteric nervous system in mammals. Recently, calbindin-immunoreactive enteric neurons have also been demonstrated in shorthorn sculpin (Myoxocephalus scorpius). In the present study, calbindin immunoreactivity has been investigated in the gut of adult and larval zebrafish (Danio rerio) and differences and similarities between the two species are discussed. Calbindin immunoreactivity is present in 40%?C50% of all enteric neurons in adult zebrafish. It first appears at 3?days post-fertilisation (dpf) and is present in all regions of the gut by 13 dpf. Calbindin-immunoreactive nerve cell bodies do not differ in size from calbindin-negative cells. Zebrafish calbindin-immunoreactive neurons are serotonin-negative, with at least some being choline acetyltransferase (ChAT)-positive, in contrast to the sculpin in which cells are generally smaller than the average enteric neuron and are serotonin-positive and ChAT-negative. These findings further emphasise the importance of comparative studies for understanding the diversity of chemical coding in the enteric nervous system of fish and other vertebrates. Improved knowledge of the role of the enteric nervous system is also essential for future studies of gut activity with regard to zebrafish being used as a model organism.     

Continuous conversion of neuron phenotype in hydra.

All neurons in adult hydra are constantly changing their location. This poses interesting problems for the maintenance of the organization of the hydra nervous system. The solutions provide a different perspective on the development of nervous systems.     

The hydra effect in predator–prey models

The seemingly paradoxical increase of a species population size in response to an increase in its mortality rate has been observed in several continuous-time and discrete-time models. This phenomenon has been termed the “hydra effect”. In light of the fact that there is almost no empirical evidence yet for hydra effects in natural and laboratory populations, we address the question whether the examples that have been put forward are exceptions, or whether hydra effects are in fact a common feature of a wide range of models. We first propose a rigorous definition of the hydra effect in population models. Our results show that hydra effects typically occur in the well-known Gause-type models whenever the system dynamics are cyclic. We discuss the apparent discrepancy between the lack of hydra effects in natural populations and their occurrence in this standard class of predator–prey models.     

Sexual differentiation of brain and behavior in the zebra finch: Critical periods for effects of early estrogen treatment

In order to determine the critical period(s) during which estrogen alters sexually dimorphic behavior and neuroanatomy in zebra finches (Poephila guttata), nestlings were injected daily 20 μg estradiol benzoate (EB) during posthatching week 1, week 2, week 3, or weeks 1, 2, and 3. At 7 months of age, birds were implanted with testosterone propionate and tested with female partners for singing, dancing, and copulatory mounting. Brains were subsequently processed for morphometry, and the volumes of the song system nuclei HVC, area X, and RA and the soma sizes and densities of neurons in RA were determined. Males given EB during week 1 failed to mount. Females given EB during week 1 were fully masculinized with respect to dancing and RA neuron soma size and density, and were partially masculinized with respect to song nuclei volumes and singing. Treatment beginning after week 1 was ineffective or less effective for all measures. Only for RA neuron measures was treatment for all three weeks more effective than week 1 treatment. Thus the first post-hatching week is the most influential period of those tested for effects of exogenous estrogen on sexual differentiation in this species, and is a period during which both masculinization of females and demasculinization of males is possible. 1994 John Wiley & Sons, Inc.     

Morphological and biochemical studies on the development of cholinergic properties in cultured sympathetic neurons. I. Correlative changes in choline acetyltransferase and synaptic vesicle cytochemistry

Under certain culture conditions, neonatal rat superior cervical ganglion neurons display not only a number of expected adrenergic characteristics but, paradoxically, also certain cholinergic functions such as the development of hexamethonium-sensitive synaptic contacts and accumulation of choline acetyltransferase (ChAc). The purpose of this study was to determine whether the entire population of cultured neurons was aquiring cholinergic capabilities, or whether this phenomenon was restricted to a subpopulation. After 1--6 and 8 wk in culture, neurons were fixed in KMnO4 after incubation in norepinephrine and prepared for electron microscopy analysis of synaptic vesicle content to determine whether vesicles were dense cored or clear. ChAc, acetylcholinesterase (AChE), and DOPA-decarboxylase (DDC) activities were assayed in sister cultures. In the period from 1 to 8 wk in culture, the average ChAc activity per neuron increased 1,100-fold, and the DDC and AChE activities increased 20- and 30-fold, respectively. After 1 wk in culture, 48 of 50 synaptic boutons contained predominantly dense-cored vesicles, but by 8 wk the synaptic vesicle population was predominantly of the clear type. At intermediate times, the vesicle population in many boutons was mixed. The morphology of the synaptic contacts on neuronal surfaces was that characteristic of autonomic systems, with no definite clustering of the vesicles adjacent to the area of contact. Increased vesicle size correlated with increasing age in culture and the presence of a dense core. Considering these data along with available physiological studies, we conclude that these cultures contain one population of neurons that is initially adrenergic. Over time, under conditions of this culture system, this population develops cholinergic mechanisms. That a neuron may, at a given time, express both cholinergic and adrenergic mechanisms is suggested by the approximately equal numbers of clear and dense-cored vesicles in the boutons found at the intermediate times.     

