GABAergic Transmission and Chloride Equilibrium Potential Are Not Modulated by Pyruvate in the Developing Optic Tectum of Xenopus laevis Tadpoles

In the developing mammalian brain, gamma-aminobutyric acid (GABA) is thought to play an excitatory rather than an inhibitory role due to high levels of intracellular Cl− in immature neurons. This idea, however, has been questioned by recent studies which suggest that glucose-based artificial cerebrospinal fluid (ACSF) may be inadequate for experiments on immature and developing brains. These studies suggest that immature neurons may require alternative energy sources, such as lactate or pyruvate. Lack of these other energy sources is thought to result in artificially high intracellular Cl− concentrations, and therefore a more depolarized GABA receptor (GABAR) reversal potential. Since glucose metabolism can vary widely among different species, it is important to test the effects of these alternative energy sources on different experimental preparations. We tested whether pyruvate affects GABAergic transmission in isolated brains of developing wild type Xenopus tadpoles in vitro by recording the responsiveness of tectal neurons to optic nerve stimulation, and by measuring currents evoked by local GABA application in a gramicidin perforated patch configuration. We found that, in contrast with previously reported results, the reversal potential for GABAR-mediated currents does not change significantly between developmental stages 45 and 49. Partial substitution of glucose by pyruvate had only minor effects on both the GABA reversal potential, and the responsiveness of tectal neurons at stages 45 and 49. Total depletion of energy sources from the ACSF did not affect neural responsiveness. We also report a strong spatial gradient in GABA reversal potential, with immature cells adjacent to the lateral and caudal proliferative zones having more positive reversal potentials. We conclude that in this experimental preparation standard glucose-based ACSF is an appropriate extracellular media for in vitro experiments

In Vivo Spike-Timing-Dependent Plasticity in the Optic Tectum of Xenopus Laevis

Spike-timing-dependent plasticity (STDP) is found in vivo in a variety of systems and species, but the first demonstrations of in vivo STDP were carried out in the optic tectum of Xenopus laevis embryos. Since then, the optic tectum has served as an excellent experimental model for studying STDP in sensory systems, allowing researchers to probe the developmental consequences of this form of synaptic plasticity during early development. In this review, we will describe what is known about the role of STDP in shaping feed-forward and recurrent circuits in the optic tectum with a focus on the functional implications for vision. We will discuss both the similarities and differences between the optic tectum and mammalian sensory systems that are relevant to STDP. Finally, we will highlight the unique properties of the embryonic tectum that make it an important system for researchers who are interested in how STDP contributes to activity-dependent development of sensory computations

Neurodevelopmental effects of chronic exposure to elevated levels of pro-inflammatory cytokines in a developing visual system

<p>Abstract</p> <p>Background</p> <p>Imbalances in the regulation of pro-inflammatory cytokines have been increasingly correlated with a number of severe and prevalent neurodevelopmental disorders, including autism spectrum disorder, schizophrenia and Down syndrome. Although several studies have shown that cytokines have potent effects on neural function, their role in neural development is still poorly understood. In this study, we investigated the link between abnormal cytokine levels and neural development using the <it>Xenopus laevis </it>tadpole visual system, a model frequently used to examine the anatomical and functional development of neural circuits.</p> <p>Results</p> <p>Using a test for a visually guided behavior that requires normal visual system development, we examined the long-term effects of prolonged developmental exposure to three pro-inflammatory cytokines with known neural functions: interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. We found that all cytokines affected the development of normal visually guided behavior. Neuroanatomical imaging of the visual projection showed that none of the cytokines caused any gross abnormalities in the anatomical organization of this projection, suggesting that they may be acting at the level of neuronal microcircuits. We further tested the effects of TNF-α on the electrophysiological properties of the retinotectal circuit and found that long-term developmental exposure to TNF-α resulted in enhanced spontaneous excitatory synaptic transmission in tectal neurons, increased AMPA/NMDA ratios of retinotectal synapses, and a decrease in the number of immature synapses containing only NMDA receptors, consistent with premature maturation and stabilization of these synapses. Local interconnectivity within the tectum also appeared to remain widespread, as shown by increased recurrent polysynaptic activity, and was similar to what is seen in more immature, less refined tectal circuits. TNF-α treatment also enhanced the overall growth of tectal cell dendrites. Finally, we found that TNF-α-reared tadpoles had increased susceptibility to pentylenetetrazol-induced seizures.</p> <p>Conclusions</p> <p>Taken together our data are consistent with a model in which TNF-α causes premature stabilization of developing synapses within the tectum, therefore preventing normal refinement and synapse elimination that occurs during development, leading to increased local connectivity and epilepsy. This experimental model also provides an integrative approach to understanding the effects of cytokines on the development of neural circuits and may provide novel insights into the etiology underlying some neurodevelopmental disorders.</p

