Co-transmission of acetylcholine and GABA regulates hippocampal states

The basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter. Yet, we find that mouse hippocampal cholinergic terminals invariably establish GABAergic synapses, and their cholinergic vesicles dock at those synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes composite postsynaptic potentials, which are mutually cross-regulated by presynaptic autoreceptors. Although postsynaptic cholinergic receptor distribution cannot be investigated, their response latencies suggest a focal, intra- and/or peri-synaptic localisation, while GABAA receptors are detected intra-synaptically. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. Therefore, the differentially regulated GABAergic and cholinergic co-transmission suggests a hitherto unrecognised level of control over cortical states. This novel model of hippocampal cholinergic neurotransmission may lead to alternative pharmacotherapies after cholinergic deinnervation seen in neurodegenerative disorders.

medial septum. These results confirmed that all hippocampal projecting cholinergic cells express vGAT.
To confirm that septo-hippocampal cholinergic fibres can synthetize GABA, we performed immunofluorescent reactions against glutamate-decarboxylase (GAD65) and eYFP on hippocampal sections of ChAT-Cre mice, in which medial septal cholinergic fibres were labelled with Cre-dependent eYFP-AAV. We confirmed the GAD65 expression in eYFP positive terminals ( Figure 2C) as well.
To confirm vGAT protein expression in cholinergic terminals we performed vGAT-eYFP and vAChT-eYFP multiple labelings, and found that vGAT was present in the majority of the eYFP positive cholinergic terminals (at least 82.9%, n=311 in 3 mice Figure 2D). We also quantified vAChT expression, which was found to be present in at least 63.6% of the eYFP positive cholinergic terminals (n=364 in 3 mice).
Using postembedding GABA-immunogold staining, we also tested, whether cholinergic terminals contain GABA itself (Figure 2 E-F). We measured postembedding GABA-immunogold labelling densities in preembedding labelled vAChT-positive terminals (n=2 mice, 24 terminals). Background level was estimated measuring gold particles on vAChT-negative, putative glutamatergic terminals that formed asymmetric synapses in the vicinity of the examined vAChTpositive terminals (n=2 mice, 34 terminals). Data from two mice were not different statistically; therefore, they were pooled. We found a significantly higher level of immunogold labelling for GABA in vAChT-positive terminals than in glutamatergic terminals (3.3 times higher density; Figure 2F), suggesting the presence of GABA in these terminals.

Supplementary Note 4: Synaptic vesicles of cholinergic terminals are highly heterogeneous and relatively large
We analysed the volume and elongation of vesicles 8,9 in cholinergic terminals along with similar data from purely GABAergic terminals ( Figure 4E). As expected, GABAergic vesicles were small and elongated (median volume: 13730 nm 3 , 11174-16434 nm 3 interquartile range; median of elongation factor: 2.90, 2.52-3.41 interquartile range; n=54 vesicles from 2 mice). However, the volume of cholinergic vesicles were significantly larger (Mann-Whitney test, p<0.001). The volume of the vesicles in cholinergic terminals showed a significantly higher variability as well (Ftest, p<0.001), ranging from the very large and round ones to the small and elongated vesicles (median volume: 23267 nm 3 , 19160-27539 nm 3 interquartile range; median elongation factor: 1.80, 1.57-2.04 interquartile range; n=140 vesicles from two mice, Figure 4E). The small and elongated vesicles in the cholinergic terminals were similar to the purely GABAergic ones from GABAergic interneuron terminals ( Figure 4E). These data suggest that cholinergic terminals contain both smaller, more elongated GABAergic vesicles and larger, rounder cholinergic vesicles, both of which are released from the same synaptic active zone. Interestingly, some vesicles that were directly labelled with vAChT-immunogold particles were rather large and round ( Figure 4E). We also observed a vAChT-labelled vesicle, fused to the synaptic membrane, likely releasing its transmitter into the synaptic cleft ( Figure 4C). In subsequent experiments, we collected further evidence that acetylcholine and GABA are filled into different vesicles allowing their separatelyregulated co-transmission.