光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶活力的影响

单细胞真核绿藻在中国水螅(Hydra sinensis)内胚层皮肌细胞中共生是有较高科研价值的特殊生物学现象。水螅宿主细胞为共生藻提供CO2、氮源及矿物质,而共生藻通过光合作用可能为宿主提供碳水化合物等有机物营养,因此水螅与共生藻间代谢流是以共生藻光合作用为中心,但基于代谢流二者间的互作机制目前尚未阐明。水螅通过营养积累进行出芽生殖,从母体脱落的芽体数量间接反映水螅营养积累的相对量。而光暴露时长能影响共生绿藻光合作用,如果共生藻的确能向水螅细胞转移光合作用产物,那光暴露时长应该能间接影响水螅的营养积累、从而进一步影响水螅无性出芽生殖。为证实该假说,本研究应用种群累积培养法,观察了光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶(SOD和CAT)活力的影响。结果显示,光周期对中国水螅种群增长具有明显的影响。培养15 d后,所有实验组水螅的种群密度均为正增长,其中8L∶16D(在一个24h周期内光暴露8 h、黑暗16 h)实验组种群密度最大、而0L∶24D(持续黑暗)实验组种群密度最小。另外,随着光暴露时长的增加,中国水螅SOD及CAT活力整体均呈下降趋势。结果表明,从光周期对中国水螅无性出芽生殖及两种抗氧化酶活力的影响来看,中国水螅对光周期的生理学响应较为敏感,这个现象可能源于共生绿藻能通过向宿主细胞转移光合作用产物的方式为水螅提供营养补充。     

Expression of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and PAC1 in the Periodontal Ligament After Tooth Luxation

Pituitary adenylate cyclase-activating peptide (PACAP) is widely distributed throughout the nervous system. PACAP not only acts as a neurotransmitter but also elicits a broad spectrum of biological action via the PACAP-specific receptor, PAC1. However, no studies have investigated PACAP and PAC1 in the periodontal ligament (PDL), so we aimed to perform this investigation in rats after tooth luxation. In the PDL of an intact first molar, there are few osteoclasts and osteoblasts. However, at days 3 and 5 after luxation, large PAC1-positive cells, thought to be osteoclasts because of their expression of the osteoclast marker, tartrate-resistant acid phosphatase, were detected in appreciable numbers. Osteoblast numbers increased dramatically on day 7 after luxation, and PAC1-positive mononuclear small cells were increased at day 14, many of which expressed the osteoblast marker, alkaline phosphatase. PACAP-positive nerve fibers were rarely detected in the PDL of intact first molars, but were increasingly evident at this site on days 5 and 7 after luxation. Double-immunofluorescence analysis demonstrated the relationship between PACAP-positive nerve fibers and PAC1-positive osteoclasts/-blasts in the PDL. At 5 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoclasts. At 7 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoblasts. These results suggest that PACAP may have effects on osteoclasts and osteoblasts in the PDL after tooth luxation and thus regulate bone remodeling after these types of injury.     

Effects of death of a single neuron on the functional organization of the neuronal ensemble in the leech

The reaction of leech nervous system after elimination of individual mechanosensory neuron by intracellular Pronase injection were studied. In the motor neurons connected with killed cells at 14-90th days after the operation there were the changes of the resting membrane potential and amplitude of postsynaptic potentials developed by the stimulation of mechanosensory neuron. It is suggested that the leech nervous system serves as the dynamic formation depending on changes of neuronal ensemble structure.     

