Astrocytes modulate thalamic sensory processing via mGlu2 receptor activation

Abstract

Astrocytes possess many of the same signalling molecules as neurons. However, the role of astrocytes in information processing, if any, is unknown. Using electrophysiological and imaging methods, we report the first evidence that astrocytes modulate neuronal sensory inhibition in the rodent thalamus. We found that mGlu2 receptor activity reduces inhibitory transmission from the thalamic reticular nucleus to the somatosensory ventrobasal thalamus (VB): mIPSC frequencies in VB slices were reduced by the Group II mGlu receptor agonist LY354740, an effect potentiated by mGlu2 positive allosteric modulator (PAM) LY487379 co-application (30 nM LY354740: 10.0 ± 1.6% reduction; 30 nM LY354740 & 30 μM LY487379: 34.6 ± 5.2% reduction). We then showed activation of mGlu2 receptors on astrocytes: astrocytic intracellular calcium levels were elevated by the Group II agonist, which were further potentiated upon mGlu2 PAM co-application (300 nM LY354740: ratio amplitude 0.016 ± 0.002; 300 nM LY354740 & 30 μM LY487379: ratio amplitude 0.035 ± 0.003). We then demonstrated mGlu2-dependent astrocytic disinhibition of VB neurons in vivo: VB neuronal responses to vibrissae stimulation trains were disinhibited by the Group II agonist and the mGlu2 PAM (LY354740: 156 ± 12% of control; LY487379: 144 ± 10% of control). Presence of the glial inhibitor fluorocitrate abolished the mGlu2 PAM effect (91 ± 5% of control), suggesting the mGlu2 component to the Group II effect can be attributed to activation of mGlu2 receptors localised on astrocytic processes within the VB. Gating of thalamocortical function via astrocyte activation represents a novel sensory processing mechanism. As this thalamocortical circuitry is important in discriminative processes, this demonstrates the importance of astrocytes in synaptic processes underlying attention and cognition.

Keywords: Astrocyte; Metabotropic glutamate receptor subtype 2; Synaptic inhibition; Thalamic reticular nucleus; Thalamus.

Fig. 1

Thalamic circuitry underlying responses to vibrissal deflection. Branching collaterals from excitatory thalamocortical and corticothalamic axons (black), which originate from functionally linked topographical areas in the thalamus/cortex, innervate the TRN (Ohara and Lieberman, 1985, Shosaku et al., 1989, Rouiller et al., 1998, Kakei et al., 2001), and the TRN sends a reciprocal inhibitory projection (grey) back to the thalamic area from which it receives its thalamocortical innervation (Jones, 1985, Salt, 1989, Shosaku et al., 1989, Pinault and Deschenes, 1998, Salt and Turner, 1998, Crabtree, 1999).

Fig. 2

The Group II mGlu receptor effect on spontaneous presynaptic quantal release events includes an mGlu2 receptor-mediated component. a Circuitry between the TRN and VB with recording site indicated. b Effects of the Group II agonist LY354740 (30 nM) alone or in conjunction with the mGlu2 PAM LY487379 (30ìM) on the total number of spontaneous mIPSC events (final 5 min bin) in the VB. Specificity of the Group II agonist effect was confirmed upon its reversal using the Group II antagonist LY341495 (100 nM). c Traces from individual neurons illustrating the mean responses of neurons to the same conditions as described in b. d Effects of the same compound application combinations on the cumulative fraction of the calculated inter-event intervals of the spontaneous mIPSCs in the VB. **p < 0.001; ***p < 0.0001; Glu – glutamate.

