APLICACIONES CORPORATIVAS

Here, we wanted to test if PRV-152 infection alters the electrophysiological proper- ties of infected neurons. We stereotactically injected 400µl of virus solution into the IC of P15 Mongolian gerbils. Brain slices for patch clamp recordings were obtained 12 hpi to ensure only 1st _{order infected neurons were recorded. Patch-clamp record-}

ings were taken from cells with different intesities of eGFP expression. Images of recorded cells were taken before patching, during on-cell mode and after whole-cell recording when the cell was filled with Alexa568 hydrazide (see fig. 4.10). Due to the exchange of intracellular solution no double fluorescence was detectable after the recording. Therefore, the comparison of the three afore mentioned images was used to confirm that recorded cells were PRV-152 infected.

All recordings were done in current-clamp mode with no pharmacological interac- tion. AP thresholds were probed using stepwise increases (50-150 pA) of an 1 ms current injection (see fig. 4.11 A, same cell as in fig 4.10). To determine the firing rate currents of 500 to 1000 ms duration were injected, which were increased in steps of 50, 100 or 200 pA (see fig. 4.11 B, same cell as in A). The input resistance was

analyzed using small current injections of 5 pA with a duration of 250 to 1000 ms (see fig. 4.11 C, same cell as in A). The responses were averaged for each cell (black trace) and exponential functions were fitted to on- and offset phases (white lines) to determine the membrane time constant. Cell capacitance was then calculated on the basis of both experimentally determined values.

While all control cells recorded in the IC were excitable, in some 1st order infected cells in the IC no APs could be evoked by using the relevant test current (see fig. 4.11 D). Most of these non-excitable cells showed an atypical, not exclusively passive response pattern to current injections (see fig. 4.11 D-F, n=5). In the example recording, the AP threshold test protocol could not elicit an AP. However, higher amplitude current injections activated a conductance that prolonged the repolarisation phase (see fig. 4.11 D). The IV-curve of this example shows a small and fast onset response (see fig. 4.11 E) with an articulated kink in the rising phase (see fig. 4.11 F). This indicates that this response to longer current injections is actually an AP, although previously, using the AP-threshold tester, no APs could be induced in this cell. One of the 1st order infected non excitable cells showed a totally passive repsonse behaviour as illustrated in figure 4.13 A and B for a 2nd

40 mV 5 ms 40 mV 100 ms 2 mV 200 ms

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Figure 4.11: Basic membrane and firing properties in 1st order PRV-152 infected IC cells I, A

AP threshold test in PRV-152 infected neurons was performed using 50-150 pA stepwise increases of a 1 ms current injection. Sub- and suprathreshold traces are depicted in black. Same cell as shown in figure 4.10B Firing rate in PRV-152 infected neurons was determined by a 500-1000 ms current injection increased in steps of 50, 100 or 200 pA. First and suprathreshold traces are depicted in black. Same cell as in ACTo determine the input resistance small current injections of 5 pA and 250- 1000 ms duration were applied. Responses were averaged for each cell (black trace) and exponential functions were fitted to on- and offset phases (white lines) to determine the membrane time constant. Same cell as in AD AP threshold test in eGFP expressing cell which did not fire APs. E Current injections of 1000 ms duration lead in the same cell to an onset AP.FExtension of onset AP in E. Arrow marks the inflection in the upstroke, which indicates that this event is actually an AP. Slices for recordings were taken 12 hpi.

Figure 4.12 A shows that 64.71 % of infected neurons (n=17) were excitable while 35.29 % of 1st _{order PRV-152 infected cells in the IC responded only passively to}

current injections (n=1) or showed responses similar to the one depicted in figure 4.11 D-F (n=5). Non eGFP expressing IC cells in the infected brain slices were recorded as control and showed an excitability of 100 %. Care was taken to choose control cells, which were located close to infected cells. To see if there are any dif- ferences in the basic membrane properties of excitable and non-excitable 1st order infected cells both cell populations were displayed separately and compared to the control population (see fig. 4.12 B-E). The average membrane resting potential was

100 80 60 40 20 0 excitable cells (%) PRV control 2.0 1.5 1.0 0.5 0.0

current threshold (nA)

PRV control 60 40 20 0 time constant (ms)

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-80 -60 -40 -20 0 E Rest (mV)

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1.0 0.5 0.0 R input (G Ω)

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200 150 100 50 0 capacitance (pF)

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Figure 4.12: Basic membrane and firing properties in 1st_{order PRV-152 infected IC cells II, A}

Excitability in percent of 1st_{order infected cells (n=17). Non fluorescent neurons located close to PRV-} 152 infected cells were recorded as a control population (n=14). BResting potential,Cmembrane time constant,Dinput resistance andEcell capacitance are shown for excitable infected cells (PRV, n=11), non-excitable infected cells (n=6) and control (n=14).FCurrent threshold is plotted for excitable infected cells and control. Bar graphs show average and SEM. None of the differences seen in basic membrane and firing properties (B-C) was significant (p > 0.05).

similar for all three cell groups. In excitable infected neurons the average membrane potential was -60.08 ± 3.43 mV (see fig. 4.12 B). It was slightly depolarized com- pared to non-excitable (-68.74 ± 4.61 mV) and control cells (-66.89 ± 1.95 mV). The membrane time constant showed also only minor differences between the cell populations. It was 16.71 ± 4.34 ms in excitable, 23.6 ± 9.44 ms in non-excitable and 13.24±2.83 ms in control cells (see fig. 4.12 C). Cells’ average input resistance ranged between 0.21 ± 0.05 GW for control and 0.45 ± 0.21 GW for non-excitable cells (see fig. 4.12 D). Negligible changes in cell capacitance (70.27 ± 8.54 pF to

82.4 ± 11.9 pF) indicated that cell size (see fig. 4.12 E) and AP threshold (see fig. 4.12 F) were also unaffected in PRV-152 infected neurons 12 hpi. None of these minor differences depicted in figure 4.12 B-F were significant (p>0.05). The only significant change in infected IC cells apparent 12 hpi was the high number of non-excitable cells (35.29 %).