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, 523 Pt 3 (Pt 3), 639-51

Opposite Changes in Synaptic Activity of Organotypic Rat Spinal Cord Cultures After Chronic Block of AMPA/kainate or Glycine and GABAA Receptors

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Opposite Changes in Synaptic Activity of Organotypic Rat Spinal Cord Cultures After Chronic Block of AMPA/kainate or Glycine and GABAA Receptors

M Galante et al. J Physiol.

Abstract

1. The well-developed cytoarchitecture of rat organotypic spinal cord culture makes it a suitable model to explore how persistent suppression of certain synaptic inputs might be compensated by increased synaptic efficacy (homeostatic plasticity). 2. Spontaneous or electrically evoked synaptic transmission of patch-clamped ventral horn interneurons was studied in control solution after blocking, for the second week in culture, AMPA/kainate receptors with CNQX or glycine and GABAA receptors with strychnine and bicuculline, or indiscriminately removing inputs with tetrodotoxin (TTX). 3. In untreated cells, spontaneous postsynaptic currents (PSCs) had fast (tau < 5 ms) or slow (tau > 10 ms) decay. A similar separation was observed when recording miniature currents (mPSCs). Slow decay PSCs were suppressed by strychnine plus bicuculline while fast decay events were eliminated by CNQX. 4. After chronic CNQX treatment the frequency of spontaneous, fast PSCs (of larger amplitude) or mPSCs was almost doubled with respect to control. These events were blocked by acutely applied CNQX, which unmasked slow PSCs. 5. After chronic TTX treatment neither the frequency nor the amplitude of spontaneous events was changed. 6. After chronic strychnine and bicuculline treatment the frequency and amplitude of all PSCs was decreased in most cells. mPSCs were also decreased in frequency. Spontaneous or electrically evoked currents acquired a larger component mediated by NMDA receptor activity. 7. The developing spinal network thus operated distinct homeostatic processes which led to strong enhancement in glutamatergic transmission after CNQX block or to broad downregulation of synaptic activity following chronic exposure to strychnine and bicuculline.

