Animal models with transgenic labels in multiple cell typessuch as neurons with expression of the calcium indicator GCaMPcould be crucial in understanding the complex effects the coating is playing . probes at similar velocities. After the processes made Rabbit Polyclonal to Actin-beta contact with the probes, microglial processes expanded to cover 47.7% of the control probes surfaces. For L1-coated probes, however, there was a statistically significant 83% reduction in microglial surface coverage. This effect was sustained through the experiment. At 6 h post-implant, the radius of microglia activation was reduced for the L1 probes by 20%, shifting from 130.0 to Muscimol 103.5 m with the coating. Microglia as far as 270 m from the implant site displayed significantly lower morphological characteristics of activation for the L1 group. These results suggest that the L1 surface treatment works in an acute setting by microglial mediated mechanisms. [98C101]. In the context of neural implants, our group has shown that covalent attachment of brain tissue derived L1 to neural probes can reduce glial Muscimol scarring, while simultaneously encouraging neuronal attachment to the probes surface for at least 2 months post-implant [61,76,77]. While these studies suggest L1 can modify the behavior of glial scars, the mechanism behind this is unclear. In the present work, we use two-photon microscopy (TPM) to study the dynamic microglial response to L1 coated microelectrodes for the first 6 h post-implant in living mice, as the first step to uncover the mechanisms. Compared to uncoated microelectrodes, there was significantly less microglial coverage of the L1 probes from 8 minC6 h post-implant, despite similar degrees of microglial process extension toward both coated and control probes. This suggests that L1s mechanism for preventing glial attachment and scarring occurs rapidly after initial contact. 2. Methods 2.1. Neural probes and L1 protein immobilization All studies were performed using four-shank NeuroNexus 16-channel, 15 m thick, 3 mm long SOI silicon probes (NeuroNexus Technologies, Ann Arbor, MI) mounted on dummy boards. For quantitative analysis, L1 immobilization was conducted along the entire shank of the probes (n = 7), and all control probes (n = 7) were pristine, uncoated arrays that were washed with ethanol and phosphate buffer solution. L1 immobilization on the silicon dioxide surface and iridium oxide electrode pads were carried out as previously described with minor modifications [77,99]. Briefly, probes were cleaned and functionalized with either HNO3 (Sigma Aldrich) or by serial washes in acetone, 50% (v/v) MeOH/ H2O, and chloroform before oxygen plasma cleaning (30W) for 1 min (Harrick Plasma, PDC-001) . Probes were silanized by immersion in 2% (3-mercaptopropyl) trimethoxysilane (Sigma Aldrich) solution with 4-maleimidobutyric acid N-hydroxysuccinimide ester (2 mM, Sigma Aldrich) for 1 h. Finally, probes were fully immersed in a 100 (g/ml solution of purified L1 protein (purified at our lab) for 1 h at 4 C, and stored in sterile 1 phosphate buffer solution (Sigma Aldrich) until implantation. In an additional validation experiment (n = 1), following silanization of the probes full surface, the probe was dipped only ~150 m in the L1 solution. This half-coating design allowed for comparison between L1 and no L1 conditions on the same probe (Supplemental Fig. 1). The L1 modified probes were stored Muscimol in saline for up to 1 h prior to implantation. Previous studies have shown that the L1 coating procedure yields a uniform 6.37 nm thick coating with 0.53 g cm?3 density and increased hydrophobicity (water contact angle: 69.8 1.7 for L1 coated v. 27.3 1.4 for unmodified control) . 2.2. Surgery and probe insertion Surgical procedures were conducted as previously described with 14 adult CX3CR1-GFP transgenic mice with GFP expression in macrophages and microglia controlled by the CX3CR1 promotor (Jackson Laboratories, Bar Harbor, ME) . A cocktail of intraperitoneally (IP) administered ketamine/xylazine (90/8 mg kg?1) was used to induce anesthesia, with depth of anesthesia assessed by monitoring the toe-pinch response, breathing, and heart rate. After animals were secured in a stereotaxic frame, scalps were shaved, cleaned with 70% ethanol, and.