The HMGA gene family in chordates: evolutionary perspectives from amphioxus.

High mobility group A proteins of vertebrates, HMGA1 and 2, are chromatin architectural factors involved in development, cell differentiation, and neoplastic transformation. Here, we characterize an amphioxus HMGA gene ortholog and analyze its expression. As a basal chordate, amphioxus is well placed to provide insights into the evolution of the HMGA gene family, particularly in the transition from invertebrates to vertebrates. Our phylogenetic analysis supports the basal position of amphioxus, echinoderm, and hemichordate HMGA sequences to those of vertebrate HMGA1 and HMGA2. Consistent with this, the genomic landscape around amphioxus HMGA shares features with both. Whole mount in situ hybridization shows that amphioxus HMGA mRNA is detectable from neurula stage onwards in both nervous and non-nervous tissues. This correlates with protein expression monitored immunocytochemically using antibodies against human HMGA2 protein, revealing especially high levels of expression in cells of the lamellar body, the amphioxus homolog of the pineal, suggesting that the gene may have, among its many functions, an evolutionarily conserved role in photoreceptor differentiation.

Review: Morphofunctional and biochemical markers of stress in sea urchin life stages exposed to engineered nanoparticles.

We describe the use of different life stages of the Mediterranean sea urchin Paracentrotus lividus for the assessment of the possible risk posed by nanoparticles (NPs) in the coastal water. A first screening for the presence of NPs in sea water may be obtained by checking their presence inside tissues of organisms taken from the wild. The ability of NPs to pass from gut to the coelomic fluid is demonstrated by accumulation in sea urchin coelomocytes; the toxicity on sperms can be measured by embryotoxicity markers after sperm exposure, whereas the transfer through the food chain can be observed by developmental anomalies in larvae fed with microalgae exposed to NPs. The most used spermiotoxicity and embryotoxicity tests are described, as well as the biochemical and histochemical analyses of cholinesterase (ChE) activities, which are used to verify toxicity parameters such as inflammation, neurotoxicity, and interference in cell-to-cell communication. Morphological markers of toxicity, in particular skeletal anomalies, are described and classified. In addition, NPs may impair viability of the immune cells of adult specimens. Molecular similarity between echinoderm and human immune cells is shown and discussed. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1552-1562, 2016.

Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath.

Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and ß subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.

Pharmacological characterization of NMDA-like receptors in the single-celled organism Paramecium primaurelia.

Paramecium primaurelia is a unicellular eukaryote that moves in freshwater by ciliary beating and responds to environmental stimuli by altering motile behaviour. The movements of the cilia are controlled by the electrical changes of the cell membrane: when the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, the ciliary beating reverses its direction, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. Here, we evaluated the effects due to the activation or blockade of N-methyl-d-aspartic acid (NMDA) receptors on swimming behaviour in Paramecium. Paramecia normally swim forward, drawing almost linear tracks. We observed that the simultaneous administration of NMDA and glycine induced a partial ciliary reversal (PaCR) leading to a continuous spiral-like swim. Furthermore, the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, was longer in NMDA+glycine-treated cells. NMDA action required the presence of Ca(2+), as the normal forward swimming was restored when the ion was omitted from the extracellular milieu. The PaCR and the enhancement of CCR duration significantly decreased when the antagonists of the glutamate site D-AP5 or CGS19755, the NMDA channel blocker MK-801 or the glycine site antagonist DCKA was added. The action of NMDA+glycine was also abolished by Zn(2+) or ifenprodil, the GluN2A and the GluN2B NMDA-containing subunit blockers, respectively. Searches of the Paramecium genome database currently available indicate that the NMDA-like receptor with ligand-binding characteristics of an NMDA receptor-like complex, purified from rat brain synaptic membranes and found in some metazoan genomes, is also present in Paramecium. These results provide evidence that functional NMDA receptors similar to those typical of mammalian neuronal cells are present in the single-celled organism Paramecium and thus suggest that the glutamatergic NMDA system is a phylogenetically old behaviour-controlling mechanism.

A neurochemical map of the developing amphioxus nervous system.

