Corelease of acetylcholine and GABA from cholinergic forebrain neurons

1For many years, neurons were thought to release only a single fast neurotransmitter. This assumption led to classifying neuronal subtypes based on released neurotransmitter, a convention which helped predict a neuron's circuit function. However, many neuronal subtypes that release multiple fast neurotransmitters have now been described. In some cases, the coreleased neurotransmitters have similar post-synaptic effects, such as inhibition mediated by GABA and glycine from spinal interneurons. In other instances, the effects of the two neurotransmitters may be different and synergistic. For example, coreleased GABA and glutamate are thought to control the balance of excitation and inhibition in the lateral habenula. In neuromodulatory systems, synaptic release of fast neurotransmitters along with slow neuromodulators has emerged as a common theme. In addition to the impact of dopamine, stimulation of dopaminergic terminals from the ventral tegmental area and substantia nigra compacta activates glutamate-mediated excitatory currents in the nucleus accumbens and GABA-mediated inhibitory currents in the striatum. Likewise, serotonergic neurons of the dorsal raphe can trigger glutamate-mediated currents in post-synaptic neurons of the ventral tegmentum and nucleus accumbens which contributes to the signaling of reward.

2Several cholinergic neuron populations also release multiple neurotransmitters. Retinal starburst amacrine cells (SACs) differentially release GABA and acetylcholine (ACh) based on the pattern of light stimulation. In the central brain, selective activation of striatal cholinergic interneurons results in cholinergic and glutamatergic responses. Similarly, the cholinergic projection from habenula excites interpeduncular neurons through glutamate and ACh. Some evidence suggests that the basal forebrain cholinergic (BFC) system, which provides the major source of ACh to cortex, may corelease GABA. The dorsal-most BFC neurons, which belong to the globus pallidus externus (GP), express molecular markers for GABA synthesis and vesicular packaging and trigger GABAA receptor currents in GP and cortex when activated. We therefore asked whether GABA corelease was a general feature of forebrain cholinergic neurons.

3To address this question, we selectively activated cholinergic fibers in the cortex, with the goal of identifying synaptic events triggered by endogenous release from forebrain cholinergic neurons. Recording from layer 1 interneurons, we observed not only the expected excitatory post-synaptic currents (EPSCs) mediated by nicotinic ACh receptors (nAChRs), but an unexpected inhibitory post-synaptic current (IPSC) mediated by GABAA receptors. IPSCs insensitive to nAChR antagonists had onset latencies slightly faster than the nicotinic EPSCs (nEPSCs), and could be directly evoked under pharmacological conditions in which action potentials were blocked, suggesting cholinergic neurons were directly releasing GABA in addition to ACh. Indeed, we found that cholinergic neurons throughout the forebrain commonly coexpressed the GABA synthetic enzyme GAD65 (Gad2), and the vesicular GABA transporter (Slc32a1), indicating that these neurons possess the necessary cell machinery for GABA transmission. Finally, we show that conditional deletion of Slc32a1 selectively in cholinergic neurons eliminates monosynaptic IPSCs while leaving nEPSCs intact, confirming the direct release of GABA from cholinergic terminals. These experiments suggest a previously overlooked capability of the cholinergic system to use GABA in synaptic signaling.

4To explore the effects that cholinergic neurons have on cortical circuitry, we used double transgenic mice to optogenetically activate neurons that express endogenous choline acetyltransferase (Chat). These mice carried a knock-in allele linking Cre recombinase expression to the Chat locus through an internal-ribosome entry site (Chat i-Cre) as well as a Cre-activated channelrhodopsin-EYFP fusion allele (Rosa26 lsl-ChR2-EYFP). Chat i-Cre; Rosa26 lsl-ChR2-EYFP mice expressed ChR2-EYFP throughout the forebrain, recapitulating known patterns of Chat expression in cortex (Ctx), striatum, globus pallidus externus (GP), and nucleus basalis (NB, Figure 1A). To test whether ChR2+ cells express endogenous Chat, we performed ChAT immunohistochemistry on sections of Chat i-Cre; Rosa26 lsl-ChR2-EYFP mice (Figure 1B). We focused on those Chat+ forebrain neurons positioned to innervate the cortex, including local Chat+ interneurons and the subcortical projections arising from the GP/NB. In both regions, ChR2+ neurons were immunopositive for ChAT, confirming our ability to selectively activate endogenous Chat+ inputs to cortex.

