Article Figures & Data
Figures
- Figure 1.
Cell-type-specific activity-dependent inhibition of layer 1 interneurons by Ach. A, The iontophoretic application of ACh (bottom trace, 100 mm, 20 ms) silences ongoing action potential firing in a NGFC. In contrast, ACh generates excitatory responses at the indicated, subthreshold holding potentials. The anatomical reconstruction of a NGFC is shown in the inset. The soma and dendrites are shown in black and the axon in gray. B, Excitation, but not inhibition of a c-AC by the iontophoretic application of ACh (bottom trace, 100 mm, 20 ms). Inset, The anatomical reconstruction of the c-AC. A, B, The action potential amplitude has been truncated for clarity. C, D, Summary of the control of ongoing action potential firing by ACh in NGFCs (C) and c-ACs (D). Data have been pooled from the indicated number of neurons to generate peristimulus time histograms of action potential firing (250 ms time bins). ACh was applied at time 0.
- Figure 2.
Pharmacology of cholinergic excitation and inhibition of NGFCs. A, The nicotinic ACh receptor antagonist mecamylamine (10 μm, right) blocks subthreshold excitatory, but not suprathreshold inhibitory responses evoked by iontophoretic application of ACh (bottom trace, 100 mm, 20 ms). B, Summary of the control of action potential firing by ACh under control and in mecamylamine (10 μm). Peristimulus time histograms (250 ms time bins) of action potential firing from pooled NGFCs (n = 4 cells) under control conditions (top) and after the bath application of mecamylamine (bottom). ACh was applied at time 0. C, The muscarinic ACh receptor antagonist telenzepine (100 nm, right) blocks suprathreshold inhibitory, but not subthreshold excitatory responses evoked by iontophoretic application of ACh (bottom trace, 100 mm, 20 ms). A, C, The action potential amplitude has been truncated for clarity. D, Summary of the control of action potential firing by ACh under control and in telenzepine (n = 5). E, ACh-evoked excitatory responses in the presence of telenzepine (100 nm) are attenuated by DHβE (500 nm) and blocked by mecamylamine (10 μm). F, G, Summary of the actions of DHβE (500 nm) on the peak amplitude (F) and decay time constant (G) of ACh-evoked excitatory responses. Values represent mean ± SEM.
- Figure 3.
Intracellular calcium controls ACh-evoked inhibition of NGFCs. A, The iontophoretic application of ACh (bottom trace, 100 mm, 20 ms) silences ongoing action potential firing of a NGFC recorded with a pipette filled with normal internal solution. This inhibitory action was abolished when the NGFC was repatched with a pipette filled with an internal solution containing the calcium-chelator BAPTA (10 mm, gray trace). B, Summary of the abolition of the inhibitory actions of ACh by BAPTA (gray bars, n = 8 cells). Data have been pooled to generate peristimulus time histograms of action potential firing (250 ms time bins). ACh was applied at time 0. C, Repatching NGFCs with pipettes containing standard intrapipette solution does not disturb the inhibitory actions of ACh (gray bars, n = 5). D, Simultaneous voltage (gray traces) recording and Oregon green BAPTA-6F (ΔF/F black traces) imaging show that ACh-evoked inhibition of action potential firing is accompanied by a rise in intracellular calcium, whereas subthreshold excitatory responses are not. ACh was delivered by iontophoresis (bottom trace, 100 mm, 20 ms). E, Linear relationship between the time course of ACh-evoked inhibition of action potential firing and calcium signaling. Data are from multiple trials in 15 NGFCs evoked by ACh iontophoresis. The line represents a linear regression. F, Area of ACh-evoked Oregon Green BAPTA-6F signals (ΔF/F · s) as a function of holding voltage (mV) for the cell shown in D (Thresh. refers to action potential firing threshold).G, Summary of the area of ACh-evoked Oregon green BAPTA-6F signals (ΔF/F · s) at RMP and threshold (Thresh; n = 16 cells). Values represent mean ± SEM. H, The broad-spectrum calcium channel antagonist cadmium (50 μm) blocks the inhibitory actions of ACh. A, D, H, The action potential amplitude has been truncated for clarity. I, Summary of the abolition of the inhibitory actions of ACh by cadmium (right, n = 7 cells). Data have been pooled to generate peristimulus time histograms of action potential firing (250 ms time bins). ACh was applied at time 0.