Figures

Figure 1
Figure 1. Spontaneous and evoked activity from a ventral interneuron in an untreated culture
A, spontaneous synaptic activity from a ventral interneuron is composed by inward currents of variable amplitude (left); single PSCs comprise events with fast or slow decay times (middle, superimposed tracings). Rise time and decay time are measured from the fast PSC average (top right: rise time = 0.8 ms, τ = 2 ms) and the slow PSC average (bottom right: rise time = 1.1 ms, τ = 22.6 ms). B, application of CNQX (left, same cell as in A) completely blocks all fast events and leaves only slow ones (middle, superimposed traces). Kinetics are measured from the average of slow decay events (right, rise time = 1.5 ms, τ = 22 ms). C, synaptic currents evoked in the same interneuron as in A and B by DRG stimulation are shown in standard Krebs (left) and in the presence of CPP (right); each panel represents the average of 7 consecutive evoked PSCs. Note that the application of CPP reduces the area subtended by the evoked PSC by 38 %. Dotted lines indicate the baseline. D, scatter plot of rise time versus half-width (same cell as in A); regression analysis reveals no linear relationship between these two parameters (r2 = 0.006, Pearson correlation coefficient = 0.07).
Figure 2
Figure 2. PSC amplitudes from control cultures or cultures chronically treated with CNQX
A, spontaneous synaptic currents in standard Krebs solution are shown in both control culture (top tracings, left) and chronically CNQX-treated sister culture (bottom tracings, left). Single PSCs are shown to the right, note that both fast and slow decay events are present in the control interneuron (top right) while in that from the chronically CNQX-treated culture only fast PSCs are detected (bottom right). B, cumulative amplitude plot for PSCs recorded in control and chronically CNQX-treated sister cultures; the entire distribution of PSC amplitudes includes larger values for CNQX-treated interneurons (see arrow). C, scatter plot of rise time against peak amplitude in control culture (same cell as in A). Note the lack of linear relationship between these parameters (r2 = 0.00003, Pearson correlation coefficient = 0.006).
Figure 3
Figure 3. Spontaneous and evoked activity from ventral interneurons in cultures chronically treated with CNQX
A, recordings from a ventral interneuron in CNQX-treated culture (left) show very intense spontaneous activity; PSCs comprise only fast decay events (middle, superimposed traces; right, average response), with τ of 2.6 ms and a rise time of 0.8 ms, measured as in Fig. 1 B,. block of AMPA/kainate receptors by CNQX (left, same cell as in A) fully abolished fast PSCs and unmasked slow τ events; middle, single events are superimposed, and kinetics are measured from the average PSC (right, τ = 29 ms, rise time = 1.6 ms). C, DRG stimulations evoke polysynaptic currents in the patched ventral interneuron. Evoked currents in standard Krebs solution (left) appear reduced (by 41 %) in area after the application of CPP (right); each trace (left and right) represents the average of 5 consecutive evoked PSCs (different cell from A and B).
Figure 4
Figure 4. Spontaneous and evoked activity from ventral interneurons in cultures chronically treated with TTX
A, left, example of spontaneous activity recorded from an interneuron in a chronically TTX-treated culture; middle, superimposed events: only fast decay events are detected; right, from their average, rise time (0.9 ms) and τ (3.2 ms) values are measured. B, addition of CNQX blocked all fast events leaving no residual activity (same cell as in A C,). evoked PSCs elicited by DRG stimulations are shown in standard Krebs solution (left) and in the presence of CPP (right); dotted line represents the baseline current. CPP produced a 44 % reduction in the of the evoked PSC. Each panel represents the average of 5 consecutive evoked PSCs (different cell from A and B). D, cumulative distribution plot for PSC data obtained after chronic TTX treatment or in control shows no statistically significant difference.
Figure 5
Figure 5. Spontaneous and evoked activity from ventral interneurons in cultures chronically treated with strychnine plus bicuculline
A, spontaneous synaptic activity from a ventral interneuron appears as bursts of inward currents (left) followed by quiescent periods during which both fast and slow PSCs (middle, superimposed traces) are detected; kinetics are measured from the average of PSCs showing both fast decay (top right: rise time = 0.9 ms, τ = 2.8 ms) and slow decay (bottom right: rise time = 1.5 ms, τ = 13.3 ms). B, application of CNQX (same cell as in A) abolished spontaneous bursting (left) and completely removed fast decay events; only slow decay events survived (middle, superimposed traces), with kinetics (right) similar to those normally found in standard Krebs solution. C, evoked synaptic currents induced by stimulation of DRG (left) appear strongly reduced in area (by 60 %) during the application of CPP (right). Each trace (left and right) is the average of 8 subsequent responses. D, cumulative probability plot of PSC amplitudes from a ventral interneuron (different cells) in control (see arrow) and in a sister culture chronically treated with strychnine plus bicuculline: the entire amplitude distribution for PSCs deviates towards smaller values for the treated interneuron.
Figure 6
Figure 6. Spontaneous mPSCs in control and chronically treated cultures
A, recordings from a ventral interneuron in the presence of TTX from a control culture (left): events are superimposed at faster speed on the right. B, miniature currents in an interneuron from a chronically CNQX-treated culture (left): note the higher frequency of mPSCs (superimposed traces, right) compared to control culture. C, spontaneous mPSCs from a chronically TTX-treated culture; note the higher frequency of mPSCs (superimposed traces, right) compared to control. D, miniature currents recorded from an interneuron in a culture chronically treated with strychnine plus bicuculline (S+B, left); note very low frequency of the miniature events (superimposed traces, right), compared to the other culture conditions (A-C). E, distribution of mPSC amplitude (left) and mPSC inter-event distribution plot (right) from the cells shown in A–D. In the righthand panel the plot for cells chronically treated with CNQX, TTX and strychnine plus bicuculline (S.B) are significantly different from control. Note almost complete overlap of CNQX data with TTX data.

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