Heterogeneity acetylcholine

Journal of Physiology (1995), 484. 1, pp. 87-105

Heterogeneity of neuronal nicotinic acetylcholine receptorsin 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 studiedusing patch clamp recording techniques.

2. Whole cell current responses to cytisine could be blocked by hexamethonium, as expectedfor nicotinic receptors. The whole cell current-voltage relations were linear at negativemembrane potentials, but showed strong inward rectification when chloride currents wereminimized.

3. When 1 mm Ca2+ (0 mM Mg2+) was present in the external recording solution, the singlechannel conductances elicited by acetylcholine or nicotine in twenty patches were in therange 39-58 pS, with a mean of 47 pS. There appeared to be at least two groups ofconductances.

4. In the open point amplitude distributions of three patches, the most common amplitudecorresponded to 41 pS (81 % of the area). In another four patches the most commonamplitude corresponded to a mean conductance of 51 pS (83% of the area). Directtransitions 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 thedistributions of open times longer than two filter rise times averaged 5'8 ms. Those patcheswith predominantly 41 pS openings averaged 14 ms. Also, for patches with predominantly51 pS openings the overall mean burst length was 5-8 ms, whereas for patches withpredominantly 41 pS openings it was 16-1 ms.

6. These observations suggest that 51 and 41 pS openings result from the activity of at leasttwo, but possibly more, different receptor subtypes. We conclude that nicotinic receptors inthe rat ventral medial habenula are heterogeneous.

Electrophysiological, radiolabelled ligand-binding and insitu hybridization studies suggest that nicotinic acetyl-choline receptors (AChRs) are widespread throughout thebrain (for reviews see Deneris, Connolly, Rogers &Duvoisin, 1991; Role, 1992). These receptors have beenmuch less well characterized than their counterparts inganglia and at the neuromuscular junction.

A recent technical development, the thin slice technique(Edwards, Konnerth, Sakmann & Takahashi, 1989), hasmeant that all regions of the brain can now be studiedwith the full range of patch-clamp techniques. Advantagesof this technique are that the cells being studied can beseen directly and that many of the synaptic connectionsof the neurones are maintained.

Within the rat brain, in situ hybridization studies haveshown that the medial habenula is particularly rich in theexpression 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 transcribedthroughout the rat medial habenula, the a3 and ,84 genesare transcribed only in the ventral areas. The a3, a4, /12and /14 subunits (but not /13) have been functionallyexpressed (in pairs) in Xenopus oocytes while a7 expressesas a homo-oligomer (Deneris et at. 1991, review; Seguela etal. 1993).

The medial habenula also contains sites of high-affinitynicotine 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.

2901

J 0. Connolly, A. J Cibb and D. Colquhoun

1985) and intracellular electrophysiological studies in theguinea-pig medial habenula have revealed the presence ofexcitatory 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 fromthe medial habenula region using patch-clamp techniques,and recorded nicotinic whole cell responses and singlechannel activity. We chose also to study nicotinic responsesin 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 thereceptor population that might be brought about by theenzyme treatment during dissociation. In this study wefind evidence that there are at least two types of nicotinicacetylcholine receptor in the medial habenula. Apreliminary report of this data has been published(Connolly & Colquhoun, 1991).

METHODSPreparation of slicesThe procedures followed were essentially the same as thosedescribed in Edwards et al. (1989). A male, or female,Sprague-Dawley rat (16-23 days) was killed by cervicaldislocation and then decapitated. The brain was removed andplaced in ice-cold physiological buffer (slicing solution) ofcomposition (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

A

medial habenula was then fixed to a Teflon block using a thin filmof liquid cyanoacrylate glue and sliced using a CampdenInstruments Vibroslice. Slices of 180-220 um thickness wereused in these experiments. The pigmented, in some placesclustered, cells of the medial habenula were clearly distinguishablefrom surrounding brain regions (Fig. 1A). Between four and sixsuch sections could be obtained, depending upon the age of theanimal.

Optical equipment and recording bathSlices were placed individually in a circular bath of about 0 7 mlvolume through which oxygenated recording solution wascontinuously perfused at a rate of 3 ml mint. The slice was heldin place by a ladder of nylon fibres glued to a circular platinumframe (Fig. 1B).

Cells within the slice were viewed using differential interferencecontrast (Nomarski) optics on the stage of a Zeiss-Jena uprightmicroscope using a Zeiss x 40, 0 75 numerical aperture, water-immersion objective lens with 1i6 mm working distance at atotal magnification of x 600. Healthy cells were identified bytheir soft, smooth appearance.

Recording conditionsWVhole cell and single channel patch clamp recordings were thenobtained using an Axopatch 1B patch clamp amplifier and thesignals recorded on a Racal Store 4 FM tape-recorder (DC to5 kHz, -3 dB). Single channel recordings were made using theoutside-out patch configuration (Hamill, Marty, Neher,Sakmann & Sigworth, 1981). Recordings were made at roomtemperature (20-24 0C) with the patch membrane potentialclamped at -60 mV. In the experiments selected for time courseanalysis, the agonist used was ACh (1-3 2 pM). Patches werechosen where the overall level of activity was low and wheredouble openings were absent or rare.

B

Figure 1. Location of the ventral medial habenula in the slice preparationA, diagrammatic outline of a coronal section through the rat brain showing the location of the medialhabenular nuclei adjacent to the walls of the third ventricle. B, the outline of the part of the slice usedfor experiments is shown with the surrounding cortical regions removed. The slice is held stably in therecording 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-walledborosilicate 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 cellrecordings were made with pipettes of 5-13 MQ resistancepulled from thin-walled glass (GC150TF, o.d. 1-5 mm, i.d.1'17 mm). VVhole cell resistances averaged 1-5 GQ2 at negativemembrane potentials and 0-45 GQ2 at positive potentials. Wholecell capacitances were in the range 6-15 pF, and seriesresistances ranged from 17 to 60 MQ2. Series resistancecompensation was not used.

SolutionsOutside-out patches. The normal external recording solutionfor 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 solutionunless 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 sodiumgluconate solution was occasionally diluted by 6% to assist sealformation.

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+ and1 mM Mg2+ in solution (ii) were replaced by 3 mm Mg2+. Allexternal solutions were freshly made to minimize glutamate or

glycine contamination. All reagents were obtained from Sigma(UK). Pipette solutions were either the 94% sodium gluconatedescribed above, an 80% sodium gluconate solution containing5 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 switchingmanually between control and drug solution reservoirs at a

control valve close to the inlet of the recording bath. Outside-outpatches were brought close to the mouth of the solution inlettube. This enabled solutions around outside-out patches to bechanged within 1-2 s and also reduced the risk of activation ofchannels in the patch by neurotransmitters leaking from theslice. When recording whole cell currents, cells were in situ in theslice, and so it took 10-30 s for the response to reach a peak,suggesting a much slower solution exchange within the sliceitself.

Whole cell I-V ramps

These were initiated by, and recorded on, a Dell 486microcomputer using a Cambridge Electronic Design 1401interface. From a holding potential of -50 mV, a 3 s voltageramp was applied. The membrane potential was first ramped to-100 mV, then to +100 mV, and finally back to the holdingpotential. The current response to this voltage ramp was filteredat 250 Hz (-3 dB, Bessel-type filter), digitized at 500 Hz, andstored 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 responseto the application of agonist (30juM ACh or 20,uM cytisine).Finally a further four control responses were obtained after theagonist had been washed out. Individual responses in control andagonist-containing solutions were then examined off-line andaccepted control responses from both before and after the agonistapplication were averaged together. The acceptable rampresponses during agonist application were also averaged.Comparison of the control responses from before and afteragonist application was used to confirm that the restingproperties of the cell had not changed during agonist application.The mean of all control responses was then subtracted from themean response obtained in the presence of the agonist.

