CHAPTER 2: MATERIALS AND METHODS 36
2.5 WHOLE-‐‑CELL PATCH CLAMPING ISOLATED CELLS 40
The preparation of isolated cells was kept in the recording chamber for the cells to settle to the bottom of the recording chamber, where they adhere to the glass surface. Unhealthy cells float and therefore are lost during the perfusion.
Electrophysiological recordings were made from isolated cells suspended in the New Ringer solution being perfused through the bath chamber (see Figure 1 for composition) at 1.5-‐‑2 ml/min. Patch pipette were made of borosilicate glass
capillaries of 1.5 mm diameter (World Precision Instruments, USA), pulled with a laser micro pipette puller (Sutton Instrument P-‐‑2000). Pipettes were differential sizes for neurons (4-‐‑5 MΩ) and astrocytes (6-‐‑8 MΩ) and filled with intracellular solution (see Figure 1 for solution composition). The AxoPatch200B patch-‐‑clamp amplifier (Axon Instruments, USA) was used to observe the currents and those were filtered at 2 kHz and digitized at 4 kHz. To control the experiments the data acquisition board PCI-‐‑6229 (NI, USA) was used. Cells with input resistance of 500-‐‑
A
B
1100 MΩ and 50-‐‑150 MΩ for neurons and astrocytes respectively, with less than 20% variation throughout recordings, were used for the analysis (Pankratov et al. 2007; Pankratov et al. 2009).
2.5.1 Cell identification: astrocytes vs neurons
Astrocytes and neurons were distinguished by their corresponding morphology and input resistances. Figure 2.2 shows examples of the typical morphologies displayed by astrocytes and neurons (A and B respectively) and the currents evoked from a series of voltage steps (C and D). Morphologically, the neuronal cell body is much larger than that of a cortical astrocyte. Upon the isolation process neurons normally keep their axons intact, whereas astrocytes tend to lose their fine projections and are left mostly with the soma.
Electrophysiologically, the main difference between neurons and astrocytes is the inward sodium current, which is absent in astrocytes (Perea and Araque 2010). Astrocytes can also be distinguished from oligodendrocytes for the same reason – oligodendrocytes display a small inward sodium current (Fields 2008). Compared to published data, the currents recorded in isolated astrocytes are smaller than reported values (Bekar 2004). This is most likely due to the loss of fine projections that contain a high number of channels, and thus correspond to the overall
current. Currents recorded from slices, however were similar in size to the published values (Pannicke et al. 2000).
Figure 2.2: Isolated neurones and astrocytes have distinctive electrophysiological and
morphological properties. A-‐‑ Isolated astrocyte (right) and neurone (left), B – typical I/V curves from isolated neurone (black) and astrocyte (blue), C – typical currents of astrocyte and neurones in response to depolarising voltage steps from holding potential -‐‑80 mV, D -‐‑ Inward sodium current of an isolated neurone.
2.5.2 Patch clamping in slice
WinFlour software (Strathclyde University, UK) was used to record the currents from layers II/III of somatosensory cortical astrocytes in coronal slices. A protocol with 9 consecutive voltage steps, from -‐‑130 mV to +30 mV, from a holding
potential of -‐‑50 mV, was used to record both the inwardly rectifying potassium current, which is most prominent at the lowest 2-‐‑3 steps of this protocol and the voltage gated potassium current, which is considered to be the dominant subtype
10μM 5μM
A
B
C
D
-150 -100 -50 50 100 -500 500 1000 1500 -80mV +40mV Astrocytic currents Neuronal currents Cu rre n t / p A Voltage / mV 0.6 ms 0.4 nA -80mVof current at the highest voltage steps of this protocol. Another protocol, with one voltage step from the holding potential of -‐‑80 mV to -‐‑70 mV was used for
calculating the capacitance and for controlling the health of the patched cell.
2.5.3 Patch clamping isolated cells
For isolated cells WinWCP (Strathclyde University, UK) was used to record from isolated neurons and astrocytes. Four protocols were used to gather electrical currents from cells.
Firstly, to watch the health of the experimental cell, a single 10 mV step protocol (from a holding potential of -‐‑80 mV to -‐‑70 mV) was recorded throughout the experiment. The capacitance of the cell and input resistance was calculated at the start of the experiment. As stated above, the cells with greater variation than 20% from the normal range (50-‐‑150 MΩ for astrocytes and 500-‐‑1100 MΩ for neurons) were not used for data analysis. The experimental leak was also estimated from this protocol; any experiments with abrupt leak changes were excluded from the data collected.
Figure 2.3: Voltage protocol used in the study. A – Protocol to record sodium current in neurones and total potassium current in astrocytes. B – Protocol to record inwardly rectifying potassium current. C – Protocol with a pre step to deactivate inwardly rectifying potassium current and measure voltage-‐‑gated current.
Figure 2.3 illustrates structure of protocols, used to record currents in this study. A series of voltage steps were used to record sodium current in neurons were also utilised to test for the absence of a sodium current in astrocytes: the holding potential was kept at -‐‑80 mV, to keep the glial cell healthy, and 12 voltage steps were recorded from -‐‑70 mV to +40 mV. The current recorded at the highest voltage steps of this protocol is referred to as total potassium current throughout this thesis.
-70 mV
+40 mV
-130 mV
+70 mV
-70 mV
+40 mV
-20 mV
-50 mV
-80 mV
C
B
K
AK
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ir-80 mV
total
A
Thirdly, a protocol that recorded both inwardly rectifying and voltage gated potassium currents consisted of 11 voltage steps from -‐‑130 mV to +70 mV from a holding potential of -‐‑50 mV. At lowest voltage steps the majority of the current is conducted through the inwardly rectifying potassium channels, whereas the highest voltage steps display mainly the voltage sensitive potassium current. To exclude the influence of the inwardly rectifying potassium channel, another protocol with a deactivating pre-‐‑step was devised. The holding potential was -‐‑80 mV and again 12 voltage steps were recorded from -‐‑70 mV to +40 mV but with a -‐‑ 20 mV pre-‐‑step.