Article Figures & Data
Figures
- Fig. 1.
U-tube configuration and photomicrographs of CA1 neurons in rat hippocampal slices. A: schematic diagram showing the configuration of a U-tube in relation to a pyramidal neuron. The largest diameter for the U-tube pore is ∼250 μm. The valve closure results in a linear flow of agonist solution through the U-tube pore driven by gravity. However, the external bath superfusion changes the agonist flow in the direction shown by the dashed lines.B–F: photomicrographs of images of biocytin-filled CA1 neurons in fixed hippocampal slices: pyramidal neuron (B), stratum oriens (SO) interneuron (C), s. pyramidal (SP) interneuron (D), s. radiatum (SR) interneuron (E), and s. lacunosum moleculare (SLM) interneuron (F). a refers to axonal branching. Calibration bar in the images = 150 μm.
- Fig. 2.
Efficacy of choline and ACh in inducing GABAergic postsynaptic currents (PSCs) in CA1 pyramidal neurons of rat hippocampal slices.A: sample neurolucida drawing of an image of a biocytin-filled CA1 pyramidal neuron from which GABAergic PSCs were recorded. Dendrites are drawn as thick lines (for simplicity, uniform thickness was applied to all dendritic branches) and axon as thin lines. B–E: sample tracings of agonist-induced GABAergic PSCs recorded under whole cell patch-clamp mode at 0 mV from 4 pyramidal neurons are shown. GABAergic PSCs were outward currents under the present experimental conditions. Choline (10 mM) or ACh (1 mM) was applied for 12 s as indicated by solid lines at thebottom of each trace. Agonists were applied to the surface of the neurons, covering cell somata and dendrites, by a stream of solution flowing through a U-tube with a pore of 250 μm. Note that summation of individual PSCs resulted in large tonic outward currents, summation being more pronounced in ACh than in choline. InB, exposure of the slice to bicuculline (10 μM) for 5–10 min decreased the spontaneous and agonist-induced PSCs. InC, exposure of the slice to methyllycaconitine (MLA, 10 nM) for 10 min completely abolished the effect of choline and exposure to dihydro-β-erythroidine (DHβE, 10 μM) abolished large portion of ACh-induced PSCs. In D, exposure of the slice to a combination of 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 10 μM) and 2-amino-5-phosphonovaleric acid (APV, 20 μM) for 10 min had minimal inhibitory effect on choline-induced PSCs. In E,exposure of the slice to TTX (0.5 μM) markedly decreased the frequency of ACh-induced PSCs. F: scatter plot shows the distribution of net charge values for each agonist calculated for individual neurons (n = 12). Note that in this and in subsequent figures, there is superimposition of the symbols when the individual values are near to each other.
- Fig. 3.
Efficacy of choline and ACh in inducing GABAergic PSCs in CA1 SO interneurons of rat hippocampal slices. A–C: sample neurolucida drawings of images of 3 biocytin-filled CA1 SO interneurons from which GABAergic PSCs were recorded. All 3 neurons had horizontally oriented dendrites of different lengths, but their axons had a tendency to terminate either in the SO (left) or SLM (middle and right). D–F: sample tracings of GABAergic PSCs recorded from the interneurons shown at the top and the effects of choline and ACh. Note that the agonist-induced PSCs remained isolated for the most part and that choline failed to increase the frequency of GABAergic PSCs in themiddle and right neurons. G: scatter plot shows the distribution of net charge values for each agonist calculated for individual neurons.
- Fig. 4.
Efficacy of choline and ACh in inducing GABAergic PSCs in CA1 SP interneurons of rat hippocampal slices. A andB: sample neurolucida drawings of images of 2 biocytin-filled CA1 SP interneurons from which GABAergic PSCs were recorded. The interneurons had several dendrites emerging from the cell somata that traversed through the SR into the SLM. Axonal branches were clustered in the SP layer. C and D: sample tracings of GABAergic PSCs recorded from the interneurons shown above. E: scatter plot shows the distribution of net charge values for each agonist calculated for individual neurons.
- Fig. 5.
Efficacy of choline and ACh in inducing GABAergic PSCs in CA1 SR interneurons of rat hippocampal slices. A andB: sample neurolucida drawings of images of 2 biocytin-filled CA1 SR interneurons from which GABAergic PSCs were recorded. The dendrites were extended either to the SR and SO (A) or to SR only (B), whereas axon branching was restricted to SR. C and D: sample tracings of GABAergic PSCs recorded from the interneurons shown at the top and the effects of choline and ACh. Note that summation of individual PSCs resulted in large tonic outward currents, the summation being more pronounced in ACh than in choline.E: scatter plot shows the distribution of net charge values for each agonist calculated for individual neurons.
- Fig. 6.
Efficacy of choline and ACh in inducing GABAergic PSCs in CA1 SLM interneurons of rat hippocampal slices. A andB: sample neurolucida drawings of images of 2 biocytin-filled CA1 SLM interneurons from which GABAergic PSCs were recorded. The dendrites were located in the SLM only (A) or to SLM and SR (B). The axon terminated either in the SLM (A) or in various layers (B).C and D: sample tracings of GABAergic PSCs and the effect of choline and ACh recorded from the respective interneurons shown at the top. Note that ACh and choline both produced PSCs that summated to form a large tonic outward current.E: scatter plot shows the distribution of net charge values for each agonist calculated for individual neurons.
