Muscarinic Modulation of Recruitment Threshold and Firing ...personal.us.es/pnunez/pdfs/5-j.neurophysiol2009.pdf · Muscarinic modulation of recruitment threshold and ﬁring rate

Muscarinic Modulation of Recruitment Threshold and Firing Rate in RatOculomotor Nucleus Motoneurons

Jose Luis Nieto-Gonzalez,* Livia Carrascal,* Pedro Nunez-Abades, and Blas TorresDepartment of Physiology and Zoology, University of Seville, Seville, Spain

Submitted 6 February 2008; accepted in final form 21 October 2008

Nieto-Gonzalez JL, Carrascal L, Nunez-Abades P, Torres B.Muscarinic modulation of recruitment threshold and firing rate in ratoculomotor nucleus motoneurons. J Neurophysiol 101: 100–111,2009. First published October 29, 2008; doi:10.1152/jn.90239.2008.Above recruitment threshold, ocular motoneurons (Mns) show a firingrate linearly related with eye position. Current hypothesis suggeststhat synaptic inputs are determinant for establishing the recruitmentthreshold and firing rate gain in these Mns. We investigated thisproposal by studying the cholinergic modulation in oculomotor nu-cleus Mns by intracellular recordings in rat brain slice preparation. Allrecorded Mns were silent at their resting membrane potential. Bathapplication of carbachol (10 �m) produced a depolarization and asustained firing that was not silenced on returning membrane potentialto the precarbachol value via DC injection. In response to similarmembrane depolarization or equal-current steps, carbachol-exposedMns produced a higher firing rate and a shorter spike afterhyperpo-larization phase with lower amplitude. The relationship betweeninjected current and firing rate (I–F) was linear in control andcarbachol-exposed Mns. The slope of these relationships (I–F gain)decreased with carbachol exposure. Bath application of agonist andantagonist of nicotinic and muscarinic acetylcholine receptors inaddition to immunohistochemical studies support the notion thatmuscarinic receptors are primarily involved in the preceding re-sponses. We conclude that muscarinic inputs play an important role indetermining the recruitment threshold and firing rate gain observed inoculomotor Mns in vivo.

I N T R O D U C T I O N

The firing rate of motoneurons (Mns) driving eye move-ments and fixations has been characterized in alert preparation(de la Cruz et al. 1989; Delgado-García et al. 1986; Fuchs et al.1988; Pastor et al. 1991; Stahl and Simpson 1995; Sylvestreand Cullen 1999). The beginning of firing is correlated with aparticular eye position. Above this threshold, the Mns dis-charge at a steady tonic rate that increases linearly with eyeposition (fixation) in the on-direction, which corresponds to thepulling direction of the muscle that the Mn innervates. Theslope of this relationship (K) indicates the eye-position sensi-tivity. The recruitment threshold and K vary from Mn to Mn,distributing over a large range that ensures a fine gradation offorce and stable generation of eye position to prevent eye driftand misalignment. Studies in alert preparation have demon-strated a covariation between recruitment threshold and K: Mnswith higher threshold have greater K (de la Cruz et al. 1989;Delgado-García et al. 1986; Fuchs et al. 1988; Pastor et al.1991). The recruitment threshold of the ocular Mns could

depend on intrinsic membrane properties and/or synaptic in-puts (Dean 1997). Using in vitro preparation, we have recentlyfound Mns whose phasic–tonic firing pattern resembles that inalert preparation and demonstrated an inverse relationshipbetween Mn threshold and tonic firing gain. According to thesefindings, we have ruled out intrinsic membrane properties asbeing the sole mechanism supporting the covariation betweenrecruitment threshold and K reported in alert studies (Nieto-Gonzalez et al. 2007). Our proposal is in agreement with thesuggestion that synaptic inputs are determinant in establishingthe recruitment threshold in ocular Mns (Hazel et al. 2002;Pastor and Gonzalez-Forero 2003). The first aim of this workwas to investigate the hypothesis that synaptic inputs play akey role in determining the recruitment threshold and firingrate gain in oculomotor nucleus Mns.

Recent studies in the gracilis nucleus have demonstrated thatcarbachol, an agonist of cholinergic receptors, reduces thecurrent threshold for spike generation by about 50% andincreases the number of spikes by about 100% in response toidentical depolarizing current pulses (Fernandez de Sevillaet al. 2006). It has been suggested that cholinergic inputs in thehypoglossal Mns contribute to the maintenance of the upperdilator muscle activity in awake animals (Chamberlin et al.2002). Neurons of some brain stem nuclei encoding eye- andhead-position signals—the medial vestibular nucleus and pre-positus hypoglossi nucleus—respond to carbachol with mem-brane potential depolarization and the evoking of action po-tentials (Navarro-Lopez et al. 2004; Sun et al. 2002). In theprepositus hypoglossi nucleus, a cholinergic synaptically trig-gered event participates in the generation of the persistentactivity (Navarro-Lopez et al. 2004), which is characteristic ofthe neurons carrying eye-position signals. Microinjections of cho-linergic antagonists in the prepositus hypoglossi nucleus of alertbehaving cats evoked a gaze-holding deficit consisting of a recen-tering drift of the eye after each saccade. Anatomical data inmonkeys (Carpenter et al. 1992), combining retrograde labelingof neurons and acetylcholine transferase immunohistochemis-try (ChAT), have demonstrated that a moderate number ofcholinergic neurons located in the caudal region of the medialvestibular nucleus project to the oculomotor nucleus, as docholinergic neurons in the paragigantocellular reticular area.Fibers showing bouton-like structures positive for ChAT andvesicular acetylcholine transporter have been reported in theneuropil of the oculomotor nucleus (Hellstrom et al. 2003;Ichikawa and Shimizu 1998). In view of these findings, we askwhether cholinergic synaptic inputs exert, via muscarinic

