Methods for culturing adult CNS neurons reveal a CNS conditioning effect

Abstract

Neuronal cultures provide a basis for reductionist insights that rely on molecular and pharmacological manipulation. However, the inability to culture mature adult CNS neurons limits our understanding of adult neuronal physiology. Here, we report methods for culturing adult central nervous system neurons in large numbers and across multiple brain regions for extended time periods. Primary adult neuronal cultures develop polarity; they establish segregated dendritic and axonal compartments, maintain resting membrane potentials, exhibit spontaneous and evoked electrical activity, and form neural networks. Cultured adult neurons isolated from different brain regions such as the hippocampus, cortex, brainstem, and cerebellum exhibit distinct cell morphologies, growth patterns, and spontaneous firing characteristics reflective of their regions of origin. Using adult motor cortex cultures, we identify a CNS "conditioning" effect after spinal cord injury. The ability to culture adult neurons offers a valuable tool for studying basic and therapeutic science of the brain.

Keywords: conditioning lesion; neural repair; primary adult neuronal cultures; regeneration.

Graphical abstract

Figure 1

Methods for culturing adult primary CNS neurons (A) Tissue is harvested rapidly under cold conditions. (B) Enzymatic-buffer solution containing DNase and papain is used with very gentle mechanical dissociation to slowly disrupt tissue. (C) Debris mostly consisting of dead cells and myelin is removed. (D) Magnetic biotinylated antibodies are added against cell surface markers of astrocytes, oligodendrocytes, microglia, and endothelial cells to deplete non-neuronal cells. (E) A cell-antibody magnetically labeled mixture is loaded onto a ferromagnetic sphere column and magnetically labeled cells remain in column while non-magnetically labeled cells (primarily neurons) are eluted from column, resulting in neuronal enrichment.

Figure 2

Adult CNS neuron cultures (A) Neurons from different CNS regions exhibit distinct phenotypic characteristics. Labeling for neuronal marker Tuj1. FezF2 (inset) identifies layer 5b glutamatergic neurons. Cerebellar Purkinje cell neuron labels for calbindin (inset) and spinal cord neuron labels for choline acetyltransferase (ChAT, inset). Neurons isolated at age 6 weeks. Scale bars, 20 μm. (B) Motor cortex neurons form distinct dendritic compartments identified by MAP2 after 5 days in vitro. Tuj1 red, MAP2 green. Neurons isolated at age 6 weeks. Scale bar, 20 μm. (C) Tau labeling (green) identifies axons in culture. At the axon tip is a growth cone. Tuj1 red, with Tau only panel shown below. Neurons isolated at age 4 weeks and cultured for 5 days in vitro. Scale bar, 20 μm. (D) Growth cone (arrow and inset) with lamellipodia is evident in cortical cultures isolated at age 4 weeks, with 7 additional days in vitro. Labeled for Tau. Scale bar, 20 μm. (E) Vesicular glutamate-1 (VGlut1) is present in soma and at bouton-like structures (arrows) indicating excitatory synapses. Tuj1 red. Neurons isolated at age 6 weeks. Scale bars, 10 and 5 μm. (F) Synaptophysin boutons present on motor cortical cell bodies and on Tuj1-labeled neurites. Neurons were isolated at age 6 weeks. Scale bar, 10 μm. (G) Post-synaptic marker Homer (green) localizes with the presynaptic marker synaptophysin (red) at the junction of two Tuj1-labeled processes (blue), indicating a synapse (arrows). Inset with Homer and synaptophysin. Neurons isolated at age 9 weeks. Scale bars, 1 and 2.5 μm. (H) FACS analysis of enriched neuronal fraction. 82.8% of cells are neuronal, 8.6% astrocytes, 3.37% oligodendrocytes, 1.18% microglia, and 2.08% endothelial cells. Flow cytometry antibodies: neurons (Tuj1), astrocytes (ACSA), oligodendrocytes (O4), microglia (CD45, CD11b), and endothelial cells (CD31). Flow cytometry dyes include APC (allophycocyanin), PE (phycoerythrin), VioBlue, VioBright-FITC, and PE-Vio770. Cells isolated at age 8 weeks. Error bars = STDEV.

