Neuron-Microglia Contact-Dependent Mechanisms Attenuate Methamphetamine-Induced Microglia Reactivity and Enhance Neuronal Plasticity

Abstract

Exposure to methamphetamine (Meth) has been classically associated with damage to neuronal terminals. However, it is now becoming clear that addiction may also result from the interplay between glial cells and neurons. Recently, we demonstrated that binge Meth administration promotes microgliosis and microglia pro-inflammation via astrocytic glutamate release in a TNF/IP₃R2-Ca²⁺-dependent manner. Here, we investigated the contribution of neuronal cells to this process. As the crosstalk between microglia and neurons may occur by contact-dependent and/or contact-independent mechanisms, we developed co-cultures of primary neurons and microglia in microfluidic devices to investigate how their interaction affects Meth-induced microglia activation. Our results show that neurons exposed to Meth do not activate microglia in a cell-autonomous way but require astrocyte mediation. Importantly, we found that neurons can partially prevent Meth-induced microglia activation via astrocytes, which seems to be achieved by increasing arginase 1 expression and strengthening the CD200/CD200r pathway. We also observed an increase in synaptic individual area, as determined by co-localization of pre- and post-synaptic markers. The present study provides evidence that contact-dependent mechanisms between neurons and microglia can attenuate pro-inflammatory events such as Meth-induced microglia activation.

Keywords: CD200; PSD95; contact-dependent; methamphetamine; neuron-to-microglia; neuroprotection.

Figure 1

Neurons exposed to Meth do not promote microglial activation through contact-independent mechanisms. (A) Experimental design I—Neuronal cell cultures from the hippocampus were incubated with 100 µM Meth for 24 h. Following that, primary microglial cell cultures were incubated for 24 h with the conditioned media obtained from neurons treated with Meth (NCM Meth) or from control neuronal cell cultures (NCM Ctrl). (B) Fluorescence imaging of microglial cells immunolabeled for iNOS (green) incubated with NCM Ctrl or NCM Meth for 24 h. Results express the iNOS intensity (mean ± SEM) of three independent cultures (NCM Ctrl—132 cells; NCM Meth—143 cells). (C) Fluorescence imaging of microglial cells immunolabeled for iba1 (red) incubated with NCM Ctrl or NCM Meth for 24 h. Results express iba1 intensity (mean ± SEM) of five independent cultures (NCM Ctrl—252 cells; NCM Meth—265 cells). (D) Fluorescence imaging of microglial cells immunolabeled for iba1 (red) incubated with microbeads (green) and treated with NCM Ctrl or NCM Meth for 24 h. Results represent the number of beads per cell (mean ± SEM) of five independent cultures (NCM Ctrl—267 cells; NCM Meth—264 cells). Statistical analysis for (B–D) was performed using a linear mixed model followed by Tukey–Kramer comparison test (* p < 0.05). Symbol colors represent the mean of each independent cell culture and the violin plots the variability of all cells quantified. Scale bar: 10 µm. (E) RT-qPCR for TNF, Il-1β, and Il-6 from microglia exposed to NCM Ctrl and NCM Meth for 24 h. (F) RT-qPCR for Il-10 and TGFβ from microglia exposed to NCM Ctrl and NCM Meth for 24 h. In both cases, results were normalized to the S18 gene and are expressed as the mean ± SEM of five independent cultures. Statistical analysis was performed using paired Student’s t-test.

Figure 2

Neurons exposed to Meth and in direct contact with microglia do not promote its activation. (A) Experimental design II—neuronal cell culture from the hippocampus was seeded in one side of a microfluidic device, and the axons were allowed to extend to the other compartment for 12 days. On DIV12, microglial cells were seeded in the axonal compartment. On DIV13, Meth 100 µM was added to the neuronal compartment for 24 h. (B) Fluorescence imaging of microglial cells cultured in a microfluidic device with neurons exposed to Meth 100 µM for 24 h. Microglial cells were immunolabeled for iNOS (green), and the results express the iNOS intensity (mean ± SEM) of four independent cultures (Ctrl—297 cells; Meth—325 cells). (C) Fluorescence imaging of microglial cells immunolabeled for iba1 (red) incubated with NCM Ctrl or NCM Meth for 24 h. Results express iba1 intensity (average ± SEM) of five independent cultures (Ctrl—199 cells; Meth—206 cells). (D) Fluorescence imaging of microglial cells cultured in a microfluidic device with neurons exposed to Meth 100 µM for 24 h. Microglia were incubated with microbeads (green) and immunolabeled for iba1 (red). Results represent the number of beads per cell (mean ± SEM) of five independent cultures (Ctrl—268 cells; Meth—211 cells). (E) Fluorescence imaging of microglial cells cultured in a microfluidic device with neurons exposed to Meth 100 µM for 24 h. Microglial cells were immunolabeled for arginase 1 (magenta), and results express the arginase 1 intensity (mean ± SEM) of six independent cultures (Ctrl—489 cells; Meth—410 cells). Statistical analysis for B, C, D, and E was performed using a linear mixed model followed by Tukey–Kramer comparison test (* p < 0.05 and **** p < 0.0001). Symbol colors represent the mean of each independent cell culture and the violin plots the variability of all cells quantified. Scale bar: 10 µm. (F) RT-qPCR for TNF, Il-1β, and Il-6 from microglia and neuron co-culture cells, cultured in a microfluidic device where neurons were exposed to Meth 100 µM for 24 h. (G) RT-qPCR for Il-10 and TGFβ from microglia and neuron co-cultured cells, in a microfluidic device where neurons were exposed to Meth 100 µM for 24 h. In both cases, results were normalized to the S18 gene and are expressed as the mean ± SEM of six independent cultures. Statistical analysis was performed using paired Student’s t-test.