* These authors contributed equally to this work.Address for reprint requests and other correspondence: B. Torres, Departa-

mento de Fisiología y Zoología, Facultad de Biología, Avenida Reina Mer-cedes 6, 41012 Sevilla, Spain (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 101: 100–111, 2009.First published October 29, 2008; doi:10.1152/jn.90239.2008.

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and/or nicotinic receptors, an influence on recruitment thresh-old and firing rate gain in oculomotor nucleus Mns.

M E T H O D S

Surgery and solutions

Experiments were carried out in juvenile Wistar rats (20–30 daysold) of both sexes. The experiments were performed in accord withDirective 86/609/CEE of the European Community Council, theSpanish Real Decreto 223/1988, and Seville University regulations onlaboratory animal care. Rats were anesthetized with sodium pentobar-bital (50 mg kg�1) and quickly decapitated. The methods to obtain theslices, recordings, and analysis are fully detailed elsewhere (Carrascalet al. 2006; Nieto-Gonzalez et al. 2007). In brief, brain slices of 300�m including the oculomotor nucleus were first incubated in achamber containing cold sucrose-artificial cerebrospinal fluid (ACSF)for 35–45 min and then transferred to a second chamber containingACSF at a temperature of 21 � 1°C. Single slices were transferred tothe recording chamber and superfused at 2 mL/min (Harvard, MPII)with ACSF bubbled with 95% O2-5% CO2 (pH 7.4; 21 � 1°C). Thecomposition of ACSF was as follows (in mM): 126 NaCl, 2 KCl, 1.25Na2HPO4, 26 NaHCO3, 10 glucose, 2 MgSO4, and 2 CaCl2. Forsucrose-ACSF solution, the 126 NaCl was substituted by 240 sucrose.

The drugs (all from Sigma–Aldrich) used in this study, dissolved indistilled water for stock solution and stored frozen at �20°C, were asfollows: carbamylcholine chloride (carbachol, 10 mM), muscarinechloride (10 mM), atropine sulfate (10 mM), pirenzepine dihydro-chloride (10 mM), methoctramine (10 mM), 1,1-dimethyl-4-phe-nylpiperazinium iodide (DMPP, 10 mM), and tetrodotoxin (TTX, 1mM). These drugs were diluted in the ACSF at different concentra-tions (see RESULTS), maintained at the same temperature and pH asthose of ACSF. The time taken to completely exchange the recordingchamber was about 50 s and bath application of drugs was always for�2 min. When drug responses were compared after repeated expo-sures or another drug application, the length of application was keptconstant. Drugs were usually applied to only one Mn per slice; if itwas necessary to apply more than one drug, the slice was washed withACSF solution for �20 min (Nunez-Abades et al. 2000).

Electrophysiological recordings and data analysis

All recorded neurons were identified as Mns by their antidromicactivation from the root of the third nerve and by the collision test (fordetails see Fig. 1 in Carrascal et al. 2006). The micropipettes used forrecordings were filled with a 3 M KCl (40–70 M�) solution. Mono-synaptic potentials were elicited by stimulating the region of themedial longitudinal fasciculus close to the boundaries of the oculo-motor nucleus. Metallic bipolar microelectrodes (tips �300 �m apart)were constructed of 25-�m-diameter stainless steel wire insulated inglass micropipettes. A cathodal monopulse of 150 �s at 2 Hz was usedand the current adjusted at subthreshold values. Recordings werestored on videotape (Neuro-Corder, NeuroData Instruments, Dela-ware Water Gap, PA) and subsequently played back and acquired witha PCI-6070E card (National Instruments, Austin, TX) for off-lineanalysis. All Mns included for analysis showed a stable restingmembrane potential of �55 mV or more negative, an action potential�60 mV, and fired repetitively in response to suprathreshold depo-larizing current steps of 1 s. With the exception of exposure to TTX,the effects of the pharmacological manipulations were reversible. Thedata for any given Mn in response to the different drugs were acceptedonly if the membrane potential and spike amplitude returned to thecontrol value (that obtained when the impaled Mn was checked justbefore drug application) after a washout period.