Fig. 3

mGlu2 receptor activation can elicit increases in astrocytic intracellular calcium levels. a Images from a slice loaded with Fluo-4-AM for calcium imaging, and SR101 for astrocyte differentiation. Identified astrocytes and neurons are indicated. b Traces displaying transient intracellular calcium elevations in an astrocyte in response to application of increasing concentrations of the Group II agonist LY354740 either alone or in conjunction with the mGlu2 PAM LY487379. Two traces on the right display ratio over time for example a neuron. c Bargraphs summarise results from a number of experiments corresponding to the illustrative traces above in b. (Astrocytes: 100 nM LY354740 alone and co-applied with 30 μM LY487379, 3 slices, n = 21; 300 nM LY354740 alone and co-applied with 30 μM LY487379, 5 slices, n = 56; 1 μM LY354740 alone and co-applied with 30 μM LY487379, 3 slices, n = 49; Neuron: 1 μM LY354740 alone and co-applied with 30 μM LY487379, 3 slices, n = 36). Compound application is indicated by the striped (LY354740) and grey (LY4872379) bars. d Bargraphs summarise results from a number of experiments demonstrating antagonism of the Group II agonist effect. The two bars on the left display ÄF% changes in calcium fluorescence upon application of 1 μM of the Group II agonist LY354740 alone and in conjunction with 1 μM of the Group II antagonist LY341495. The two bars on the right display ÄF% changes in calcium fluorescence upon application of 1 μM of the Group II agonist LY354740 alone and in conjunction with 5 μM MPEP and 100 μM suramin. e Upper traces display fluorescence over time for four example astrocytes from a slice from a wild-type (IP3R2+/+) mouse with responses to Group II agonist (1 μM) and glutamate (100 μM). Traces below show responses from astrocytes in a slice from an IP3R2(-/-) knock-out mouse. Bargraphs to the right summarise a number of experiments. Bars in c and d represent the mean % response (±SEM) of the fluorescence, **p < 0.01, ***p < 0.001.

Fig. 4

Astrocyte inactivation attenuates the maintained component of VB neuron responses without affecting responses to NMDA. a Circuitry between the TRN and VB with recording site indicated. b Raster displays and peristimulus time histograms (PSTHs) of responses of a VB neuron (CVB142c) to either train stimulation of a single vibrissa (50 ms bins, 8 trials) or iontophoretic application of NMDA (15 nA; 1s bins, 2 trials) under normal conditions and in the presence of fluorocitrate (20 nA; 5 min). c Bars represent the mean % response (±SEM) under normal conditions (100%) and in the presence of fluorocitrate to train stimulation (total, initial and maintained) of single vibrissae (n = 16) and NMDA (n = 11). ***p < 0.001.

Fig. 5

Astrocyte inactivation attenuates the mGlu2 component of the Group II effect on sensory inhibition in the VB. a Raster displays and PSTHs of responses of a VB neuron (CVB138a) to train stimulation (50 ms bins, 6 trials) of a single vibrissa under normal conditions and in the presence of fluorocitrate (20 nA; 10 min) during a control period, upon iontophoretic application of either LY487379 (50 nA, 2 min) or LY354740 (50 nA, 2 min), and during recovery. Abscissa indicated on the bottom left raster and PSTH plot applies to all plots. b Bars represent the mean % of control (±SEM) of responses to train stimulation of single vibrissae (n = 6) to application of either LY487379 or LY354740 under normal conditions and in the presence of fluorocitrate. *p < 0.05.

Fig. 6

Summary diagram of Group II mGlu receptor localizations in the VB, and their effects upon synaptic transmission. Using selective pharmacological compounds, we have been able to show that mGlu2 receptors are likely located on astrocytic processes surrounding the TRN-VB synapse, whilst mGlu3 receptors are likely located on the TRN terminals themselves in the VB. Activation of astrocytic mGlu2 receptors likely facilitates elevations in intracellular calcium levels (indicated by a green plus), which may lead to presynaptic modulation of the TRN-VB synapse, whilst neuronal mGlu3 receptor activation is thought to decrease GABAergic transmission (indicated by the red minus signs). Both of the Group II mGlu receptor subtypes are likely activated via glutamate spillover from the synapse formed between the sensory afferent and the VB proximal dendrite upon physiological sensory stimulation (Copeland et al., 2012). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)