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Co-transmission of acetylcholine and GABA regulates hippocampal states

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Takács, Virág T., Cserép, Csaba, Schlingloff, Dániel, Pósfai, Balázs, Szőnyi, András, Sos, Katalin E., Környei, Zsuzsanna, Dénes, Ádám, Gulyás, Attila I., Freund, Tamás F., Nyiri, Gábor·2018·Nature Communications
DOI 10.1038/s41467-018-05136-1PMC PMC6054650

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Abstract

Abstract 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 GABA A 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.

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Fig. 1 All cholinergic terminals establish synapses, express GABAergic markers and innervate pyramidal cells or interneurons. a Three-dimensional EM reconstructions show that hippocampal cholinergic fibres form synapses (arrows) frequently. Axon A is labelled for ChAT in a WT mouse. Axon B is an AAV-eYFP virus-labelled septo-hippocampal fibre in a ChAT-Cre mouse. Insets show two typical terminals with synapses (blue). The plasma membrane was made partially transparent to reveal mitochondria (mito, green). Gold labellings of NL2 (axon A, synapse 1–4) or gephyrin (axon B, synapse 5–7) were used to recognise synapses (black dots and arrows in the insets). EM images show terminal boutons (b) of the reconstructed axonal segments establishing synapses 1–7 (arrowheads, indicated by the same numbers on the left) on dendrites (d) and a spine (s). Next to synapse 1, a ChAT-negative, putative GABAergic terminal bouton (bneg) forming a NL2-positive synapse (arrow) is also shown. b–n EM images reveal the presence of gephyrin (arrowheads, gold; b–d; g–j) and GABAA γ2 receptor subunits (arrowheads, gold; e–f; k–n) postsynaptically in synapses established by vAChT-positive terminals in WT mice (b–f; DAB, b) or by AAV-eYFP-labelled septo-hippocampal terminals in ChAT-Cre mice (g–n; DAB, b). Images of consecutive sections are separated by thin black lines. Terminals innervate dendrites (d) or spines (s). In j, the postsynaptic target is an interneuron dendrite (INd) that receives type-I synapses as well (arrows). Synapses are from str. ori. (a, b–e, g–j, l, m), str. rad. (k, n) and str. l-m (f). Scale bar is 200 nm for all EM images. o Postsynaptic target selectivity of reconstructed cholinergic axonal segments from str. oriens and radiatum. Spine: 27.1%, pyramidal cell dendrite: 62.6%, interneuron dendrite: 4.7%, unidentifiable: 5.6%. p Comparison of the number of synapses per 100 µm cholinergic axonal segments in CA1 and S1 cortex and GABAergic fibres in CA1
Fig. 1 All cholinergic terminals establish synapses, express GABAergic markers and innervate pyramidal cells or interneurons. a Three-dimensional EM reconstructions show that hippocampal cholinergic fibres form synapses (arrows) frequently. Axon A is labelled for ChAT in a WT mouse. Axon B is an AAV-eYFP virus-labelled septo-hippocampal fibre in a ChAT-Cre mouse. Insets show two typical terminals with synapses (blue). The plasma membrane was made partially transparent to reveal mitochondria (mito, green). Gold labellings of NL2 (axon A, synapse 1–4) or gephyrin (axon B, synapse 5–7) were used to recognise synapses (black dots and arrows in the insets). EM images show terminal boutons (b) of the reconstructed axonal segments establishing synapses 1–7 (arrowheads, indicated by the same numbers on the left) on dendrites (d) and a spine (s). Next to synapse 1, a ChAT-negative, putative GABAergic terminal bouton (bneg) forming a NL2-positive synapse (arrow) is also shown. b–n EM images reveal the presence of gephyrin (arrowheads, gold; b–d; g–j) and GABAA γ2 receptor subunits (arrowheads, gold; e–f; k–n) postsynaptically in synapses established by vAChT-positive terminals in WT mice (b–f; DAB, b) or by AAV-eYFP-labelled septo-hippocampal terminals in ChAT-Cre mice (g–n; DAB, b). Images of consecutive sections are separated by thin black lines. Terminals innervate dendrites (d) or spines (s). In j, the postsynaptic target is an interneuron dendrite (INd) that receives type-I synapses as well (arrows). Synapses are from str. ori. (a, b–e, g–j, l, m), str. rad. (k, n) and str. l-m (f). Scale bar is 200 nm for all EM images. o Postsynaptic target selectivity of reconstructed cholinergic axonal segments from str. oriens and radiatum. Spine: 27.1%, pyramidal cell dendrite: 62.6%, interneuron dendrite: 4.7%, unidentifiable: 5.6%. p Comparison of the number of synapses per 100 µm cholinergic axonal segments in CA1 and S1 cortex and GABAergic fibres in CA1CC BY · PubMed Central