Amplitude measurementsFor single channel I-V curves, and for most estimates ofconductance in Fig. 6, measurements of current amplitudes wereobtained from single channel records plotted on a UV writingchart recorder (Medelec) and by cursor fitting using amodification (by S. F. Traynelis, Emory University, Atlanta,GA, USA) of the Axotape data acquisition programme. At leastten amplitude measurements were made for each conductance oramplitude value quoted. The amplitudes which contributed tothe open point amplitude histograms were measured as part ofthe time course fitting method of analysis of the single channelrecords (Colquhoun & Sigworth, 1995). When estimatingconductances from amplitudes, the reversal potentials wereassumed to be -8-6 mV when CsCl was the internal pipettesolution (based on the intercepts of single channel I-Vrelations),and -4-6 and -3-2 mV, respectively, when 100 or 94% sodiumgluconate was in the pipette solution (based on the calculatedreversal potential for Na+ ions).

Time course analysis of single channel recordsSingle channel currents were stored on FM tape (Racal Store 4,DC to 5 kHz) after filtering at 10 kHz (-3 dB, 8 pole Besselresponse). Data records were replayed from FM tape, amplifiedand filtered at from 1 to 4 kHz (-3 dB, 8 pole Bessel), andcontinuously sampled at 10-40 kHz onto a PDP 11/73 computerhard disk using a CED502 interface (Cambridge ElectronicDesign). The duration of open and closed periods in the datarecord was measured using the method of time course fitting(Colquhoun & Sigworth, 1995), after first measuring theamplitude of each channel opening using manually controlledcursors placed on the data display. An idealized record of theduration and amplitude of every detectable event in the datarecord was obtained by imposing a fixed resolution for opentimes and closed times on the data before construction and fittingof distributions. This resolution was 80-140 ,us for open timesand 70-120 ,us for closed times.

Histograms of the distribution of open times and shut times, andproperties of bursts of openings were constructed for display andevaluation of the data. In most cases the distribution oflog(duration) is used for display purposes (Blatz & Magleby, 1986;Sigworth & Sine, 1987), with a square root transformation of theordinate (Sigworth & Sine, 1987).

Distributions were fitted with the sum of several exponential,Gaussian or geometric components where appropriate. Theindividual observations of open times, shut times, etc. were usedfor 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 levelsSubconductance levels were identified during analysis if anamplitude change of greater than 10% of the open channelcurrent was observed. Since it is not possible to estimateaccurately the amplitude of very brief channel openings,amplitude histograms contained only values measured fromopenings of duration at least 2tr, where tr is the filter rise time ofthe recording system (e.g. Colquhoun & Sigworth, 1995). Foropenings shorter than 2tr their duration was measured afterassuming their amplitude to be equal to the mean amplitude ofthe clearly resolved openings in the data record. Shortattenuated closings were assumed to be complete closures unlessthere was clear evidence that the transition was to asubconductance level. Direct transitions between different openlevels were identified when no shutting longer than the shuttime resolution could be detected between two open periods, bothopen periods being at least 2tr in duration.

Distributions conditional on event amplitude were constructedusing a critical amplitude value, Acrit, which was calculated usingthe fitted Gaussian parameters of the amplitude distribution soas to minimize the overlap between different components in theamplitude distribution (Howe, Cull-Candy & Colquhoun, 1991).

Definition of burstsBursts of openings were defined as openings separated by closedtimes shorter than a critical length t,. This was calculated so as to

A 20 FM cytisine

2s-

100pA L2 S

C 100 /M hexamethonium+

20 FSM cytisine

100 pA

2 s

make the percentage of long closed times that were misclassifiedas within bursts equal to the percentage of short closed times thatwere misclassified as being between bursts (Colquhoun &Sakmann, 1985). Closed time distributions were fitted with fourexponential components. For each patch t0 was calculated fromthe best-fit parameters of the closed time distribution treatingcomponents two and three as being 'within bursts' and 'betweenbursts' closed times, respectively.

RESULTSWhole cell current responsesFigure 2 shows the reponses of two cells in the medialhabenula to nicotinic agonists. The response of the firstcell to 20 uM cytisine (Fig. 2A and B) was blocked by theco-application of 100 /uM hexamethonium (Fig. 2C). Theresponse of a different cell to the application of acetyl-choline is shown in Fig. 2D. The magnitude of theresponses to acetylcholine was very variable, with somecells giving no clear response to acetylcholine while othersgave responses of the magnitude shown in Fig. 2D. Themean peak response at -50 mV to 30 ,uM ACh from sixof the positively responding cells was 98 + 24 pA(mean + S.E.M.), while the mean response to 20 /SM

B 20 FM cytisine

100 pA

2 s

D10 gM ACh

400 pA

2 s

Figure 2. Examples of nicotinic whole cell currents recorded from rat medial habenula cellsA, response of a cell to bath perfusion of the nicotinic agonist cytisine (20 #M). B, a second response tocytisine evoked from the same cell as in A 10 min later. C, same cell as in A and B. The bath wasperfused with cytisine (20 /SM) plus the nicotinic antagonist hexamethonium (100 /M). The response tocytisine is completely blocked. The example responses shown in A, B and C were kindly provided byDr 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 medialhabenula cells to acetylcholine was very variable, with some cells giving no response, and some, as inthis example, giving a large response.

.. -i 0 i la 009m-- I ---v

F-- I I

T--,

J. Phy8iol. 484.1


cytisine from eight cells was 160 + 22 pA. In contrast tothe inconsistency of the response to acetylcholine, all cellstested in the ventral medial habenula responded tocytisine. The responses to acetylcholine were obtained inthe absence of anticholinesterases and therefore hydrolysisof acetylcholine may contribute to the inconsistency ofthe current responses.

A

CL4-c2cJ

Whole cell I-V relationsOne well-established difference between neuronal nicotinicreceptors and those at the neuromuscular junction is thatlittle outward whole cell current can be detected atpositive potentials in neurones, whereas outward currentsare easily elicited in muscle. To see which category thenicotinic responses of cells in the ventral medial habenula

B

Membrane potential (mV)

Time (1 03 ms)

D

0 1 2 3

Time (103 ms)

4

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) ofcytisine (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 currentat -50 mV from -40 to -160 pA. Since the cytisine responses slowly wane during superfusion of thedrug (Fig. 2), and may also run down, the absolute value of the averaged cytisine response at -50 mVmay vary from one set of ramps to another depending upon the time after initial drug application atwhich 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 largedifference between the control and cytisine current traces at positive potentials. Thus, when the controlrecords obtained before cytisine application and after cytisine washout were averaged and thensubtracted from the cytisine records to give the nicotinic response I-V relations shown in Fig. 3B, therewas very little evidence of rectification. However, panel C shows a trace from the same cell but afterreplacing most of the chloride in the external solution with isethionate. Under these conditions, thetrace of the outward current at positive potentials in the presence of cytisine was very similar to thecontrol trace. The subtracted I-V relations in isethionate solution (Fig. 3D) show strong inwardrectification of the nicotinic response. Indeed there is so little outward current that the reversalpotential is poorly defined. The results suggest that an inward Cl- movement may be carrying theoutward current seen in panel B. Experiments by Mulle and co-workers (Mulle et al. 1992a) suggestthat this Cl- current is activated by Ca2+ influx through the neuronal nicotinic channels.