Figure 3

Spontaneous neural growth and electrical activity (A) Neuritic outgrowth from adult cortical neurons over 5 subsequent days. Dendrites are visible after 3 days (inset). Tuj1 labeling. Scale bar, 50 μm. Neurons isolated from 6-week-old animals. Image is a composite. (B) Sholl analysis of neuritic “crossings” of Tuj1-labeled processes at 1-μm interval distances from centroid. Data represent 50 neurons measured at every time point in culture from motor cortex ± SEM. (C) Phalloidin labeling (red) of cortical growth cone in culture (red) and Tuj1 (green). Phalloidin only panel below. Scale bar 5 μm. (D) Corticospinal neurons identified by CTB retrograde tracer injections into C8 spinal cord. Motor cortex cultures harvested at age 9 weeks and maintained in vitro 5 days. Scale bars, 20 μm. (E) Reticulospinal neurons identified by CTB retrograde tracer injections into C8. Brainstem cultures isolated at 9 weeks. Tuj1 red, CTB green. Scale bars, 25, 50, and 100 μm. (F) Electrophysiological activity in cultured adult cortical and hippocampal neurons. Neurons isolated from 6-week-old mice, plated onto microelectrode arrays and cultured for 10 days in vitro. Spontaneous electrical activity is evident. (G) Evoked activity is present after stimulation at 500 mV for 400 μs every 5 s. Recordings from other cells simultaneously indicate network firing patterns. Dashed line represents minimum five spikes/100 ms. (I) Spontaneous bursting in cultured cortical neurons. Synchronous bursting (defined as at least 25% of electrodes firing simultaneously) occurs (pink arrows); 16 electrodes per well. A mean of 12.2 electrodes (SD 1.87) contributed to network bursts of mean duration 13.3 ms. (J) Hippocampal neurons exhibit greater spontaneous activity than cortical cultures over 10 min. A mean of 12.5 electrodes (SD 2.26) participated in network bursts with average duration 15.3 ms among hippocampal neurons.

Figure 4

Conditioning lesions significantly enhance CNS cortical neurite growth (A–E) The spinal cord was lesioned at C8; the motor cortex was then removed and cultured either (A) 1 h, (B) 6 h, (C) 24 h, (D) 72 h, or (E) 7 days later. All cells were then maintained for 5 days in culture. An increase in neurite length is particularly appreciable in cultures prepared 24 h after conditioning. Tuj1 label for neurons. Scale bar, 20 μm. Images of neurons were placed onto equal-sized background panels for presentation purposes. (F and G) Total neurite length and (G) longest neurite length measured using Tuj1 labeling. N = 3 replicates, ± SEM There is a significant conditioning effect beginning 1 h after spinal cord injury and lasting 24 h. (H) RNA sequencing of conditioned neuronal cultures. Spinal cord lesions were made 24 h before plating cells, and RNA sequencing was performed after 1, 2, 3, 5, and 7 days in culture. Number of differentially expressed genes (DEGs) compared with non-conditioned cultures sampled at the same number of days in culture are shown. FDR ≤0.1. (I) Heatmap of top 1,000 differentially expressed genes (FDR ≤0.1, Benjamini-Hochberg adjusted p values) of conditioned relative to non-conditioned neurons at 1, 2, 3, 5, and 7 days in culture, arranged by hierarchical clustering. Red, increased expression; green, reduced expression. Intensity of color reflects degree of gene differential expression compared to non-conditioned cultures. (J) Pathway analysis of differentially expressed genes over days 1 to 7 in culture. The most significant clusters of transcription factor genes were related to embryonic development, cell morphology, and cell growth and proliferation, suggesting conversion of the cell to an immature growth state. State of activity of signaling pathway is represented by Z score (right-tailed Fisher exact test). Red, activation; green, inhibition. Scaling parameters listed at bottom right. n = 3 biological replicates for each day and condition. (K) Pathway analysis supports the emergence of a stem cell-like transcriptional state after 5–7 days in culture. (L) On the very earliest days of culture, cells exhibit marked elevations in cell metabolic, anti-oxidant and glycolytic pathways, indicating an abrupt transition to a state that may foster cell survival after removal from the brain. This state rapidly diminishes. (M), General signaling pathways show early reductions in many trophic and guidance pathways, followed by their recovery starting approximately 3 days after culture as cells extend neurites and form synapses.