Fig. 2 Cholinergic cells express the molecular machinery required for GABA release. a, b The cholinergic neurons of the MS are GABAergic. White box in a contains area enlarged in b. Images show neurons stained for ChAT in red, while the green labelling marks the vGAT-expressing neurons in vGAT-ZsGreen reporter mouse. c, d AAV-eYFP virus-traced septo-hippocampal fibres express GAD65 (c). AAV-eYFP virus-traced septo-hippocampal fibres express vGAT and vAChT (d). Insets show xz and yz projections of the terminal labelled with an arrow. Arrowhead points to another terminal. Green line marks the fibre outline. (Scale bar on d is 210, 14, 2 and 1 μm for a, b, c and d, respectively.) e, f Hippocampal cholinergic terminals contain GABA. Three consecutive EM sections of a vAChT-positive terminal (e, red pseudocolor) are shown. vAChT was visualised by pre-embedding immunogold method (the first panel of e, silver-intensified gold particles, large arrows), whereas on the next ultrathin sections (the second and third panels of e) postembedding GABA immunostaining was performed (smaller gold particles, thin arrows, some GABA molecules penetrate into mitochondria during fixation). vAChT signal is absent in postembedding images, because of the etching procedure. Scale bar is 200 nm for all EM images. f Cholinergic terminals contained significantly higher immunogold signal than glutamatergic ones (p < 0.05) suggesting the presence of GABA in cholinergic terminals (median and interquartile ranges, Supplementary Note 8)
Fig. 2 Cholinergic cells express the molecular machinery required for GABA release. a, b The cholinergic neurons of the MS are GABAergic. White box in a contains area enlarged in b. Images show neurons stained for ChAT in red, while the green labelling marks the vGAT-expressing neurons in vGAT-ZsGreen reporter mouse. c, d AAV-eYFP virus-traced septo-hippocampal fibres express GAD65 (c). AAV-eYFP virus-traced septo-hippocampal fibres express vGAT and vAChT (d). Insets show xz and yz projections of the terminal labelled with an arrow. Arrowhead points to another terminal. Green line marks the fibre outline. (Scale bar on d is 210, 14, 2 and 1 μm for a, b, c and d, respectively.) e, f Hippocampal cholinergic terminals contain GABA. Three consecutive EM sections of a vAChT-positive terminal (e, red pseudocolor) are shown. vAChT was visualised by pre-embedding immunogold method (the first panel of e, silver-intensified gold particles, large arrows), whereas on the next ultrathin sections (the second and third panels of e) postembedding GABA immunostaining was performed (smaller gold particles, thin arrows, some GABA molecules penetrate into mitochondria during fixation). vAChT signal is absent in postembedding images, because of the etching procedure. Scale bar is 200 nm for all EM images. f Cholinergic terminals contained significantly higher immunogold signal than glutamatergic ones (p < 0.05) suggesting the presence of GABA in cholinergic terminals (median and interquartile ranges, Supplementary Note 8)CC BY · PubMed Central