C

CLc

e03

J. Physiol. 484.1 91

J C. Connolly, A. J Gibb and D. Colquhoun

belonged to, their current-voltage relations wereinvestigated as described in the Methods.

The initial results were obtained with 1 mm Ca2+ and118 mM NaCl in the external solution and sodiumgluconate in the intracellular solution. TTX (0 5 FM) wasused to block Na+ currents. These early results werevariable, sometimes showing rectification and sometimesnot. Even more remarkable is the observation that thedegree of rectification seen in repeated ramps during asingle, continuous application of ACh also varied, therectification being apparently reduced as the number oframps applied increased. There was also some evidence ofhysteresis in the I-V relations. This suggested that theapplication of ACh induces an additional outwardcurrent. Since the internal solution used during theseramp experiments contained gluconate as the main anion,the reduced rectification could be caused by an inwardchloride movement at positive membrane potentials.Therefore one explanation could be that an inward Ca2+current through the AChRs activates a chloride currentfrom within the cell.

Figure 3 shows the I-V relations for a cell in NaCl (slicingsolution) and in low-Cl- (sodium isethionate) solution.Figure 3A shows the averaged current responses to avoltage ramp, plotted against time. In this experiment,Cl- was the major anion in the extracellular bathingsolution. The outward current at positive potentials in thepresence of cytisine (thicker trace) is seen to be muchgreater than that in the absence of agonist. When thecontrol I-V relations are subtracted from the I-Vrelations obtained in the presence of agonist there is verylittle evidence of rectification (Fig. 3B). However, inFig. 3C, sodium isethionate was used to replace much ofthe NaCl in the external solution bathing the same cellfrom which the data in panels A and B were obtained. Itis immediately apparent that the difference betweencontrol and agonist responses at positive potentials wasgreatly reduced. Consequently, when the averagedcontrol I-V relations were subtracted from the averagedI-V relations in the presence of 20 FM cytisine (Fig. 3D),strong rectification was revealed. A chloride current wasinvolved in masking the rectification of the nicotinicwhole cell current.

Nicotinic receptors in outside-out patchesOutside-out patches were obtained from a variety of cells,ranging in diameter from 8-20 ,um, located in the ventralregion of the medial habenula. Outside-out patches fromabout 60% (45/75) of cells tested showed clear and rapidresponses to nicotinic agonists. Cells with nicotinicreceptors were present throughout the rostral-caudal axisof the ventral medial habenula. They were also observedover the entire age range (16-21 days) of animals tested.Figure 4 shows an example of such activity in response to

It was possible to obtain repeated responses toacetylcholine from outside-out patches (Fig. 4A and C),and the patches were quiescent during the interveningwashing periods (Fig. 4B). Figure 4D-F shows singlechannel activity at the point of application and washingout of acetylcholine, but at a faster time scale than tracesA-C. The rapid onset and washout clearly demonstratesthat the single channel activity is specifically associatedwith the agonist application. Unfortunately the activitytended to run down, often becoming difficult to detectwithin 5-10 min of the formation of the outside-outpatch. A similar time course for run-down has beenobserved by Lester & Dani (1994) in dissociated neuronesfrom rat medial habenula.

Pharmacological properties of the single channelactivityAtropine (0 5 uM) did not block the single channelcurrents activated by 1-10 /M acetylcholine (n = 34) or

1-10 /M nicotine (n = 3), so the evoked channel activityis nicotinic rather than muscarinic.

NMDA receptor channels also have similar single channelconductances and, despite the precautions described in theMethods, could still have been activated. However, theresponses were not affected by the presence of up to 25 uMAP5, a selective NMDA antagonist (Fig. 4A and C). Also,at negative holding potentials, there was no evidence ofchannel 'flickering' with 100 /M Mg2+ in the externalsolution, nor block when 1 mM Mg2+ was included. Thechannel activity is therefore not due to activation ofNMDA receptors by endogenous glutamate. Nevertheless,5-25 FM AP5 was included in most experimental solutionsas an additional precaution against NMDA receptoractivity. In two patches, 10 #M picrotoxin was also foundnot to eliminate the channel activity, confirming thatthese were not GABA receptor channels.

These experiments demonstrate that the single channelactivity we have observed is mediated by nicotinic acetyl-choline receptors.

Cation selectivityInitial observations of the inward currents carried by thechannels were obtained at negative holding potentialswith CsCl solution inside the pipette. When sodiumgluconate replaced CsCl as the pipette solution, similarinward currents were still obtained in response to theapplication of acetylcholine. This indicates that thechannels were not permeable to chloride ions, but insteadwere permeable to Na+ ions, which were the main cationicspecies in the external solution.

Channels were also active at positive holding potentials inoutside-out patches, but with much lower frequency thanat negative potentials. The openings at positive potentialsoccurred when either Cs+ or Na+ are the main cations in

the application of 3-16 AM ACh.

J. Phy8iol. 484.192

the pipette solution. Therefore, as with other nicotinic


AChRs, the single channel currents can be reversed andthe channels are permeable to Cs+ ions (Fig. 5B and C).

Single channel current-voltage relationshipsIn Fig. 5A, the single channel current-voltagerelationships obtained in two different outside-outpatches with CsCl solution inside the pipette are shown.At negative holding potentials, the slope conductance ofthe larger amplitude channel was 56 pS (reversalpotential -11 mV), while that of the smaller one was46 pS (reversal potential -8 mV). Run-down, the lowfrequency of single channel events at positive holdingpotentials and decreasing patch stability above +60 mVmade slope conductances at positive membrane potentialsdifficult to measure.

A

B

C

Heterogeneity of nicotinic single channel currentsThe difference between the two negative slopeconductances shown in Fig. 5 was sufficiently large tosuggest that there may be more than one class of nicotinicconductance present in the medial habenula. Figure 6shows a histogram of the mean conductances of channelsin twenty patches taken from individual cells in theventral medial habenula. The data were obtained innormal recording solution, at -60 mV holding potential,and with either CsCl or sodium gluconate internal pipettesolutions (corrections for reversal potentials are given infigure legend). The single channel conductances elicited bythe application of acetylcholine or nicotine were in therange 36-58 pS with a mean of 46'9 + 14 pS

D

1] t 1 11 11 !

E

F

--

2 pA

24 s

,-WI,11t"11,11hN.

2 pA

50 ms

Figure 4. Examples of single channel currents recorded from outside-out patches isolatedfrom ventral medial habenula cellsA, slow time scale recording showing the patch response to the application of 3-16CM ACh. Thecontinuous line above each trace shows the period during which the agonist was washing through thebath. A delay of around 2 s occurs between switching the solution tap and the arrival of the agonist inthe bath. The agonist and bath solution contained, in addition to the normal extracellular solution,1 FM atropine, 100 fiM Mg2+ and 25 AM AP5 to block muscarinic and NMDA receptors. B, wash-off ofthe ACh response in A after 15 s in the presence of ACh. C, second application of ACh (316 fiM) to thesame patch as illustrated in A and B. The first application of agonist generally evoked multiplesuperimposed single channel currents. However, subsequent applications of agonist generally gavesmaller responses, suggesting some run-down of the nicotinic response in outside-out patches. Panels Dand F show, on an expanded time scale, the first few channel openings which occurred on changing to ACh-containing solution. Panel E shows the section of the trace in B immediately afterchanging to agonist-free solution.

