Xanomeline restores endogenous nicotinic acetylcholine receptor signaling in mouse prefrontal cortex

Cholinergic synapses in prefrontal cortex are vital for attention, but this modulatory system undergoes substantial pre- and post-synaptic alterations during adulthood. To examine the integrated impact of these changes, we optophysiologically probe cholinergic synapses ex vivo, revealing a clear decline in neurotransmission in middle adulthood. Pharmacological dissection of synaptic components reveals a selective reduction in postsynaptic nicotinic receptor currents. Other components of cholinergic synapses appear stable, by contrast, including acetylcholine autoinhibition, metabolism, and excitation of postsynaptic muscarinic receptors. Pursuing strategies to strengthen cholinergic neurotransmission, we find that positive allosteric modulation of nicotinic receptors with NS9283 is effective in young adults but wanes with age. To boost nicotinic receptor availability, we harness the second messenger pathways of the preserved excitatory muscarinic receptors with xanomeline. This muscarinic agonist and cognitive-enhancer restores nicotinic signaling in older mice significantly, in a muscarinic- and PKC-dependent manner. The rescued nicotinic component regains youthful sensitivity to allosteric enhancement: treatment with xanomeline and NS9283 restores cholinergic synapses in older mice to the strength, speed, and receptor mechanism of young adults. Our results reveal a new and efficient strategy to rescue age-related nicotinic signaling deficits, demonstrating a novel pathway for xanomeline to restore cognitively-essential endogenous cholinergic neurotransmission.


Animals
Our colony was maintained by breeding on a C57BL/6 background. Male and female mice were weaned at postnatal day (P)21, separated by sex, and group housed (2-4 mice per cage) in plastic cages with corn cob bedding, houses for environmental enrichment, with ad libitum access to food and water on a 12hr light/dark cycle with lights on at 7:00AM.
For whole-cell patch-clamp electrophysiology, brain slices were transferred to a chamber mounted on the stage of a BX51WI (Olympus) microscope and perfused with oxygenated ACSF at 30˚C at 3-4mL/min. Layer 6 pyramidal neurons were patched in accordance with their size, morphology, and proximity to white matter, as visualized using infrared differential interference contrast microscopy. Recording electrodes were filled with patch solution containing 120mM Kgluconate, 5mM MgCl2, 4mM K-ATP, 0.4mM Na2-GTP, 10mM Na2-phosphocreatine, and 10mM HEPES buffer adjusted to pH 7.33 with KOH. Data were acquired and low-pass filtered at 20kHz with an Axopatch 200b amplifier (Molecular Devices) and Digidata 1440 digitizer and pClamp10.3 acquisition software (Molecular Devices).

Analysis
Analysis was performed in Clampfit 10.3 (Molecular Devices) and Axograph. Raw traces were used for calculating rising slope of nicotinic current response within 50ms of opto-ACh onset to measure fast-onset kinetics. Downsampled traces were used to fit triple exponentials to cholinergic responses.

Supplemental Table S1
Capacitance Input resistance Neuronal intrinsic properties by age group. Table shows mean ± SEM for each intrinsic property in neurons from younger and older groups of mice, as well as the results of the unpaired t-tests.

Supplemental Figure S2
Endogenous regulation of opto-ACh responses by autoinhibition and acetylcholine metabolism is similar across the age groups. (A) Antagonist for M2 autoreceptor increases opto-ACh responses in both age groups: example paired responses before and after muscarinic M2 inhibition with AF-DX116 in a younger and older neuron. (B) Blocking acetylcholinesterase activity increases opto-ACh responses in both age groups: example paired responses before and after acetylcholinesterase inhibition with galantamine in a younger and older neuron. For quantitative investigation into these effects, see Figure 1.

Supplemental Figure S3
Opto-ACh responses are increased by combination treatment of xanomeline + NS9283. Graph shows opto-ACh response of older mice with amplitude normalized to mean response amplitude of the younger mice. Either NS9283 (NS) or xanomeline (xano) alone do not substantially improve older responses (changes are not significant as measured by multiple comparisons analysis), but combination of xanomeline + NS9283 substantially increases older responses, even beyond levels observed in younger mice (multiple comparisons **P < 0.01, ***P < 0.001, ****P < 0.0001).