Supplementary Note 5: Acetylcholine and GABA are released at the same active zone in cholinergic terminals
We also tested, whether the two transmitter systems use the same or distinct active zones. We performed multiple immunofluorescent labelling experiments on virus labelled (eYFP) cholinergic fibres in ChAT-Cre mice for gephyrin, vAChT and eYFP, followed by confocal fluorescent imaging. We observed gephyrin-labelled puncta opposed to eYFP-positive terminals ( Figure 4G, H), identifying the postsynaptic active zones of these fibres. vAChT-labelling was clearly concentrated opposite to the gephyrin-puncta, proving a tight association to the synaptic active zones ( Figure 4G, H). Scale-free analysis confirmed that the likelihood of vAChT labelling was the highest at the synaptic active zones ( Figure 4I; n=32 synapses from two mice). To directly examine the existence of a mixed cholinergic/GABAergic vesicle pool, we labelled brain slices for vGAT, vAChT and eYFP, and performed correlated fluorescent confocal laser scanning microscopy (CLSM) and superresolution STORM imaging ( Figure 4J). The superresolution images confirmed that vAChT-and vGAT-labelled vesicle pools overlap, and were localized to the same small, confined portions of the eYFP septo-hippocampal terminals.

Supplementary Note 6: Acetylcholine and GABA are released from different vesicles in cholinergic terminals
Although a previous study in rat has suggested that GABA-containing synaptic vesicles do not contain acetylcholine 10 , using a highly specific method, we confirmed that GABA and acetylcholine vesicular transporters are localized on different vesicles in mouse cortical axon terminals. We used isolated synaptic vesicles to test whether acetylcholine and GABA are packed into the same vesicles. Isolation from neocortex and hippocampus was performed according to Mutch et al. (Supplementary Figure 4A,11 ). Isolated vesicles were investigated by flow cytometry for synaptophysin (SYP) expression. Labelling with a specific SYP antibody resulted in an about two orders of magnitude higher mean fluorescent intensity of vesicle preparations compared to the labelling with the secondary antibody alone (Supplementary Figure 4B), suggesting a highly purified preparation. After fixation and dehydration of vesicle preparations, we confirmed the presence of synaptic vesicles surrounded by lipid bilayer on electron microscopic images (Supplementary Figure 4E). The analysis confirmed that the diameter of the isolated vesicles was 37.55 nm (median, 33.78-40.21 nm interquartile range, n=100 vesicles; Supplementary Figure 4C), in accordance with literature data 12 . Next, we performed immunolabelling experiments on isolated synaptic vesicles fixed onto coverslips. Prior to CLSM imaging, we labelled the samples for SYP, vGAT, vAChT and vesicular glutamate transporter (VG1). As expected, we observed well separated fluorescent dots (point-spread functions, PSF) of the fluorophores in one single focal plane (Supplementary Figure 4F), but most PSFs showed vesicular co-localization of one of the vesicular transporters and SYP. Control experiments of the immunolabelling confirmed the lack of unspecific staining (Supplementary Figure 4I). In the absence of vesicle suspension, no PSFs were found in the CLSM scans, and the exclusion of any primary antibody led to the selective disappearance of PSFs in the corresponding channel. We also tested the distribution of fluorescent PSFs on the CLSM images. SYP-labelled vesicles were usually more than 1 µm away from each other as nearest-neighbor analysis of PSF centroids confirmed (median: 1.16 µm, 0.82-1.59 µm interquartile range, 0.46-3.92 min-max, n=149 vesicles; Supplementary Figure 4D). When PSFs in different channels colocalized, their centroids were never farther away from each other than 0.130 µm (median: 0.03 µm, 10-50 µm interquartile range, 0-0.13 min-max, n=92 vesicles; Supplementary Figure 4D). These experiments confirmed that co-localizing PSFs correspond to a single vesicle. Next, we analysed co-localizations of the PSFs in different channels (Supplementary Figure 4G) and found that 29.2% of vesicles were labelled only for SYP, 44.5% were doublelabelled for VG1 and SYP, 14.3% were double-labelled for vGAT and SYP, 11.1% were doublelabelled for vAChT and SYP. Only a negligible amount of vesicles (0.9%) were triple labelled with any combinations, whereas only a sub-fraction of these vesicles (0.14% of all) were co-labelled for vAChT and vGAT. Only 0.98% of all vGAT/SYP positive vesicles were labelled for vAChT, and only 1.26% of all vAChT/SYP positive vesicles were labelled for vGAT. These numbers are in the range of false positive labelling as confirmed in the control experiments, where primary antibodies were omitted (Supplementary Figure 4I). These data suggest that vesicular transporters for glutamate, GABA and acetylcholine are expressed by distinct vesicle populations in cortical samples (Supplementary Figure 4H; n=353 vesicles). Therefore, acetylcholine and GABA may be released at the same active zones, but from different vesicles.

