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Heterogeneity acetylcholine

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Heterogeneity of neuronal nicotinic acetylcholine receptors in thin slices of rat medial habenula
J. G. Connolly *, A. J. Gibb and D. Colquhoun
Department of Pharmacology, University College London, Cower Street, London WC1E 6BT, UK
1. Neuronal nicotinic acetylcholine receptors in slices of rat medial habenula were studied using patch clamp recording techniques.
2. Whole cell current responses to cytisine could be blocked by hexamethonium, as expected for nicotinic receptors. The whole cell current-voltage relations were linear at negative membrane potentials, but showed strong inward rectification when chloride currents were minimized.
3. When 1 mm Ca2+ (0 mM Mg2+) was present in the external recording solution, the single channel conductances elicited by acetylcholine or nicotine in twenty patches were in the range 39-58 pS, with a mean of 47 pS. There appeared to be at least two groups of conductances.
4. In the open point amplitude distributions of three patches, the most common amplitude corresponded to 41 pS (81 % of the area). In another four patches the most common amplitude corresponded to a mean conductance of 51 pS (83% of the area). Direct transitions between open levels were rare.
5. Channel closed times were not significantly different for the two conductance groups. However, for the four patches with predominantly 51 pS openings, the means of the distributions of open times longer than two filter rise times averaged 5'8 ms. Those patches with predominantly 41 pS openings averaged 14 ms. Also, for patches with predominantly 51 pS openings the overall mean burst length was 5-8 ms, whereas for patches with predominantly 41 pS openings it was 16-1 ms.
6. These observations suggest that 51 and 41 pS openings result from the activity of at least two, but possibly more, different receptor subtypes. We conclude that nicotinic receptors in the rat ventral medial habenula are heterogeneous.
Electrophysiological, radiolabelled ligand-binding and in situ hybridization studies suggest that nicotinic acetyl- choline receptors (AChRs) are widespread throughout the brain (for reviews see Deneris, Connolly, Rogers & Duvoisin, 1991; Role, 1992). These receptors have been much less well characterized than their counterparts in ganglia and at the neuromuscular junction.
A recent technical development, the thin slice technique (Edwards, Konnerth, Sakmann & Takahashi, 1989), has meant that all regions of the brain can now be studied with the full range of patch-clamp techniques. Advantages of this technique are that the cells being studied can be seen directly and that many of the synaptic connections of the neurones are maintained.
Within the rat brain, in situ hybridization studies have shown that the medial habenula is particularly rich in the expression of neuronal AChR subunit RNAs (Wada et al. 1988; Deneris et al. 1988; Duvoisin, Deneris, Patrick & Heinemann, 1989; Seguela, Wadiche, Dinely-Miller, Dani & Patrick, 1993). However the distribution is not uniform. While the neuronal a7, a4, fl2 and /l3 genes are transcribed throughout the rat medial habenula, the a3 and ,84 genes are transcribed only in the ventral areas. The a3, a4, /12 and /14 subunits (but not /13) have been functionally expressed (in pairs) in Xenopus oocytes while a7 expresses as a homo-oligomer (Deneris et at. 1991, review; Seguela et al. 1993).
The medial habenula also contains sites of high-affinity nicotine binding (Clarke, Schwartz, Paul, Pert & Pert,
* To whom correspondence should be sent at the following address: Department of Physiology and Pharmacology, University of Strathclyde, Royal College, 204 George Street, Glasgow GI 1XW, UK.
J 0. Connolly, A. J Cibb and D. Colquhoun
1985) and intracellular electrophysiological studies in the guinea-pig medial habenula have revealed the presence of excitatory nicotinic acetylcholine responses (McCormick & Prince, 1987). Furthermore, Mulle and co-workers (Mulle & Changeux, 1990; Mulle, Vidal, Benoit & Changeux, 1991; Mulle, Choquet, Korn & Changeux, 1992a; Mulle, Lena & Changeux, 1992b) have examined dissociated cells from the medial habenula region using patch-clamp techniques, and recorded nicotinic whole cell responses and single channel activity. We chose also to study nicotinic responses in cells in the medial habenula, but made use of the thin- slice technique to allow us to study the receptors in situ, and to avoid the possibility of inducing changes in the receptor population that might be brought about by the enzyme treatment during dissociation. In this study we find evidence that there are at least two types of nicotinic acetylcholine receptor in the medial habenula. A preliminary report of this data has been published (Connolly & Colquhoun, 1991).
