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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 Xenopus laevis tadpolevisual system, a model frequently used to examine the anatomical and functional development of neural circuits. 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)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha. 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-alpha on the electrophysiological properties of the retinotectal circuit and found that long-term developmental exposure to TNF-alpha 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-alpha treatment also enhanced the overall growth of tectal cell dendrites. Finally, we found that TNF-alpha-reared tadpoles had increased susceptibility to pentylenetetrazol-induced seizures. Taken together our data are consistent with a model in which TNF-alpha 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.
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20067608
???displayArticle.pmcLink???PMC2819242 ???displayArticle.link???Neural Dev ???displayArticle.grants???[+]
Figure 1. Visual avoidance is impaired by chronic cytokine treatment. (A) Control tadpoles and tadpoles reared in IL-1β, IL-6 and TNF-α were tested for normal visual avoidance behavior. This behavior requires tectal processing. Tadpoles were placed in a testing chamber with no stimulus for 30 s. The dot pattern was then presented for 30 seconds, such that the dots were drifting on one half of the tank and stationary on the other. After the stimulus, the number of tadpoles on the stationary side was counted to calculate percent avoidance (see Methods for details). All cytokine-reared groups showed impaired avoidance behavior. (B) Control tadpoles and tadpoles reared in IL-1β, IL-6 and TNF-α were tested for an optomotor response (OMR). This behavior does not require tectal processing. Tadpoles were placed in a testing chamber with no stimulus for 30 s. The drifting grating was presented for 30 s. After the stimulus, the number of tadpoles that swam in the direction of the grating was counted to calculate the percent correct (see Methods for details). All groups showed a significant OMR, although the OMR in the IL-6 group was slightly reduced. For P-values, see Results.
Figure 2. Chronic cytokine treatment does not result in abnormalities in the gross projection pattern of central visual pathways. (A) Diagram of the Xenopus tadpolebrain visual projections. Visual inputs enter the tectum via the optic nerve (ON) and primarily project contralaterally to the optic tectum (Tec). Other projections are to the contralateral pre-tectal neuropil (PT), the thalamus (Th) and the basal optic neuropil (BO). Rostral and caudal directions are indicated as is the location of the hindbrain (HB). (B) Two separate examples of the visual projection are shown from Isl-2b:GFP transgenic tadpoles reared under different conditions. The top images were taken with a conventional fluorescent microscope and show the different areas. In IL-6 the thalamic projection is shown as an inset since it was outside of the field of view of the original image. The bottom images are maximum projections of confocal stacks focusing on the tectum. None of the cytokine treatments appeared to cause any gross morphological abnormalities. At least five different brains were imaged for each of the rearing conditions.
Figure 3. Acute effects of TNF-α application on voltage-gated currents and spontaneous synaptic transmission in tectal neurons. (A) I-V curves for Na+ and K+ currents in control tectal cells and cells acutely (approximately 1 h) treated with TNF-α. (B) Maximum peak amplitudes (pA) of Na+ and K+ currents from both groups show no significant differences. (C) Spontaneous EPSCs recorded from control neurons and neurons acutely (approximately 1 h) treated with TNF-α. (D) TNF-α-treated tadpoles exhibited a higher frequency of sEPSCs than controls but showed no difference in amplitude. Asterisk indicates p < 0.05, for actual P-values see Results.
Figure 4. Long-term developmental exposure to TNF-α does not alter intrinsic membrane currents of tectal neurons. (A) Family of current traces recorded in response to a series of depolarizing steps from -60 to +30 mV. The fast inward current is a voltage-gated Na+ current while the outward current is a voltage-gated K+ current. (B) I-V curves for Na+ and K+ currents in control tectal cells and cells from TNF-α reared tadpoles. (C) Maximum peak amplitudes of Na+ and K+ currents from both groups show no significant differences. For P-values see Results. N.S., not significant.
Figure 5. Long-term developmental exposure to TNF-α enhances AMPAR-mediated synaptic transmission. (A) Spontaneous EPSCs recorded from a tectal neuron in a control and a TNF-α reared tadpole. (B) TNF-α treated tadpoles exhibited a higher frequency of sEPSCs than controls but showed no difference in amplitude. (C) Sample traces showing glutamatergic EPSCs evoked by direct optic nerve stimulation and recorded at -60 and +60 mV. At -60 mV only the AMPAR-mediated response is observed, while at +60 mV both outward AMPAR- and NMDAR-mediated responses are seen. (D) The AMPA/NMDA ratio (see Methods) was significantly increased in tectal cells from tadpoles reared in TNF-α, consistent with an increase in AMPAR-mediated synaptic transmission. Asterisk indicates p < 0.05. For P-values see Results.
Figure 6. Long-term developmental exposure to TNF-α results in a decrease of NMDAR-only synapses. (A, B) Left: superimposed responses evoked by minimal stimulation recorded at -60 and +60 mV from control and TNF-α-treated animals. Right: histogram of all minimal stimulation AMPA and NMDA responses from control and TNF-α-treated animals. (C) Failure rates of NMDA currents were significantly lower in control animals, whereas they were not different in drug-treated animals, consistent with a decreased number of silent synapses. (D) Estimation of the percent of silent synapses in the stimulated projection was also significantly different. Asterisk indicates p < 0.05. For P-values see Results.
Figure 7. Long-term developmental exposure to TNF-α results in altered tectal circuitry. (A) Sample retinotectal synaptic currents in response to minimal and maximal electrical stimulation of the optic nerve. (B) The number of retinotectal (RT) inputs innervating a single tectal cell was estimated by dividing the maximal response by the minimal response. TNF-α-treated tadpoles show an increased number of retinotectal inputs per cell, consistent with lack of developmental refinement. (C) Maximal stimulation of the optic chiasm results in a prolonged barrage of polysynaptic activity driven by local intratectal circuits. The first peak of the response is the monosynaptic retinotectal input. Two sample traces are shown for both control and TNF-α reared tectal neurons. Notice that the duration and distribution of the recurrent activity is longer and slower in the TNF-α reared tadpoles. (D) Quantification of the time course of the polysynaptic activity. Synaptic charge was measured over the first three 50-ms bins following the onset of the response. Data were plotted as percent of the total synaptic charge during the entire time period. Grouped data were then fitted to a line using linear regression. Neurons from TNF-α reared tadpoles show significantly slower decay of the polysynaptic response, indicating increased intratectal interconnectivity. Asterisk indicates p < 0.05. For P-values see Results.
Figure 8. Long-term developmental exposure to TNF-α results in enhanced growth of tectal cell dendrites. (A) Representative images of tectal cell dendrites imaged before drug treatment and after chronic treatment with TNF-α. The TNF-α cells are compared with sham-treated controls. Asterisk indicates location of cell body. (B) TNF-α results in significantly enhanced growth rate of tectal cell dendrites. Asterisk indicates p < 0.05. For P-values see Results.
Figure 9. Long-term developmental exposure to TNF-α results in increased seizure susceptibility. (A) Tadpoles were exposed to 10 mM of the convulsant agent PTZ in their rearing media and the latency to seizure onset was calculated (see Methods). TNF-α reared tadpoles consistently showed a faster seizure onset time than their control clutchmates. (B) Cumulative probability distribution of seizure onset times for individual tadpoles in both groups. Asterisk indicates p < 0.05. For P-values see Results.
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