Supplementary Note 7: Identification of basal forebrain cholinergic fibres in the hippocampus: control experiments
We either used immunolabelling against the vesicular acetylcholine transporter (vAChT), or performed anti-eYFP staining on sections from ChAT-Cre mice, the medial septal areas of which have previously been injected with Cre-dependent eYFP-adeno associated virus (AAV). Both of these methods had to be verified for selectivity and specificity, thus we completed a comprehensive set of control experiments. The cholinergic innervation of the hippocampus is reported to originate exclusively from the basal forebrain. Although the presence of a local cholinergic cell population in the mouse hippocampus was reported to be an artefact 13 we also tested for it. We injected Cre-dependent eYFP-AAV into the hippocampi of ChAT-Cre mice (Supplementary Figure 2C, inset), and stained hippocampal sections for eYFP, vAChT and vGAT (Supplementary Figure 2E, F). We found a few eYFP positive cells in the hippocampus. They were extremely rare and resembled dentate gyrus granule cells and CA3 pyramidal cells. We also found some sparsely distributed eYFP positive fibres originating from them, but vAChT or vGAT immunoreactivity was never found in these eYFP positive terminals (0 out of 323 terminals, from 2 mice, Supplementary Figure 2F). We also tested vAChT positive terminals in the same samples, and never found any eYFP-positivity in them (0 out of 3673 from 2 mice, Supplementary Figure  2F). Thus, we confirmed that there are no cholinergic cells in the hippocampus, only some extremely rare ectopic expression of the Cre enzyme. These results also confirmed that we can reliably label the septo-hippocampal cholinergic fibres with vAChT labelling.
To verify the other approach, we injected eYFP-AAV into the medial septal areas of ChAT-Cre mice (Supplementary Figure 2C), and performed PV/ChAT/eYFP triple labelings (Supplementary Figure 2D). 97.6% of all tested eYFP positive cells in the MS were also positive for ChAT (the few % of false negative cells are likely due to not perfectly efficient antibody penetration), but none of them were positive for PV (n=212 in 2 mice). We also tested the fibres of these cells in the hippocampus, and performed a PV/vAChT/eYFP triple labelling ( Supplementary Figure 2A, B). We found that eYFP positive terminals colocalized with vAChTlabelling, but were never positive for PV (n=252 terminals from 2 mice). These results confirmed that eYFP positive fibres in these animals originate exclusively from cholinergic cells. 6 Figure 2F: Medians (columns) and interquartile ranges (bars) of immunogold densities of GABA labelling in glutamatergic (Glut, median: 3.5 gold particles/ µm2, interquartile ranges: 1.5-5.3) and in VAChT-positive terminals (VAChT, median: 11.5 gold particles/ µm2, interquartile ranges: 6.8-22.7). Asterisk indicates significant difference (Mann-Whitney Test: p< 0.05). vAChTnegative terminals forming type I synapses were considered to be glutamatergic. Figure 3H: Amplitude, 20-80% rise time and decay time of unitary GABAergic IPSCs from pyramidal cells (n=5) and inhibitory neurons (n=16). Box plots represent median values, with interquartile ranges, whiskers represent min/max values. Amplitude in pA: PCs: 37.28 (20.      For decades, the predominant form of cholinergic communication was thought to be a form of "non-synaptic volume transmission" [14][15][16][17][18][19][20] 29 ]. Although acetylcholine esterase (AChE) was known to be highly effective in terminating extracellular cholinergic signal, the presence of certain extrasynaptic acetylcholine receptors ("receptor mismatch", 26,30 ) suggested that extracellular diffusion of acetylcholine occurs. Micro-dialysis experiments 31 and the localization of AChE, distant from cholinergic terminals, also seemed to support a non-synaptic "volume" transmission hypothesis 14 . However, later, highly sensitive microelectrodes showed faster, phasic changes in extracellular acetylcholine levels that facilitated cue detection and cortical information processing 20,32-35 . Basal forebrain cholinergic neurons were also shown to respond to reward and punishment with extremely high speed and precision 36 , and recent data suggested that cholinergic cells may regulate cortical information processing with a remarkable, millisecond-scale temporal precision 34,[37][38][39] . However, such a delicate temporal precision is hard to imagine without synapses and it remained inconclusive, whether the mode of acetylcholine signalling is synaptic "wired" transmission or "non-synaptic volume" transmission by ambient acetylcholine 26 .

