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. 2005 Jan 1;562(Pt 1):165-82.
doi: 10.1113/jphysiol.2004.070300. Epub 2004 Nov 4.

Somato-dendritic nicotinic receptor responses recorded in vitro from the medial septal diagonal band complex of the rodent

Zaineb Henderson  1 , András Boros, Gergely Janzso, Andrew J Westwood, Hannah Monyer, Katalin Halasy
Affiliations

Somato-dendritic nicotinic receptor responses recorded in vitro from the medial septal diagonal band complex of the rodent

Zaineb Henderson et al. J Physiol. .

Abstract

The medial septal diagonal band area (MS/DB), made up of GABAergic and cholinergic neurones, plays an essential role in the generation and modulation of the hippocampal theta rhythm. To understand the part that the cholinergic neurones might play in this activity, we sought to determine whether postsynaptic nicotinic receptor responses can be detected in slices of the rodent MS/DB by puffing on acetylcholine (ACh). Neurones were characterized electrophysiologically into GABAergic and cholinergic neurones according to previous criteria. Responses of the MS/SB neurones to ACh were various combinations of fast depolarizations (1.5-2.5 s), fast hyperpolarizations (3-4 s) and slow depolarizations (20-30 s), the latter two being blocked by atropine. The fast depolarizations were partially or not blocked with cadmium and low calcium, tetrodotoxin, and antagonists of other ionotropic receptors, and were antagonized with 25 microm mecamylamine. Pharmacological investigation of the responses showed that the alpha 7* nicotinic receptor type is associated with cholinergic neurones and 10% of the GABAergic neurones, and that non alpha 7* nicotinic receptor subtypes are associated with 50% of the GABAergic neurones. Pharmacological dissection of evoked and spontaneous postsynaptic responses, however, did not provide evidence for synaptic nicotinic receptor transmission in the MS/DB. It was concluded that nicotinic receptors, although prevalent on the somatic and/or dendritic membrane compartments of neurones in the MS/DB, are on extrasynaptic sites where they presumably play a neuromodulatory role. The presence of alpha 7* nicotinic receptors on cholinergic neurones may also render these cells specifically vulnerable to degeneration in Alzheimer's disease.

