Astrocytes play a critical role in transient heterosynaptic depression in the rat hippocampal CA1 region

Abstract

Active synapses can reduce the probability of transmitter release at neighbouring synapses. Depending on whether such heterosynaptic depression is mediated by intersynaptic diffusion of transmitter or by release of gliotransmitters, astrocytes should either hinder or promote the heterosynaptic depression. In the present study we have examined the developmental profile and astrocytic involvement in a transient heterosynaptic depression (tHeSD) in the CA1 region of the rat hippocampal slice preparation. A short stimulus burst (3 impulses at 50 Hz) to one group of synapses elicited a depression of the field EPSP evoked in another group of synapses that amounted to about 25% 0.5 s after the conditioning burst. This tHeSD was associated with an increase in the paired-pulse ratio of about 30%. The tHeSD was not present in slices from rats younger than 10 postnatal days and developed towards the adult magnitude between postnatal days 10 and 20. The tHeSD was totally prevented by the glia-specific toxin fluoroacetate (FAC), by carbenoxolone, a general blocker of connexin-based channels, and by endothelin, an endogenous peptide that has been shown to block astrocytic connexin-based channels. Antagonists to GABA(B) receptors and group II/III metabotropic glutamate receptors (mGluRs) abolished the tHeSD whereas antagonists to NMDA- and adenosine A1 receptors, and to group I mGluRs, did not affect the tHeSD. These results suggest that the tHeSD relies on GABA(B) receptors, group II/III mGluRs and on gliotransmitter release from functionally mature astrocytes.

Figure 1. The transient heterosynaptic depression is associated with increased paired-pulse ratio

A, schematic illustration of the placement of stimulation and recording electrodes in the stratum radiatum of the CA1 region. B, sweeps illustrating the long-interval conditioning (5 s, upper) used as control and the short-interval conditioning (0.5 s, lower) used to elicit the tHeSD. The conditioning input is activated with 3 impulses at 50 Hz as shown expanded in the left inset. The test input is activated with 2 impulses at 20 Hz as shown expanded in the right inset. Ca, summation test for synaptic pathway independence. The sum (C + T, left) of the fEPSP evoked from the conditioning input (C, upper left sweep) and the fEPSP evoked from the test input (T, upper right sweep) is compared to the fEPSP obtained when activating the two inputs simultaneously (C & T). Since the sum of the two fEPSPs (C + T) is not larger than the simultaneously evoked fEPSP (C & T) this result indicates that the two synaptic pathways do not share common synapses. Cb, paired-pulse test for synaptic pathway independence. The fEPSPs evoked from the test input (T) are shown when activated with (T, left) and without (T, right) the activation of the conditioning input (C) 50 ms earlier. The absence of a larger (facilitated) test fEPSP with a preceding conditioning stimulation indicates that the two synaptic pathways do not share common synapses. D, an experiment illustrating the reversible depression of the fEPSP (filled circles) and the associated increase in paired-pulse ratio (open squares). Note that fibre volley remains stable (grey circles). Shaded area indicates short-interval conditioning (0.5 s). Values of fEPSP initial slope and paired-pulse ratio are normalized to the average value during the long-interval conditioning. Average fEPSPs taken from the long-, short- and long-interval conditioning, respectively, are shown on top (n = 18 sweeps for each interval). E, graph summarizing 56 experiments such as that shown in A from 21- to 50-day-old-rats.

Figure 2. tHeSD is absent in young animals

A, an experiment from a 6-day-old rat illustrating the absence of transient heterosynaptic depression. Shaded area indicates short-interval conditioning (0.5 s). Values of fEPSP initial slope and paired-pulse ratio are normalized to the average value during the long-interval conditioning. Average fEPSPs taken from the long-, short- and long-interval conditioning, respectively, are shown on top (n = 18 sweeps from each interval). B, graph summarizing 26 experiments such as that shown in A from 5- to 10-day-old rats. C, developmental profile of the transient heterosynaptic depression. Average change in fEPSP initial slope (filled circles) and paired-pulse ratio (open squares) during the short-interval conditioning are plotted versus postnatal age (n = 5–15 slices per group). Sigmoidal fits (dashed lines) are included for visual guidance.