OR A 1911111 kwM Fm Trxxn-

-7 -",-I

- ----I I-i

J. Phy8iol. 484.1 93

-Firl7-1 --f-IT - -[i

I 'I7"T'

1! I

J C. Connolly, A. J Gibb and D. Colquhoun

A

Membrane potential (mV)

I I I I-100 -80 -60 -40

B4

3

2

-1

+60 mV

I 1 pA

25 ms

20 40 60 80

--1

--2 <

C

--3 :30

- -4

C-60 mV

I1 pA

25 ms

Figure 5. Current-voltage relations for single channel currents activated by ACh in twoseparate outside-out patchesAt negative voltages, the conductance levels in the two patches were different: 46 (0) and 56 pS (0).The single channel currents are reversible. However, because of the very low frequency of channelopenings evident at positive potentials, only data for the smaller conductance is shown at positivepotentials where the conductance was 58 pS (0). The slight outward rectification of this response isprobably due to the presence of 150 mm Cs+ and the low Ca2+ concentration in the internal pipettesolution. Panels B and C show examples of single channel currents which passed through the smallerconductance receptor channel recorded at +60 and -60 mV, respectively.

FTW30 35 41

urn0 45 50Conductance (pS)

Figure 6. Histogram of single channel conductances measured from twenty-one outside-outpatchesAll twenty-one of these recordings were obtained in normal extracellular recording solution. Estimatedreversal potentials of -8'6 + I 1 mV ( mean + S.E.M., n = 4 patches), and -4-6 or -3-2 mV (calculated,see Methods) were used in calculating conductances depending upon whether the internal pipettesolution was CsCl, 100 or 94% sodium gluconate solution, respectively. It has been assumed that thesubstitution of Na+ for Cs+ in the internal solution will not affect the single channel current measuredat -60 mV. The histogram shows the wide spread of conductances observed in these recordings andsuggests that there may be at least two discrete populations of larger and smaller nicotinicconductances present in cells of the ventral medial habenula.

4-

3-

0)c

' 2-

Ez

1 -

0 - F I55 60

J. Physiol. 484.194


(mean + S.E.M.). This range of conductances is far widerthan would be expected to result from experimentalerrors or open-channel noise. The conductances are clearlyheterogeneous.

The results in Fig. 6 could be interpreted as suggestingthat there are at least two major classes, though there aretoo few observations for this to be at all certain. If therewere indeed only two major classes, the spread of valueswithin each would still be unusually large. Regardless ofhow this wide range of conductances is interpreted, it wasobserved that, in any given patch, the activity wasusually dominated by only one conductance (see below).However, in most patches, even if at a very lowfrequency, more than one amplitude level was observed.Patches dominated by either the larger or smallerconductances were similarly abundant and both types ofpatch could be obtained from cells within the same slice.Also, there was no significant correlation between theages of the animals and the occurrence of a particular classof amplitudes. Thus, it seems unlikely that the two patchtypes represent different stages of development, althougha study over a wider age range may reveal such an effect.

A51 pS

-- --- ----- --- --- --- ---

4 pA

20 ms

B

-1~~~~~~~~, 1l"V ,III

---------l--~~-------~~

4 pA

20 ms

Representative examples of single channel activity inlarger and smaller conductance patches are shown inFig. 7. ACh (1 uM) was applied, and the patches were heldat -60 mV. Figure 7A-C shows examples of the singlechannel activity in a patch dominated by the largerconductance while Fig. 7D-F shows examples of thesmaller channel activity.

Figure 7A shows an apparently direct transition betweena full opening of 3 0 pA and a substate of 2-1 pA. Also,Fig. 7E-F shows evidence of transitions from a main openstate of 2-3 pA to substates of 1-8 and 1.0 pA. However,direct transitions from the main state to substates wererare events. The vast majority of events, in both the largeand small amplitude classes, involve openings to a singleconductance level. To test further for heterogeneity, thesingle channel properties of these receptors were analysedin more detail.

Distributions of measured amplitudesFor the seven patches analysed in detail, distributions ofopen channel amplitudes were fitted with three Gaussiancomponents in six cases, and two components in one case.