BACKGROUND: Amphioxus, representing the most basal group of living chordates, is the best available proxy for the last invertebrate ancestor of the chordates. Although the central nervous system (CNS) of amphioxus comprises only about 20,000 neurons (as compared to billions in vertebrates), the developmental genetics and neuroanatomy of amphioxus are strikingly vertebrate-like. In the present study, we mapped the distribution of amphioxus CNS cells producing distinctive neurochemicals. To this end, we cloned genes encoding biosynthetic enzymes and/or transporters of the most common neurotransmitters and assayed their developmental expression in the embryo and early larva. RESULTS: By single and double in situ hybridization experiments, we identified glutamatergic, GABAergic/glycinergic, serotonergic and cholinergic neurons in developing amphioxus. In addition to characterizing the distribution of excitatory and inhibitory neurons in the developing amphioxus CNS, we observed that cholinergic and GABAergic/glycinergic neurons are segmentally arranged in the hindbrain, whereas serotonergic, glutamatergic and dopaminergic neurons are restricted to specific regions of the cerebral vesicle and the hindbrain. We were further able to identify discrete groups of GABAergic and glutamatergic interneurons and cholinergic motoneurons at the level of the primary motor center (PMC), the major integrative center of sensory and motor stimuli of the amphioxus nerve cord. CONCLUSIONS: In this study, we assessed neuronal differentiation in the developing amphioxus nervous system and compiled the first neurochemical map of the amphioxus CNS. This map is a first step towards a full characterization of the neurotransmitter signature of previously described nerve cell types in the amphioxus CNS, such as motoneurons and interneurons.

Evidence for ectopic aerobic ATP production on C6 glioma cell plasma membrane.

Extracellular ATP plays a pivotal role as a signaling molecule in physiological and pathological conditions in the CNS. In several glioma cell lines, ATP is a positive factor for one or more characteristics important for the abnormal growth and survival of these cells. This work presents immunofluorescence and biochemical analyses suggesting that an aerobic metabolism, besides mitochondria, is located also on the plasma membrane of C6 glioma cells. An ATP synthesis coupled to oxygen consumption was measured in plasma membrane isolated from C6 cells, sensitive to common inhibitors of respiratory chain complexes, suggesting the involvement of a putative surface ATP synthase complex. Immunofluorescence imaging showed that Cytochrome c oxydase colocalized with WGA, a typical plasma membrane protein, on the plasma membrane of glioma cells. Cytochrome c oxydase staining pattern appeared punctuate, suggesting the intriguing possibility that the redox chains may be expressed in discrete sites on C6 glioma cell membrane. Data suggest that the whole respiratory chain is localized on C6 glioma cell surface. Moreover, when resveratrol, an ATP synthase inhibitor, was added to culture medium, a cytostatic effect was observed, suggesting a correlation among the ectopic ATP synthesis and the tumor growth. So, a potential direction for the design of new targets for future therapies may arise.

The GABAergic-like system in the cyprid of Balanus amphitrite (=Amphibalanus amphitrite) (Cirripedia, Crustacea).

In the present study, biochemical and immunochemical methods were used to investigate the presence and distribution of GABA, glutamate decarboxylase (GAD), GABA(B)R1 and GABA(A) gamma2 subunit receptors and the vesicular GABA transporter (vGAT) in the cyprid of Balanus amphitrite (=Amphibalanus amphitrite). GAD(65/67) immunoreactive neuron cell bodies and nerve fibers were detected in the central nervous system. Paired GAD(65/67) immunoreactive nerves running from the posterior ganglion to the body and limb muscles were detected. Thin GABA-immunoreactive nerve terminals were present on striated muscular fibers and in the antennules. Furthermore, GABA, GAD(65/67), GABA(B)R1 and GABA(A)gamma2 subunit receptors and vGAT were observed in the lateral compound eyes, and GABA(A)gamma2 subunit receptor immunoreactivity was seen in the naupliar eye. These results suggest a neurotransmitter/neuromodulatory role for GABA in thoracic muscle contraction and regulatory functions in compound eyes and antennules of B. amphitrite cyprids.

Neutrophil Extracellular Traps Profiles in Patients with Incident Systemic Lupus Erythematosus and Lupus Nephritis.