Sexual experience and testosterone during adolescence alter adult neuronal morphology and behavior

Steroid hormones released immediately before and after birth provoke sexual differentiation of neural circuits. Further, steroid hormones secreted during adolescence also exert long lasting effects on the nervous system. Hormones secreted during development may act through two distinct pathways: (1) hormones can directly affect neuron and synapse elimination and (2) endocrine changes in the nervous system may occur secondary to changes in social behaviors. Therefore, a critical period for organization of the nervous system by steroid hormones during adolescence may also be a sensitive period for the effects of social experience. The overall goal of this experiment was to determine whether the opportunity to mate with a sexually receptive female during this adolescent critical period would have enduring effects on behavior and neuronal morphology into adulthood. A second question was to determine the extent to which testosterone mediated the effects of these social interactions on adult outcomes. Compared to sexually inexperienced hamsters and those that experienced sex for the first time in adulthood, hamsters that experienced adolescent sexual experience displayed increased anxiety- and depressive-like behavioral responses. Adolescent sexual experiences decreased the complexity and length of dendrites on prefrontal cortical neurons and increased the expression of the pro-inflammatory cytokine interleukin 1β (IL-1β) in the PFC. In a second experiment, administration of testosterone during the adolescent period largely recapitulated the effects of adolescent sexual experience. These data support the overall hypothesis that a sensitive period extends into adolescence and that salient social stimuli during this time can significantly and persistently alter adult phenotype.     

Colocalized FMRFamide-related neuropeptides in the nervous system of the colorado potato beetle,Leptinotarsa decemlineata (say) (Coleoptera : Chrysomelidae) demonstrated immunohistochemically with mono- and polyclonal antibodies

The distribution of—still chemically undefined—peptide epitopes in subclasses of peptidergic neurons in the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera : Chrysomelidae) was studied in preparation for peptide isolation by immunological methods. Two monoclonal antibodies, MAC-13 and MAC-3, were selected for their capacity to reveal selected neuron populations by immunohistochemical methods. They partially mimic the immunostaining pattern of a polyclonal anti-FMRFamide antiserum. Immunoreactive neurons, containing FMRFamide-related antigens, were demonstrated with high specificity in the CNS as well as in the visceral and peripheral nervous system. Their total number is large, compared with other subclasses of peptidergic neurons, approximately 230 (peripheral neurons not included). Axons of these neurons run either to the corpora cardiaca for release of peptides into the bloodstream, or to distant target organs, such as the digestive tract, reproductive organs, and somatic muscles. Large, isolated peripheral neurons occur in the vicinity of the ventral nerve cord and release products through terminals located in or on the sheath of nerves emanating from the thoracic ganglia. Different antigens may be colocalized in neuron subclass-specific combinations and then always in the same secretory granules. These observations make clear that groups of peptidergic neurons, considered earlier as a homogeneous population, are much more diversified than hitherto suspected. We may anticipate the occurrence, in subsequent isolation procedures, of at least 3 classes of FMRFamide-related peptides. Compartments in the central nervous system containing high concentrations of single or mixed antigens have been identified.     

Ontogenesis of the snail,Helix aspersa: Embryogenesis timetable and ontogenesis of GABA-like immunoreactive neurons in the central nervous system

Late stages of embryogenesis in the terrestrial snail Helix aspersa L. were studied and a developmental timetable was produced. The distribution of gamma-aminobutyric acid-like immunoreactive (GABA-ir) elements in the CNS of the snail was studied from embryos to adulthood in wholemounts. In adults, approximately 226 GABA-ir neurons were located in the buccal, cerebral and pedal ganglia. The population of GABA-ir cells included four pairs of buccal neurons, three neuronal clusters in the pedal ganglia, two clusters and six single neurons in the cerebral ganglia. GABA-ir fibers were observed in all ganglia and in some nerves. The first detected pair of GABA-ir cells in the embryos appeared in the buccal ganglia at about 63–64% of embryonic development. Five pairs of GABA-ir cell bodies were observed in the cerebral ganglia at about 64–65% of development. During the following 30% of development three more pairs of GABA-ir neurons were detected in the buccal ganglia and over fifteen cells were detected in each cerebral ganglion. At the stage of 70% of development, the first pair of GABA-ir neurons was found in the pedal ganglia. In the suboesophageal ganglion complex, GABA-ir fibers were first detected at about 90% of embryonic development. In the posthatching period, the quantity of GABA-ir neurons reached the adult status in four days in the cerebral ganglia, and in three weeks in the pedal ganglia. In juveniles, transient expression of GABA was found in the pedal ganglia (fourth cluster).