Fig. 3 Optogenetic stimulation of cholinergic fibres elicits composite GABAergic and cholinergic postsynaptic responses. a Medial septum (MS) of ChAT-Cre mice were injected with Cre-dependent AAV containing Channelrhodopsin-2 (ChR2). Using whole-cell patch-clamp in horizontal hippocampal slices, we recorded voltage or current response from hippocampal neurons upon optical excitation of septo-hippocampal cholinergic fibres. NMDA and AMPA receptors were blocked with AP5 (50 µM) and NBQX (20 µM) in all experiments presented in Figs. 3 and 5. b Representative post-hoc visualised CA1 pyramidal cell (magenta) and the surrounding cholinergic fibres (green) with putative contacts (inset, white arrowheads). c Blue light pulses elicited a composite membrane potential response from str. lacunosum-moleculare inhibitory neurons (green, average of 50 stimulations). Inhibition of GABAARs (10 µM gabazine) abolishes the hyperpolarising component (nine from nine tested cells), resulting in a putative cholinergic excitatory potential (black). Inhibition of all AChRs (1 µM MLA, 1 µM DHβE, 10 µM atropine, four from four tested cells) blocks the remaining depolarising response (magenta). d Conversely, first blocking the AChRs, resulted in a putative GABAergic IPSP (magenta), which was blocked by GABAARs inhibitor gabazine (black). The increase of IPSP amplitude for AChR block is addressed in Fig. 5. e Magnified cholinergic EPSP (black) from c, and GABAergic IPSP from d (magenta) demonstrate their short latency (see also Supplementary Figure 3). f A representative recorded pyramidal cell (PC, top) and an inhibitory neuron (IN, bottom) post-hoc visualised in magenta and ChR2 positive cholinergic axons in green. Insets: Immunostaining for gephyrin (white) identify their putative synapses (white arrows). g We blocked both nicotinic and muscarinic AChRs (1 µM MLA, 1 µM DHβE, 10 µM atropine) and recorded inhibitory postsynaptic currents (IPSC) evoked by cholinergic fibre illumination. Traces show IPSC response of a PC and an IN to five light pulses (1 ms) at increasing stimulation frequencies (2, 5, 10, 20 Hz, cells were recorded in VC@0 mV). h Amplitude and decay time of unitary GABAergic IPSCs from pyramidal cells (n = 5) and inhibitory neurons (n = 16) were statistically different (p < 0.05), while their rise time was not different (median values, interquartile ranges and min/max values, see Supplementary Note 8). i Averages of IPSC amplitudes for the five pulses presented on g shows strong short-term depression (STD) of GABAergic transmission evoked by stimulating cholinergic fibres (for details see Supplementary Note 8 and Supplementary Figure 3)
Fig. 3 Optogenetic stimulation of cholinergic fibres elicits composite GABAergic and cholinergic postsynaptic responses. a Medial septum (MS) of ChAT-Cre mice were injected with Cre-dependent AAV containing Channelrhodopsin-2 (ChR2). Using whole-cell patch-clamp in horizontal hippocampal slices, we recorded voltage or current response from hippocampal neurons upon optical excitation of septo-hippocampal cholinergic fibres. NMDA and AMPA receptors were blocked with AP5 (50 µM) and NBQX (20 µM) in all experiments presented in Figs. 3 and 5. b Representative post-hoc visualised CA1 pyramidal cell (magenta) and the surrounding cholinergic fibres (green) with putative contacts (inset, white arrowheads). c Blue light pulses elicited a composite membrane potential response from str. lacunosum-moleculare inhibitory neurons (green, average of 50 stimulations). Inhibition of GABAARs (10 µM gabazine) abolishes the hyperpolarising component (nine from nine tested cells), resulting in a putative cholinergic excitatory potential (black). Inhibition of all AChRs (1 µM MLA, 1 µM DHβE, 10 µM atropine, four from four tested cells) blocks the remaining depolarising response (magenta). d Conversely, first blocking the AChRs, resulted in a putative GABAergic IPSP (magenta), which was blocked by GABAARs inhibitor gabazine (black). The increase of IPSP amplitude for AChR block is addressed in Fig. 5. e Magnified cholinergic EPSP (black) from c, and GABAergic IPSP from d (magenta) demonstrate their short latency (see also Supplementary Figure 3). f A representative recorded pyramidal cell (PC, top) and an inhibitory neuron (IN, bottom) post-hoc visualised in magenta and ChR2 positive cholinergic axons in green. Insets: Immunostaining for gephyrin (white) identify their putative synapses (white arrows). g We blocked both nicotinic and muscarinic AChRs (1 µM MLA, 1 µM DHβE, 10 µM atropine) and recorded inhibitory postsynaptic currents (IPSC) evoked by cholinergic fibre illumination. Traces show IPSC response of a PC and an IN to five light pulses (1 ms) at increasing stimulation frequencies (2, 5, 10, 20 Hz, cells were recorded in VC@0 mV). h Amplitude and decay time of unitary GABAergic IPSCs from pyramidal cells (n = 5) and inhibitory neurons (n = 16) were statistically different (p < 0.05), while their rise time was not different (median values, interquartile ranges and min/max values, see Supplementary Note 8). i Averages of IPSC amplitudes for the five pulses presented on g shows strong short-term depression (STD) of GABAergic transmission evoked by stimulating cholinergic fibres (for details see Supplementary Note 8 and Supplementary Figure 3)CC BY · PubMed Central
Fig. 4 Separate vesicular release of acetylcholine and GABA from the same synaptic active zones. a–c About 0.5 nm-thick virtual sections from electron tomographic reconstructions of cholinergic terminals. Terminal is orange, mitochondria are yellow, synaptic vesicles are blue. Black arrowheads point to the edges of the synapse. Scale bar is 200 nm. b Active zone is enlarged from a. The omega-shaped vesicle is being fused with the terminal membrane (blue arrowhead). c Two serial virtual sections showing a portion of the active zone of another terminal. A fused synaptic vesicle (blue arrowhead) is labelled for vesicular acetylcholine transporter (black immunogold particle). Scale bar for b and c is 100 nm. d Three-dimensional models of reconstructed terminals. Terminal membranes are orange, mitochondria are yellow, synaptic vesicles are blue and synapses are green. Green arrows point to the synapses. Scale bar is 200 nm. The association of the vesicle pools to the active zones is evident. e Scatterplot shows the relationship of vesicle size (volume) and elongation factor in the sampled vesicles. Vesicles in the cholinergic terminals had a broader size-distribution than vesicles of purely GABAergic terminals, which were smaller and more elongated (Supplementary Note 4). Some vesicles (black markers) could be identified as vAChT-positive because immunogold particles clearly labelled them. f The density of the vesicles within a 60-nm-wide band from the membrane is plotted as a function of distance from the terminal membrane at synaptic (green) and extra-synaptic (black) areas. Vesicle density was 6.5 times higher (in the first 60 nm from the membrane) in the synaptic active zone than extrasynaptically (n = 183 vesicles from 8 terminals in 2 mice). Docked (closer to the membrane than 5 nm) or fused (undergoing exocytotic fusion) vesicles were absent extrasynaptically, but were present in the active zones. g, h vAChT labelling was confined to synaptic active zones in cholinergic fibres. CLSM images show terminals of virus-traced septo-hippocampal fibres (red pseudocolor) containing vAChT-positive vesicle pools (yellow), localised to the synaptic active zones that are identified by gephyrin labelling (blue). Scale bar is 500 nm for g and h. i Scale-free analysis confirms that the likelihood of vAChT labelling is the highest at the synaptic active zones (n = 32 synapses, 2 mice). j Correlated CLSM-STORM super-resolution microscopy images show that vAChT (yellow) and vGAT (cyan) vesicle pools are overlapping and are confined to small portions within AAV-eYFP virus-traced septo-hippocampal fibres (red pseudocolor)