METHODS Preparation of slices The procedures followed were essentially the same as those described in Edwards et al. (1989). A male, or female, Sprague-Dawley rat (16-23 days) was killed by cervical dislocation and then decapitated. The brain was removed and placed in ice-cold physiological buffer (slicing solution) of composition (mM): NaCl, 125; KCl, 2-5; NaHCO3, 26; NaH2PO4, 1P25; glucose, 25; CaCl2, 2; MgCl2, 1; bubbled with 95% 02-5% C02, (pH 7 4). A coronal section of brain which included the
medial habenula was then fixed to a Teflon block using a thin film of liquid cyanoacrylate glue and sliced using a Campden Instruments Vibroslice. Slices of 180-220 um thickness were used in these experiments. The pigmented, in some places clustered, cells of the medial habenula were clearly distinguishable from surrounding brain regions (Fig. 1A). Between four and six such sections could be obtained, depending upon the age of the animal.
Optical equipment and recording bath Slices were placed individually in a circular bath of about 0 7 ml volume through which oxygenated recording solution was continuously perfused at a rate of 3 ml mint. The slice was held in place by a ladder of nylon fibres glued to a circular platinum frame (Fig. 1B).
Cells within the slice were viewed using differential interference contrast (Nomarski) optics on the stage of a Zeiss-Jena upright microscope using a Zeiss x 40, 0 75 numerical aperture, water- immersion objective lens with 1i6 mm working distance at a total magnification of x 600. Healthy cells were identified by their soft, smooth appearance.
Recording conditions WVhole cell and single channel patch clamp recordings were then obtained using an Axopatch 1B patch clamp amplifier and the signals recorded on a Racal Store 4 FM tape-recorder (DC to 5 kHz, -3 dB). Single channel recordings were made using the outside-out patch configuration (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Recordings were made at room temperature (20-24 0C) with the patch membrane potential clamped at -60 mV. In the experiments selected for time course analysis, the agonist used was ACh (1-3 2 pM). Patches were chosen where the overall level of activity was low and where double openings were absent or rare.
Figure 1. Location of the ventral medial habenula in the slice preparation A, diagrammatic outline of a coronal section through the rat brain showing the location of the medial habenular nuclei adjacent to the walls of the third ventricle. B, the outline of the part of the slice used for experiments is shown with the surrounding cortical regions removed. The slice is held stably in the recording chamber using a ladder of nylon fibres glued to a platinum ring (Edwards et al. 1989).
J. Phy8iol. 484.188
Nicotinic receptors in rat medial habenula
Patch electrodes were pulled from filamented, thick-walled borosilicate glass (Clark Electromedical GC150F-7 5; o.d. 1 5 mm, i.d. 0-86 mm), coated to within 100 ,sm of the tip with Sylgard® resin (Dow Corning 184) and fire polished on a Narashige (MF-83) microforge to a final tip resistance of 8-20 MQ. Whole cell recordings were made with pipettes of 5-13 MQ resistance pulled from thin-walled glass (GC150TF, o.d. 1-5 mm, i.d. 1'17 mm). VVhole cell resistances averaged 1-5 GQ2 at negative membrane potentials and 0-45 GQ2 at positive potentials. Whole cell capacitances were in the range 6-15 pF, and series resistances ranged from 17 to 60 MQ2. Series resistance compensation was not used.
Solutions Outside-out patches. The normal external recording solution for outside-out patches was (mM): NaCl, 118; NaHCO3, 30; NaH2PO4, 1; CaCl2, 1; KCl, 2-5; bubbled with 95% 02-5% CO2 (pH 7 4). Atropine (05juM) and AP5 (D-aminophosphono- valerate, 5-25 uM) were routinely added to this control solution unless stated otherwise. The usual internal recording solutions (mM) were: (i) CsCl, 140; NaCl, 10; Na-EGTA, 11; Hepes-NaOH, 10 (pH 7 4); and (ii) gluconolactone, 150; NaOH, 150; EGTA, 11; Hepes-NaOH, 10 (pH 7 4). As indicated in the text, the sodium gluconate solution was occasionally diluted by 6% to assist seal formation.
Whole cell recordings. Three different external solutions were
used: (i) slicing solution (described above), (ii) low-Cl- solution (composition (mM): sodium isethionate, 110; NaCl, 15; NaHCO3, 26; NaH2PO4, 1-25; KCl, 2-5; CaCl2, 2; MgCl2, 1; glucose, 25), and (iii) low-Cl-, 0 mm Ca2+ solution in which 2 mm Ca2+ and 1 mM Mg2+ in solution (ii) were replaced by 3 mm Mg2+. All external solutions were freshly made to minimize glutamate or
glycine contamination. All reagents were obtained from Sigma (UK). Pipette solutions were either the 94% sodium gluconate described above, an 80% sodium gluconate solution containing 5 mM BAPTA and 5 mm EGTA instead of 10 mm EGTA, or a
KCl solution containing (mM): KCl, 140; MgCl, 1; CaCl, 1; EGTA, 10; Hepes-NaOH, 10; ATP, 2 (pH 7 3).