We focused our analysis on Mns (n � 54) showing a phasic–tonicdischarge in response to suprathreshold current steps (Carrascal et al.2005; Nieto-Gonzalez et al. 2007). The carbachol effects (10 �M) on

the membrane potential and firing frequency for each Mn werequantified before exposure (control condition), during perfusion, andafter washout with ACSF. The carbachol effects on membrane poten-tial were measured by the difference between resting membranepotential and spike threshold at a steady tonic rate. To assess whetherthe membrane potentials during DC current injection were correct, weroutinely monitored bridge balance through the whole experimentalsession (details in Nunez-Abades et al. 1993). To determine whethercarbachol led to changes in input resistance, TTX (1 �M) was addedto the extracellular solution to block voltage-gated Na� channels and,thereby, the spikes. The input resistance was determined by passingnegative current pulses (0.2 nA, 500 ms, 1 Hz) in the controlcondition, then applying TTX, and finally in a carbachol–TTX solu-tion. The instantaneous firing frequency was calculated for the wholerecording as the reciprocal of the interspike-interval (ISI) duration.The dots in Figs. 1 and 7 plot the instantaneous frequency valuessampled at a rate of 1 in 100 (i.e., the frequency of the first ISI, nextthe frequency of the ISI number 101,. . .), with the exception ofpirenzepine exposure and atropine exposure, which were sampled at arate of 1/50. During repetitive discharge, the afterhyperpolarization(AHP) phase amplitude was measured as the voltage differencebetween the spike threshold and the most negative value reachedduring this phase. The AHP duration was calculated as the timespent from when the voltage value in the repolarizing phasereached the level of the spike threshold to the threshold of the nextspike. The spike threshold was the value of the membrane potentialat which the first derivative surpassed 10 V/s (Carrascal et al.2006). The repetitive-firing frequency was evoked by depolarizingcurrent steps (1 s, 0.5 Hz) with 0.1-nA increments. The steady-state firing frequency was the average of the instantaneous fre-quencies during the last 500 ms of the current step. For each Mn,the relationship between the steady-state firing frequency andinjected current was represented (I–F plot) to calculate the slope,termed I–F gain. We defined current threshold as the intensity ofstimulating current capable of eliciting maintained repetitive firingat 20 spikes s�1 (Nieto-Gonzalez et al. 2007).

All statistical analyses and exponential fits were carried out on theraw data. Significant differences between the Mns before (control) andduring bath application of carbachol were determined by using aone-way ANOVA test. The significance level was established at P �0.05. All data are reported as means � SE.

Immunoblotting and immunohistochemistry

Immunohistochemical experiments were carried out to demon-strate the presence of muscarinic receptors (M1 and M2) in theoculomotor nucleus. The specificity of the antibodies was tested byWestern blotting (for details, see Disney et al. 2006). The recordedbrain slices (300 �m) were fixed in a solution of 4% formaldehydein 0.05 M phosphate buffer (pH 7.4) at 4°C and then placed into asolution containing 10% sucrose in phosphate buffer (4 – 6 h at4°C). To facilitate resectioning, slices were transferred to a solu-tion consisting of two parts phosphate buffer saline (PBS) and onepart optimum cutting temperature embedding compound (TissueTek, Miles Ames Division, Elkhart, IN) for �24 h (Johnson andBywood 1998). The brain slices were cut in a cryostat (LeicaCM1850) at 40-�m thickness. Two distinct immunohistochemicalprocedures were carried out to reveal M1 and M2 receptors bybright-field (biotin-conjugated donkey anti-rabbit, Jackson Immu-noResearch) and confocal laser (fluorescein isothiocyanate[FITC]– conjugated goat anti-rabbit, Sigma–Aldrich) microscopy.For bright field, free-floating brain slices were washed in PBS andendogenous peroxidase activity was blocked with ethanol. Sectionswere incubated for 1 h at room temperature in 3% normal donkeyserum and 0.15% Triton X-100 in PBS, and then in the dilutedprimary antibody (1:100 M1; 1:500 M2) overnight at 4°C. Thesections were incubated with biotinylated donkey anti-rabbit IgG

101MUSCARINIC MODULATION OF OCULAR Mn EXCITABILITY

J Neurophysiol • VOL 101 • JANUARY 2009 • www.jn.org

(1:2,000; Jackson ImmunoResearch) overnight at 4°C and with thestandard avidin-biotinylated peroxidase complex (ABC kit, VectorLaboratories) for 1 h at room temperature. Peroxidase activity wasrevealed by 0.02% diaminobenzidine with 0.01% H2O2 and en-hanced with 0.04% nickel ammonium sulfate. For immunofluores-ence, free-floating brain slices were washed in PBS and incubatedfor 1 h at room temperature in 3% normal goat serum and 0.15%Triton X-100 in PBS. They were incubated with polyclonal pri-mary antibodies raised against M1 (1:100) or M2 (1:500) receptorsovernight at 4°C and washed in PBS; all sections were incubatedwith secondary antibody labeled with FITC (1:200) for 2 h at roomtemperature. Finally, the sections were placed on gelatinized glassslides and coverslipped with fluorescent mounting medium (DakoCytomation).

R E S U L T S

All recorded Mns (n � 54) of the oculomotor nucleus weresilent at their resting membrane potential (�61.6 � 2.1 mV)and required suprathreshold depolarizing current steps to evokea sustained firing. Bath application of carbachol (10 �M), acholinergic agonist that is not metabolized by acetylcholines-terase, evoked in all recorded Mns a membrane potentialdepolarization and a sustained firing discharge. Figure 1, A andB illustrates the effect of carbachol on the membrane potentialfor a representative Mn. Carbachol induced a depolarizationthat triggered action potentials at about 15 mV and continueddepolarizing (�18 mV) until the Mn reached a steady firing.