5To identify the synaptic signaling mechanisms engaged by activation of the cortical cholinergic system, we targeted layer 1 interneurons for whole-cell voltage-clamp recordings in acute brain slices. Layer 1 is strongly innervated by ChAT+ cells of the basal forebrain across species including in Chat i-Cre; Rosa26 lsl-ChR2-EYFP mice, where ChR2-EYFP is expressed in a dense plexus (Figure 1C,D). As expected, in a subset of interneurons (n = 41 of 58 cells from 9 mice), we observed robust excitatory postsynaptic currents (EPSCs) at −70 mV in response to brief pulses of blue light (2–7 ms, Figure 1E, left). These EPSCs were not blocked by NBQX and CPP, ruling out a glutamatergic source, but were blocked by the nicotinic ACh receptor (nAChR) antagonists DHβE, MLA, and MEC, confirming their cholinergic identity (nEPSCs). nEPSCs displayed a typical biphasic response, with a fast component, likely mediated by synaptic receptors containing the low-affinity α7 nAChR subunit, and a slow component, likely mediated by non-synaptic receptors expressing the high-affinity non-α7 subunits.

6In addition to the expected nEPSCs recorded at −70 mV, we also observed barrages of outward inhibitory postsynaptic currents (IPSCs) at 0 mV, indicative of signaling through GABA receptors (n = 28 of 58 cells, Figure 1E, center). One possible explanation for these IPSCs could be ACh-mediated feed-forward activation of local interneurons, resulting in disynaptic release of GABA. Indeed, when nAChR antagonists were applied, the delayed outward IPSCs disappeared. However, in a subset of recorded cells IPSCs remained (n = 9 of 58 cells, Figure 1F, right), suggesting that these PSCs were not dependent on nAChR signaling.

7To test if nAChR-resistant IPSCs are caused by direct release of GABA from cholinergic fibers, we bath applied the voltage-gated sodium channel antagonist TTX, which blocked light-evoked IPSCs (Figure 1F,G). In the presence of TTX, IPSCs could be rescued by enhancing ChR2-mediated depolarization with the voltage-gated potassium channel blocker 4AP. Rescued IPSCs were subsequently blocked by the GABAA receptor antagonist SR95531 (n = 5 cells from 4 mice). Moreover, nAChR-resistant ‘direct’ IPSCs had faster average onsets than both nEPSCs and nAChR-sensitive ‘indirect’ IPSCs (nEPSCs, 4.0 ± 0.2 ms, n = 41 cells; direct IPSCs, 2.5 ± 0.2, n = 9 cells; indirect IPSCs, 11.8 ± 2.3, n = 19 cells from 9 mice, Figure 1H). These data suggest direct IPSCs are independent of nAChR signaling and mediated by GABAA receptors, consistent with monosynaptic release of GABA by cholinergic neurons. In support of this possibility, gene expression analyses have suggested some populations of Chat+ subcortical neurons contain the synthetic machinery for GABA.

