3.1. Astroglia-Induced Homeostatic Synaptic Scaling in Cultured Neurons
A conventional approach to test molecular mechanisms of homeostatic synaptic plasticity is exposure of primary neuronal cultures to conditions of chronic inhibition or enhancement of neuronal firing, e.g., 24 h-long incubation with tetrodotoxin or picrotoxin [9
]. To probe the specific role of astrocytes, we modified this approach and selectively enhanced astroglial Ca2+
-signalling with TFLLR, an agonist of PAR-1 receptors [20
]. The efficiency and specificity of PAR-1 receptor-mediated activation of astrocytes and lack of such action in neurons have been verified previously [20
The 24 h-long incubation with TFLLR (3 µM) caused a considerable increase in the amplitude of mEPSCs in wild-type hippocampal pyramidal neurons (Figure 1
A) suggesting that enhancement of astrocytic signalling can induce synaptic scaling. The average mEPSC amplitude increased from 14.01 ± 2.77 pA (n
= 11) to 26.7 ± 6.55 pA (n
= 9, p
To confirm that the observed synaptic scaling was BDNF-dependent, we incubated wild-type cultured neurones in TFLLR with Cyclotraxin-B (CTX-B, 10 nM), a selective and potent TrkB receptor inhibitor which has been shown to block the actions of BDNF [28
]. Addition of the TrkB receptor inhibitor blocked the TFFLR-induced enhancement of mEPSCs (Figure 1
A). Furthermore, the glia-induced synaptic scaling was impaired in cell cultures derived from MSK1 KD mice (Figure 1
B,C) indicating the crucial role for BDNF-MSK1 pathway in this phenomenon.
Analysis of amplitude distributions of mEPSCs (Figure 1
B) showed that the increase in the average amplitude occurred due to an increase in the unitary quantal size of synaptic response (manifested as the main peak of the amplitude distribution). The incubation with TFLLR alone caused a robust increase in the quantal size of mEPSCs recorded in wild type neurons, whereas there was no significant change in mEPSC frequency (Figure 1
B,C). These results imply that glia-induced BDNF-dependent homeostatic synaptic scaling occurs mainly via postsynaptic mechanisms, similar to previous reports on various forms of homeostatic synaptic plasticity.
3.2. Astroglia-Induced Homeostatic Changes in Synaptic Morphology
An enhancement of strength of excitatory synapses at postsynaptic locus can occur via two principle pathways: an increase in efficacy of neurotransmitter receptors (i.e., permeability and open time of glutamate receptor-associated ion channels) or an increase in their surface expression. These functional alterations are often accompanied by changes in synaptic morphology, in particular an increase in the size of dendritic spines. Brain-derived neurotrophic factor has been previously implicated in the regulation of synaptic morphology via induction of Arc/Arg3.1
To elucidate whether this cascade is involved in the TFFLR-induced synaptic scaling, we assessed the alterations in the size and shape of postsynaptic sites using SICM. This technique is based on decrease in the ionic current passing through a glass microelectrode in close proximity of cells (or any other obstacles). The SICM method enables the imaging of live cells at nanoscale resolution and adequately maps the shape and size of small subcellular structures, such as dendrites, axons and synaptic boutons [25
]. This technique has been successfully used before in the structure–function studies of synaptic networks of cultured hippocampal neurons and mechano-sensory stereocilia of cochlear hair cells [25
To test for the effects of astroglia-derived BDNF on synaptic morphology, we examined live neurons from wild-type mice hippocampal cultures incubated with TFLLR alone, TFLLR with the inhibitor of TrkB receptors, or BDNF (Figure 2
A). To identify functional synapses, neurons were labelled with FM1-43, an activity-dependent marker of synaptic vesicles, prior to SICM imaging (Figure 2
A). Whenever a FM1-43 signal was observed, varicosities could be mapped at the SICM images; with the size and shape of varicosities consistent with the geometry expected of synaptic boutons. We targeted fluorescently-labelled varicosities located on distal dendrites to increase the probability of encountering excitatory synapses. Upon 3D-mapping of identified synaptic boutons (Figure 2
B), their volume and effective size were evaluated as described in Methods (Figure 2
Consistent with our observations of astroglia-induced increase in synaptic strength, 24 h-incubation with TFLLR caused significant, up to 60%, increase in the average size of synapses (from 0.798 ± 0.223 µm to 1.248 ± 0.345 µm, p < 0.01). Correspondingly, the average volume of synaptic boutons showed >2.5-fold increase, from 0.218 ± 0.208 µm3 to 0.569 ± 0.489 µm3 (n = 31, p < 0.01).
The effect of TFLLR was effectively antagonized by Cyclotraxin-B (average size increased by just 10%) and reproduced by incubation with BDNF (Figure 3
Combined with data on the changes in mEPSC amplitude (Figure 1
), these results demonstrate that activation of astrocytes can engage the BDNF-dependent molecular cascade, which is instrumental for homeostatic regulation of synaptic strength.
3.3. Astrocytes Participate in Homeostatic Plasticity In Vivo
BDNF-mediated homeostatic synaptic scaling has been implicated in the positive effects of environmental enrichment on synaptic transmission [9
]. There is also evidence that an increase in BDNF can underlie the beneficial effects of a low-calorie diet on brain function [12
]. Previously, we demonstrated that environmental enrichment and caloric restriction can enhance Ca2+
-signalling in neocortical astrocytes [19
]. Hence, one might predict that an increase in astroglial Ca2+
-dependent BDNF release plays a key role in the beneficial effects of EE and CR on synaptic transmission in the aging brain. To explore the role of this signalling mechanism in a behavioural model of experience-dependent synaptic plasticity, we examined the effect of environmental enrichment and caloric restriction on excitatory and inhibitory synaptic transmission in the neocortex.