Fig. 4 Separate vesicular release of acetylcholine and GABA from the same synaptic active zones. a–c About 0.5 nm-thick virtual sections from electron tomographic reconstructions of cholinergic terminals. Terminal is orange, mitochondria are yellow, synaptic vesicles are blue. Black arrowheads point to the edges of the synapse. Scale bar is 200 nm. b Active zone is enlarged from a. The omega-shaped vesicle is being fused with the terminal membrane (blue arrowhead). c Two serial virtual sections showing a portion of the active zone of another terminal. A fused synaptic vesicle (blue arrowhead) is labelled for vesicular acetylcholine transporter (black immunogold particle). Scale bar for b and c is 100 nm. d Three-dimensional models of reconstructed terminals. Terminal membranes are orange, mitochondria are yellow, synaptic vesicles are blue and synapses are green. Green arrows point to the synapses. Scale bar is 200 nm. The association of the vesicle pools to the active zones is evident. e Scatterplot shows the relationship of vesicle size (volume) and elongation factor in the sampled vesicles. Vesicles in the cholinergic terminals had a broader size-distribution than vesicles of purely GABAergic terminals, which were smaller and more elongated (Supplementary Note 4). Some vesicles (black markers) could be identified as vAChT-positive because immunogold particles clearly labelled them. f The density of the vesicles within a 60-nm-wide band from the membrane is plotted as a function of distance from the terminal membrane at synaptic (green) and extra-synaptic (black) areas. Vesicle density was 6.5 times higher (in the first 60 nm from the membrane) in the synaptic active zone than extrasynaptically (n = 183 vesicles from 8 terminals in 2 mice). Docked (closer to the membrane than 5 nm) or fused (undergoing exocytotic fusion) vesicles were absent extrasynaptically, but were present in the active zones. g, h vAChT labelling was confined to synaptic active zones in cholinergic fibres. CLSM images show terminals of virus-traced septo-hippocampal fibres (red pseudocolor) containing vAChT-positive vesicle pools (yellow), localised to the synaptic active zones that are identified by gephyrin labelling (blue). Scale bar is 500 nm for g and h. i Scale-free analysis confirms that the likelihood of vAChT labelling is the highest at the synaptic active zones (n = 32 synapses, 2 mice). j Correlated CLSM-STORM super-resolution microscopy images show that vAChT (yellow) and vGAT (cyan) vesicle pools are overlapping and are confined to small portions within AAV-eYFP virus-traced septo-hippocampal fibres (red pseudocolor)CC BY · PubMed Central
Fig. 5 Co-transmission of GABA and acetylcholine is cross-regulated by autoreceptors and mediated by distinct calcium channels. a Optical stimulations of cholinergic fibres trigger composite membrane potential responses (average of 50 traces) of an inhibitory neuron recorded in str. lacunosum-moleculare (green). Cells were held at approximately −50 mV to record hyperpolarising and depolarising components concurrently. Early hyperpolarization was confirmed to be of GABAergic origin by bath application of the GABAAR blocker gabazine (magenta, 10 µM, which abolished quick hyperpolarization in 9 out of 9 tested cells), while the late depolarisation was confirmed to be cholinergic by the application of the alpha4-type nicotinic acetylcholine receptor blocker DHβE (black, 1 µM, which abolished depolarisation in 5 out of 5 tested cells). Cells with identifiable nicotinic responses were used to further investigate the properties of GABA and acetylcholine release from cholinergic fibres. b Increase in GABAergic response after AChR block (Fig. 3d) suggested that presynaptic muscarinic receptors cross-modulated GABA (and possibly ACh) release from cholinergic fibres. Therefore, putative presynaptic muscarinic receptors were selectively inhibited via bath application of atropine (n = 6), which led to a significant increase in amplitude of both the GABAergic hyperpolarization and nicotinic depolarisation (green, see Supplementary Note 8). c Bath application of the selective M2 antagonist AFDX-116 (10 µM, n = 6) resulted in changes similar to that of atropine application (for data see Supplementary Note 8). d Blocking GABABRs with CGP-55845 (1 µM, n = 7) also resulted in increased GABAergic and cholinergic responses suggesting a dual modulatory role (for data see Supplementary Note 8). e, f Testing roles of distinct voltage-dependent calcium channels (VDCC) in release of GABA and ACh . e Blockade of P/Q-type VDCC, using selective antagonist ω-agatoxin (1 µM, puff application), reliably decreased GABAergic response component but did not alter cholinergic component. At control conditions, 5 short (1 ms) light pulses at 10 Hz evoked a composite event of GABAergic hyperpolarization and cholinergic depolarisation (green, average of 20 traces). In response to ω-agatoxin application (magenta, n = 9), the GABAergic component (measured as IPSP amplitude) decreased robustly (magenta arrowhead) and the cholinergic component (measured as EPSP area) showed no change (for details see Supplementary Note 8). f Blockade of N-type VDCC, using selective blocker ω-conotoxin (1 µM), reliably reduced cholinergic response component (n = 6) but left GABAergic component unchanged (for details see Supplementary Note 4)
Fig. 5 Co-transmission of GABA and acetylcholine is cross-regulated by autoreceptors and mediated by distinct calcium channels. a Optical stimulations of cholinergic fibres trigger composite membrane potential responses (average of 50 traces) of an inhibitory neuron recorded in str. lacunosum-moleculare (green). Cells were held at approximately −50 mV to record hyperpolarising and depolarising components concurrently. Early hyperpolarization was confirmed to be of GABAergic origin by bath application of the GABAAR blocker gabazine (magenta, 10 µM, which abolished quick hyperpolarization in 9 out of 9 tested cells), while the late depolarisation was confirmed to be cholinergic by the application of the alpha4-type nicotinic acetylcholine receptor blocker DHβE (black, 1 µM, which abolished depolarisation in 5 out of 5 tested cells). Cells with identifiable nicotinic responses were used to further investigate the properties of GABA and acetylcholine release from cholinergic fibres. b Increase in GABAergic response after AChR block (Fig. 3d) suggested that presynaptic muscarinic receptors cross-modulated GABA (and possibly ACh) release from cholinergic fibres. Therefore, putative presynaptic muscarinic receptors were selectively inhibited via bath application of atropine (n = 6), which led to a significant increase in amplitude of both the GABAergic hyperpolarization and nicotinic depolarisation (green, see Supplementary Note 8). c Bath application of the selective M2 antagonist AFDX-116 (10 µM, n = 6) resulted in changes similar to that of atropine application (for data see Supplementary Note 8). d Blocking GABABRs with CGP-55845 (1 µM, n = 7) also resulted in increased GABAergic and cholinergic responses suggesting a dual modulatory role (for data see Supplementary Note 8). e, f Testing roles of distinct voltage-dependent calcium channels (VDCC) in release of GABA and ACh . e Blockade of P/Q-type VDCC, using selective antagonist ω-agatoxin (1 µM, puff application), reliably decreased GABAergic response component but did not alter cholinergic component. At control conditions, 5 short (1 ms) light pulses at 10 Hz evoked a composite event of GABAergic hyperpolarization and cholinergic depolarisation (green, average of 20 traces). In response to ω-agatoxin application (magenta, n = 9), the GABAergic component (measured as IPSP amplitude) decreased robustly (magenta arrowhead) and the cholinergic component (measured as EPSP area) showed no change (for details see Supplementary Note 8). f Blockade of N-type VDCC, using selective blocker ω-conotoxin (1 µM), reliably reduced cholinergic response component (n = 6) but left GABAergic component unchanged (for details see Supplementary Note 4)CC BY · PubMed Central
