Control of Brain State Transitions with a Photoswitchable Muscarinic Agonist

Abstract

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.

Keywords: brain states; light-mediated control; muscarinic acetylcholine receptors; neuromodulation; photopharmacology.

Figure 1

Activation of mAChRs with non subtype‐specific agonist Iperoxo (IPX) evokes neuronal hyperexcitability in cortical slices. A) On the left, the experimental setup: 16‐channel multielectrode array (MEA); WM, white matter; L1‐L6, layer 1–6. On the right, raw local field potential (LFP) traces illustrating network activity showing the increase in oscillatory frequency corresponding to the spectrogram of panel B with 100  × 10^–9 m IPX. B) Spectrogram from the same time recording of LFP traces on panel A: control, 100  × 10^–9 m and periods of seizure‐like discharges. C) Oscillatory frequency (Hz) and FR (a.u.) during the Up‐states in control (ctrl) conditions and after IPX (1, 10, 100  × 10^–9 m; n = 7 ferret brain slices). Muscarinic activation of the brain slice with 100  × 10^–9 m IPX produces a significant increase in oscillatory frequency compared to control conditions, and no significant changes in the firing rate (FR) during Up‐states. Data are reported as mean ± standard error of mean (SEM). Analyses were performed with one‐way ANOVA test (Brown–Forsythe and Welch test, unpaired t with Welch's correction). *p‐value <5 × 10⁻². D) Averaged power spectral density (PSD) of oscillatory activity showing low (<1 Hz), delta (1–4 Hz) and alpha (7–12 Hz) frequency component.

Figure 2

Effect on SO of mAChRs activation by trans‐PAI (dark‐relaxed) and cis‐PAI (pre‐illuminated with UV). A) Chemical structures of trans‐ and cis‐PAI. B) Subtype selectivity of trans‐PAI (3, 10 × 10^–12 m) was studied by comparing the amplitude of fluorescence calcium imaging responses of cells expressing M1 (3 × 10^–12 m, n = 232 cells; 10 × 10^–12 m, n = 232 cells; IPX, n = 258) or M2‐GqTOP (3 × 10^–12 m, n = 70 cells; 10 × 10^–12 m, n = 200 cells; IPX, n = 300) mAChRs, using the procedure described in Riefolo et al. 2019^{[ 37 ]} and by pre‐incubating cells with OGB‐1AM (10 × 10^–6 m for 30 min) as calcium indicator. M2 mAChR transfected cells gave a significantly higher response than M1 mAChR expressing cells (33% for M2 compared to 8% for M1). Data were normalized over the maximum response obtained with the nonselective orthosteric agonist IPX in saturation conditions at 30 × 10^–12 m. These results in cell lines are in agreement with previous reports (Riefolo et al., 2019),^{[ 37 ]} in which higher concentrations were required for cardiac assays in vivo. Bars are mean ± SEM and compared using a t‐test of two samples assuming equal variances. *p < 5 × 10⁻³. C) Raw local field potential (LFP) example recordings showing the different ability of trans‐ and cis‐PAI to increasing the oscillatory frequency. Note that trans‐PAI is a stronger agonist of M2 mAChR than cis‐PAI.^{[ 37 ]} D) Raster plots showing the FR during the Up‐states (color coded) under control conditions and different trans‐ and cis‐PAI concentrations. E) oscillatory frequency (Hz) and FR during the Up‐states (a.u.) of the two different PAI isomers, trans‐ (blue, n = 6 ferret brain slices) and cis‐PAI (pink, n = 6 ferret brain slices) at different concentrations. Data are reported as mean ± SEM. Analyses were performed with multiple t‐test (Mann‐Whitney). Significant differences between cis‐ and trans‐PAI (**p‐value < 10⁻²) are observed in the oscillatory frequency at 100  × 10^–9 m and in the FR at 1  × 10^–6 m. These experiments are aimed at estimating the concentration of effective photoswitching of cortical oscillations, which was set at 200  × 10^–9 m in subsequent experiments (see Figures 3 and 4).

Figure 3

Photocontrol of brain waves in vitro using PAI and direct illumination with white light. A) Representative local field potential (LFP) traces (top) and raster plots of firing rate (FR) during the Up‐states under control conditions, 200  × 10^–9 m of cis‐PAI and 200  × 10^–9 m trans‐PAI after photoconversion with white light (WL) (n = 17 ferret slices) (bottom). B) Representative spectrogram under control condition, 200  × 10^–9 m of cis‐PAI and 200  × 10^–9 m trans‐PAI (WL). C) Oscillatory frequency (Hz) and FR during the Up‐states (a.u.) at 200  × 10^–9 m PAI after pre‐illumination with 365 nm (cis‐PAI), and photoswitching with WL (trans‐PAI). Data are reported as mean ± SEM. Analyses were performed with one‐way ANOVA test (Brown–Forsythe and Welch test, unpaired t with Welch's correction). **p‐value < 10^–2; ***p‐value < 10^–3, ****p‐value < 10^–4. D) Averaged power spectral density (PSD) of oscillatory activity under control conditions, 200  × 10^–9 m of cis‐PAI and 200  × 10^–9 m trans‐PAI after WL activation (color code).

Figure 4

In vivo photomodulation of brain waves. A) Representative raw traces of local field potential (LFP) (top, in mV) and multiunit activity (bottom, in arbitrary units), showing the differences in oscillatory frequency and firing rate (FR) during the Up‐states between the control, 1 x 10^–6 m cis‐PAI (pre‐illuminated with 365 nm), and trans‐PAI after photoswitching with white light (WL). B) Individual (left) and mean (right) quantification of oscillatory frequency (Hz) (n = 8 mice), showing a significant increase upon illuminating cis‐PAI with WL. Data are reported as mean ± SEM. Analyses were performed with one‐way ANOVA test (repeated measures [RM], Geisser–Greenhouse correction—no sphericity—and uncorrected Fisher's LSD). **p‐value < 10^–2; ***p‐value < 10^–3, ****p‐value < 10^–4. *p‐value < 5 × 10⁻². C) The mean quantification of FR during the Up‐states (a.u.) is not significantly affected by illumination of cis‐PAI (n = 8). D) Averaged power spectral density (PSD) of oscillatory activity at different concentrations displays significant enhancement at delta, theta, alpha, and gamma frequency bands after WL activation (left). Mean ± SEM quantification of PSD from D‐left panel (n = 8) (right). Analyses were performed with Friedman test and the Wilcoxon post‐hoc tests corrected for multiple comparisons. *p‐value < 5 × 10⁻².