8GABA corelease has been observed in other neuromodulatory systems, namely from dopaminergic neurons of the substantia nigra. In those neurons, GABA is co-packaged with dopamine into vesicles by the transporter VMAT2 (Slc18a2), instead of by the typical vesicular transporter for GABA (VGAT, encoded by Slc32a1), which is necessary for packaging in most GABAergic neurons. To assess whether cholinergic neurons could use VGAT to package GABA into vesicles, we tested for Slc32a1/ChAT co-expression throughout the brain, including in cortex, GP, NB, diagonal band of broca (DBB)/medial septum (MS), and pedunculopontine nucleus (PPN, Figure 2A, top). We labeled cells expressing endogenous Slc32a1 using double-transgenic mice that link Cre recombinase expression to the Slc32a1 locus (Slc32a1i-Cre) and carry a zsGreen Cre reporter allele (Rosa26 lsl-zsGreen). Subsequent ChAT immunolabeling on sagittal and coronal sections from Slc32a1i-Cre; Rosa26 lsl-zsGreen mice was used to examine coexpression. Since zsGreen accumulates in somata and does not diffuse throughout the cytoplasm, this strategy allowed the clear identification of ChAT+ soma free from background fluorescence caused by Cre+ axons and dendrites. In cortex, GP, NB, and MS/DBB, nearly all of ChAT+ cell were Slc32a1+ (zsGreen+/ChAT+, from 3 mice: Ctx, 628/628; GP, 238/243; NB, 598/624; MS/DBB; 560/601, Figure 2A,B). In contrast, ChAT+ cells of the PPN very rarely expressed Slc32a1 (3/157, Figure 2B). These data suggest that in both cortical and subcortical forebrain regions, ChAT+ cells express the canonical molecular machinery to package GABA into vesicles. However, since ChAT+ neurons in the brainstem PPN do not express Slc32a1, this GABAergic marker is not a ubiquitous feature of the central cholinergic system.

9GABA is synthesized by one of two GABA synthetic enzymes, GAD65 or GAD67, encoded by the genes Gad2 or Gad1, respectively. GAD67 is expressed largely in cell bodies and is thought to be responsible for synthesizing GABA for general metabolic cell functions, whereas GAD65 expression is more prominent in axon terminals and is thought to mediate the majority of synthesis of synaptic GABA. We examined if cholinergic neurons expressed GAD67 by immunostaining for ChAT in brain sections from knock-in mice where GFP replaces the first exon of the Gad1 gene (Gad1GFP). We detected only minor overlap between neurons that stained positive for ChAT and those that expressed GFP in the NB, GP, MS/DBB, or PPN, except for cortical ChAT+ interneurons, where we observed significant overlap (GFP+/ChAT + neurons: Ctx: 122/136; MS/DBB, 1/573; NB, 0/439; GP, 7/413; PPN, 12/246, Figure 3—figure supplement 1). This suggests that within the major subcortical cholinergic projections, GAD67-mediated GABA synthesis does not occur in cholinergic neurons. To test for coexpression of ChAT and GAD65, we immunostained sections of knock-in mice where Cre recombinase was targeted to the endogenous Gad2 gene (Gad2 i-Cre) and visualized Cre expression with the zsGreen Cre reporter. In contrast to Gad1, in cortex, GP, NB, and MS/DBB of Gad2 i-Cre; Rosa26 lsl-zsGreen mice, most ChAT+ neurons were Gad2+ (zsGreen+/ChAT+, from 4 mice: Ctx, 518/519; GP, 273/372; NB, 860/934; MS/DBB, 673/685, Figure 3A,B). In the brainstem, however, few of the ChAT+ cells were Gad2+ (PPN, 6/110, Figure 3A,B). This ChAT co-expression pattern for Gad2 is similar to Slc32a1, suggesting that forebrain cholinergic neurons possess the necessary cellular machinery to both synthesize and package synaptic GABA.

10Though the fast onset and persistence of GABAergic IPSCs in the presence of nAChR antagonists and TTX/4AP argues strongly for direct release of GABA from cholinergic terminals, we wished to confirm this finding using conditional genetics. To determine if GABA release is indeed monosynaptic, we took advantage of the observation that cholinergic neurons express Slc32a1. If GABA is released directly from cholinergic neurons, then conditional knock-out of Slc32a1 selectively in these cells should abolish GABA release. We therefore bred triple transgenic mice which carried conditional (floxed) Slc32a1 alleles in addition to Chat i-Cre and Rosa26 lsl-ChR2-EYFP. These mice allowed us to compare the optogenetic stimulation of wild-type cholinergic neurons (Slc32a1+/+) to those lacking Slc32a1 (Slc32a1fl/fl) in acute brain slices. In voltage-clamp recordings from layer 1 interneurons, we observed fast onset IPSCs in 31% of cells recorded in Chat i-Cre; Slc32a1+/+ mice (n = 5 of 16 from 2 mice), but no direct IPSCs in Chat i-Cre; Slc32a1fl/fl mice (n = 0 of 34 cells from 4 mice, Figure 4A,B). In contrast, the proportion and average peak amplitude of nAChR responses remains unchanged between Slc32a1+/+ and Slc32a1fl/fl mice (Figure 4A,B). These data demonstrate that GABA but not ACh release from cholinergic neurons relies on cell autonomous Slc32a1, ruling out a disynaptic mechanism.