- Figure 4.
Calcium release from internal stores controls cholinergic inhibition of NGFCs. A, Blockade of ACh-evoked silencing of action potential firing by thapsigargin (10 μm). Action potential amplitude has been truncated for clarity. B, Peristimulus time histogram of action potential firing under control and after bath application of thapsigargin (bottom; 250 time bins; n = 4 cells). ACh was applied at time 0. C, Average voltage (Vm) and Oregon Green BAPTA-6F signals (ΔF/F) evoked by the iontophoretic application of ACh in TTX (1 μm; top) are reduced by bath application of 2-APB (bottom). Single traces are shown in gray. D, E, Summary of the effects of 2-APB on ACh-evoked voltage responses (D) and Oregon green BAPTA-6F signals (E; n = 4). Values represent mean ± SEM.
- Figure 5.
Depletion and refilling of internal calcium stores controls cholinergic inhibition of NGFCs. A, Illustration of the experimental paradigm. ACh was iontophoretically applied (1 m, 20 ms) after 7 s of repetitive action potential firing and evoked a pronounced hyperpolarization (gray trace) together with an Oregon green BAPTA-6F signal (black trace). To investigate the role of action potential-dependent calcium store refilling, the membrane voltage was either stepped back to resting membrane potential (depletion) or the NGFCs were allowed to fire action potentials for a further 7 s (refilling). In both cases, the inhibitory actions of ACh were re-examined at times following the reintroduction of repetitive action potential firing (test responses). B, In the depleted case (left), an ACh test response generated 50 ms after the reintroduction of repetitive action potential firing did not silence neuronal output (gray trace) and evoked a small amplitude Oregon green BAPTA-6F signal (black trace). In contrast, after 5 s of repetitive action potential firing the inhibitory actions of ACh were restored. In the refilling case (right), the inhibitory actions of ACh were time-independent. A, B, The action potential amplitude has been truncated for clarity. C, D, Time-dependent recovery of ACh-evoked Oregon green BAPTA-6F signals (C; gray symbols) and the time course of ACh-evoked silencing of action potential firing following depletion (D; gray symbol), but time-independent Oregon green BAPTA-6F signals (C; black symbols) and inhibition following refilling (D; black symbols; n = 5 NGFCs). Values represent mean ± SEM.
- Figure 6.
Activity-dependent cholinergic inhibition of NGFCs is mediated by SK potassium channels. A, Action potential firing transforms ACh signaling. The top trace shows the transformation of ACh signaling from excitatory to inhibitory following a period of action potential (AP) firing (100 APs evoked at 17 Hz). ACh was applied iontophoretically (bottom trace, 100 mm, 20 ms). The middle voltage trace shows the sequential generation of excitatory responses by ACh in the absence of AP firing. B, Pooled data describing the voltage integral of ACh responses evoked before (open bar) and after (gray bar) AP firing. Values represent mean ± SEM. C, ACh-evoked silencing of AP firing is prevented by the blockade of SK potassium channels with apamin (100 nm). ACh delivered by iontophoresis (bottom trace, 100 mm, 20 ms). A, C, The action potential amplitude has been truncated for clarity. D, Summary of the control of ongoing AP firing under control and in the presence of apamin (right). Data have been pooled to generate peristimulus time histograms of AP firing (250 ms time bins). ACh was applied at time 0.
- Figure 7.