GABAergic markers in cholinergic cells
Basal forebrain cholinergic cells share a common developmental origin with different populations of cortical, striatal and basal forebrain GABAergic neurons [40][41][42][43] . Previous studies have suggested that less than 2% of cholinergic cells express GABAergic markers 44,45 , while about 8% of ChAT-positive boutons in the cat striate cortex was shown to contain GABA 46 . The recognition of GABAergic signalling in the BF cholinergic system may have been hampered by its lack of GAD67 47 and GABA transporter 1 48 . Although the associations of cholinergic terminals with gephyrin 49 and NL2 1 suggested the capability of GABAergic signalling from these terminals. While, for example, GABA is released together with glutamate or aspartate in the hippocampus, or with dopamine in periglomerular cells 50,51 , and acetylcholine is released with glutamate in striatum 52 ; GABA and acetylcholine were also shown to be released together in retina and frontal cortex 45,47,53,54 . However, the precise architecture, the mechanism of the dual cholinergic/GABAergic transmission and their hippocampal synaptic physiological and network effects have not yet been investigated.

Supplementary Figure 3, GABAergic short-term depression is a presynaptic property of cholinergic fibers.
A: Average membrane potential response of an inhibitory neuron recorded in str. lacunosum-moleculare for cholinergic fiber stimulation (top). Latency from stimulation start to PSP onset was calculated as the time when the signal crosses 3 times standard deviation of baseline (orange line and grey shaded area represent mean and 3 times STD of the baseline). Inhibition of GABAARs (10 µM gabazine) blocks hyperpolarization (bottom) and the latency of the cholinergic response can be calculated similarly as well. B: Responses are magnified in time (from panel A). Blue arrowheads mark photoelectric artefacts evoked by 1 ms optical stimulus (blue bar). Note the short latency to rise (dotted line) in both the GABAergic IPSP and cholinergic EPSP. C: Latency from stimulus start to PSP onset (top) and PSP peak (bottom) are shown. IPSP time to onset (n = 7, in ms): 2.8 (2.2, 3.1), time to peak: 13.8 (12.7, 14.9). EPSP time to onset (n = 6, in ms): 7.4(7.0, 11.7), time to peak: 92.0 (80.5, 98.0). D: Channelrhodopsin 2 expression in axon terminals could change the short-term plasticity of the examined synapse by illumination driven calcium entry through the light activated channels. To exclude this possibility, with the help of a digital micro-mirror device (DMD), we have illuminated only axons running towards the measured cell, but not the axon terminals themselves. E: We have recorded from inhibitory neurons in str. lacunosum moleculare (n=5), and illuminated the slice far (~500 µm) from the cells. Similarly as in Figure 3, we applied 5 pulses at different frequencies (2, 5, 10 and 20 Hz). Averaged traces from one cell are shown. Wilcoxon-sign rank test: 1 st -2 nd : p<0.05, 2 nd -3 rd : p<0.05, 3 rd -4th and 4 th -5 th : not significant. In some cells, transmission probability remained stable despite the observed STD in IPSC amplitude, suggesting that multiple contacts were excited and "averaged" by optical illumination. These results support our hypothesis that GABAergic short-term depression emerges presynaptically, and not the result of channelrhodopsin-2 expression or postsynaptic chloride loading. J: Short-term dynamics of cholinergic EPSPs recorded from inhibitory neurons in str. lac.-mol. in response to 5 light pulses at different frequencies (top; 2, 5, 10, 20 Hz, n = 7). Cholinergic events overlap, not allowing reliable EPSP peak detection. Therefore, STP was quantified as the integral of the evoked events. K: The relative change in EPSP integral for the stimulations at different frequencies (n=7, average is green). Unlike the GABAergic component (Figure 3   a : WT3-6: indicates 4 wild type mice, ChAT-Cre1-3: indicates 3 different mice, ori: stratum oriens, pyr: stratum pyramidale, rad: stratum radiatum, l-m: stratum lacunosum-moleculare; b : We did not identify the cell types that established the dendritic shafts in CA1 lacunosum-moleculare and S1 (see Supplemental Experimental Procedures).