D41 pS

4 pA

20 ms

E

~~~~~, TI - - ---

4 pA

20 ms

F

4 pA

100 ms

Figure 7. Examples of single channel currents activated by 1 /SM ACh recorded at -60 mVfrom two different outside-out patches in identical solutionsPanels A, B and C show examples of openings from a single patch which displayed predominately largeconductance openings. Panels D, E and F show examples of channel openings from a different patch inwhich there were predominantly smaller conductance openings. The current levels which correspond toconductance levels of 41 and 51 pS are indicated by the dashed lines. On average, the smallerconductance openings were of much longer duration than the larger openings. In both types ofrecording occasional direct transitions between conductance levels were observed but these were

generally rare. Records were filtered at 2 kHz (-3 dB) and sampled at 20 kHz.

C

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4 pA

100 ms

J. Phy8iol. 484.1

96 J G. Connolly, A. J

Figure 8A and B shows two examples of point amplitudedistributions (Colquhoun & Sigworth, 1995). Distributionswere fitted with the standard deviation constrained to bethe same for each component. The mean amplitudes of eachcomponent (and relative areas) for all patches were1P17+0-04pA (6+2%), 2-05+0-11pA (20+8%) and2-68 + 0 11 pA (74 + 10%). These components correspondto channel conductances of 21, 37 and 48 pS (assuming a-3-9 mV reversal potential). However, these data includeresults from three patches in which the most commonamplitude level averaged 2-28 + 0-06 pA (41 pS) andaccounted for 81 + 5-8% of the open amplitudedistribution. In the other four patches the most commonamplitude was 2-86 + 0-12 pA (51 pS), which averaged83 + 2-4% of the area of the open amplitude distribution.

A

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Gibb and D. Colquhoun J. Physiol. 484.1

Since these means refer only to this small group of patchesanalysed in great detail, they cannot be compared directlywith the median values of the larger groups which aredepicted in Fig. 6.

The fact that different patches have different proportionsof the various single channel amplitudes suggests thatmore than one type of receptor is present in patches fromcells in the medial habenula. In Fig. 8B for example, thelarge conductance channel is virtually undetectable.Furthermore, because direct transitions from one openlevel to another were very rare in these experiments,there is no evidence from this analysis to suggest that thedifferent conductance levels observed originate fromactivation of a single type of AChR (though this possibilitycannot be ruled out entirely).

4

0 1 2 3 4-Amplitude (pA): open/shut point

Figure 8. Point amplitude distributions from two patches with predominantly largeconductance (A) or lower conductance (B) channel openingsMembrane potential -60 mV. For these distributions each individual data point within open periodsand measured closed periods longer than 2tr contributed a single observation to the distribution. Forconvenience the single channel amplitudes are shown as positive. In each case the distributions were

fitted with the sum of four Gaussian components to include the distribution of closed channel current.The fit is shown as a continous line. The parameters of the fitted lines were (mean + S.D., percentagearea): in A, 0 0 + 0-13 pA (48%), 0-64 + 0 35 pA (1-5%), 2-22 + 0-22 pA (5 2%) and 2-98 + 0-27 pA(45%); in B, 0 0 + 0-24 pA (40%), 0 79 + 0O16 pA (2 7%), 1t23 + 016 pA (2 5%) and 2-19 + 045 pA(55%).

0 1 2 3

Amplitude and kinetic stability plotsOne possible explanation for the different classes ofconductance observed could be that a particular channel isoscillating between different 'modes', i.e. long lastingperiods during which the channel operates with analternative kinetic or conductance behaviour. Switchesbetween 'mode', unless they are very rare, should beapparent in stability plots (Weiss & Magelby, 1989) of thechannel characteristics during the time course (asrepresented by interval number) of the experiment. Suchplots are shown in Fig. 9. There was no evidence of modechanges in any of the seven patches studied in detail. Theamplitude stability plots in Fig. 9A (larger channel) and B(smaller channel) show no evidence that there isamplitude instability during the experiment. Similarly

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the open time stability plots in Fig. 9A and B show noevidence that any substantial change in the kineticbehaviour of the channels took place during a recording.

In Fig. 9A, however, there is an increase in the mean shuttime during the recording, resulting in a gradual decreasein the Popen (the probability of the channel being open)over the time course of the record. This 'run-down' ofreceptor activity was a problem that occurred in manyoutside-out patches, though not in all. Some patchesseemed to show sustained activity, though somewhatreduced from the initial response, for periods of up to40 min. The extent of run-down in any particular patchdid not obviously depend on the predominant conductancelevel of the channels. The addition of 4 mm Mg-ATP tothe pipette solution did not prevent run-down.

B

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Figure 9. Stability plot analysis of single channel parametersPanels A and B show results for the same two patches as illustrated in Fig. 8. A, a patch withpredominantly large conductance openings. B, a patch with predominantly smaller conductanceopenings. Although there is a fairly wide variation in channel amplitudes, there was no evidence forchanges in the single channel amplitudes measured during the course of either recording. The lowerparts of each panel show stability plots of the measured mean shut time, mean open time, and mean

Popen These traces show a running mean of the measured parameter which is plotted on a log scale on

the y-axis. In each case forty observations were averaged with an increment of twenty observationsbetween each mean. Also shown for reference is the mean of each parameter for the whole data recordshown as dashed line. In panel A these means were 170 ms for shut times, 5-5 ms for open times and0 03 for Popen In panel B these means were 105 ms for shut times, 20-6 ms for open times and 0-16 forPopen The data illustrate that although there were no time-dependent changes in the single channelamplitudes in either patch, the patch illustrated in panel A showed some run-down of channel activity(as evidenced by a decrease in Popen and an increase in mean shut time during the recording), whereasthe patch illustrated in panel B showed no significant run-down. For both recordings the resolution wasset to 100 ,us for both open times and closed times. Approximately 2 min of data were used in compilingeach of the sets of plots in A and B.

J. Physiol. 484.1 Nicotinic receptors in rat medial habenula

0-1

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J G. Connolly, A. J Gibb and D. Colquhoun

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Figure 10. Dwell time distributionsDwell time distributions for two patches which were predominantly of large conductance (A, B and C) orsmall conductance openings (D, E and F). A, distribution of all closed times greater than 90 ps in duration(n = 385 gaps) recorded from the same patch as illustrated in Fig. 8A. The histogram shows the distributionof log time versus the square root of bin frequency on the abscissa. This transformation converts the normalexponential distribution into a peaked function. Each peak is close to the time constant of each exponentialcomponent. The distribution is shown fitted with the sum of four exponential components with timeconstants (and relative areas) of: 99 jAs (14%), 0 75 ms (16%), 16 9 ms (19%) and 445 ms (51 %). The fitpredicts that there were 429 gaps. The resolution was 110 jus for open times and 90 /zs for closed times. Therecord was filtered at 2 kHz (-3 dB). B, distribution of the length of 420 apparent open times between110 /ts and 100 ms long. The distribution is fitted with the sum of three exponential components withparameters of 0 28 ms (55%), 3-16 ms (36%) and 10 4 ms (9%). The fit predicts that there were 517 openingsin the data record. C, distribution of burst lengths for the same patch as illustrated in A and B. Critical gaplength was 1P74 ms, 303 bursts were identified between 110 ,#s and 100 ms long. The distribution is shownfitted with the sum of three exponential components. These had time constants (and relative areas) of:0-23 ms (45%), 2-82 ms (45%) and 17-7 ms (11%). The fitted curve predicts a total of 374 bursts.D, distribution of closed times greater than 80 us in duration (n = 389 gaps) recorded from the smallconductance patch illustrated in Fig. 8B. The sum of four exponential components was fitted to thedistribution with time constants (and relative areas) of: 43 ,us (46%), 0 49 ms (15%), 17-4 ms (9 9%) and479 ms (29%). The predicted number of gaps from the fit was 657. The resolution was 100 ,sS for open timesand 80 ,us for closed times. The record was filtered at 2 kHz (-3 dB). E, distribution of the length of 433apparent open times between 100 #ss and 200 ms long. The distribution is fitted with the sum of threeexponential components with parameters of 0 68 ms (33%), 6 31 ms (40%) and 30 ms (28%). The fit predictsthat there were 457 openings in the data record. F, distribution of burst lengths for the same patch asillustrated in D and E. Critical gap length was 1 3 ms. 258 bursts were identified between 100 Its and 700 mslong. The distribution is shown fitted with the sum of three exponential components. These had timeconstants (and relative areas) of: 0-46 ms (25%), 4 38 ms (39%) and 31 ms (36 %). The fitted curve predicts atotal of 274 bursts.