Figure 3. Astrocytes are necessary for the tHeSD

A, the graph illustrates the results of matched comparisons of tHeSD before and after pharmacological interventions. The filled bars compare the effect on the fEPSP slope and the open bars compare the effect on the paired-pulse ratio. 100% means the same tHeSD as in the control situation and 0% means absence of tHeSD. Typical control (‘5 s’, upper) and test (‘0.5 s’, lower) sweeps are shown above the bars. Dashed line indicates the amplitude of the control fEPSP. ‘Rep. sequence’ experiments showing no change in the tHeSD when the experimental protocol is repeated in the absence of any drug. ‘Fluoroacetate’ shows effect of the astrocyte-specific metabolic inhibitor fluoroacetate (10 mm) on the tHeSD (n = 6). ‘Carbenoxolone’ shows effect of the connexin-based channel inhibitor carbenoxolone (100 μm) on the tHeSD (n = 6). ‘Endothelin-1’ shows the effect of the connexin-based channel inhibitor endothelin-1 (1 μm) on the tHeSD (n = 6). B, average (5 min binned) 1st fEPSP initial slope (dark grey circles), paired-pulse ratio (open squares) and volley (light grey circles) after application of fluoroacetate (FAC) expressed as percentage of 1st fEPSP initial slope, paired-pulse ratio and volley before application and plotted against minutes in FAC (n = 8).

Figure 4. GABA_B receptors, but not NMDA and adenosine A1 receptors, are involved in the transient heterosynaptic depression

The graph illustrates the results of matched comparisons of tHeSD before and after pharmacological interventions. The filled bars compare the effect on the fEPSP slope and the open bars compare the effect on the paired-pulse ratio. 100% means the same tHeSD as in the control situation and 0% means absence of tHeSD. Typical control (‘5 s’, upper) and test (‘0.5 s’, lower) example sweeps are shown above the bars. Dashed line indicates the amplitude of the control fEPSP. ‘CGP52432’ shows the effect of GABA_B receptor antagonist CGP52432 (4 μm) on the tHeSD (n = 4). B, ‘d-AP5’ shows the effect of the NMDA receptor antagonist d-AP5 (50 μm) on the tHeSD (n = 6). C, ‘DPCPX’ shows the effect of adenosine A1 receptor antagonist DPCPX (0.2 μm) on the tHeSD (n = 4).

Figure 5. Metabotropic glutamate receptors are necessary for the transient heterosynaptic depression

The graph illustrates the results of matched comparisons of tHeSD before and after pharmacological interventions. The filled bars compare the effect on the fEPSP slope and the open bars compare the effect on the paired-pulse ratio. 100% means the same tHeSD as in the control situation and 0% means absence of tHeSD. Typical control (‘5 s’, upper) and test (‘0.5 s’, lower) example sweeps are shown above the bars. Dashed line indicates the amplitude of the control fEPSP. ‘LY 341495’ shows the effect of the group I, II and III metabotropic glutamate receptor antagonist LY 341495 (50 μm) on the tHeSD (n = 6). ‘CPCCOet’ shows the effect of the group I metabotropic glutamate receptor antagonist CPCOOet (150 μm) on the tHeSD (n = 4). ‘APCD’ shows the effect of the group II metabotropic glutamate agonist APCD (50 μm) on the tHeSD (n = 8). ‘l-AP4, 50 μm’ shows the effect of the group III metabotropic glutamate agonist l-AP4 (50 μm; mGluR4, 6, 8) on the tHeSD (n = 4). ‘l-AP4, 1.2 (mm)’ shows the effect of the group III metabotropic glutamate agonist l-AP4 (1.2 mm; mGluR4, 6, 7, 8) on the tHeSD (n = 8).

Figure 6. Working model for the transient heterosynaptic depression

Schematic illustration of two active (left) synapses (one glutamatergic and one GABAergic) and one inactive (right) glutamatergic synapse with part of an astrocyte in between. The astrocyte is equipped with GABA_BRs and group II/III mGluRs, whose activation can increase intracellular calcium which can spread via autologous gap junctions. The activated astrocyte releases the gliotransmitter glutamate, which activates presynaptic group II/III mGluRs and inhibits release probability at glutamatergic terminals.