- Fig. 7.
Comparison of the net charge of GABAergic PSCs induced by choline and ACh in pyramidal neurons and interneurons of the CA1 region.A: the net charge flowing through GABAAreceptors during a 12-s application of choline (representing the magnitude of α7 nAChR activation) and ACh (representing the magnitude of α7 and α4β2 nAChR activation combined) was calculated and plotted as the mean ± SE for each neuron type. To assess the contribution of α4β2 nAChRs alone, we subtracted the net charge induced by choline from that induced by ACh and plotted it separately in B. The neuron sample size was 12 in the stratum pyramidal pyramidal neuron (SPP), 8 in SO, 7 in SPI, 12 in SR, and 10 in SLM. The magnitude of choline-induced PSCs in A is significantly different between various neuron groups by Dunnett's multiple comparison test: SPP vs. SO (P < 0.01); SPP vs. SPI (P < 0.05); SO vs. SLM (P < 0.001); SPI vs. SLM (P < 0.001); SR vs. SLM (P < 0.01). The magnitude of ACh-induced PSCs in A is significantly different between various groups by Dunnett's multiple comparison test: SPP vs. SO (P < 0.05); SPP vs. SPI (P < 0.01); SO vs. SLM (P < 0.01); SPI vs. SLM (P < 0.001). In A, the mean values for ACh are significantly different (P < 0.01 byt-test) from those for choline in SPP, SR, and SLM. InB, the mean value for SPI is significantly lower than that in SPP (P < 0.001), SR (P< 0.05), and SLM (P < 0.05) by Dunnett's multiple comparison test.
- Fig. 8.
Focal application of ACh triggers GABAergic PSCs in SR interneurons.A: continuous recording of GABAergic PSCs from an SR interneuron. ACh (1 mM) was pressure-ejected (●) close to the cell somata for 25 ms (1st 2 pulses) or 50 ms (3rd and 4th pulse).B: trace shows sample of spontaneous GABAergic PSCs.C: trace (corresponding to the 4th pulse inA) showing the PSCs induced by 50 ms pulse of ACh.D: trace showing the effect of a 50-ms pulse of ACh after exposure of the slice for 2 min to 0.5 μM TTX. Calibration bar applies to traces in B, C, and D. E: plot of the distribution of the events from the trace shown in A.
- Fig. 9.
A low concentration of ACh induces GABAergic PSCs but does not evoke action potentials. A, top: a cell-attached recording from a SR interneuron shows action currents in response to U-tube applied choline. Pipette potential = −60 mV.Bottom: a whole cell recording from the same neuron in which application of a similar pulse of choline evokes nicotinic inward current. Holding potential = −68 mV. Vertical calibration: 25 pA for top trace and 50 pA for bottom trace. B, top: a cell-attached recording in which U-tube applied ACh (10 μM) failed to evoke action currents.Bottom: a whole cell current recording from the same neuron at 0 mV and reveals that same pulse of ACh triggers a burst of GABAergic PSCs. C: cumulative distribution plots of GABAergic PSCs under control conditions and during a 5-min bath exposure to ACh (10 μM) in another SR interneuron. ACh-induced changes in the distribution of the amplitudes and interevent intervals were found to be statistically significant (P < 0.001 by Kolmogorov-Smirnov analysis).
- Fig. 10.
Schematic representation of hippocampal CA1 neurocircuitry showing the impact of nAChR-mediated GABAergic inhibition. One pyramidal neuron (P) and 4 interneurons (I) in different laminae are shown. Excitatory glutamatergic afferents (blue lines) and inhibitory GABAergic afferents (red lines) are shown. The prevalent nAChR subtypes expressed on the somatodendritic region of the interneurons are indicated near each neuron. The strength of inhibition exerted onto each category of neurons arising from nAChR-mediated GABA release is shown on an arbitrary scale of 1+ to 5+, with 1+ representing a low-level inhibition and 5+ a high-level inhibition.
Tables
- Table 1.
ACh/choline ratio in inducing GABAergic PSCs
Parameter SPP SO SPI SR SLM Mean 4.20 ± 0.67 4.04 ± 0.64 1.36 ± 0.35 4.40 ± 0.60 1.83 ± 0.17 Range 1.9–9.6 2.1–5.6 0.7–3.1 1.3–7.4 1.0–2.6 n 12 5 6 12 10 P* <0.01 <0.05 >0.05 <0.01 >0.05 -
↵* Data were compared to a hypothetical mean ratio of 2.0 for significance using the one-sample t-test. An ACh/choline ratio of 2.0 would be expected if there was equal contribution from α7 and α4β2 nicotinic ACh receptors (nAChRs) in ACh-induced postsynaptic currents (PSCs). SPP, stratum pyramidal pyramidal neuron; SO, stratum oriens; SPI, s. pyramidal interneuron; SR, s. radiatum; SLM, s. lacunosum moleculare.
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