Drug solutions were perfused through the bath by switching manually between control and drug solution reservoirs at a
control valve close to the inlet of the recording bath. Outside-out patches were brought close to the mouth of the solution inlet tube. This enabled solutions around outside-out patches to be changed within 1-2 s and also reduced the risk of activation of channels in the patch by neurotransmitters leaking from the slice. When recording whole cell currents, cells were in situ in the slice, and so it took 10-30 s for the response to reach a peak, suggesting a much slower solution exchange within the slice itself.
Whole cell I-V ramps
These were initiated by, and recorded on, a Dell 486 microcomputer using a Cambridge Electronic Design 1401 interface. From a holding potential of -50 mV, a 3 s voltage ramp was applied. The membrane potential was first ramped to -100 mV, then to +100 mV, and finally back to the holding potential. The current response to this voltage ramp was filtered at 250 Hz (-3 dB, Bessel-type filter), digitized at 500 Hz, and stored directly on the computer hard disk.
In each experiment, a series of four control ramp responses were
obtained before the application of agonist. A second set of four to
eight responses were obtained during the steady-state response to the application of agonist (30juM ACh or 20,uM cytisine). Finally a further four control responses were obtained after the agonist had been washed out. Individual responses in control and agonist-containing solutions were then examined off-line and accepted control responses from both before and after the agonist application were averaged together. The acceptable ramp responses during agonist application were also averaged. Comparison of the control responses from before and after agonist application was used to confirm that the resting properties of the cell had not changed during agonist application. The mean of all control responses was then subtracted from the mean response obtained in the presence of the agonist.
Amplitude measurements For single channel I-V curves, and for most estimates of conductance in Fig. 6, measurements of current amplitudes were obtained from single channel records plotted on a UV writing chart recorder (Medelec) and by cursor fitting using a modification (by S. F. Traynelis, Emory University, Atlanta, GA, USA) of the Axotape data acquisition programme. At least ten amplitude measurements were made for each conductance or amplitude value quoted. The amplitudes which contributed to the open point amplitude histograms were measured as part of the time course fitting method of analysis of the single channel records (Colquhoun & Sigworth, 1995). When estimating conductances from amplitudes, the reversal potentials were assumed to be -8-6 mV when CsCl was the internal pipette solution (based on the intercepts of single channel I-Vrelations), and -4-6 and -3-2 mV, respectively, when 100 or 94% sodium gluconate was in the pipette solution (based on the calculated reversal potential for Na+ ions).
Time course analysis of single channel records Single channel currents were stored on FM tape (Racal Store 4, DC to 5 kHz) after filtering at 10 kHz (-3 dB, 8 pole Bessel response). Data records were replayed from FM tape, amplified and filtered at from 1 to 4 kHz (-3 dB, 8 pole Bessel), and continuously sampled at 10-40 kHz onto a PDP 11/73 computer hard disk using a CED502 interface (Cambridge Electronic Design). The duration of open and closed periods in the data record was measured using the method of time course fitting (Colquhoun & Sigworth, 1995), after first measuring the amplitude of each channel opening using manually controlled cursors placed on the data display. An idealized record of the duration and amplitude of every detectable event in the data record was obtained by imposing a fixed resolution for open times and closed times on the data before construction and fitting of distributions. This resolution was 80-140 ,us for open times and 70-120 ,us for closed times.
Histograms of the distribution of open times and shut times, and properties of bursts of openings were constructed for display and evaluation of the data. In most cases the distribution of log(duration) is used for display purposes (Blatz & Magleby, 1986; Sigworth & Sine, 1987), with a square root transformation of the ordinate (Sigworth & Sine, 1987).
Distributions were fitted with the sum of several exponential, Gaussian or geometric components where appropriate. The individual observations of open times, shut times, etc. were used for fitting by the method of maximum likelihood (Colquhoun & Sakmann, 1985; Colquhoun & Sigworth, 1995).
J. Phy8iol. 484.1 89
J C. Connolly, A. J: Gibb and D. Colquhoun
Identification of channel conductance levels Subconductance levels were identified during analysis if an amplitude change of greater than 10% of the open channel current was observed. Since it is not possible to estimate accurately the amplitude of very brief channel openings, amplitude histograms contained only values measured from openings of duration at least 2tr, where tr is the filter rise time of the recording system (e.g. Colquhoun & Sigworth, 1995). For openings shorter than 2tr their duration was measured after assuming their amplitude to be equal to the mean amplitude of the clearly resolved openings in the data record. Short attenuated closings were assumed to be complete closures unless there was clear evidence that the transition was to a subconductance level. Direct transitions between different open levels were identified when no shutting longer than the shut time resolution could be detected between two open periods, both open periods being at least 2tr in duration.