J. Physiol. 484.198


Overall, the stability plots re-emphasize the differences inamplitude between the larger and smaller conductanceclasses and support the idea that the two types ofbehaviour we have observed arise from different receptortypes.

Shut time distributionsWhen the closed time distributions for patches withpredominantly 41 pS openings were analysed, the meantime constants (relative areas) were 130 + 60 Acs(21 + 11%), 0-85 + 0-2 ms (19 + 8%), 13 + 2 ms(22 + 10%) and 291 + 103 ms (38 + 11 %). For the fourpatches with predominantly 51 pS channels, the shuttime distribution components averaged 36 + 7 #s(42+17%), 0 70+0-19ms (19+5%), 13+P14ms(9 + 2%) and 211 + 65 ms (31 + 10%). Apart from thefastest time constant, there was no obvious differencebetween the shut times from these two types of patches.The shortest shuttings seem to be much briefer for thelarger conductance channels (as for muscle receptors), butthe variability was substantial and the values were tooshort to be measured very reliably with the resolutionachieved in these experiments. Examples of shut timedistributions for the two types of patches are shown inFig. 10 (A and D).

Distributions of open timesOpen time distributions were fitted with three exponentialcomponents with mean time constants (relative areas) forall seven patches of 0-51 + 0-13 ms (32 + 71 %), 4.93± 1-44 ms (44-6 + 5-6%) and 19-6 + 4-9 ms (23-4 + 2-2%).The overall mean was 7-44 + 2'1 ms. Of these threecomponents, only openings underlying the longer twocomponents will have clearly resolved amplitudes(Fig. 10B and E). Therefore, when constructingdistributions of openings conditional on the amplitude ofthe openings, only openings longer than 2tr (tr being thefilter rise time of the recording apparatus) were used.Only two exponential components were evident indistributions of open times longer than 2tr. For allopenings in each data record these had mean timeconstants (relative areas) of 2-6 + 0-6 ms (49 + 6%) and15-8 + 3-2 ms (51 + 6%). These distributions had a meanof 9-4 + 2-0 ms. For the four patches with predominantly51 pS openings averaged, the values (for all open timeslonger than 2tr) averaged 5-8 + 0 9 ms, whereas thosepatches with predominantly 41 pS openings averaged14-1 + 2-7 ms.

Distributions of open times conditional onamplitudeIn order to compare the duration of openings at eachamplitude level, distributions of open times conditionalon the amplitude of the openings were constructed. Thiswas done by selecting openings within a particular patchrecord according to whether they were larger or smaller

than a critical amplitude level (Acrit) calculated todistinguish between the largest two amplitude levels, thusdividing the openings into two classes: large and small.For all patches, the open time distribution means for largeopenings averaged 11 2 + 3 0 ms while small openingsaveraged 5'0 + 2-2 ms. However, when patches weresubdivided according to the main conductance observed,the patches with predominantly 51 pS openings had amean open time for large openings was 6-5 + 1P1 ms andthe mean open time for small openings was 2-7 + 0-8 ms.In contrast, for the patches with predominantly 41 pSopenings the mean open time for large openings was18 + 5 ms and for small openings (subconductance levels)it was 8-0 + 4*9 ms. Thus, in the four patches withpredominantly 51 pS openings, the mean open time forboth large and small conductance openings wassignificantly shorter than the mean open time in patcheswith predominantly 41 pS openings. The most likelyexplanation for this seems to be that the receptors inpatches with predominantly 41 pS openings are differentfrom those in patches where the predominant conductanceis about 51 pS.

Burst length distributionsBursts of channel openings were identified using a criticalgap length t,, which was calculated for each patch fromthe fitted parameters of the second and third componentsof the shut time distribution. For all patches t, averaged1P5 + 0-2 ms. Burst length distributions were fitted withthree exponential components (Fig. 10C and F). Theseaveraged 0 40 + 0-12 ms (24 + 7%), 4-1 + 1'4 ms(41 + 3 %) and 24 + 5 ms (35 + 8%). The overall meanwas 10 2 + 2-8 ms. For patches with predominantly 51 pSopenings the burst length components were 0 20 + 0 04 ms(25 + 13%), 2-1 + 0-6 ms (39 + 5%) and 15 + 2 ms(36 + 13%). The overall mean was 5f8 + 1-7 ms. Forpatches with predominantly 41 pS openings the burstlength components averaged 0-66 + 0-2 ms (22 + 4%),6-8 + 2 ms (44 + 3%) and 37 + 5 ms (34 + 6%) with anoverall distribution mean of 16 + 4*5 ms. Thus, the burstlengths were considerably longer for those patches wherethe predominant single channel conductance was around41 pS.

Total open time per burstDistributions of total open time per burst were verysimilar to the burst length distributions, largely becausethe probability of the channel being open during the burst(burst Popen) was very high. The mean total open time perburst for all patches was 10-1 + 2-8 ms. This was dividedby the mean burst length for all patches giving a meanburst Popen of 0'99 + 0 003. For patches with mainly51 pS openings the mean total open time per burst was5-8 + 1-7 ms and for patches with predominantly 41 pSopenings this was 15-9 + 4-5 ms. Thus, the burst Popen forboth types of patch was very high (0 99).

J. Physiol. 484.1 99


Number of openings per burstDistributions of the number of openings per burst werefitted with two geometric components. For all patchesthese had component means (and relative areas) of1-15 + 0 05 (70 + 5 9%) and 2-13 + 0-17 (30 + 5 9%)with an overall mean of 1P54 + 0-17 openings per burst.When these are selected according to the main channelamplitude present in the recording, patches withpredominantly 51 pS openings had components of1-21 + 0 07 (81 + 4%) and 2-29 + 0-21 (19 + 4%), whilethe patches with predominantly 41 pS openings hadcomponents of 1-08 + 0 05 (55 + 5-2%) and 1-92 + 0-28(45 + 5-2%).

DISCUSSIONWe have used the thin-slice technique to carry out patchclamp studies on nicotinic acetylcholine receptors in theventral region of the rat medial habenula. Evidence hasbeen found that these cells express more than one type ofnicotinic receptor. In this study, two main classes of singlechannel conductance were observed. In patches givingmostly large conductance openings (referred to below as

51 pS openings), the open times were 2-3 times shorterthan in patches giving mostly smaller conductanceopenings (referred to below as 41 pS openings). The shuttime distributions in both types of patches were similar,though the briefest closures may be shorter for the largerconductance channels.

Previous reports have suggested that the cells of the ratmedial habenula contain only one major nicotinic singlechannel conductance, though comparison of values (seebelow) is hindered by the different calcium concentrationsused in different studies. Many genes encoding subunits ofneuronal nicotinic AChRs are transcribed in the ratmedial habenula, (Wada et al. 1988; Deneris et al. 1988;Deneris, Boulter, Swanson, Patrick & Heinemann, 1989;Duvoisin et al. 1989; Wada, McKinnon, Heinemann,Patrick & Swanson, 1990; Seguela et al. 1993), so it isentirely plausible that more than one sort of AChR may

be present. However, it is also possible that although themRNAs are expressed in the medial habenula, thereceptor proteins may be transported along axons to theinterpeduncular nucleus (Mulle et al. 1991). In the ventralregion, these include genes encoding the a3, a4, a7, /12, /B3and /14 subunits. Several combinations of these subunits are

known to be capable of forming functional nicotinic AChRswhen their mRNAs are expressed in Xenopus oocytes. Forrat subunits these include a3,82, a4-1/12 and a4-2,82(Boulter et al. 1987; Deneris et al. 1988; Connolly et al.1992), a.314, ca4-1/14 (Duvoisin et al. 1989) and a7 (Seguelaet al. 1993). Parallel results have also been obtained withchick AChR subunits (see Role, 1992, for review). Furthercombinations of these subunits are also possible. We

present evidence that more than one type of nicotinicreceptor is indeed present in the medial habenula.

Whole cell nicotinic responses and current-voltagerelationsWhole cell current responses were obtained to bothcytisine and acetylcholine. Cytisine was more potent thanACh (mean responses were 98 pA with 30 uM ACh and160 pA with 20 /M cytisine). In dissociated cells, Mulle &Changeux (1990) and Mulle et al. (1991) found the sameorder of potency. However, these results were obtained inthe absence of anticholinesterases so that potency of AChmay be underestimated. Also in agreement with Mulleand co-workers, we found that the agonist responses couldbe blocked by hexamethonium.