OBJECTIVE: Neutrophil extracellular traps (NET) expose modified antigens for autoantibodies in vasculitis. Little is known about levels and removal pathways of NET in systemic lupus erythematosus (SLE), especially in lupus nephritis (LN). We determined circulating levels and defined NET removal in large subsets of patients with incident SLE (iSLE), some of whom had new-onset nephritis. METHODS: Serum levels of NET (ELISA), DNase1/DNase1L3 (ELISA), and DNase activity (functional assay) were determined in 216 patients with iSLE [103 had incident LN (iLN)], in 50 patients with other primary glomerulonephritis, and in healthy controls. Ex vivo NET production by neutrophils purified from a random selection of patients was quantified as elastase/DNA release and by immunofluorescence techniques. RESULTS: Serum NET levels were very high in iSLE/iLN compared to all groups of controls and correlated with anti-dsDNA, C3-C4, and proteinuria; iLN had the highest levels. DNase activity was decreased in iLN compared to SLE (20% had one-half DNase activity) despite similar serum levels of DNase1/DNase1L3. In these cases, pretreatment of serum with protein A restored DNase efficiency; 1 patient was homozygous for a c.289_290delAC variant of DNASE1L3. Ex vivo NET production by neutrophils purified from LN, SLE, and normal controls was similar in all cases. CONCLUSION: Patients with iLN have increased circulating NET and reduced DNase activity, the latter being explained by the presence of inhibitory substances in circulation and/or by rare DNase1L3 mutations. Accumulation of NET derives from a multifactorial mechanism, and is associated and may contribute to disease severity in SLE, in particular to renal lesions. (Clinical trial registration: The Zeus study was registered at ClinicalTrials.gov, study number NCT02403115).

Biophysical effects of the natural product euplotin C on the Paramecium membrane.

The effect of euplotin C--a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus--on the voltage-dependent Ca(2+) channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca(2+) concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca(2+) influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca(2+) channel blockers, we demonstrate that euplotin C modulates Ca(2+) channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca(2+) channel functions through the reduction of cell membrane fluidity.

Homocysteine and A2A-D2 Receptor-Receptor Interaction at Striatal Astrocyte Processes.

The interaction between adenosine A2A and dopamine D2 receptors in striatal neurons is a well-established phenomenon and has opened up new perspectives on the molecular mechanisms involved in Parkinson's disease. However, it has barely been investigated in astrocytes. Here, we show by immunofluorescence that both A2A and D2 receptors are expressed in adult rat striatal astrocytes in situ, and investigate on presence, function, and interactions of the receptors in the astrocyte processes-acutely prepared from the adult rat striatum-and on the effects of homocysteine on the A2A-D2 receptor-receptor interaction. We found that A2A and D2 receptors were co-expressed on vesicular glutamate transporter-1-positive astrocyte processes, and confirmed that A2A-D2 receptor-receptor interaction controlled glutamate release-assessed by measuring the [3H]D-aspartate release-from the processes. The complexity of A2A-D2 receptor-receptor interaction is suggested by the effect of intracellular homocysteine, which reduced D2-mediated inhibition of glutamate release (homocysteine allosteric action on D2), without interfering with the A2A-mediated antagonism of the D2 effect (maintained A2A-D2 interaction). Our findings indicate the crucial integrative role of A2A-D2 molecular circuits at the plasma membrane of striatal astrocyte processes. The fact that homocysteine reduced D2-mediated inhibition of glutamate release could provide new insights into striatal astrocyte-neuron intercellular communications. As striatal astrocytes are recognized to be involved in Parkinson's pathophysiology, these findings may shed light on the pathogenic mechanisms of the disease and contribute to the development of new drugs for its treatment.

Evidence for ciliary pigment localization in colored ciliates and implications for their photosensory transduction chain: a confocal microscopy study.

In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal microscopy investigation. Optical sectioning confocal microscopy has been used for studying the spatial distribution of the pigment in the cell body, surprisingly showing that, besides its expected presence in the cortical region immediately below the cell membrane, it is located in the cilia too. In order to ascertain possible differences in the pigment fluorescence properties along the cell body, we have measured emission spectra from different parts of it (anterior, posterior, and cilia). Our results clearly indicate that in all cases the spectra are the same, within experimental errors. Finally, we have evaluated the pigment relative fluorescence efficiency of these ciliates. In an ordered scale from lower to greater efficiency, we have S. coeruleus, B. japonicum blue, B. japonicum red, and F. salina. The possible implications of our findings for the process of photosensory transduction are discussed.

The GABAergic-like system in the marine demosponge Chondrilla nucula.

