Scopolamine rapidly increases mammalian target of rapamycin complex 1 signaling, synaptogenesis, and antidepressant behavioral responses

Abstract

Background: Clinical studies report that scopolamine, an acetylcholine muscarinic receptor antagonist, produces rapid antidepressant effects in depressed patients, but the mechanisms underlying the therapeutic response have not been determined. The present study examines the role of the mammalian target of rapamycin complex 1 (mTORC1) and synaptogenesis, which have been implicated in the rapid actions of N-methyl-D-aspartate receptor antagonists.

Methods: The influence of scopolamine on mTORC1 signaling was determined by analysis of the phosphorylated and activated forms of mTORC1 signaling proteins in the prefrontal cortex (PFC). The numbers and function of spine synapses were analyzed by whole cell patch clamp recording and two-photon image analysis of PFC neurons. The actions of scopolamine were examined in the forced swim test in the absence or presence of selective mTORC1 and glutamate receptor inhibitors.

Results: The results demonstrate that a single, low dose of scopolamine rapidly increases mTORC1 signaling and the number and function of spine synapses in layer V pyramidal neurons in the PFC. Scopolamine administration also produces an antidepressant response in the forced swim test that is blocked by pretreatment with the mTORC1 inhibitor or by a glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonist.

Conclusions: Taken together, the results demonstrate that the antidepressant actions of scopolamine require mTORC1 signaling and are associated with increased glutamate transmission, and synaptogenesis, similar to N-methyl-D-aspartate receptor antagonists. These findings provide novel targets for safer and more efficacious rapid-acting antidepressant agents.

Keywords: Acetylcholine; GABA; depression; glutamate; ketamine; synaptic plasticity.

Figure 1. Muscarinic receptor antagonist administration increases mTORC1 signaling in rat frontal cortex

Rats were administered scopolamine (25 µg/kg, i.p.) or telenzapine (3 mg/kg, s.c.) and levels of mTORC1 signaling were determined 1 or 2 hr as indicated. Levels of the phosphorylated and activated forms of mTORC1, ERK, Akt, S6K were determined by western blot analysis. Levels of total mTORC1 and GAPDH were determined to control for loading differences. (a) Representative western blot images for each signaling protein are shown. (b) Results were quantified and are the mean ± SEM, percent of control (n = 6 animals; *P < 0.05, compared to control, Student’s t-test).

Figure 2. Scopolamine administration rapidly increases EPSC responses in PFC layer V pyramidal cells

Rats were administered scopolamine and 24 hr later slices of PFC were prepared for whole-cell recordings followed by neurobiotin labeling. (a) Sample whole cell voltage-clamp traces of 5-HT and hypocretin-induced EPSCs in slices from saline control or scopolamine treated rats (24 hr post drug treatment). (b) Frequency of 5-HT- and hypocretin-induced EPSCs (*p < 0.05 and **p < 0.01 Student’s t-test) (c) Cumulative probability distributions showing that scopolamine administration exposure increases the amplitude of EPSCs (P < 0.001 for 5-HT, Kolmogorov-Smirnov-test z value =3.65; p < 0.001 for Hcrt, Kolmogrov-Smirnov-test z value = 3.83) (n = 18 neurons for 5-HT group; n = 15 neurons for Hcrt group).

Figure 3. Scopolamine administration increases spine density and maturation in PFC layer V pyramidal cells

After whole-cell recordings slices were subjected to post hoc two-photon microscopy image of the neurobiotin-labeled layer V pyramidal cells. (a) Representative images are shown of high magnification Z-stack projections of distal and proximal segments of the layer V pyramidal cell apical tuft dendrites (Scale: 10 µm). (b) The density of spines was analyzed using automated software (MBF Bioscience, Neurolucida V10/Autospine) and the results are the mean ± SEM (12 cells from 5 rats for control and 14 cells from 6 rats for scopolamine). (c, d) Quantification of distal and proximal spine head diameter, and cumulative fraction curves; note the increase in population of large diameter, mushroom spines in the scopolamine group as compared to the saline controls *p < 0.05; **p < 0.01; Student’s t-test).

Figure 4. Behavioral actions of scopolamine in the FST require mTORC1 signaling and glutamate-AMPA receptors

Rats were administered scopolamine or telenzapine at the doses indicated and immobility in the FST was determined 24 hr later. (a) Both scopolamine and telenzapine significantly decreased immobility in the FST at the same doses that increased mTORC1 signaling and synaptogenesis. Values represent mean ± SEM (n = 3 per group (Sco) and n=6 per group (Tel); *P < 0.05 compared to control. (b) Pretreatment with the selective mTORC1 inhibitor rapamycin (0.2 nmol, i.c.v., 30 min before scopolamine) completely blocked the antidepressant effects of scopolamine (25 µg/kg, i.p.). Values represent mean ± SEM (n = 7 per group; *P < 0.05 compared to scopolamine alone (ANOVA). (c) Pretreatment with the selective AMPA receptor antagonist NBQX (10 mg/kg, i.p. 30 min before scopolamine) completely blocked the antidepressant effects of scopolamine (25 µg/kg, i.p.). Values represent mean ± SEM (n = 9; *P < 0.05 compared to control by ANOVA.

Figure 5. Model depicting the cellular mechanisms for the synaptogenic actions of scopolamine

Scopolamine rapidly increases the number, maturation, and function of spine synapses in pyramidal neurons in the medial PFC. This could occur via increased glutamate transmission, or a glutamate burst resulting in a long-term potentiation (LTP) like synaptogenic effect. The induction of glutamate could occur via blockade of muscarinic receptors located on GABAergic interneurons, acetylcholine (Ach) terminals, or even glutamate terminals. In addition, postsynaptic muscarinic receptors that couple to protein kinase C (PKC) can produce long-term depression (LTD), and blockade of these receptors could enhance the synaptogenic response to glutamate. Scopolamine also increases mTORC1-p70S6K (S6K) signaling resulting in increased translation of synaptic proteins, including AMPA receptors, which are required for the expansion and stabilization of spines. The antidepressant behavioral responses to scopolamine require activation of the mTORC1 pathway (demonstrated by blockade with rapamycin). The actions of ketamine, another rapid acting antidepressant, require BDNF-TrkB-Akt signaling, and we hypothesize that scopolamine-induction of mTORC1 signaling also requires release of this neurotrophic factor, although this has not yet been tested. The model also shows that depression relapse, as well as stress, are associated with a reduction in spine synapses, due to failure of synaptic homeostasis. This could result from genetic mutations or environmental factors such as sustained stress, and decreased expression of BDNF.