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FIG. 1. Effect of carbachol (10 �M) onthe membrane potential and firing frequencyin the motoneurons (Mns) of the rat oculo-motor nucleus. A: response evoked by bathapplication of carbachol in a representativeMn. The framed area is magnified in B toillustrate the time course of the membranepotential depolarization. C: plot representingthe firing frequency evoked by carbachol.The dashed line indicates that the increase infiring frequency fitted the single exponential.C1 is a representation of the onset in spikingof the Mn. The numbers (1–3) indicate theprogressive increase in frequency. C2 showsthe firing at the steady-state level. The timeof carbachol application is indicated by thesolid line.

102 NIETO-GONZALEZ, CARRASCAL, NUNEZ-ABADES, AND TORRES


Washing carbachol out led to recovery of the resting value. Theeffect of carbachol on membrane depolarization was similar inall Mns quantified, with a depolarization level of 16.5 � 0.68mV (n � 34; those preexposed to TTX, atropine, or DMPPwere not included). Figure 1C shows the effect on the firingfrequency evoked by bath application of carbachol in a repre-sentative Mn. The duration of the first ISIs decreased progres-sively, leading to an increase in the instantaneous firing fre-quency (Fig. 1C1), until it reached a stable level (Fig. 1C2). Thetime course of this response was well fitted to a single expo-nential (r � 0.9) with a time constant of 0.66 min and a firingin the steady state of 27 spikes s�1 (Fig. 1C). The exponentialincrease in the firing frequency evoked by carbachol was ageneral feature for all Mns quantified (n � 34), showing a timeconstant of 0.59 � 0.1 min and a steady firing of 23.3 � 1.8spikes s�1. After the carbachol was washed out, the firingfrequency decreased progressively until it ceased (Fig. 1C). Inaddition, nonsignificant changes were found in the input resis-tance between the Mns (n � 6) in control condition (65.9 � 5.6M�), control condition–TTX (62.4 � 7.1 M�), and thenexposed to carbachol–TTX (71.2 � 6.2 M�) (not illustrated).

To elucidate the postsynaptic nature of the effects of carba-chol on oculomotor Mns, the brain slices were exposed to TTX(1 �M). This drug abolishes action potentials and thus thesynaptic transmission from premotor circuit interneurons tar-geting the Mns. Bath application of carbachol evoked a depo-larization of 16.5 mV for a representative Mn, which slightlydecreased by 1.8 mV when the slices were also exposed toTTX (Fig. 2A). We also checked the effect of the premotorcircuits in the present study, first measuring the level ofdepolarization (15.2 mV) evoked by carbachol in anotheroculomotor nucleus Mn and then washing with normal ACSF.The same Mn was exposed to TTX, which did not produce anymodification in the resting membrane potential, and then tocarbachol (Fig. 2B), evoking a depolarization of similar mag-nitude (13.4 mV). Similar findings were recorded in other Mns

(n � 3). These experiments demonstrated that carbachol pro-duced its effects through the activation of cholinergic receptorslocated in the oculomotor nucleus Mns.

The electrical microstimulation of the medial longitudinalfasciculus region close to the boundaries of the oculomotornucleus (Fig. 3A) evoked an excitatory postsynaptic potential(EPSP) in the recorded Mns (n � 5). The increase of currentintensity led to a graded increase in the amplitude of EPSPs.EPSPs showed a delay of 2.3 � 0.26 ms in relation to thestimulus onset (Fig. 3B). These EPSPs were accepted asmonosynaptic based on the criteria of brief (�3 ms) andconstant latencies, irrespective of the intensity of the electricalstimulation. Bath application of atropine, an antagonist ofmuscarinic receptors, produced a mean decrement of 34.5 �4.9% in the amplitude of EPSPs in all studied Mns (Fig. 3C),showing that this excitatory monosynaptic potential evoked inthe oculomotor nucleus Mns was in part cholinergic.

As shown in Fig. 1, carbachol evoked both a membranepotential depolarization and a sustained firing discharge. Wewondered to what extent firing discharge was dependent onmembrane potential depolarization. Figure 4A illustrates arecord of a representative Mn exposed to carbachol and then,when depolarization and firing frequency were stable, injectedwith hyperpolarizing DC until the value of resting membranepotential was recovered. The Mn was silent (as was each onebefore carbachol exposure) at its resting membrane potential(Fig. 4, A and B, part 1). During bath application of carbachol,the membrane potential was depolarized and evoked a repeti-tive sustained firing of about 21 spikes s�1 (Fig. 4, A and B,part 2). When the Mn was brought to its previous membranepotential, the firing continued, although the frequency waslower (�10 spikes s�1; Fig. 4, A and B, part 3). All Mnsstudied in this procedure (n � 14) showed a similar response.They were silent at their resting potential before carbacholapplication, but discharged at 14.2 � 0.9 spikes s�1 on return-ing membrane potential to the precarbachol values. These data

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FIG. 2. Effects of tetrodotoxin (TTX) onthe electrophysiological response evoked bycarbachol (10 �M) in the Mns of the ratoculomotor nucleus. A: repolarization of themembrane potential after application of TTX(1 �M) in an Mn exposed to carbachol.B: membrane potential depolarization afterapplication of carbachol in an Mn previouslyexposed to TTX. This Mn was previouslyexposed to carbachol and showed a depolar-ization level of 15.2 mV. Drugs were addedas indicated by the solid lines.



demonstrated that carbachol exposure diminished recruitmentfiring threshold.