Fig. 6 GABA release from cholinergic terminals controls healthy and pathological hippocampal network activity. a To understand the functional impact of GABA and acetylcholine co-transmission onto cortical network oscillations, local field potential (LFP) was recorded with standard patch pipettes from the CA1 area of thick hippocampal slices. These slices generated healthy and pathological in vivo like activity patterns. Cholinergic fibres expressing ChR2 were illuminated with blue light in the presence, or in the absence of AChR blockers parallel with LFP recordings. b A spontaneous sharp wave-ripple (SWR) LFP signature (bottom), and its wavelet transform (top) highlighting the characteristic ripple-band frequency component (~200 Hz, white arrowhead). c, d LFP recording from hippocampal CA1 in the absence of AChR blockers (n = 9). Orange raster indicate SWR peaks for three consecutive stimulation periods. Optical stimulation decreased SWR rate significantly (d), then SWR rate increased temporarily after the cessation of optical stimulation (median values, interquartile ranges and min/max values, for data see Supplementary Note 8). e, f After we blocked AChRs (1 µM MLA, 1 µM DHβE, 10 µM atropine), LFP recording from CA1 (n = 9) showed that GABA release from cholinergic fibres alone could decrease SWR rate even more effectively (f). There was no SWR rate increase after the cessation of optical stimulation (median values, interquartile ranges and min/max values, for data see Supplementary Note 8). g Epileptiform activity was evoked by elevating K+ concentration in the ACSF to 8.5 mM in slices. LFP (bottom) and its wavelet transform (top) shows the signature of a pharmacologically evoked epileptiform events. Note the robust differences in amplitude, length and frequency components compared to physiological SWR activity (Fig. 6b) that is consistent with literature data41. h, i Epileptic activity recorded in CA1 in the absence of AChR blockers (n = 7). Orange raster indicate detected epileptic activity peaks for three consecutive stimulation periods. Illumination of cholinergic fibres reduced the rate of epileptic events (i), which recovers after the cessation of optical stimulation (for data see Supplementary Note 8). j, k Epileptic activity recorded in CA1 in the presence of AChR blockers (n = 8). Even in the presence of AChR blockers, illumination of cholinergic fibres reduced the rate of epileptic discharges (k), which recovers after the stimulation (median values, interquartile ranges and min/max values, for data see Supplementary Note 8), suggesting a crucial role for GABA release in controlling epileptiform activity
Fig. 6 GABA release from cholinergic terminals controls healthy and pathological hippocampal network activity. a To understand the functional impact of GABA and acetylcholine co-transmission onto cortical network oscillations, local field potential (LFP) was recorded with standard patch pipettes from the CA1 area of thick hippocampal slices. These slices generated healthy and pathological in vivo like activity patterns. Cholinergic fibres expressing ChR2 were illuminated with blue light in the presence, or in the absence of AChR blockers parallel with LFP recordings. b A spontaneous sharp wave-ripple (SWR) LFP signature (bottom), and its wavelet transform (top) highlighting the characteristic ripple-band frequency component (~200 Hz, white arrowhead). c, d LFP recording from hippocampal CA1 in the absence of AChR blockers (n = 9). Orange raster indicate SWR peaks for three consecutive stimulation periods. Optical stimulation decreased SWR rate significantly (d), then SWR rate increased temporarily after the cessation of optical stimulation (median values, interquartile ranges and min/max values, for data see Supplementary Note 8). e, f After we blocked AChRs (1 µM MLA, 1 µM DHβE, 10 µM atropine), LFP recording from CA1 (n = 9) showed that GABA release from cholinergic fibres alone could decrease SWR rate even more effectively (f). There was no SWR rate increase after the cessation of optical stimulation (median values, interquartile ranges and min/max values, for data see Supplementary Note 8). g Epileptiform activity was evoked by elevating K+ concentration in the ACSF to 8.5 mM in slices. LFP (bottom) and its wavelet transform (top) shows the signature of a pharmacologically evoked epileptiform events. Note the robust differences in amplitude, length and frequency components compared to physiological SWR activity (Fig. 6b) that is consistent with