Gamma-amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. The presence of GABA in the marine demosponge Chondrilla nucula was verified using immunofluorescence detection and high-pressure liquid chromatography. A strong GABA-like immunoreactivity (IR) was found associated with choanocytes, exopinacocytes, endopinacocytes lining inhalant, and exhalant canals, as well as in archaeocytes scattered in the mesohyl. The capacity to synthesize GABA from glutamate and to transport it into the vesicles was confirmed by the presence in C. nucula of glutamate decarboxylase (GAD) and vesicular GABA transporters (vGATs), respectively. GAD-like and vGAT-like IR show the same distribution as GABA-like IR. Supporting the similarity between sponge and mammalian proteins, bands with an apparent molecular weight of about 65-67 kDa and 57 kDa were detected using antibodies raised against mammalian GAD and vGAT, respectively. A functional metabotropic GABA(B)-like receptor is also present in C. nucula. Indeed, both GABA(B) R1 and R2 isoforms were detected by immunoblot and immunofluorescence. Also in this case, IR was found in choanocytes, exopinacocytes, and endopinacocytes. The content of GABA in C. nucula amounts to 1225.75 +/- 79 pmol/mg proteins and GABA is released into the medium when sponge cells are depolarized. In conclusion, this study is the first indication of the existence of the GABA biosynthetic enzyme GAD and of the GABA transporter vGAT in sponges, as well as the first demonstration that the neurotransmitter GABA is released extracellularly.

Effect of the bioactive metabolite euplotin C on phagocytosis and fluid-phase endocytosis in the single-celled eukaryote Paramecium.

The effect of euplotin C -- a lipophilic bioactive metabolite produced by the ciliate Euplotes crassus -- on the kinetics of both phagocytosis of latex particles and fluid-phase uptake of dextran, was studied in the single-cell ciliate Paramecium primaurelia. The inhibition of food vacuole formation was concentration- and time-dependent (p<0.001), even if euplotin C did not completely block the phagocytosis. Following a 15 min treatment with a euplotin C (0.5 microg/ml), the latex particle uptake was inhibited up to 25%. Furthermore, the pretreatment of cells with taxol strongly counteracted euplotin C effect. The amount of extracellularly provided dextran, which is internalized exclusively by fluid-phase uptake, was quantified in cells whose phagocytic activity was blocked by trifluoperazine. The amount of the internalized dextran was about 50% of that in controls after 15 min incubation in the presence of euplotin C. Fluorescence confocal images showed that no endosomes were formed on the surface of these cells. The effect of euplotin C on the food vacuole formation and fluid-phase endocytosis is apparently mediated by a modification of microtubule network.

Distribution of FMRFamide-like immunoreactivity in the alimentary tract and hindgut ganglia of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

In this study, the presence and distribution of FMRFamide-like immunoreactivity in the alimentary tract of barnacle Balanus amphitrite were investigated. A net of nerve fibers strongly immunoreactive to FMRFamide-like molecules was localized in the posterior midgut and hindgut. Positive varicose nerve terminals were also localized close to the circular muscle cells and, in the hindgut, close to the radial muscular fibers. Besides this nerve fibers network, one pair of contralateral ganglia was localized in the hindgut, each of them constituted by two strongly FMRFamide-labeled neurons and one nonlabeled neuron. Their immunoreactive axons directed toward the hindgut and posterior midgut suggest an involvement of FMRFamide-like substances in adult B. amphitrite gut motility. The hindgut associated ganglia of barnacles seem to correspond to the terminal abdominal ganglia of the other crustaceans. Since they are the only residual gut ganglia in the barnacle's reduced nervous system, we can hypothesize that gut motility needs a nervous system regulation partially independent of the central nervous system.

Distribution of choline acetyltransferase immunoreactivity in the alimentary tract of the barnacle Balanus amphitrite (Cirripedia, Crustacea).

To date only peptidergic innervation has been described in the alimentary tract of barnacles. In the present work the presence and distribution of choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, was investigated by immunohistochemistry in the alimentary tract of the adult barnacle Balanus amphitrite. Numerous ChAT-immunoreactive (IR) cells and a net of ChAT-IR cytoplasmic processes were localized inside the epithelium of the posterior midgut, close to the basement membrane; no IR nerve endings were detected in the midgut longitudinal and circular muscle bundles. Epithelial neurons or endocrine cells in the gut epithelium have been described in some invertebrate species belonging to different taxa and their peptidergic features are reported in the literature. Our results point out the presence of neuroepithelial cells also in the gut epithelium of barnacles; moreover, for the first time, a cholinergic feature is suggested for this cell type. These data seem to indicate the involvement of ACh in the gut functions of barnacle and suggest that the barnacle alimentary tract is more complex than previously thought and requires further study.