Distributions conditional on event amplitude were constructed using a critical amplitude value, Acrit, which was calculated using the fitted Gaussian parameters of the amplitude distribution so as to minimize the overlap between different components in the amplitude distribution (Howe, Cull-Candy & Colquhoun, 1991).
Definition of bursts Bursts of openings were defined as openings separated by closed times shorter than a critical length t,. This was calculated so as to
A 20 FM cytisine
100 pA
2 s
make the percentage of long closed times that were misclassified as within bursts equal to the percentage of short closed times that were misclassified as being between bursts (Colquhoun & Sakmann, 1985). Closed time distributions were fitted with four exponential components. For each patch t0 was calculated from the best-fit parameters of the closed time distribution treating components two and three as being 'within bursts' and 'between bursts' closed times, respectively.
RESULTS Whole cell current responses Figure 2 shows the reponses of two cells in the medial habenula to nicotinic agonists. The response of the first cell to 20 uM cytisine (Fig. 2A and B) was blocked by the co-application of 100 /uM hexamethonium (Fig. 2C). The response of a different cell to the application of acetyl- choline is shown in Fig. 2D. The magnitude of the responses to acetylcholine was very variable, with some cells giving no clear response to acetylcholine while others gave responses of the magnitude shown in Fig. 2D. The mean peak response at -50 mV to 30 ,uM ACh from six of the positively responding cells was 98 + 24 pA (mean + S.E.M.), while the mean response to 20 /SM
B 20 FM cytisine
400 pA
2 s
Figure 2. Examples of nicotinic whole cell currents recorded from rat medial habenula cells A, response of a cell to bath perfusion of the nicotinic agonist cytisine (20 #M). B, a second response to cytisine evoked from the same cell as in A 10 min later. C, same cell as in A and B. The bath was perfused with cytisine (20 /SM) plus the nicotinic antagonist hexamethonium (100 /M). The response to cytisine is completely blocked. The example responses shown in A, B and C were kindly provided by Dr Frances Edwards (Department of Pharmacology, University of Sydney, NSW, Australia). D, response to acetylcholine (10 uM) in a different cell from that in A, B or C. The response of medial habenula cells to acetylcholine was very variable, with some cells giving no response, and some, as in this example, giving a large response.
.. -i 0 i la 009m- - I ---v
F-- I I
Nicotinic receptors in rat medial habenula
cytisine from eight cells was 160 + 22 pA. In contrast to the inconsistency of the response to acetylcholine, all cells tested in the ventral medial habenula responded to cytisine. The responses to acetylcholine were obtained in the absence of anticholinesterases and therefore hydrolysis of acetylcholine may contribute to the inconsistency of the current responses.
CL4- c 2 cJ
Whole cell I-V relations One well-established difference between neuronal nicotinic receptors and those at the neuromuscular junction is that little outward whole cell current can be detected at positive potentials in neurones, whereas outward currents are easily elicited in muscle. To see which category the nicotinic responses of cells in the ventral medial habenula
Figure 3. Rectification of the nicotinic conductance in medial habenula cells
A, averaged whole cell current responses shown in the absence (thin trace) and presence (thick trace) of cytisine (20 /M) during voltage ramp protocols where the membrane potential is ramped during a 3 s
period (shown by the duration of the step in the top trace) from - 50 to -100 mV, then from -100 to +100 mV and then back to -50 mV. Application of cytisine causes an increase in the holding current at -50 mV from -40 to -160 pA. Since the cytisine responses slowly wane during superfusion of the drug (Fig. 2), and may also run down, the absolute value of the averaged cytisine response at -50 mV may vary from one set of ramps to another depending upon the time after initial drug application at which the ramp protocol is applied. These responses were obtained in slicing solution, where Cl- was
the main anion and 85% sodium gluconate was the intracellular pipette solution. There is a large difference between the control and cytisine current traces at positive potentials. Thus, when the control records obtained before cytisine application and after cytisine washout were averaged and then subtracted from the cytisine records to give the nicotinic response I-V relations shown in Fig. 3B, there was very little evidence of rectification. However, panel C shows a trace from the same cell but after replacing most of the chloride in the external solution with isethionate. Under these conditions, the trace of the outward current at positive potentials in the presence…

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