Multivariate analysis of electrophysiological diversity of Xenopus visual neurons during development and plasticity

Abstract Biophysical properties of neurons become increasingly diverse over development, but mechanisms underlying and constraining this diversity are not fully understood. Here we investigate electrophysiological characteristics of Xenopus tadpole midbrain neurons across development and during homeostatic plasticity induced by patterned visual stimulation. We show that in development tectal neuron properties not only change on average, but also become increasingly diverse. After sensory stimulation, both electrophysiological diversity and functional differentiation of cells are reduced. At the same time, the amount of cross-correlations between cell properties increase after patterned stimulation as a result of homeostatic plasticity. We show that tectal neurons with similar spiking profiles often have strikingly different electrophysiological properties, and demonstrate that changes in intrinsic excitability during development and in response to sensory stimulation are mediated by different underlying mechanisms. Overall, this analysis and the accompanying dataset provide a unique framework for further studies of network maturation in Xenopus tadpoles

Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>It is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.</p> <p><b>Methods</b></p> <p>We used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).</p> <p><b>Results</b></p> <p>METH induced microglial cell death in a concentration-dependent manner (EC₅₀ = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.</p> <p><b>Conclusions</b></p> <p>These findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.</p

Multisensory Integration in Mesencephalic Trigeminal Neurons in Xenopus Tadpoles

Mesencephalic trigeminal (M-V) neurons are primary somatosensory neurons with somata located within the CNS, instead of in peripheral sensory ganglia. In amphibians, these unipolar cells are found within the optic tectum and have a single axon that runs along the mandibular branch of the trigeminal nerve. The axon has collaterals in the brain stem and is believed to make synaptic contact with neurons in the trigeminal motor nucleus, forming part of a sensorimotor loop. The number of M-V neurons is known to increase until metamorphosis and then decrease, suggesting that at least some M-V neurons may play a transient role during tadpole development. It is not known whether their location in the optic tectum allows them to process both visual and somatosensory information. Here we compare the anatomical and electrophysiological properties of M-V neurons in the Xenopus tadpole to principal tectal neurons. We find that, unlike principal tectal cells, M-V neurons can sustain repetitive spiking when depolarized and express a significant H-type current. M-V neurons could also be driven synaptically by visual input both in vitro and in vivo, but visual responses were smaller and longer-lasting than those seen in principal tectal neurons. We also found that the axon of M-V neurons appears to directly innervate a tentacle found in the corner of the mouth of premetamorphic tadpoles. Electrical stimulation of this transient sensory organ results in antidromic spiking in M-V neurons in the tectum. Thus M-V neurons may play an integrative multisensory role during tadpole development

Spatial gradient of GABAR reversal potential in the OT.

<p><b>A.</b> All cells that had their position within OT recorded (n = 105), for both stages and ACSF formulations, shown projected onto open right OT outline. Rostral direction is up, caudal is down, medial is left, lateral is right. Circles stand for cells recorded in control ACSF at stage s45; squares – control ACSF s49; down triangles – pyruvate-containing ACSF s45; up triangles – pyruvate-containing ACSF s49. Color of the marker encodes E_GABA measured in each of the cells, with blue corresponding to more negative, and red – to more positive values (see color-bar on the right). Subset of cells further referred to as the “Rostral group” is encircled with a dashed circle. <b>B.</b> E_GABA observed in OT cells as a function of distance from the center of the “Rostral group” shown on the left. Blue for control ACSF; green for pyruvate-containing ACSF; marker shapes follow same conventions as in the left panel. The threshold distance limiting the “Rostral group” is shown as a dashed line.</p

Measurement of GABAR reversal potential.

<p><b>A.</b> Example of currents evoked by local GABA application at different command potentials (stage 45, control ACSF, central third of OT). <b>B.</b> IV-curve of GABA-evoked currents from panel A, with amplitudes shown against the command potential, polynomial fit of these amplitudes, and an estimation for GABAR reversal potential E_GABA. <b>C.</b> E_GABA values observed in all cells recorded (n = 122), shown separately for stage 45 (left) and stage 49 (right) animals; control ACSF (“c”, blue) and pyruvate-containing ACSF (“p”, green). Horizontal bars show average E_GABA values across all cells in a data group.</p