The whole cell current-voltage relations of the nicotinicAChR response showed strong inward rectification atpositive potentials, similar to that seen in sympatheticneurones (Mathie, Colquhoun & Cull-Candy, 1990).However, this was not immediately obvious. With asodium gluconate solution inside the pipette and in anexternal solution which included mainly NaCl and 1 mMCa2+, the whole cell current-voltage relations werealways linear at negative potentials. In the positive rangethe relations were much more variable, often showingvery little evidence of rectification. When most of theexternal NaCl was substituted by sodium isethionate, theoutward current response between 0 and +100 mV wasvery much dimininshed. Mulle et al. (1992b) saw strongrectification, even when 4 mm Ca2+ was present in theexternal NaCl solution and caesium glutamate waspresent inside the cell. In PC12 cells Ifune & Steinbach(1990) also found that the current-voltage relationshipfor acetylcholine-elicited currents was inwardlyrectifying when the pipette solution contained 1-3 mMfree Mg2+, and isethionate (to eliminate Cl- currents) wasthe main anion in the extracellular bath solution.

Vijayaraghavan, Pugh, Xhang, Rathouz & Berg (1992),Vernino, Amador, Luetje, Patrick & Dan (1992) and Mulleet al. (1992a) have shown that neuronal nicotinic AChRsare very permeable to Ca2 . Mulle and co-workers alsofound that Ca2+ entry through nicotinic AChRs activateda Cl- current in cells dissociated from the medial habenula(one consequence of the elevated Ca2+ influx was apersistent inhibition of the GABA response in these cells).The suggestion that a Cl- current is directly activated byCa2+ entering through the nicotinic AChR ion channels incells of the rat ventral medial habenula is also inagreement with our results.

Single channel activityBath-applied nicotine or acetylcholine rapidly activatedsingle channel activity in outside-out patches. Thisactivity could be elicited repeatedly from the same patch,

J. Physiol. 484.1100


and was not blocked by 10 /LM picrotoxin, 1 /M atropine,1 mM Mg2+ or 25 /SM AP5. These results indicate that theactivity is mediated by nicotinic receptors and notmuscarinic, GABA or NMDA receptors. The channelswere found to be cationic, being permeable to both Na+and Cs+, but not to CF.

In contrast to the whole cell responses, single channelactivity was present at positive potentials although atreduced frequency. Mathie et al. (1990) found, in ratsympathetic ganglion neurones, that nicotinic AChRchannels were active at positive potentials and were

blocked by internal Mg2+, but that openings occurredmuch less frequently than at negative potentials. Mulle etal. (1992b) also saw activity at positive potentials in cellsdissociated from the medial habenula, but detected no

single channel activity at positive potentials whenstudying nicotinic receptors in the interpeduncularnucleus (Mulle et al. 1991). In their analysis of rectificationin PC12 cells, Ifune & Steinbach (1990) noted that if Mg2+was included in the pipette solution, they observed a

voltage-dependent block of outward single channelcurrents. They also found that the mean burst duration ofthe AChR channels was voltage dependent, increasinge-fold over -123 mV. It was proposed that the voltagedependence of the burst duration caused rectification ofinward whole cell currents, but at positive potentials theshape of the current-voltage relationship was dominatedby Mg2+ block of outward single channel currents. In our

studies of the whole cell and single channelcurrent-voltage relationship Mg2+ was not added to theinternal pipette solution.

Multiple conductance classesWe previously reported a single channel conductance ofabout 50 pS with 1 mm Ca2+ and 0 mM Mg2+ (Connolly &Colquhoun, 1991). Mulle & Changeux (1990) reported26 pS channels (with 1 mm Ca2+ externally), but, using a

different protocol for dissociation of cells, Mulle et al.(1991, 1992a) later reported a conductance of 42 pS with1 mM Ca2+, 1 mM Mg2+ externally. In a separate study,Mulle and co-workers (1992b) determined the singlechannel conductance of the receptors to be 61 pS in theabsence of divalent cations, and 37 pS when 4 mm Ca2+was present externally. In three patches in a solutioncontaining 1 mM Ca2+ and 0 Mg2+, the conductanceobserved was 57 pS. Although this latter solution was

similar to that used here, the age range of animals used(10-15 days) was younger than those used in the presentexperiments. The data were interpreted as providingevidence of one class of conductances. Lester & Dani(1994) have also recently studied nicotinic acetylcholinereceptors in dissociated rat habenular neurones. In a

solution containing 5 mm Ca2+, they observed singlechannel activity with conductances of 30 pS and, less

frequently, approximately 20 pS. They compared thiswith the 37 pS conductance observed by Mulle et al.(1992b), but were unable to resolve whether or not thesmaller conductance was a substate of the mainconductance that they had observed.

In the present work, when 1 mm Ca2+ was present as theonly divalent cation in the external recording solution,the averaged single channel conductances elicited byacetylcholine or nicotine in twenty patches were in therange 36-58 pS, with a mean of 46-9 + 1P4 pS(mean + S.E.M.). The spread of values was wide, but, as afirst approximation, it appeared that individual patcheswere dominated by either a larger or a smallerconductance level, the levels being centred around 41 and51 pS. On average more than 80% of the openings in apatch appeared to fall into a single amplitude componentthat fell roughly into one of these conductance classes.The question arises whether all these conductances resultfrom the activity of one type of channel which could opento several different conductances, or whether the activitywas produced by two or more different channel subtypes.

If there was only one channel type then one might expectto see some direct transitions between the larger andsmaller conductance levels. Such transitions were veryrare and did not correspond accurately with the two mostcommon conductance levels. Also, the smaller conductanceclass had excursions to sublevels which were not visitedby the larger conductance class. These results cannot ruleout entirely the possibility that all the activity arisesfrom one channel type, but the different patterns ofsublevel activity associated with the two most commoncurrent amplitudes can be more easily explained byassuming that they originate from molecularly distinctchannel subtypes.

One alternative possibility is that we are observing asingle channel subtype which is oscillating between twolong-lasting 'modes' of behaviour. Amplitude stabilityplots show no evidence for switches between 'modes'during the lifetime of a single patch. In fact they reinforcethe distinctions between the amplitude groups. It is alsopossible that the reason direct transitions were rarebetween conductance levels was because a proportion of asingle population of channels was stably modified (e.g. byphosphorylation) before the outside-out patches wereformed.

A variety of conductances have been observed in studiesusing other neuronal tissues of rat origin. Nicotinicreceptors with similar conductances to those observedhere have been found in 17- to 21-day-old ratsympathetic neurones: range 26-48 pS, mean 36f8 pS in1 mM Ca2+ and 0 mM Mg2+, and about 51 pS in divalentcation-free solution (Mathie, Cull-Candy & Colquhoun,1991). In superior cervical ganglion cells conductances of

J. Physiol. 484.1


20 and 50 pS were observed in 2-5 mm Ca2+ and 1 mMMg2+ (Derkach, North, Selyanko & Skok, 1987). In PC12cells conductances of 22, 31 and 39 pS have been reportedin 1 mm Ca2+ and 1 mM Mg2+ (Bormann & Matthaei,1983), 49 pS in 0 mM Mg2+, 0 mm Ca2+ (Ifune & Steinbach,1990), and 48 pS was reported in 2 mm Ca2+ and 0 mMMg2+ (Sands & Barish, 1992). In retinal ganglion cells aconductance of 48 pS has been reported in 2-5 mm Ca2+and 0 5 mM Mg2+ (Lipton, Aizenmann &Loring, 1987).

Kinetic characteristics of the receptorsIn these experiments, distributions of channel closedtimes were fitted with four exponential components. Forthe seven patches analysed in detail, the mean timeconstants (and relative areas) were 85 ,ss (32-5%), 0-76 ms(19%), 12-6 ms (15%) and 245 ms (34%). The overalldistribution means were 95 ms. Mulle et al. (1992b)estimated that the time constant of short closures withinbursts was 0-6 ms when 5 sM ACh was applied to medialhabenular cells, which is similar to our 0-76 ms component.Using 1-5 /M ACh, Mathie et al. (1991) found fourexponential components in the shut times observed in ratsympathetic neurones. The mean time constants were36 #ss (78%), 0 73 ms (6 6%), 40 ms (5 3%) and 1704 ms(11 %). Although the range of conductances seen in the ratsympathetic ganglion cells overlaps with the range ofconductances seen here, the mean single channelconductance (in 1 mm Ca) was 37 PS, considerably lowerthan the mean value of 46-9 + 1P4 pS observed here.