We have previously classified the Mns with sustained dis-charge into types IA and IB, based, at least in part, on thecharacteristics of the AHP (Nieto-Gonzalez et al. 2007). The IAMns showed a single deep AHP (Fig. 5A, inset), whereas IBMns showed an early fast AHP and delayed slow AHP. Inaddition, an afterdepolarization phase was observable in IBMns (Fig. 5B, inset). The firing frequency and AHP of the IA(n � 7) and IB (n � 5) Mns were compared at similar levels ofmembrane potential depolarization evoked by either intracel-lular current steps (control) or carbachol (Fig. 5, A–D). Themembrane potential response of IA and IB Mns to carbacholapplication was similar (IA � 17.2 � 0.8 mV; IB � 16.3 � 1.2mV). Bath application of carbachol evoked similar sustainedspiking in IA and IB Mns and both were higher (IA � 19.3 �2.9 spikes s�1; IB � 21.9 � 3.8 spikes s�1) than those evoked

by intracellular current steps (IA � 12.9 � 1.5 spikes s�1; IB �13.2 � 0.8 spikes s�1). The effects of the drug were alsosimilar on AHP duration and amplitude into IA and IB Mns(Fig. 5, E and F), in which the values of duration and amplitudeof the AHP were significantly higher in the control condition(78.6 � 5.4 ms and 8.2 � 0.6 mV) than those during exposureto carbachol (53.8 � 6.7 ms and 5.9 � 0.8 mV).

The repetitive-firing properties evoked by equal depolarizingcurrent steps before (control) and during bath application ofcarbachol (in this latter condition, the Mns were hyperpolar-ized to restore the membrane potential prior to carbacholtreatment) were also quantified. As shown in Fig. 6A for arepresentative Mn, the number of spikes was higher in pres-ence of carbachol. Thus in the case illustrated in response to0.2 nA of injected current, the Mn exhibited 11 spikes in thecontrol condition and 20 spikes when exposed to carbachol. Infact, the firing frequency was significantly higher in the studied

A

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FIG. 3. Stimulation sites and recording ofevoked excitatory postsynaptic potential(EPSP) in the rat oculomotor nucleus Mns.A: photomicrograph of a transverse sectionshowing the locations of the bipolar elec-trodes to elicit the antidromic activation (St1)and EPSP (St2) of the Mns within the OCM.B and C: monosynaptic EPSP in controlcondition and exposed to atropine, respec-tively. MLF, medial longitudinal fasciculus;OCM, oculomotor nucleus; PAG, periaque-ductal gray; SC, superior colliculus; IIIn,third nerve root.

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FIG. 4. Membrane potential contributionto the firing frequency in Mns of the ratoculomotor nucleus exposed to carbachol(10 �M). A: electrophysiological recordingof an Mn exposed to carbachol and thenrepolarized to its resting membrane potentialby hyperpolarizing DC. B: details of 3 inter-vals (1, 2, and 3) indicated in A. It should benoted that the same Mn was silent at restbefore (1) carbachol application and spikedduring carbachol exposure at rest (3).



Mns (n � 12) exposed to carbachol than in the controlcondition, irrespective of the current intensity (Fig. 6B). Ad-ditionally, the I–F plots in both control and carbachol-exposurecondition showed that, above recruitment threshold, all Mnsexhibited a good linear relationship (for the representative Mnillustrated in Fig. 6C, r � 0.99, P � 0.0001). These plots alsoshow—consistent with the above-cited data—that Mns ex-posed to carbachol had a lower recruitment threshold (seearrow) and fired at higher frequency than control through thewhole range of current stimulation. The I–F gain values werelower when the Mns were exposed to carbachol (Fig. 6C).Because we did not find any significant difference in currentthreshold and I–F gain between IA and IB Mns, all data were

pooled. Figure 6D shows I–F relationships of all studied cases:carbachol-exposed Mns showed a lower current threshold(0.1 � 0.02 nA) than that of control (0.41 � 0.03 nA) andlower I–F gain (carbachol � 54.8 spikes s�1 nA�1; control �66.1 spikes s�1 nA�1). These differences were significant.

Since the recordings were carried out at room temperature(21 � 1°C) and could yield qualitatively different results atmore physiological temperatures, an additional set of experi-ments was performed at 33 � 1°C (n � 6). In this condition,carbachol evoked a membrane potential depolarization of12.8 � 1.5 mV and a sustained firing of 15.9 � 2.8 spikes s�1.When the membrane potential values of these Mns wererecovered by hyperpolarizing DC, the firing continued at a

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FIG. 5. Firing frequency and afterhyper-polarization phase (AHP) in types IA and IB

Mns depolarized by carbachol (10 �M) orcurrent steps of the rat oculomotor nucleus.A–D: response in firing frequency for an IA

(A and C) and an IB (B and D) Mn similarlydepolarized by either an intracellular depo-larizing current step (control) or carbacholapplication. The insets show that the AHPwas monophasic for IA Mn (arrow) and bi-phasic in IB Mn (indicated by 2 arrows). Thislatter Mn also exhibits an afterdepolarizationphase (asterisk). E–F: comparison of theAHP for IA and IB Mns illustrated in A–D.



frequency of 11.2 � 2.5 spikes s�1. Carbachol-exposed Mnsalso showed a lower current threshold (0.2 � 0.04 nA) thanthat of control (0.47 � 0.03 nA) and lower I–F gain (carba-chol � 38.5 � 4.2 spikes s�1 nA�1; control � 45.7 � 3.5spikes s�1 nA�1). In short, the results were not qualitativelydifferent from those obtained at room temperature.

