The overall goal of this protocol is to induce a general state of cerebral inactivity in vivo to study single neurons integrated properties in the absence of synaptic inputs. The method we present here can help to answer key questions in neurophysiology, such as assessing the impact of the network activity on single cells computation or study the responsiveness of neuron in comatose states. The main advantage of this technique is that neuronal properties can be studied in isolation from the network in an intact in vivo preparation.
To begin this procedure, place a fully anesthetized rat on a heating blanket. Insert a rectal probe in the animal to maintain core temperature around 37 degrees Celsius. To place a catheter in the peritoneal cavity to facilitate subsequent injection of anesthetic agents, first remove the hair over a small area on the stomach's lower right or left quadrant using depilatory cream.
After injecting the local anesthetic, sterilize the surgical site and make a two to three millimeter incision on the skin with sharp scissors. Using blunt scissors, remove the fat and muscle layers until the peritoneal cavity is visible. Then insert about one to two centimeters of a small catheter in the cavity and close the wound with surgical glue.
Make a loop and suture the catheter to the skin to secure it in place. To install a tracheal tube to control ventilation during artificial respiration, remove the hair, sterilize the site and incise the skin over the trachea right above the manubrium. Blunt dissect the first layer of fat and muscles.
Move the salivary gland aside and expose the trachea by gently dissecting the last layer of muscles. Carefully isolate the trachea and slide a thread below it using small forceps. Then make a loose surgeons knot with the thread.
Incise the trachea transversely between two cartilaginous rings. Next, insert a tracheal tube with the appropriate diameter and tighten the thread's knot in order to secure the tube. For additional stability, the thread can be further attached to a higher point of the trachea tube.
Suture or use surgical staples to close the wound. Subsequently, transfer the animal to a stereotaxic frame and apply eye ointment to avoid desiccation. Then place appropriate probes to monitor its physiological variables such as the electrocardiogram, the blood oxygen saturation and the end tidal carbon dioxide pressure.
After that, remove the hair over the scalp and sterilize the surgical site. Make a two centimeters longitudinal incision and resect the connective tissues overlying the skull using a scalpel. Then apply surgical glue on the skull and tissues to stabilize the preparation.
Then make a small craniotomy of 1.5 millimeter diameter over the region of interest with a dental drill. Afterward, use a pair of extra fine forceps and vena scissors to gently make a small hole in the dura. Reserve of 0.5 millimeter region within the cranial trepanation to place the electrocorticographic electrode.
Be sure to keep the cortex moist with 0.9%sodium chloride solution, or aCSF. Next, place a reference electrode on the contralateral scalp muscle, and place a low impedance electrocorticographic electrode on the dura. Adjust the artificial ventilation system so that the respiratory frequency and volume are similar to those of the rat's spontaneous breathing.
If all the physiological variables and electrocorticographic patterns are within the normal values, inject gallamine triethiodide in each leg to paralyze the animal. Then connect the mechanical ventilation to the tracheal tube and verify the proper thoracic cage inflation on both sides. In this step, pull a glass micropipette with a 0.2 micrometer tip and fill it with two molar potassium acetate solution.
Place the pipette in a specific holder with a silver/silver chloride wire to connect the pipette solution to an intracellular amplifier. Then place a silver/silver chloride reference electrode on the rat's neck muscles. Place the pipette above the region of interest.
Slowly insert the pipette into the brain down to the desired depth using the buzz button of the amplifier to clear the pipette if needed. Compensate for the pipette resistance by zeroing the voltage drops in response to current steps using the bridge circuit of the amplifier. Next, use cotton swabs or synthetic absorption triangles to dry the craniotomy, and cover it with silicone elastomer or 4%agarose to reduce brain movements.
Afterward, lower the pipette in one or two micrometer steps until it's resistance increases when approaching a cell. Use the buzz function of the amplifier to penetrate into the neuron. Once the recording is stable, compensate for the electrode resistance by adjusting the bridge parameter while small steps of currents are injected into the neuron.
Then perform the appropriate experimental protocol, such as injecting current intracellularly to extract intrinsic properties of the neuron or perform sensory stimulations. Next, inject a high but infralethal dose of sodium pentobarbital through the intraperitoneal line. Within 15 to 20 minutes, the intracellular and electrocorticographic activities should slow down with intermittent electrical silences to transiently reach the so-called burst suppression profile which progressively collapses to a complete isoelectric state.
It is expected that the heart rate significantly slows down but the SPO2 and EtCO2 should stay relatively steady. Then repeat the stimulation protocol to compare the impact of the network dynamics on the recorded neurons integrative properties. After the cessation of anesthetic injections, the electrical brain activity should fully recover within there to four hours.
This figure shows spontaneous intracellular and electrocorticogram activities in two anesthetic conditions, mimicking the cortical dynamics endogenously generated during the early stages of sleep, or during waking. The sleeplike state is induced by sodium pentobarbital injection, and the waking-like state by fentanyl. In both cases, the subsequent injection of a high dose of sodium pentobarbital resulted in a complete abolition of spontaneous activity in the ECOG and the simultaneously recorded neurons, hence the term isoelectric.
Suppressing the ongoing synaptic activity resulted in a significant study hyperpolarization of the neuronal membrane potential. Current evoked responses can be generated before and after the induction of the isoelectric state. This allows for the comparison of different cerebral activities on the input output transfer function of neurons, expressed as the main evoked firing rate in response to current steps of increasing intensity.
While attempting this procedure, it's important to remember to keep the animal in the best physiological conditions to obtain good quality recording associated with our reliable cerebral dynamics. This approach can be further employed as a model to study neuron's responsiveness in irreversible or reversible deep comatose states.
