Amitriptyline Decreases GABAergic Transmission in Basal Forebrain Neurons Using an Optogenetic Model of Aging

Abstract

The antidepressant drug amitriptyline is used in the treatment of clinical depression and a variety of neurological conditions such as anxiety, neuropathic pain disorders and migraine. Antidepressants are associated with both therapeutic and untoward effects, and their use in the elderly has tripled since the mid-1990s. Because of this widespread use, we are interested in testing the acute effects of amitriptyline on synaptic transmission at therapeutic concentrations well below those that block voltage-gated calcium channels. We found that 3 μM amitriptyline reduced the frequency of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) and reduced quantal content in mice at ages of 7–10 mo. and 23–25 mo., suggesting a presynaptic mechanism of action that does not diminish with age. We employed a reduced synaptic preparation of the basal forebrain (BF) and a new optogenetic aging model utilizing a bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R specific for GABAergic neurons [VGAT-ChR2(H134R)-EYFP]. This model enables optogenetic light stimulation of specific GABAergic synaptic terminals across aging. Age-related impairment of circadian behavior was used to confirm predictable age-related changes associated with this model. Our results suggest that low concentrations of amitriptyline act presynaptically to reduce neurotransmitter release and that this action is maintained during aging.

Late-Onset, Short-Term Intermittent Fasting Reverses Age-Related Changes in Calcium Buffering and Inhibitory Synaptic Transmission in Mouse Basal Forebrain Neurons

“…Male wild-type (WT) C57 Bl/6 mice and VGAT-ChR2(H134R)-EYFP optogenetic mice of two ages, young (four to eight months), and aged (18-25 months) were used for all late-onset, short-term IF experiments. Breeding pairs were purchased from The Jackson Laboratory (stock #014548) and an aging colony was maintained for both genotypes as previously described (Bang et al, 2021). Mice were identified as WT or vesicular GABA transporter (VGAT) by standard tail-clip genotyping methods after weaning (EZ tissue/tail PCR genotyping kit, EZ BioResearch) as described previously (Bang et al, 2021).…”

“…Breeding pairs were purchased from The Jackson Laboratory (stock #014548) and an aging colony was maintained for both genotypes as previously described (Bang et al, 2021). Mice were identified as WT or vesicular GABA transporter (VGAT) by standard tail-clip genotyping methods after weaning (EZ tissue/tail PCR genotyping kit, EZ BioResearch) as described previously (Bang et al, 2021). In aged animals, IF was initiated later in life.…”

“…Reduced synaptic preparation using enzyme-free, acutely dissociated neurons to study synaptic transmission across aging We used a reduced synaptic preparation to examine synaptic transmission in acutely dissociated BF neurons across aging (Griffith et al, 2014;Bang et al, 2021). In this preparation, there is a very low frequency of spontaneous glutamatergic EPSCs which show rapid kinetics with decay time constant (t d = ;5 ms).…”

“…For the quantal content analysis, 50-100 consecutive traces of oIPSCs were recorded, and minimal light stimulation was used to ensure a sufficient number of failures. Using the method of failures [m = ln(N/N0)] in the Poisson model, quantal content (m) was calculated where, N is the total number of stimuli, and N0 is the number of failures where stimulation did not evoke a synaptic response (Bang et al, 2021).…”

“…We have extended these studies to mice using a bacterial artificial chromosome (BAC) transgenic line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R (VGAT-ChR2 (H134R)-EYFP) specific for GABAergic neurons (Zhao et al, 2011;Bang et al, 2021). We used acutely dissociated BF neurons and a reduced synaptic preparation (Griffith et al, 2014;Bang et al, 2021), to examine the effect of late-onset short-term IF diet (24-h alternate-day fasting for four to six weeks) on age-related changes in inhibitory synaptic transmission and Ca 21 buffering in behaviorally characterized mice. This optogenetic aging model enabled us to selectively stimulate GABAergic nerve terminals on voltage-clamped BF neurons, and calculate quantal content (m) of inhibitory neurotransmitter release.…”