Cholinergic receptors involved in firing modulation

Carbachol is a general agonist of cholinergic receptors(muscarinic and nicotinic); therefore we carried out someexperiments to determine the contribution of the differentcholinergic receptors to the firing rate of Mns. To elucidate theeffect of muscarinic receptors, we performed five differentexperiments (n � 15). The Mns (n � 3) were exposed tocarbachol and then we applied atropine (1.5 �M) as a musca-rinic antagonist. Even though the brain slices were exposed tocarbachol, bath application of atropine completely blocked thecarbachol-evoked depolarization and the repetitive firing(Fig. 7A). The blockage of firing with atropine was shorter-lasting than that produced by washing carbachol out withACSF. Second, we treated the brain slices with atropine andthen carbachol (n � 3; not illustrated). In these assays, the Mnsmaintained the level of the resting membrane potential andremained silent. Third, in some cases (n � 3; not shown), we

studied the response to bath application of DMPP (10 �M), anicotinic agonist, and found that Mns remained silent. How-ever, when the same Mns were exposed to carbachol, thisevoked a similar response to that shown in Fig. 1. Fourth, wecompared (n � 3) the firing discharge evoked by carbacholwith that produced by muscarine (10 �M) in the same Mn. Asshown in Fig. 7B, the two procedures evoked indistinguishableresponses: both led the Mn to discharge at about 25 spikes s�1

in its steady state. In addition, the temporal courses of the risein firing frequencies were exponentially fitted (r � 0.9) withsimilar time constants in response to carbachol (0.74 min) andmuscarine (0.76 min). Fifth, bath application of atropine (n �3) similarly blocked the muscarine-evoked firing discharge tocarbachol (Fig. 7B). Together, these results demonstrated thatin the oculomotor nucleus Mns, the cholinergic receptorsinvolved in the modulation of the firing rate are muscarinic.

The presence of muscarinic receptors M1 and M2 wasinvestigated. The procedure was bath application of carbachol,after which we added either pirenzepine (2 �M), as an antag-onist of M1 receptors, or methoctramine (5 �M), as an antag-onist of M2 receptors. As control, before and after applicationof these antagonists, the firing frequency evoked by carbacholwas routinely determined. Figure 7C illustrates the firing-frequency plot of an Mn exposed to carbachol and thenpirenzepine. As shown, the firing discharge evoked by carba-

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FIG. 6. Carbachol effects in the firingfrequency and I–F gain in Mns of the ratOCM. A: firing discharge evoked by equalcurrent steps in an Mn in control conditionand then exposed to carbachol (10 �M).B: histogram showing the firing frequencyevoked by equal-current steps in control andcarbachol-exposed Mns. Asterisks indicatesignificant differences. C: firing frequency–current (F–I) relationship in a representativeMn in control condition and then exposed tocarbachol. Arrow indicates that carbacholdecreased the recruitment threshold of theMn, which discharged at rest with a rate of12 spikes s�1. D: linear relationships be-tween firing frequency and current obtainedin Mns before (control) and during carbacholapplication. Fgain and Ithr are the mean of F–Igain and current threshold, respectively, forall recorded Mns.



chol was completely blocked by pirenzepine application. Asimilar response was obtained in two additional Mns; i.e., thefiring discharge (23.3 � 2.8 spikes s�1) in carbachol-exposedMns became silent by pirenzepine. We also studied the role ofthe M2 receptors in the firing of the oculomotor nucleus Mns.The mean firing discharge of three Mns tested was 20.5 � 3.1spikes s�1 and decreased to 15.5 � 3.3 spikes s�1 followingmetoctramine application (Fig. 7D). These two results indicatea greater role of M1 receptors in the modulation of repetitivefiring in the oculomotor nucleus Mns.

The combination of electrophysiological and pharmacolog-ical studies indicated the presence of M1 and M2 receptors in

the oculomotor nucleus Mns; we performed experiments toreveal them by immunohistochemical procedures. The speci-ficity of the antibodies was tested by Western blot. Proteinsseparated from rat brain samples containing the oculomotornucleus and incubated in the solution containing the primaryantibodies stained a single protein band at about 66 kDa inblots for either M1 or M2 receptors (Fig. 8A, lanes 1 and 4).The specificity of these antibodies was validated using positivecontrol (lanes 2 and 5) with a colon tumoral cell line thatoverexpresses M1 and M2 acetylcholine receptors acting in anautocrine manner (Eglen 2006). As negative control (lanes 3and 6), the antibodies were preadsorbed with the appropriate

