[Behavioral-electrophysiological observation of the involvement of dopamine D1 receptor of the rat anterior cingulate cortex in the regulation of pain-related emotion].

The present study was aimed to explore the involvement of dopamine D1 receptor of the anterior cingulate cortex (ACC) in the regulation of chronic inflammatory pain-related emotion. On the first day, the rats were acclimated to the environment and the baseline indices were measured. On the second day, the rats were administered with the dopamine D1 receptor antagonist SCH-23390 or agonist SKF38393 in the ACC, and then they were subcutaneously injected with complete Freund's adjuvant (CFA, 0.08 mL) in the left hind paw to establish conditioned place avoidance (CPA) response after pairing with specific environment. On the third day, the CPA response and the firing frequency of ACC neurons were observed synchronously, and the open-field behavior, mechanical pain behavior and paw withdrawal latency (PWL) tests were also observed subsequently. In other experiments, rats were given subcutaneous injection of normal saline (NS) on the left hind paw after SCH-23390 or SKF-38393 was administered in the ACC, and then the same observations were performed. The results showed that: (1) Compared with the control group, the PWL and mechanical pain thresholds of rats injected with CFA on the left hind paw were significantly decreased (P < 0.05); (2) The residence time of rats injected with CFA in the "pain environment" and open field center was significantly shortened (P < 0.05); (3) Pre-injection of antagonist SCH-23390 in ACC (10 µg) alleviated the anxiety-like negative behavior response induced by CFA (P < 0.05) and reversed CFA-induced increases of discharge frequency of ACC neurons (P < 0.05); (4) Pre-injection of agonist SKF-38393 in the ACC (10 µg) induced CPA-like behavioral response in rats injected with NS in the left hind paw, and increased the firing frequency of ACC neurons (P < 0.05); (5) Immunofluorescence detection showed that dopamine D1 receptor and NMDA receptor were co-expressed in the same neuron. These results suggest that inhibition of dopamine D1 receptor in ACC can alleviate the negative emotional response induced by persistent pain.

[A comparative study of microwire electrode array with built-in and external reference electrodes].

Objective: To compare the difference between the built-in and external reference electrode of microwire electrode array in the process of recording rat brain neuron firings, optimizing the production and embedding of the microwire electrode array, and providing a more affordable and excellent media tool for multi-channel electrophysiological real-time recording system. Methods: A 16 channel microwire electrode array was made by using nickel chromium alloy wires, circuit board, electrode pin and ground wires (silver wires). The reference electrode of the microwire electrode array was built-in (the reference electrode and electrode array were arranged in parallel) or external (the reference electrode and ground wire were welded at both ends of one side of the electrode), and the difference between the two electrodes was observed and compared in recording neuronal discharges in ACC brain area of rats. Experimental rats were divided into built-in group and external group, n=8-9. The test indicators included signal-to-noise ratio (n=8), discharge amplitude (n=380) and discharge frequency (n=54). Results: The microwire electrode array with both built-in and external reference electrodes successfully recorded the electrical signals of neurons in the ACC brain region of rats. Compared with the external group, the electrical signals of neurons in built-in group had the advantages of a higher signal-to-noise ratio (P＜0.05), a smaller amplitude of background signals and less noise interference, and a larger discharge amplitude(P＜0.05); there was no significant difference in spike discharge frequency recorded by these two types of electrodes (P＞0.05). Conclusion: When recording the electrical activity of neurons in the ACC brain region of rats, the microwire electrode array with built-in reference electrode recorded electrical signals with higher signal-to-noise ratio and larger discharge amplitude, providing a more reliable tool for multi-channel electrophysiology technology.

[A new method of microglia sorting and functional characteristics of spinal microglia in aged rats].

Objective: To establish an improved method of separating microglia from aged rats and to observe the biological characteristics of spinal microglia of aged rats. Methods: Young SD rats (2 months) were used as control group. Single cell suspension of rat microglia were prepared by trypsin, trypsin substitutes or mechanical net rubbing method. Then, by assessing the purity and survival rate of cells, and observing the morphological characteristics and analyzing the inflammatory functional characteristics, we optimized the isolation and purification method of microglia from aged rats (20 months old) , and observed the functional characteristics of spinal microglia in aged rats. Results: The survival rate of cells digested by pancreatic enzyme was low(young rats 83%, aged rats 60%). Although the survival rate of mechanical net rubbing method was higher than that of pancreatic enzyme digest methods (95%), the cell acquisition rate was lower(young rats(0.207±0.020)×106, aged rats(0.243±0.023)×106). Trypsin substitute dissociation combining density gradient centrifugation method was the best way to get abundant, active and higher survival microglia, and the purity reached more than 85%. We used this method to separate microglia from spinal cord of rats. Compared with the young rats, the spinal cord tissue of old rats was larger, the digestive fluid volume was higher, but the digestion time was shorter. Compared with the young rats, the aged rat spinal microglia had larger and rounder cell body, fewer and shorter protrusions, it tended to be activated morphologically, the level of proinflammatory cytokine IL-1ß of microglia in aged rats was lower, and the level of antiinflammatory factor IL-10 was higher. Conclusion: The method of trypsin substitute dissociation combined with density gradient centrifugation was successfully established to isolate and purify microglia from spinal cord of rats, the spinal microglia of old rats showed anti-inflammatory phenotype.

[A method of marking neurons on brain slices using patch clamp technique].

Objective: To introduce a method of marking neurons using patch clamp technique. Methods: The brain slices of the target area was cut with a vibrating microtome. The glass microelectrode was perfused with the electrode liquid containing NeurobiotinTM Tracer, and the whole-cell patch-clamp recording was performed. After recording, the brain slices were fixed and rinsed with 4% paraformaldehyde. After stained in phosphate buffer with Streptavidin-Texas Red and Triton X-100 for at least 2 hours, the neurons can be observed under a fluorescence microscope. Results: The cell membrane voltage was clamped at -70 mV, and the neuron showed a gradually increasing membrane current after step stimulation. When recording in the current clamp mode, the step stimulus caused the neuron to depolarize to the threshold potential and then burst into action potentials. The morphology of intact neurons with clear cell body and protrusions of a neuron could be observed under a fluorescence microscope. Conclusion: This method is suitable for observing the morphological features of the recorded neuron after patch clamp experiments, which is easy to operate, and the image is intuitive and clear.