Institute of Neuroscience, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China
Chao-Yi Li, Institute of Neuroscience, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China. Email: cyli{at}ion.ac.cn.
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Abstract |
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Introduction |
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Two groups have successfully applied the technique in recordings from neurons of visual cortex in vivo (Pei et al., 1991; Ferster and Jagadeesh, 1992
). Essentially, their approach was to keep the patch-clamp electrode relatively clean of tissue debris by applying a continuous positive pressure during penetration. With the tip of the electrode closely resting on the cell membrane, the positive pressure is released and a small negative pressure is applied to the electrode. The shortcoming of their approach lies in the extensive procedures necessary to minimize respiratory-induced movements of the brain before recordings can be made. These procedures include mounting a hydraulic microelectrode holder on the skull, creating a bilateral pneumothorax, suspending the animal from the thoracic or cervical vertebrae, cementing a metal chamber over the craniotomy, and removing the dura above the cortex. These invasive procedures are not only time-consuming, but may incidentally result in a deterioration of the brain or indeed the general condition of the animal.
We describe a simple method for obtaining stable, in vivo whole-cell recordings in the cat visual cortex. The procedures used in the new method are as simple as those applied for making conventional extracellular recordings and all the additional steps described above, for providing stability of recording, become superfluous. The main element of the new approach is to prevent brain pulsation by retaining the dura. After being treated with an enzyme (collagenase), the dura is shown to be soft enough for the micropipette to penetrate with negligible damage to the tip. With this method, we demonstrate that high-quality intracellular recordings and staining can be obtained for neurons in all cortical layers, including the small cortical neurons in the superficial layers.
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Materials and Methods |
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Experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health, USA (revised 1987). The animal preparation was similar to that used in our extracellular recording experiments (Li and Li, 1994; Li et al., 1999
). Briefly, cats weighing 23 kg were anesthetized with Ketanest (30 mg/kg, i.m.) for initial surgical procedures. The trachea and the femoral vein were cannulated and a bilateral cervical sympathectomy was carried out. Lidocaine was applied to all wound margins and pressure points. To provide anesthesia, paralysis and physiological balance, intravenous infusion of gallamine triethiodide (10 mg/kg per h), urethane (20 mg/kg per h) and glucose (200 mg/kg per h) in Ringer's solution (1.5 ml/kg per h) was administered during the experiment. The animal's head was fixed in a stereotaxic frame. The animal was artificially respired and vital signs (ECG, heart rate, end-tidal CO2, rectal temperature) were continuously monitored. The nictitating membranes were retracted, the pupils dilated, the corneas protected by contact lenses, and refractive errors were corrected using spectacle lenses. Artificial pupils, 3 mm in diameter, were placed in front of the eyes.
Enzymatic Treatment of the Dura Using Collagenase
Our objective was to prevent brain pulsation by keeping the dura intact, and to allow the tip of a micropipette to penetrate the dura without being damaged and record intracellularly from the cortical cells. Collagenase was used to degrade collagen fibers, which make up most of the dura. A small hole (1.5 mm diameter) was made in the skull over the recording site in the striate cortex (area 17) and the exposed dura was treated with purified collagenase (Sigma Chemical Co., St Louis, MO). The enzyme was applied with a small piece of filter paper (the same size as the hole) saturated with the enzyme solution (50 mg/ml) on top of the dura for 2030 min. The collagenase-containing filter paper was then removed, and the area was rinsed thoroughly with physiological saline. At this stage, thinning of the dura became apparent. Multiple or consecutive recording sites can then be studied. Holes can be filled with wax or agar upon completion of recording for those sites.
Electrode Preparation and Penetration of the Dura
Using a programmable micropipette puller (APP-1, Stoelting Co.), electrodes were pulled to a tip size of 11.5 µm with a resistance of between 5 and 10 M, using thin-walled borosilicate glass capillaries containing fibers (1.0 mm o.d. x 0.78 mm i.d., GC100TF-10, Clark Electromedical Instruments). It is recommended that the electrodes are prepared just before carrying out the experiment. They are prepared with a 10 s pre-pull in advance of the application of the pull force. During this period, the glass is heated to a stable equilibrium temperature, in order to achieve constant results. The electrode tip produced in this way, which proved to be most suitable for in vivo whole-cell patch recordings, is illustrated in Figure 1
. Electrodes (with an inner filament) were back-filled with a solution buffered to pH 7.4 containing (in mM) KCl 90, NaCl 10, potassium EGTA 5 and HEPES buffer 10. For subsequent intracellular staining of a neuron, the solution also contained 24% biocytin.
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To avoid damage to the fine tip, it is important that insertion of the electrode should be perpendicular to the surface of the cortex, and the agar should be placed in position before the electrode touches the dura. When the dura was sufficiently digested, the electrode could pierce it without difficulty. In our experiment, only one or two electrodes might be damaged at the beginning of the first penetration. Repeated damage of the electrode tip indicates insufficient digestion of the dura. We checked the tip of the recording electrode under microscope before and after each penetration. As a rule, neither debris build-up nor damage of the tip was seen after the successful recordings.
Whole-cell Recordings In Vivo
While maintaining a positive pressure (35 kPa), the electrode pipette was advanced slowly into the cortex to search for visually responsive cells. When the large action potentials changed from being bipolar to unipolar, the positive pressure was reduced to 12 kPa, and the resistance was continuously monitored with current pulses (0.1 nA, 10 ms, 1 pulse/s). Close contact with a neuron is recognized by an increase in the resistance of the electrode and/or a sudden increase in the spontaneous discharge rate. At this point, the positive pressure is released and a small negative pressure (0.51.0 kPa) is applied. This often results in a gradual entry into the cell interior, as is indicated by a slow increase in membrane potential. When the membrane potential becomes stable, at a value between 30 and 60 mV, the negative pressure is released, in order to avoid sucking in the intracellular content. Sometimes, single positive current pulses (100 µS, 510 nA) were used to aid the penetration of the cell membrane. On average, intracellular recordings were usually maintained for more than 1 h, the longest recording time lasted for 3 h which was ended by intracellular injection. The experiment normally lasted 45 days, and three to five cells could be recorded, labeled and identified subsequent to the experiment, after the biocytin injection.