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FIG. 7. Involvement of muscarinic recep-tors in the firing frequency of rat OCM Mns.A: plot showing the firing frequency of anMn exposed to carbachol (10 �M) and thesame Mn exposed to carbachol and atropine(1.5 �M). As shown, atropine blocked thefiring discharge for less time than washed-out carbachol. B: comparison of the firingfrequency evoked by bath application of car-bachol, muscarine (10 �M), and muscarine-atropine in the same Mn. C and D: plotsshowing the effects of pirenzepine (2 �M;C), an antagonist of M1 receptors, or methoc-tramine (5 �M; D), an antagonist of M2

receptors, on the firing frequency evoked bycarbachol in 2 different Mns. Note that firingfrequency was blocked by pirenzepine,whereas it decreased by about 20% follow-ing methoctramine exposure. Drugs wereadded as indicated by the solid lines.



immunogen before proceeding as described earlier. A largenumber of cell bodies confined to the oculomotor nucleus wereintensely positive for M1 receptors, the tissue background ofwhich was scarce. Both confocal laser and bright-field micros-copy showed that such labeling was chiefly intrasomatic (Fig.8, B and C). In contrast to the case of M1 staining, the numberof cells immunoreactive for M2 was small; the labeling out-lined cell somata (Fig. 8, D–F, arrowheads) and abundantrounded and filiform structures that could have been dendritessectioned in the transverse and sagittal planes, respectively(Fig. 8, D and E, arrows).

D I S C U S S I O N

We have demonstrated for the first time that physiologicalproperties of the oculomotor nucleus Mns are under the controlof cholinergic inputs. Bath application of carbachol producedin these Mns membrane potential depolarization, an increase infiring frequency, and a decrease in both recruitment thresholdand I–F gain. These effects were mediated via muscarinicreceptors. We will discuss these results in relation to thosereported in other neuronal pools, including the conductances

underlying such muscarinic modulation, and will interpretthem in the context of the discharge pattern reported in alertpreparation.

Technical considerations of the method

Temperature lower than physiological is routinely used forexperimentation in vitro (Cameron et al. 2000; Carrascal et al.2006; Magarinos-Ascone et al. 1999; Nunez-Abades et al.2000; Sawczuk et al. 1995; present data). Reduced temperaturemay enhance slice viability, but cooling slows ionic conduc-tance kinetics (Thompson et al. 1985). Since our recordingswere carried out at room temperature, we assessed such effectwith a few recordings at 33 � 1°C. In this latter condition, theresults reported here remained qualitatively unaltered.

Excitability of oculomotor nucleus Mns dependson cholinergic inputs

Bath application of the cholinergic agonist carbachol yieldeda membrane potential depolarization in all recorded Mns.Blockage of synaptic transmission by TTX supported the

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FIG. 8. Immunohistochemistry to revealM1 or M2 receptors of the rat OCM neurons.A: Western blot controls for antibodiesagainst M1 and M2 acetylcholine receptors.Lanes 1 and 4 show a single band at about 66kDa for M1 and M2, respectively; lanes 2and 5 illustrate a band at 66 kDa for a colontumoral-cell line that overexpresses musca-rinic receptors, as positive control; lanes 3and 6 show that preadsorption of the anti-bodies abolished these bands. B–F: confocallaser and bright-field microscopy of trans-verse sections of the OCM, showing immu-nostained neurons against M1 (B, C) or M2

(D–F) receptors. The insets show magnifiedviews of labeled neurons. Positive neuronsin A, D, and E are indicated by asterisks,whereas in C and F they are indicated byblack arrows. In D and E, arrowheads showthe perisomatic labeling observed and whitearrows point to dendrite-like structures.



notion that this response was attributable to the activation ofcholinergic receptors of the Mns. Depolarization in response tocholinergic agonists is a common characteristic reported inhypoglossal and spinal Mns (Alaburda et al. 2002; Cheval-lier et al. 2006; Lape and Nistri 2000; Miles et al. 2007) andneurons of different nuclei (Klink and Alonso 1997a,b;McQuiston and Madison 1999; Navarro-Lopez et al. 2004;Nunez et al. 1997; Yajeya et al. 1997). Membrane depolar-ization has been attributed to suppression of various K� cur-rents (Krnjevic 1993), activation of Ca2�-dependent or -inde-pendent nonselective cationic conductance (Fisahn et al. 2002;Honda et al. 2003; Klink and Alonso 1997b; Yajeya et al.1999), and activation of the mixed Na�/K� current Ih (Fisahnet al. 2002). In addition, the blocking of a conductance respon-sible for an outward current and the activation of a conductanceresponsible for an inward current could contribute to maintain-ing the values of input resistance (McQuiston and Madison1999). Similar mechanisms could also be underlying the re-sponses in oculomotor nucleus Mns exposed to carbachol, butthe current data do not permit assessment of the relativecontributions of any of these potential mechanisms.

Since the decrement in recruitment threshold in carbachol-exposed Mns was not led by an increase in the input resistance,we suggest that the activation of cholinergic receptors (mus-carinic in nature; see following text) modulates conductancesunderlying the threshold of spike generation. In agreementwith this proposal, it has been reported in other neuronal poolsthat a cholinergic agonist reduces the threshold of the currentrequired for spike generation (Fernandez de Sevilla et al. 2006)and could modulate action potential generation by modificationin the fast and persistent Na� currents underlying spiking(Cantrell and Catterall 2001; Franceschetti et al. 2000). On theother hand, firing frequency increased in the presence ofcarbachol in oculomotor nucleus Mns. Similar results havebeen reported in other populations (Alaburda et al. 2002;Brown and Yu 2000; Chevallier et al. 2006; Fernandez deSevilla et al. 2006; Miles et al. 2007). There is generalconsensus that activation of muscarinic receptors produces anincrease in firing frequency by diminishing calcium-dependentpotassium conductance and IM current modifying AHP. Theseconductances could be participating to determine the firingdischarge of the oculomotor nucleus Mns.