11Here, we provide evidence that the cortical cholinergic system is capable of GABAergic neurotransmission. In response to optogenetic stimulation of neurons expressing endogenous Chat, we observed PSCs mediated by both nAChRs and GABAA receptors in layer 1 interneurons. A subset of the evoked IPSCs appeared to be monosynaptic, based on latency and pharmacological analyses. In support of their GABAergic nature, the ChAT+ neurons which innervate cortex—local interneurons and subcortical projections arising from the GP/NB—express Gad2 and Slc32a1, the canonical molecular machinery for GABA synthesis and vesicular packaging. Indeed, conditional knock-out of Slc32a1 selectively in cholinergic neurons eliminates light-evoked monosynaptic IPSCs. These genetic results confirm that cholinergic neurons release GABA directly. Cholinergic GABA release is likely to be a feature of most, but not all, central cholinergic neurons: the ChAT+ neurons of the MS and DBB—which innervate the hippocampus—also express Gad2 and Slc32a1, whereas those of the midbrain PPN do not.

12The mode of neurotransmitter corelease can vary across neuron classes. In some instances, both neurotransmitters are released from the same synaptic vesicles. This is the case for GABA/glutamate corelease onto neurons of the lateral habenula, where individual miniature EPSCs can be observed with dual GABA/glutamate components. Copackaging in individual vesicles is also the case when the same vesicular transporter loads both neurotransmitters. For example, GABA is packaged in dopaminergic neurons by VMAT2, which also packages dopamine. Similarly, both GABA and glycine packaging in spinal interneurons rely on VGAT. However, corelease from separate pools of synaptic vesicles also occurs. In retinal SACs, release of GABA and ACh can be functionally separated through patterned light stimulation or pharmacology. In Chat+ GP axons in cortex, GABA and ACh appear to be released from distinct vesicular pools which can be located within the same or neighboring pre-synaptic terminals. Though the precise mechanism by which each forebrain cholinergic population coreleases GABA and ACh remain unclear and further experiments are merited to explore how GABA/ACh cotransmission is regulated, the differences in the proportion of layer 1 interneurons showing ACh and GABA responses suggest some form of segregated release.

13The cholinergic system's function in promoting attention, alertness, and learning has classically been attributed to acetylcholine and its action as a diffuse volume transmitter, affecting cortical activity at relatively slow time scales. This model is supported by anatomical evidence showing widespread distribution of cholinergic fibers through all cortical layers with significant separation between the sites of release and ACh receptors. In addition, many in vitro pharmacological experiments have shown that ACh receptors can shape the signaling of other neurotransmitter systems, by altering properties of presynaptic release, synaptic plasticity, or the intrinsic excitability of targeted neurons. However, more recent work has focused on the participation of ACh in rapid, wired neurotransmission, acting at tightly apposed synapses. Behaviorally relevant sensory cues can cause a fast, time-locked spike in ACh concentration, suggesting that ACh may mediate detection of that cue. In addition, fast onset currents mediated by nAChRs can be recorded in cortical interneurons following optogenetic activation of cholinergic fibers. Our finding that cholinergic neurons also elicit fast-onset synaptic GABAA responses lends further support to the notion that the cholinergic system can rapidly affect cortical computations by acting at classical synapses.