literature data41. h, i Epileptic activity recorded in CA1 in the absence of AChR blockers (n = 7). Orange raster indicate detected epileptic activity peaks for three consecutive stimulation periods. Illumination of cholinergic fibres reduced the rate of epileptic events (i), which recovers after the cessation of optical stimulation (for data see Supplementary Note 8). j, k Epileptic activity recorded in CA1 in the presence of AChR blockers (n = 8). Even in the presence of AChR blockers, illumination of cholinergic fibres reduced the rate of epileptic discharges (k), which recovers after the stimulation (median values, interquartile ranges and min/max values, for data see Supplementary Note 8), suggesting a crucial role for GABA release in controlling epileptiform activityCC BY · PubMed Central
Fig. 7 Illustration of cholinergic terminals and their synaptic architecture. Summary illustration of some of the findings. All cholinergic terminals establish synapses, they are fully equipped with GABAergic–cholinergic co-transmission signalling machinery. GABAergic and cholinergic vesicles are regulated by different VDCCs. Although postsynaptic cholinergic receptor distribution cannot be investigated, their response latencies (that are at least and order of magnitude faster that typical non-synaptic transmission) suggest a focal, intra- and/or peri-synaptic localisation, while GABAA receptors are detected intra-synaptically. Synapses are established on both dendritic shafts and spines in hippocampus
Fig. 7 Illustration of cholinergic terminals and their synaptic architecture. Summary illustration of some of the findings. All cholinergic terminals establish synapses, they are fully equipped with GABAergic–cholinergic co-transmission signalling machinery. GABAergic and cholinergic vesicles are regulated by different VDCCs. Although postsynaptic cholinergic receptor distribution cannot be investigated, their response latencies (that are at least and order of magnitude faster that typical non-synaptic transmission) suggest a focal, intra- and/or peri-synaptic localisation, while GABAA receptors are detected intra-synaptically. Synapses are established on both dendritic shafts and spines in hippocampusCC BY · PubMed Central

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  1. Paxinos, G. & Franklin, K. B. J. Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates (Academic Press, São Paulo, 2012).

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Authors
Takács, V. T., Cserép, C., Schlingloff, D., Pósfai, B., Szőnyi, A., Sos, K. E., Környei, Z., Dénes, Á., Gulyás, A. I., Freund, T. F., & Nyiri, G.
Affiliations
  • HUN-REN Institute of Experimental Medicine (HU)
  • Semmelweis University (HU)
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-07-20
Volume
9
Issue
1
Pages
2848-2848
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-05136-1
PMC
PMC6054650
OpenAlex
W2883601426
Cited by
169
References
122
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en
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  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
EC | European Research Council (ERC-2011-ADG-294313, SERRACO)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Memory and Neural Mechanisms
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Neural dynamics and brain function
Keywords
co-transmission, acetylcholine, GABA, basal forebrain cholinergic system, GABAergic synapses, vesicle docking, calcium channels, presynaptic autoreceptors, postsynaptic potentials, GABAA receptor, sharp-wave ripples, epileptiform activity, cortical state, neurodegenerative diseases
MeSH terms
Acetylcholine, Animals, Calcium, Dendrites, Female, gamma-Aminobutyric Acid, Hippocampus, Male, Mice, Inbred C57BL, Synaptic Transmission, Perfusion, Receptors, GABA-A, Synapses, Synaptic Vesicles, Neurotransmitter Agents, Neurodegenerative Diseases, Imaging, Three-Dimensional, Mice, Synaptic Potentials

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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