First, we verified that EE and CR can enhance Ca2+
-signalling in neocortical astrocytes of MSK1 KD mice. Astroglial Ca2+
-signalling was monitored using multiphoton fluorescence microscopy as described previously [20
]. We measured spontaneous cytosolic Ca2+
transients in the branches and soma of neocortical astrocytes of 6–12 week-old (young adults) and 9–15 months old (old) mice (Figure 3
). There was no significant difference in Ca2+
-signalling between neocortical astrocytes of old wild-type and MSK1 KD mice (Figure 3
). Similar to our previous reports, the amplitude and frequency of spontaneous astrocytic Ca2+
-transients in old WT and MSK1 KD mice raised in standard housing (SH) were significantly lower than those measured in the young SH WT mice. Most importantly, EE and CR significantly increased spontaneous astroglial Ca2+
-signalling in the old WT and MSK1 KD mice to a similar extent (Figure 3
We also assessed the responses of astrocytes to exogenous activation of NA and ATP receptors since these neurotransmitters are released during enhanced neuronal and physical activity [21
] and potentially can mediate a link between environmental enrichment and the function of astrocytes. There is also growing evidence of the importance of α1AR and P2Y receptors for astrocytic Ca2+
-signalling and glia–neuron interactions [21
]. EE and CR had a moderate effect on the amplitudes of astrocytic responses to NA (3 µM) and ATP (30 µM) in the young mice, but caused a significant enhancement of astroglial responses at the older age, both in the WT and MSK1 KD mice (Figure 3
These results have verified that Ca2+
-signalling in astrocytes, and especially the effects of EE and CR, are not affected by MSK1 KD knock-in. We have previously demonstrated that astroglial Ca2+
-signalling is not altered in the dnSNARE mice either [19
]. Hence, if any difference in the impact of EE and CR on synaptic transmission in the dnSNARE and MSK1 KD mice were observed, they should be attributed not to the deficit in astroglial Ca2+
-elevation, but rather to the impairment of downstream signalling cascades, namely astroglial exocytosis and BDNF-mediated regulation of neuronal synaptic strength.
To test this, we recorded AMPA receptor-mediated mEPSCs in neocortical pyramidal neurons in the presence of the GABAA
receptor antagonist picrotoxin (100 µM), and the P2 Receptor antagonist PPADS (10 µM). Exposure of wild-type mice to EE from birth to 6–12 weeks increased the average amplitude of mEPSCs to 18.6 ± 6.04 pA (n
= 12), as compared to 21.3 ± 5.97 (n
= 13) in the SH mice. The difference between the EE and SH mice was even larger for the older mice (Figure 4
A,B), which might be related to the age-related decline in the amplitude of excitatory synaptic currents. In the older mice, the mEPSC amplitude increased from 10.86 ± 3.55 pA (SH, n
= 10) to 14.34 ± 3.47 pA (EE, n
Exposure to CR had similar positive effect on mEPSCs (Figure 4
B). The observed effects of both EE and CR were, most likely, of postsynaptic origin since they were accompanied by a significant increase in quantal size, whereas the frequency of mEPSCs did not undergo considerable changes (Figure 4
In stark contrast to the WT mice, mEPSCs recorded from MSK1 KD mice undergo the same age-related decline, but did not exhibit the EE- or CR-induced increase in amplitude (Figure 4
A,B). In line with data obtained in the MSK1 KD mice, the dnSNARE mice showed only modest EE- and CR-induced increase in the average mEPSCs amplitude. The difference in the quantal size of mEPSCs in the WT and dnSNARE mice was statistically significant only for the EE in the younger age (p
< 0.05). The incomplete inhibition of EE-induced plasticity in the young dnSNARE mice could be attributed to the mosaic (50–60% of cells) expression of dnSNARE and therefore incomplete loss of glial exocytosis. In addition, neuronal release of BDNF cannot be excluded.
The dependence of EE- and CR-induced upscaling of excitatory synaptic currents on the MSK1 signalling pathway and astrocytic exocytosis closely agrees with our results obtained in cell cultures (Figure 1
and Figure 2
). Combined together, our in vitro and ex vivo data strongly support the crucial importance of astroglial release of BDNF for homeostatic plasticity of excitatory synaptic transmission.
Finally, we explored the experience-dependent plasticity of inhibitory synaptic signalling. We recorded GABA receptor-mediated miniature inhibitory synaptic currents (mIPSCs) at a membrane potential of −80 mV in the presence of glutamate and P2X receptor antagonists (DNQX and PPADS, respectively). The pattern of age- and environment-related alterations in the GABAergic synaptic currents was different to the changes exhibited by mEPSCs. Firstly, both the quantal size and frequency of mIPSCs undergo dramatic increase in older WT and the MSK1 KD mice (Figure 5
). In addition, GABAergic currents were upregulated in the dnSNARE mice of both age groups as compared to their WT-littermates (Figure 5
A,B); this result is in line with our previous observations [19
]. Secondly, exposure of the wild-type mice to EE and CR efficiently downregulated inhibitory synaptic signalling, especially in the neurons of older mice (Figure 5
A,B). In contrast to mEPSCs, the EE- and CR-induced downscaling of mIPSCs was absent in the dnSNARE but not in the MSK1 KD mice. This might be explained by participation of other gliotransmitters, in particular ATP [19
], in astroglial-driven modulation of GABA receptors.
Combined, our data strongly support the importance of astrocytic exocytosis and BDNF/MSK1-mediated signalling for the beneficial effects of EE and CR on synaptic transmission in the ageing brain.