14GABA corelease from cholinergic forebrain neurons may affect cortical function in several ways. First, at the circuit level, GABA release could act in a manner that reinforces the emerging concept that the cholinergic system disinhibits cortical firing. ACh release from basal forebrain neurons excites layer 1 and VIP+ interneurons, which in turn inhibit local interneurons that target principle neurons of the cortex. Depending on the timing and targeted cell types, GABA corelease could conceivably enhance this effect by inhibiting local interneurons, thereby promoting cortical activity. Second, nAChRs can regulate both pre and post-synaptic GABAergic signaling. For example, in hippocampal interneurons, post-synaptic nAChRs are present in inhibitory synapses and when activated, reduce GABAA receptor-mediated IPSCs in a Ca2+ and PKC-dependent manner. Thus coreleased ACh and GABA could interact to modulate local synaptic signaling. Lastly, experiments poisoning or stimulating the basal forebrain cholinergic system have demonstrated that activity within this projection is necessary and sufficient for plasticity in sensory cortices. While ACh alone can induce functional changes in cortical circuits, GABA may also contribute to synaptic rewiring in vivo. Addressing these questions experimentally will benefit from future work to clarify the basic synaptic anatomy and biochemical regulation of ACh/GABA corelease. Given the presence of GABA signaling machinery throughout the distinct forebrain cholinergic systems, corelease likely has a significant and fundamental effect on brain activity and cognition.

15Cre recombinase was targeted to specific cell types using knock-in mice to drive Cre expression under endogenous gene-specific regulatory elements using an internal ribosome entry site. Cre knock-in mice for choline acetyltransferase (Chat) and vesicular GABA transporter (Slc32a1) were provided by Brad Lowell (Beth Israel Deaconess Medical Center) and are available from the Jackson Labs (Bar Harbor, ME; Chat i-Cre, stock #006410; Slc32a1i-Cre, stock #016962). Gad2 i-Cre mice were purchased from Jackson Labs (stock #010802). Gad1GFP knock-in mice replace elements of Gad1 coding sequence with GFP. We did not distinguish between mice hetero or homozygous for transgenic alleles except where indicated. All experimental manipulations were performed in accordance with a protocol (#03551) approved by the Harvard Standing Committee on Animal Care following guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.

16Mice aged post-natal day 25–124 were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (1× PBS). Brains were post-fixed for 1–3 days, washed in 1× PBS, and sectioned (40–50 μm) coronally or sagittally using a Vibratome (Leica Microsystems, Buffalo Grove, IL). For ChAT or NeuN immunohistochemistry, slices were incubated in a 1× PBS blocking solution containing 5% normal horse serum and 0.3% Triton X-100 for 1 hr at room temperature. Slices were then incubated overnight at 4°C in the same solution containing anti-choline acetyltransferase antibody (AB144P; 1:100; Millipore, Billerica, MA) or NeuN (MAB377; 1:100; Millipore). The next morning, sections were washed three times for five minutes in 1× PBS and then incubated for 1 hr at room temperature in the blocking solution containing donkey anti-goat Alexa 647 or Alexa 594 (for ChAT) or anti-mouse Alexa 647 (for NeuN) (1:500; Molecular Probes, Eugene, OR). Slices were then mounted on slides (Super Frost). After drying, slices were coverslipped with ProLong antifade mounting media containing DAPI (Molecular Probes) and imaged with an Olympus VS110 slide scanning microscope using the 10× objective. Confocal images (1–2 μm optical sections) were acquired with an Olympus FV1000 laser scanning confocal microscope (Harvard Neurobiology Imaging Facility) through a 60× objective. Colabeling quantification was carried on images obtained from the Olympus VS110 slide scanning microscope using ImageJ.

17Acute brain slices were obtained from mice aged post-natal day 30–128 using standard techniques. Mice were anesthetized by isoflurane inhalation and perfused through the heart with ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 KCl, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, and 11 glucose (∼308 mOsm·kg−1). Cerebral hemispheres were removed, placed in ice-cold choline-based cutting solution (consisting of [in mM]: 110 choline chloride, 25 NaHCO3, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 glucose, 11.6 ascorbic acid, and 3.1 pyruvic acid), blocked, and transferred into a slicing chamber containing ice-cold choline-based cutting solution. Sagittal slices (300–350 μm thick) were cut with a Leica VT1000s vibratome and transferred to a holding chamber containing ACSF at 34°C for 30 min and then subsequently at room temperature. Both cutting solution and ACSF were constantly bubbled with 95% O2/5% CO2.