Figure 3

Neurons partially prevented microglia activation via Meth-exposed astrocytes. (A) Experimental design III—primary cell cultures of astrocytes were incubated with Meth 100 µM for 24 h. Neuronal cells from the hippocampus were seeded in one side of a microfluidic device, and the axons were allowed to extend to the other compartment for 12 days. On DIV12, microglial cells were seeded in the axonal compartment. The conditioned media obtained from astrocytes treated with Meth (ACM Meth) or from control astrocyte cultures (ACM Ctrl) were added to the microglia/axonal compartment for 24 h. (B) Fluorescence imaging of microglial cells co-cultured with neurons and exposed to the ACM Ctrl or ACM Meth for 24 h. Microglial cells were immunolabeled for iNOS (green), and results express the iNOS intensity (mean ± SEM) of five independent cultures (ACM Ctrl—405 cells; ACM Meth—329 cells). (C) Fluorescence imaging of microglial cells immunolabeled for iba1 (red) incubated with NCM Ctrl or NCM Meth for 24 h. Results express iba1 intensity (mean ± SEM) of four independent cultures (Ctrl—151 cells; Meth—184 cells). (D) Fluorescence imaging of microglial cells co-cultured with neurons and incubated with ACM Ctrl or ACM Meth for 24 h. Microglia were incubated with microbeads (green) and immunolabeled for iba1 (red). The results represent the number of beads per cell (mean ± SEM) of five independent cultures (ACM Ctrl—183 cells; ACM Meth—248 cells). (E) Fluorescence imaging of microglial cells co-cultured with neurons and exposed to ACM Ctrl or ACM Meth for 24 h. Microglial cells were immunolabeled for arginase 1 (magenta), and results express the arginase 1 intensity (mean ± SEM) of six independent cultures quantified (ACM Ctrl—310 cells; ACM Meth—287 cells). Statistical analysis for B, C, D, and E was performed using a linear mixed model followed by Tukey–Kramer comparison test (**** p < 0.0001). Symbol colors represent the mean of each independent cell culture and the violin plots the variability of all cells quantified. Scale bar: 10 µm. (F) RT-qPCR for TNF, Il-1β, and Il-6 from microglia and neuron co-culture cells, cultured in a microfluidic device where neurons were exposed to Meth 100 µM for 24 h. (G) RT-qPCR for Il-10 and TGFβ from microglia and neurons co-culture cells, cultured in a microfluidic device with microglia exposed to ACM Ctrl or ACM Meth for 24 h. In both cases, results were normalized to the S18 gene and are expressed as mean ± SEM of five independent cultures. Statistical analysis was performed using paired Student’s t-test.

Figure 4

Neurons increased self-protection through contact-dependent mechanisms. RT-qPCR for ligand–receptor immunoregulatory pairs from microglia and neurons co-cultured in microfluidic devices, where microglial cells were exposed to ACM Ctrl or ACM Meth for 24 h. (A) Results for CD200 and CD200r, (B) CD22 and CD45, (C) CX3CL1 and CX3CR1, and (D) CD95. The results were normalized to the S18 gene and are expressed as the mean ± SEM of five independent cultures. Symbol colors represent the mean of each independent cell culture. Statistical analysis was performed using paired Student’s t-test (* p < 0.05). Quantification of (E) CD95 and (F) CD200r on microglia by flow cytometry, presented as median fluorescence intensity (MFI). Results are expressed as mean ± SEM of three independent cultures. Symbol colors represent the mean of each independent cell culture. Statistical analysis was performed using paired Student’s t-test. Representative histograms are shown; orange histogram represents unstained cells, gray the ACM Ctrl group, and red the ACM Meth group.

Figure 5

Synaptic plasticity was concomitantly increased. (A) Fluorescence imaging of microglial cells co-cultured with neurons and exposed to ACM Ctrl or ACM Meth for 24 h. Cells were immunolabeled for PSD95 (red), and results represent the PSD95 puncta (B) number, (C) area, and (D) intensity. (E) Fluorescence imaging of microglial cells co-cultured with neurons and exposed to ACM Ctrl or ACM Meth for 24 h. Microglial cells were immunolabeled for vGlut1 (green), and results represent the Vglut1 puncta (F) number, (G) area, and (H) intensity. (I) Fluorescence imaging of microglial cells co-cultured with neurons and exposed to ACM Ctrl or ACM Meth for 24 h. Cells were immunolabeled for PSD95 (red) and VGlut1 (green), and results represent the (J) PSD95 plus vGlut1-positive puncta number, (K) puncta area, and (L) puncta intensity. Symbol colors of each graph represent the mean of each independent cell culture (n = 5) and the violin plots the variability of all neuronal segments quantified (ACM Ctrl—149; ACM Meth—111 segments). Statistical analysis was performed using a linear mixed model followed by Tukey–Kramer comparison test. Scale bar: 10 µm. * p < 0.05, ** p < 0.01, and **** p < 0.0001.