Intracellular Injection of a Dye
On completion of functional tests with intracellular recording, the cell was injected with biocytin (Horikawa and Armstrong, 1988) by passing negative current pulses, 0.51.0 nA in a 100 ms on/200 ms off cycle, for a period ranging from 10 to 30 min. During current injection, the normal responsiveness of the cell was continuously monitored using a visual stimulus. It is important to retain the normal responsiveness of the cell during the injection process. Then the electrode was withdrawn slowly from the cell interior, and the responsiveness of the cell was checked again extracellularly. For the next penetration, a new electrode should be used and the recording site should be placed at least 1 mm away from the previous penetration.
Histological Procedures
At the end of the experiment, the animal was deeply anesthetized and perfused through the heart; first with 0.9% saline and then with 2% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.3. Blocks of tissue containing the injected cells were removed and post-fixed overnight by submersion in cold (4°C) 4% paraformaldehyde in PBS. The tissue was sectioned on a vibratome at a thickness of 80 µm. The sections were thoroughly washed in PBS followed by Tris-buffered saline (TBS), and were pretreated for 6 h in a 0.5% solution of Triton X100 in PBS. Injected cells were identified by incubating the tissue overnight in the avidinbiotinHRP complex (4°C) in dilution 1:2500 in PBS. The enzymatic reaction was revealed with diaminobenzidine (DAB) (0.06%) and H2O2 (0.003%) in 0.15 M Tris buffer (40°C) for 15 min. The sections were thoroughly rinsed and mounted onto gelatin-coated slides.
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Results |
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Clearly, collagenase digestion appeared to have little effect on the parenchyma of the cortex. As shown, the morphology of cortical neurons appeared to be normal. Moreover, at the site of entry of the electrode, there was also no evidence of damage to neural tissue by the dimpling effect on the entry of the electrode.
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Discussion |
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By applying collagenase locally over the dura at the recording site, we have shown that the dura can be transformed from an impermeable barrier into a permeable tissue for the inserted micropipette. The effect is caused by the enzymatic digestion of the dura resulting in a change in the tissue laxity shown as tissue expansion at the site of application in the histological sections. The method, therefore, effectively eliminates not only the need for opening the dura, but also surgical procedures that were either associated with the open-dura approach or used to reduce the tissue pulsations. The retention of the dura resulted in a significantly improved stability, and the rate of successes, in obtaining high-quality whole-cell recordings while still allowing the dura to act as a physical barrier and to keep the integrity of the cerebralspinal fluids and microenvironment surrounding the brain tissue.
Collagenase is known to be a proteinase with a very high degree of specificity for its substrates which comprise only collagen and gelatin (Nagai, 1961). This enzyme has been widely used with in vitro studies for primary disaggregation of adrenal and brain tissue. Cells can withstand prolonged exposure to this enzyme apparently without causing significant damage (Lasfargues and Moore, 1971
; Freshney, 1987
). Despite the fact that collagenase is known to be generally non-cytotoxic, there are still concerns that the highly concentrated enzyme may cause damage to the brain tissue. With regard to cell morphology, earlier studies have demonstrated that isolated neurons from rat cerebral cortex retained their synaptic boutons on the plasma membrane in the presence of collagenase in the perfusion medium (Huttner et al., 1979
). In addition, presynaptic terminals with mitochondria, vesicles and synaptic cleft could also be observed. These isolated neurons of the mammalian central nervous system have maintained well-preserved morphology and electrochemical membrane properties of the voltage-dependent channels after the enzymatic treatment (Kaneda et al., 1988
).
We believe that in our study the adverse effect by the applied enzyme on the cortical tissue, if there is any, can be considered insignificant. This is supported by the fact that the cells that have been recorded and stained from the most superficial layers, which are supposed to be most vulnerable to the enzymatic action, appeared to be normal both morphologically and functionally. As illustrated in Figures 3, 4 and 5, they exhibited long dendrites with numerous processes and spines, and extensively projected horizontal axons that could be traced for several millimeters within the superficial layer. In addition, normal responsiveness and stimulus selectivity can also be shown from these cells. Similar to what has been shown in the previous studies, our method is also effective for recording of cells in the deeper layers, by applying a continuous positive pressure to the micropipette during penetration.
With some of the surgical procedures such as bilateral pneumothorax being avoided and the dura kept intact, the physiological condition of the cortical tissue can be maintained relatively stable over a longer period of time to allow continuous intracellular recordings to be carried out on the animal for up to 5 days. The benefit of being able to sustain a stable condition over a long period of time for intracellular recording of cortical neurons in an in vivo preparation is especially significant when intracellular injection of dyes is among the study objectives. Adequate time can be taken to allow the dyes to diffuse up to the smallest axon collaterals. Our results show that cells could be labeled and well identified, with a success rate of 35% on average, 14 days after intracellular injection of biocytin. In addition, cells sampled over a period of 5 days showed apparently normal tuning characteristics.
To summarize, our method greatly simplifies preparatory procedures, while at the same time improving the general condition of the cortex, as a result of the retention of the protective dural membrane. While the method was developed for the specific purpose of making intracellular recordings from the visual cortex, it can be applied more generally to other cortical structures. The method therefore has a broad potential for application in neurophysiological research.
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Acknowledgments |
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References |
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