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. 2017 Oct 13;6:e29808. doi: 10.7554/eLife.29808

Figure 1. PSAM-mediated chemogenetic activation of LC neurons in vivo.

(A) Strategy using direct stereotaxic injection of lentiviral vector to express the excitatory ionophore PSAM in noradrenergic LC neurons. (B) Selective transduction of LC demonstrated by immunohistochemistry (IHC) for EGFP and dopamine β-hydroxylase (DBH) with 690 EGFP+ neurons per LC of which 98% were DBH+ (scale bar 100 µm). (C) PSAM expression was demonstrated using IHC for the HA tag. (scale bar 15 µm) (D) Schematic of PSEM308-mediated excitation of transduced neurons expressing PSAM. Patch clamp recordings from EGFP+ LC neurons in acute pontine brain slices. Perfusion of PSEM308 evoked concentration-dependent inward currents. (E) PSEM308 (3 µM) increased rate of firing of transduced LC neurons. Inset shows 16 overlaid action potentials. Group data shows increase in firing produced by PSEM308 (3 μM). (F) Extracellular recordings from LC neurons in anaesthetised rats using multi-barrel recording electrodes allowing local pressure ejection of PSEM308/saline/pontamine sky blue (PSB). Traces show graded excitation of an identified LC neuron by PSEM308. Recording sites were subsequently histologically identified by the PSB staining within the LC (transduced cells identified by IHC for EGFP (black arrowheads) and DBH (white arrowheads). The response to local PSEM308 was categorised as excitation or inhibition if it changed firing rate by more than 3 SD from the baseline rate. Application of PSEM308 produced an excitation in 50% of LC neurons. We found a second group of neurons that showed no response, presumably as they were not transduced. A third group showed an inhibition of spontaneous firing in response to local PSEM308 application. (G) Kinetics of the excitatory response to systemic PSEM308 administration (10 mg/kg i.p). (H) Timeline of conditioned place aversion protocol to assess influence of chemogenetic activation of LC neurons on behaviour. In PSAM expressing rats, PSEM308 (10 mg/kg) caused conditioned place aversion but had no effect on control animals. Representative heat maps of rat position in the pre-test and post-test after PSEM308 with bilateral LC transduction with LVPRS-EGFP-2A-PSAM HA or control LVPRS-EGFP All data analysed with repeated measures ANOVA (one or two way as appropriate) with Bonferroni’s post hoc testing (*p<0.05, **p<0.01). (See also Figure 1—figure supplements 1, 2 and 3)

Figure 1—source data 1. Figure 1 source data.
elife-29808-fig1-data1.xlsx (25.9KB, xlsx)
DOI: 10.7554/eLife.29808.006

Figure 1.

Figure 1—figure supplement 1. Generation of a traceable version of the PSAM in an expression cassette with enhanced fluorescence.

Figure 1—figure supplement 1.

(A) Schematic of the PSAM (PSAML141F,Y115F:5HT3 HC) and EGFP co-expression plasmid. Top row shows 4 EGFP+ transfected and 10 EGFP- PC12 cells. The bottom row shows [Ca2+]i increases in the transduced cells to the selective agonist PSEM89s. [Ca2+]i measured by Fura2 340:380 fluorescence ratio - F2R. (B) [Ca2+]i responses to PSEM89s in 4 EGFP+ and 5 EGFP- PC12 cells. (C) The maximum Ca2+ transient evoked by PSEM89s is sensitive to the length of C-terminal tag on PSAM. 18 amino acids remain at the C-terminal end of PSAM when the 2A peptide is downstream of PSAM. This C-terminal residue reduced the maximum PSEM89s-evoked response. There was no difference between the original PSAM-IRES-EGFP plasmid and the HA-tagged expression cassette EGFP-2A-PSAMHA (p<0.0001 Kruskal Wallis test with Bonferroni’s multiple comparison NS p>0.05, **p<0.01, ****p<0.0001). (D) The C-terminal HA tag does not interfere with the concentration response relationship (p=0.6384 sum-of-squares F test). EC50: PSAM-IRES-EGFP - 3.37 ± 0.04 vs EGFP-2A-PSAMHA - 3.86 ± 0.09 µM. (E) The upstream EGFP-2A sequence produces a significantly brighter fluorescent signal than the IRES linker (Mann-Whitney test p<0.0001). Calcium transients are shown as mean ± SEM and fluorescence data as median with interquartile intervals. N equals the number of cells analysed from at least three replicated experiments.
Figure 1—figure supplement 2. Transduction with PSAM does not alter LC neuronal electrophysiological properties.

Figure 1—figure supplement 2.

(A) Whole cell current clamp recordings from LC neurons in pontine slices 2 weeks after in vivo transduction with LVPRS-EGFP-2A-PSAM. (B) Schematic of action potential analysis. All spike measurements are taken with reference to the AP threshold defined as the point where velocity of depolarisation exceeded 7.5Vs−1. Data from 25 action potentials were averaged per cell. Input resistance was measured in voltage clamp from the average of 10 current responses to a 10 mV membrane hyperpolarization (C) No quantitative difference was observed in the intrinsic electrical properties between transduced (N = 7) and non-transduced (N = 8) LC neurons (unpaired t-test).
Figure 1—figure supplement 3. Simultaneous recording of one excited and one inhibited LC neuron in vivo with focal PSEM308 (1 mM) pressure application.

Figure 1—figure supplement 3.