NGFCs drive powerful postsynaptic inhibition. A, Current-evoked single action potential firing of a NGFC evokes unitary IPSPs in a postsynaptic layer 2/3 pyramidal neuron that are substantially inhibited by a GABAB receptor antagonist (red trace; CGP 55845, 10 μm). B, Cumulative probability distribution of the peak amplitude of single action potential evoked unitary IPSPs in NGFC–layer 2/3 pyramidal neuron pairs (n = 437 IPSPs, n = 19 connections). C, Summary of the actions of CGP 55845 (10 μm) on the area of single action potential-evoked unitary IPSPs (n = 7 connections). D, Repetitive current-evoked NGFC action potential firing evokes summating IPSPs in a postsynaptic layer 2/3 pyramidal neuron that are sensitive to GABA receptor antagonist (CGP 55845, 10 μm; SR-95531, 10 μm). E, Current-evoked NGFC IPSPs silence action potential firing in a postsynaptic layer 2/3 pyramidal neuron (n = 14 overlain traces). Tonic action potential firing of the pyramidal neuron was evoked by the delivery of positive current (130 pA). F, ACh-evoked depolarization drives single action potential firing of NGFCs and postsynaptic IPSPs in layer 2/3 pyramidal neurons (red traces), with the same properties as current-evoked action potentials (black traces). ACh was delivered by iontophoresis (100 mm, 20 ms). G, Cumulative probability distribution of ACh- and current-evoked unitary IPSPs (n = 60 IPSPs, n = 5 connections). H, Muscarinic receptor-mediated ACh inhibition silences current-evoked action potential firing and the inhibitory synaptic output of NGFCs. Control responses show current-evoked NGFC action potential firing (top black trace) which drives postsynaptic inhibition (bottom black trace). The iontophoretic delivery of ACh (100 mm, 20 ms) in the presence of the nACHR antagonist mecamylamine (10 μm) leads to the generation of a hyperpolarization, which inhibits current-evoked action potential firing and postsynaptic inhibition (red traces). E, H, The action potential amplitude has been truncated for clarity. I, Pooled data describing the normalized peak amplitude of IPSPs evoked by bursts of action potentials in NGFC—layer 2/3 pyramidal neuron pairs (black symbols), which were silenced by the iontophoretic delivery of ACh (red symbols; 100 mm, 20 ms). Data have been pooled from n = 4 connections. C, I, Values represent mean ± SEM.
- Figure 8.
Activity-dependent silencing of the synaptic output of NGFCs by ACh. A, Continuous voltage record (top trace) showing the repeated generation of current-evoked (middle trace) action potential firing of a NGFC. At the beginning of the sequence ACh-evoked a small excitatory response (ACh delivered by iontophoresis, 100 mm, 20 ms, bottom trace, blue label, test response). In contrast, after the generation of 18 bursts of action potential firing, ACh evoked a hyperpolarization, which silenced current-evoked action potential firing (red label, test response). B, High gain representation of the voltage record shown in A, showing NGFC voltage responses (top traces), the pattern of driving current (middle traces), and the inhibitory synaptic output recorded from a layer 2/3 pyramidal neuron (bottom traces). C, Summary data showing the activity-dependent silencing of the synaptic output of NGFCs by ACh. IPSP amplitude has been normalized to the first response. Data have been pooled from n = 7 connections. D, Pooled data describing the inhibition of the inhibitory synaptic output of NGFCs by ACh. Postsynaptic impact refers to the area of the postsynaptic response measured from layer 2/3 pyramidal neurons over similar time periods for test (blue) and conditioned ACh responses (red). Data pooled from n = 7 connections. C, D, Values represent mean ± SEM.
Tables
NGFCs (n) c-ACs (n) Resting membrane potential −77.0 ± 0.5 mV (72) −75.9 ± 0.73 mV (31) Apparent input resistance 109.3 ± 3.8 MΩ (72) 123.2 ± 6.0 MΩ (31) Action potential accommodation index 0.42 ± 0.06 (72) −0.42 ± 0.07 (34) Action potential after-hyperpolarization −24.9 ± 0.4 mV (68) −15.2 ± 0.9 mV (28) Dendritic field area 0.013 ± 0.006 mm2 (5) 0.041 ± 0.007 mm2 (7) Axonal density 0.191 ± 0.036 μm−1 (5) 0.064 ± 0.014 μm−1 (7) Analysis revealed that interneuronal classes could be divided electrophysiologically by statistically significant differences in apparent input resistance (measured in response to a −20 pA current step; Students t test: T = 1.99, p = 0.0483), action potential accommodation index (calculated as the ratio of the first and last interspike interval, in response to positive current steps of magnitude set to the rheobase for the generation of repetitive action potential firing; Mann–Whitney test: U = 73.5, p < 0.0001), and the amplitude of the action potential after-hyperpolarization (measured from single action potential generated at rheobase; Students t test: T = 12.28, p < 0.0001). Morphological analysis revealed that the dendritic field area (Students t test: T = 2.76, p = 0.02) and axonal density (total axonal length/axonal field area; Students t test: T = 3.70, p = 0.0041) of interneuronal classes were significantly different. Values represent mean ± SEM.
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