18Individual slices were transferred to a recording chamber mounted on a custom built two-photon laser scanning microscope (Olympus BX51WI) equipped for whole-cell patch-clamp recordings and optogenetic stimulation. Slices were continuously superfused (3.5–4.5 ml·min−1) with ACSF warmed to 32–34°C through a feedback-controlled heater (TC-324B; Warner Instruments). Cells were visualized through a water-immersion 60× objective using differential interference contrast (DIC) illumination. Epifluorescence illumination was used to identify those layer 1 interneurons surrounded by ChR2-EYFP processes. Patch pipettes (2–4 MΩ) pulled from borosilicate glass (G150F-3; Warner Instruments) were filled with a Cs+-based low Cl–internal solution containing (in mM) 135 CsMeSO3, 10 HEPES, 1 EGTA, 3.3 QX-314 (Cl–salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-Phosphocreatine (pH 7.3 adjusted with CsOH; 295 mOsm·kg−1) for voltage-clamp recordings. Series resistance (<25 MΩ) was measured with a 5-mV hyperpolarizing pulse in voltage-clamp and left uncompensated. Membrane potentials were corrected for a ∼7 mV liquid junction potential. In some cases after the recording was complete, cellular morphology was captured in a volume stack using 740 nm two-photon laser light (Coherent). To activate ChR2 in acute slices from ChAT i-Cre; Rosa26 lsl-ChR2-EYFP mice, 473 nm laser light (Optoengine) was focused onto the back aperture of the 60× water immersion objective to produce collimated whole-field illumination. Square pulses of laser light were delivered every 20 s and power (2–7 ms; 4.4 mW·mm−2) was quantified for each stimulation by measuring light diverted to a focal plane calibrated photodiode through a low-pass dichroic filter. Following bath application of TTX and 4AP, in some cases, light power or duration was increased slightly to recover currents (e.g., changing the duration from 2 to 4 ms).

19Drugs (all from Tocris, United Kingdom) were applied via bath perfusion: SR95531 (10 μΜ), tetrodotoxin (TTX; 1 μΜ), 4-aminopyridine (4AP; 500 μM), scopolamine (10 μΜ), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 10 μM), R,S-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP; 10 μM), N,2,3,3-Tetramethylbicyclo[2.2.1]heptan-2-amine, (MEC; 10 μM), [1α,4(S),6β,14α,16β]-20-Ethyl-1,6,14,16-tetramethoxy-4-[[[2-(3-methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]oxy]methyl]aconitane-7,8-diol (MLA; 0.1 μM), (2S,13bS)-2-Methoxy-2,3,5,6,8,9,10,13-octahydro-1H,12H-benzo[i]pyrano[3,4-g]indolizin-12-one (DHβE; 10 μM). CPP and NBQX were combined to make a cocktail of antagonists to target ionotropic glutamate receptors, while MEC, MLA, and DHβE were combined to make a cocktail to antagonize nicotinic receptors.

20Membrane currents and potentials were recorded using an Axoclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) filtered at 3 kHz and digitized at 10 kHz using National Instruments acquisition boards and ScanImage (available at: scanimage.org) written in MATLAB (Mathworks, Natick, MA). Electrophysiology and imaging data were analyzed offline using Igor Pro (Wavemetrics, Lake Oswego, OR), ImageJ (NIH, Bethesda, MD) and GraphPad Prism (GraphPad Software, La Jolla, CA). In figures, voltage-clamp traces represent the average waveform of 3–6 acquisitions. Peak current amplitudes were calculated by averaging over a 1 ms window around the peak. For pharmacological analyses, 3–7 consecutive acquisitions (20 s inter-stimulus interval) were averaged following a 3-min wash-in period for NBQX and CPP or a 4-min wash-in period for MEC, MLA, and DHβE. For TTX and 4AP conditions, current averages were composed of the acquisitions following full block or first-recovery of ChR2 evoked currents, respectively. Data (reported in text and figures as mean ± sem) were compared statistically using the Mann–Whitney test. p values smaller than 0.05 were considered statistically significant.