Open time distributions were fitted with three exponentialcomponents with mean time constants (relative areas) forall seven patches of 0-51 + 0-13 ms (32 + 7 %), 4.9 + 1P4 ms(45 + 6 %) and 20 + 5 ms (23 + 2 %). These values are notcorrected for missed brief shuttings. Mulle et al. (1992b)fitted their open times with two exponentials, excludingall openings of less than 2 ms duration or greater thanaround 100 ms. They obtained time constants of 1P7 and19-8 ms in 0 mm Ca2+ and 3 uM ACh. Differences in theanalysis methods and recording resolution make itdifficult to compare directly their results with the presentstudy, but the open times observed by Mulle et al. (1992 b)are similar to those obtained here when fittingdistributions of openings longer than 2tr (2-6 + 0-6 and15-8 + 3-2 ms). Mathie et al. (1991) found time constantsof 0 43 ms (40%) and 5 3 ms (60%) for apparent openings.These values are similar to the two shorter time constantsthat were obtained in our studies in the medial habenula.

Grouping of the open time distributions according to thesize of the most commmon conductance in the patchesrevealed that for open times longer than 2tr , the overallmean for the four patches with predominantly 51 pSopenings was 5-8 ms, whereas that for patches withpredominantly 41 pS openings was 14-1 ms. Distributions

of open times conditional on the amplitude of theopenings were also calculated. Within any given patch, itwas found that sublevels had shorter openings than largeramplitudes within that same patch. Substates of eitherthe 41 or 51 pS channels therefore tend to have shorteropen times than the parent amplitude. It seems unlikely,therefore, that the 41 pS channel is a substate of the 51 pSchannel, since if it were, it would be expected to have ashorter mean open time than the 51 pS channels, not alonger one. A more likely explanation is that theobservations resulted from the activity of at least twodifferent receptor subtypes.

Burst length distributions followed a similar pattern tothat observed with open times. They were fitted withthree exponential components. These averaged 0 40 ms(24%), 4-1 ms (41 %) and 24 ms (35%). The overall meanwas 10-2 ms. Mulle et al. (1992b) reported values of 1-5 ms(33%) and 23-6 ms (67%) in cells dissociated from ratmedial habenula, while Mathie et al. (1991) obtained valuesof 0-41 ms (48%) and 12 2 ms (52%) in sympatheticganglion cells. While the results of Mulle et al. (1992 b) aresimilar to those observed here, the results in thesympathetic ganglia are most comparable to the resultswe have obtained in '51 pS patches'. For patches withpredominantly 51 pS openings the overall mean burstlength was 5-8 ms. Patches with predominantly 41 pSopenings had an overall mean of 16 ms. Thus the burstlengths were considerably longer for those patches inwhich the predominant single channel conductance wasaround 41 pS. The burst Popen for both types of patch wasvery high (0 99), so despite its lower conductance,activation of the smaller channels leads to about twice asmuch charge being passed at micromolar concentrations ofacetylcholine.

Distributions of the number of openings per burst werefitted with two geometric components. For all patchesthese had component means (and relative areas) of 1-15(70%) and 2-13 (30%) with a mean of 1'54 openings perburst. The overall means of the 41 and 51 pS patches arevery similar, suggesting that the difference in burstlength between the two types of patches is attributable todifferences in open times, both types of patch having asimilar number of openings per burst and similar within-burst shut times.

The kinetic stability plots illustrated in Fig. 10demonstrate that the mean open time of both large andsmall channels did not vary much during the time courseof the experiments. In those patches selected for detailedanalysis the shut times were generally stable. Where run-down occurred this was evident in the shut times but notin the open times. The differences in the open timedistributions between the 41 and 51 pS channels are not,therefore, likely to be caused by kinetic instability.

102 J. Physiol. 484.1

J. Physiol. 484.1 Nicotinic receptors in rat medial habenula 103

It is noteworthy that in the seven patches analysed indetail, more than 80% of the activity corresponds to oneamplitude. This raises the question of whether differentcells predominantly express only one type of receptor, orwhether more than one type of receptor can be expressedin an individual cell, possibly in clusters. Attempts weremade to obtain a second outside-out patch from individualcells in order to see whether the second patch had thesame type of channels as the first. Unfortunately, thequality and stability of the second patches were not goodenough to allow amplitude distributions and kineticanalyses to be carried out. However Moss & Role (1993)have obtained multiple patches from individual neuronesin the lumbar sympathetic ganglia of embryonic chick.Before innervation, four classes of conductance werepresent in most individual cells. Following innervation,many neurones expressed only one of the subtypes.Where more than one subtype was expressed, they werefound in segregated, high activity patches. Immuno-chemical evidence for a similar segregation of receptorsubtypes within individual cells of embryonic chick ciliaryganglia has been furnished by Vernallis, Conroy & Berk(1993). Also, in cultured postnatal rat hippocampalneurones, Zorumski, Liu Lin, Isenberg & Clifford (1992)found that most cells produced only one of two kineticallyand pharmacologically distinct nicotinic responses.Although in some cells the responses observed byZorumski et al. were mixed, in general it seemed thatreceptor subtypes were segregated on different cells. Theexact spatial distribution of the 51 and 41 pS receptortypes in cells in the ventral medial habenula thereforeremains to be determined. There could be additionalfunctional distinctions between these receptor classes,such as their Ca2+ permeability, which remain to bestudied. In addition, it would be interesting to considerwhether the proportions or properties of these twochannel types in the medial habenula change duringdevelopment.

Subunit composition of the receptorsSince several subunits of nicotinic AChRs are expressed inthe medial habenula, it is not possible to predict what thesubunit composition of these receptors might be. Theproperties of these receptors do not completely match anyof those which have been expressed in oocytes from clonedreceptor subunits, although there are some interestingpoints of comparison. Leutje & Patrick (1991) showed thatreceptors containing the ,84 subunit in combination witheither a2, a3 or a4 are more sensitive to cytisine than toacetylcholine, whereas receptors containing /12 as the onlynon-a subunit show the opposite order of agonistsensitivity. The whole cell current results we haveobtained here, and those reported by Mulle et al. (1990),suggest that cytisine is a more potent agonist on these

cells than acetylcholine. Hence our receptors may include/14 subunits. However, the rat (Seguela et at. 1993)homomeric a7 receptors also show a greater sensitivity tocytisine than to acetylcholine, so on their own theseresults cannot be used to identify receptors containing the/14 subunit.

As mentioned above, there is some similarity between theproperties of the 51 pS channels recorded here and thoseobserved in studies of ganglionic AChRs (Mathie et at.1991) and receptors in PC12 cells (Ifune & Steinbach,1990). Nicotinic subunit RNAs and proteins present inPC12 cells include a3, a5, /12, /13, /14 but not a2 or a4(Boulter et al. 1990; Rogers et at. 1992). Immunochemicalstudies have shown that in chick ciliary ganglia the a5subunit is associated with a receptor containing the a3and ,B4 subunits (Conroy, Vernallis & Berg, 1992). Morerecently it has been found that this receptor type issynaptically located in the chick (Vernallis et al. 1993),while a second type is extrasynaptic and contains a7subunits. Given the abundance of /12 subunit RNA andprotein in ganglionic neurones, it seems likely that thereare additional subtypes present in ganglia. It should benoted that the a5 subunit gene is not transcribed in themedial habenula (Boulter et al. 1990) and therefore the ac3type of receptor first described by Conroy et at. (1992) isnot likely to occur there, although the a7 subtype could.

Heterogeneity of nicotinic receptors in the medialhabenulaFrom the preceding Results and Discussion, there areseveral arguments for considering the nicotinic receptorsof the medial habenula to be a non-uniform population:the grouping of single channel amplitudes intoapproximately 41 and 51 pS classes; the infrequency ofdirect transitions between the observed amplitude levels;and the association of smaller conductance channels(41 pS group) with a long open time and the largerconductance channels (51 pS group) with a shorter opentime. Thus, there is evidence of nicotinic receptorheterogeneity in cells of the rat ventral medial habenula.

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AcknowledgementsThis work was supported by grants from the Medical ResearchCouncil. We thank Dr Frances Edwards for generously providingdata on the block of the cytisine response by hexamethonium, DrSue Wonnacott for helpful discussions, and Liz Ensor forexcellent technical assistance. J.G. C. would also like to thankDrs Chris Frost and Fran Fullerton for accommodation andhospitality during the later stages of this work.

Received 24 November 1993; accepted 7 September 1994.

J. Phy8iol. 484.1

Heterogeneity acetylcholine

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