Multi-photon excitation microscopy.

Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

Evaluation of the Acquisition of the Aerobic Metabolic Capacity by Myelin, during its Development.

Our previous reports indicate that the electron transfer chain and FoF1-ATP synthase are functionally expressed in myelin sheath, performing an extra-mitochondrial oxidative phosphorylation (OXPHOS), which would provide energy to the nerve axon. This supports the idea that myelin plays a trophic role for the axon. Although the four ETC complexes and ATP synthase are considered exquisite mitochondrial proteins, they are found ectopically expressed in several membranous structures. This study was designed to understand when and how the mitochondrial OXPHOS machinery is embedded in myelin, following myelinogenesis in the rat, which starts at birth and continues until the first month of age. Rats were sacrificed at different time points (from day 5 to 90 post birth). Western blot, immunofluorescence microscopy, luminometric, and oximetric analyses show that the isolated myelin starts to show OXPHOS components around the 11th day after birth and increases proportionally to the rat age, becoming similar to those of adult rat around the 30-third day. Interestingly, WB data show the same temporal relationship between myelinogenesis and appearance of proteins involved in mitochondrial fusion and cellular trafficking. It may be speculated that the OXPHOS complexes may be transferred to the endoplasmic reticulum membrane (known to interact with mitochondria) and from there through the Golgi apparatus to the forming myelin membrane.

Support of Nerve Conduction by Respiring Myelin Sheath: Role of Connexons.

Recently, we have demonstrated that myelin conducts an extramitochondrial oxidative phosphorylation, hypothesizing a novel supportive role for myelin in favor of the axon. We have also hypothesized that the ATP produced in myelin could be transferred thought gap junctions. In this work, by biochemical, immunohistochemical, and electrophysiological techniques, the existence of a connection among myelin to the axon was evaluated, to understand how ATP could be transferred from sheath to the axoplasm. Data confirm a functional expression of oxidative phosphorylation in isolated myelin. Moreover, WB and immunohistochemistry on optic nerve slices show that connexins 32 and 43 are present in myelin and colocalize with myelin basic protein. Interestingly, addition of carbenoxolone or oleamide, two gap junction blockers, causes a decrease in oxidative metabolism in purified myelin, but not in mitochondria. Similar effects were observed on conduction speed in hippocampal Schaffer collateral, in the presence of oleamide. Confocal analysis of optic nerve slices showed that lucifer yellow (that only passes through aqueous pores) signal was found in both the sheath layers and the axoplasma. In the presence of oleamide, but not with oleic acid, signal significantly decreased in the sheath and was lost inside the axon. This suggests the existence of a link among myelin and axons. These results, while supporting the idea that ATP aerobically synthesized in myelin sheath could be transferred to the axoplasm through gap junctions, shed new light on the function of the sheath.

GABAB receptor intracellular trafficking after internalization in Paramecium.

The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome-lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively.

A role for GABAA receptors in the modulation of Paramecium swimming behavior.

The presence in Paramecium of gamma-aminobutyric acid A-type receptors (GABA(A)) and the capability of the protozoon to synthesize and release the GABA neurotransmitter into the environment have already been demonstrated. This study investigates the involvement of the GABA(A) complex in the swimming control of the ciliated protozoon. The GABA(A) receptors were pharmacologically activated by the selective agonist muscimol and the effect on Paramecium primaurelia swimming behavior was analyzed. Paramecium normally swims forward, but the activation of GABA(A) receptors induced a peculiar response, characterized by alternate periods of whirling and forward swim. This effect was inhibited by the GABA(A) selective antagonists bicuculline and picrotoxin in a dose-dependent manner. Moreover, the application of benzodiazepines did not enhance the agonist action but only reduced it. Response to muscimol was also suppressed by nimodipine, a selective antagonist of dihydropyridine-sensitive calcium channels. This inhibition suggests that an inflow of calcium ions through L-type channels mediates the muscimol-induced swimming behavior.