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Figures

Figure 1
Figure 1. Examples of different types of responses to puffed ACh
A, a fast firing cell recorded at –50, –53 and –60 mV, showing changes in amplitudes of the fast depolarization (1), fast hyperpolarization (2) and slow depolarization (3) with variation in the membrane potential. The filled triangle in this figure and in all subsequent figures, indicates when the brief (10–60 ms) application of ACh was made. B, responses of the same fast firing cell as in A are shown, this time as recordings in voltage-clamp mode at –50 and at –70 mV. The equivalents to the fast depolarization (1) and slow depolarization (3) are visible as inward currents, while the equivalent to the fast hyperpolarization (2) is visible as an outward current. C, a fast firing cell, voltage clamped at –70 mV, showing block of the outward current response to ACh by 5 μm atropine (the previous trace, shown in grey, is superimposed). D, bar chart showing the different types of responses of characterized MS/DB neurones in rat and mouse to puffed ACh. The symbols α7 and non-α7 refer to responses attributed to the presence of α7*-type nicotinic and non-α7*-type nicotinic receptors, respectively, as determined from pharmacological investigation and kinetics of the responses. EGFP-positive neurones are those that correspond to parvalbumin-containing cells in the mouse.
Figure 2
Figure 2. Examples of α7* nicotinic receptor-type responses of MS/DB cholinergic and GABAergic neurones to puffed ACh
A, a slow firing cell showing a fast depolarization response that produced a single action potential plus a slow depolarization that was blocked by 5 μm atropine. B, a voltage-clamp recording, at –60 mV, from a slow firing cell responding to ACh, showing partial block of the inward current response by 100 nmα-bungatotoxin. The remaining response had a slower rise time than the original. C, a voltage-clamp recording, at –70 mV, from a slow firing cell responding to ACh, showing the effects of the bath application of 20 μm NBQX, 50 μmd-AP5 and 20 μm bicuculline, followed by 20 nm methyllycaconitine (MLA) with recovery, then 25 μm mecamylamine (MCA). D, a fast firing neurone responding to ACh, showing the effects of application of 20 μm NBQX, 50 μmd-AP5 and 20 μm bicuculline, followed by 0.6 μm TTX, 5 μm atropine, 2.5 μm mecamylamine (MCA) and then 20 nm methyllycaconitine (MLA). E, bar chart showing the 20–80% rise times of characterized ACh responses of identified cells. Abbreviations: α7 and non-α7 are responses attributed to the presence of α7*-type nicotinic and non-α7*-type nicotinic receptors, respectively; S, slow firing neurone type; F, fast firing neurone type; B, burst firing neurone type. The symbol * indicates that there was a statistically significant difference between the rise times of α7*-type nicotinic and non-α7*-type nicotinic receptor responses. F, bar chart showing relative changes in the amplitude the α7* nicotinic receptor subtype responses in slow firing neurones after application of various agents. Error bars indicate s.d.
Figure 3
Figure 3. Examples of non-α7* nicotinic receptor-type responses of MS/DB GABAergic neurones to puffed ACh
A, a fast firing neurone showing a fast depolarization response that produced a burst of spikes, followed by a slow depolarization that was blocked with 5 μm atropine. B, a fast firing cell, showing block of the fast depolarization response to ACh by 25 μm mecamylamine (MCA), with recovery after washout of the antagonist. C, a fast firing neurone with an ACh response, showing the effects of application of 10 nm methyllycaconitine (MLA), followed by 0.1 μm DHβE, then recovery of the response after washout of the antagonist. D, a fast firing neurone with an ACh response, showing the effects of the application of 10 nm methyllycaconitine (MLA) followed by 0.1 μm DHβE then 2.5 μm mecamylamine, with recovery of the response after washout of the antagonist.
Figure 4
Figure 4. Effects of various agents on non-α7* nicotinic receptor-type responses of MS/DB GABAergic neurones to puffed ACh
A, a voltage-clamp recording, at –70 mV, from a fast firing neurone showing the effects of application of cadmium and low calcium, followed by 0.6 μm TTX, then 25 μm mecamylamine (MCA). B, a fast firing neurone with an ACh response, showing the effects of application of 20 μm NBQX, 50 μmd-AP5 and 20 μm bicuculline, followed by 25 μm mecamylamine (MCA). C, bar chart showing relative changes in the amplitudes of the non-α7* nicotinic receptor-type responses after application of non-nicotinic ionotropic receptor antagonists, TTX and cadmium and low calcium. Error bars indicate s.d.
Figure 5
Figure 5. Immunocytochemical staining for the α7, α4 and β2 nicotinic receptor subunits in the MS/DB
A–C, staining for receptor subunit immunoreactivity with the DAB method showing the distribution of α7, α4 and β2 nicotinic receptor subunits, respectively, in somata as viewed with bright field optics. Calibration bar = 100 μm. D–G, staining using a dual immunofluorescence method for parvalbumin (D, FITC filter) and the α4 nicotinic receptor subunit (E, rhodamine filter), or for VAChT (F, FITC filter) and the α4 nicotinic receptor subunit (G, rhodamine filter), as viewed by confocal microscopy. Double-labelled neurones are indicated by a ‘1’ and single labelled neurones by a ‘2’. Calibration bar = 50 μm.
Figure 6
Figure 6. Staining using a dual immunofluorescence method for parvalbumin (A and C, FITC filter) and the β2 nicotinic receptor subunit (B and D, rhodamine filter), or for VAChT (E and G, FITC filter) and the β2 nicotinic receptor subunit (F and H, rhodamine filter) and as viewed by confocal microscopy
Double-labelled neurones are indicated by a ‘1’ and single-labelled neurones by a ‘2’. Calibration bar = 50 μm.
Figure 7
Figure 7. Bar charts showing a quantitative assessment of the double immunofluorescence labelling for the nicotinic receptor subunits α4 and β2, and the neurochemical markers parvalbumin (PV) and VAChT, shown in Figs 5 and 6.
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