The neuromodulators can alter both the I–F gain and itsthreshold for repetitive firing and the distinction between thesetwo mechanisms is blurred (Binder et al. 1993). In oculomotornucleus Mns, the I–F gain was lower in the carbachol condi-tion; the diminishing in this parameter would lead to a decreasein excitability. In contrast, the effect of carbachol on recruit-ment threshold and putatively on AHP would lead to anincrease of excitability. Since the firing rate is the output of theMns—and it was higher in response to equal-current steps inthe carbachol condition—we conclude that activation of cho-linergic inputs leads to a net balance that increases the excit-ability in oculomotor nucleus Mns.

Acetylcholine could exert control over excitability via mus-carinic and/or nicotinic receptors and their involvement de-pends on the neuronal population (Alaburda et al. 2002; Cham-berlin et al. 2002; Chevallier et al. 2006; Lape and Nistri 2000;Miles et al. 2007; Zaninetti et al. 1999). Muscarinic excitatoryactions were mediated by M1 receptors in neurons of theprepositus hypoglossi nucleus (Navarro-Lopez et al. 2004),

whereas M2 receptors increased the excitability of medialvestibular nucleus neurons and spinal Mns (Miles et al. 2007;Sun et al. 2002). According to present data, acetylcholine actson the oculomotor nucleus Mns via muscarinic (in particularM1) receptors.

Muscarinic control of the oculomotor nucleus Mns:functional implications

Alert studies have reported that ocular Mns show a covaria-tion between recruitment threshold and eye-position sensitivity(K), which could depend on intrinsic membrane properties,synaptic inputs, or a combination of the two (Dean 1997). Therecruitment order has been strongly correlated with size inspinal Mns (the so-called size principle; see Mendell 2005 fora recent review). The size principle dictates the order ofrecruitment, small Mns being recruited before large ones, andthe crucial property determining systematic differences in re-cruitment is the input resistance. In trochlear Mns of the turtle,it has been reported that spiking in Mns of higher inputresistance is recruited earlier (Jones and Ariel 2008). In ocu-lomotor nucleus Mns of mammals (Nieto-Gonzalez et al.2007), we have found a poorly fitted linear regression betweencell size and input resistance and a weak relationship betweeninput resistance and I–F gain. These results are consistent withthose of alert studies, which reported a weak relationshipbetween size—inferred from the latency to elicit a spikeantidromically—and slope K (Delgado-García et al. 1986;Pastor and Gonzalez-Forero 2003) or did not find any relation-ship (Fuchs et al. 1988). Therefore we conclude that the sizeprinciple has to be tempered in its contribution to firing rategain.

We assume that in the slice preparation, the oculomotornucleus Mns are primarily deafferented and tonic synapticinputs do not exert any significant influence. We also assumethat I–F gain mimics K and that the threshold of the current toevoke a steady tonic response in slice preparation mimicseye-position threshold, in which the Mn begins to discharge ina sustained manner in alert preparation. In slice preparation, wehave previously found an inverse relationship between currentthreshold and I–F gain; i.e., Mns with lower current thresholdhave higher gain (Nieto-Gonzalez et al. 2007). These resultsare opposite to those reported in alert preparation. Thereforewe ruled out intrinsic membrane properties as determiningalone the covariation between recruitment threshold and Kreported in behaving animals. In the present work, the tonicactivation of cholinergic inputs produced a decrease in currentthreshold and I–F gain relative to control, even though inputresistance remained unaltered. These responses resemble thephysiological evidence obtained in alert preparation showingthat Mns with lower recruitment threshold also have lowereye-position sensitivity. In conclusion, present results supportthe notion that recruitment threshold and I–F gain dependprimarily on synaptic inputs in ocular Mns.

Contribution of the cholinergic synaptic inputsto tonic discharge

Short electrical stimulations applied near the boundaries ofthis nucleus, close to the medial longitudinal fasciculus, evokean excitatory postsynaptic potential (EPSP) in the Mns. This



EPSP decreased by about 30% in amplitude when atropine wasapplied. Therefore acetylcholine cannot be considered the onlyneurotransmitter involved in the EPSP and probably excitatoryamino acids like glutamate and/or aspartate are also involved(Carpenter et al. 1992; Nguyen and Spencer 1999; Spencer andWang 1996). Binder and colleagues (1993) raised the query ofhow the concurrent activation of more than one synaptic inputsystem affects motor output. Present findings lead to a similarquestion in the ocular Mns. The question is whether thedepressive effects of cholinergic inputs on I–F gain would besufficient to explain the covariation reported in awake prepa-ration between eye-position threshold and K. Applying carba-chol, we obtained a variation from 66.1 spikes s�1 nA�1

control to 54.8 spikes s�1 nA�1; this change seems to be weakwith respect to the equivalent reported in vivo (in most of thedata the range was about fivefold in monkeys; Fuchs et al.1988). According to this comparison, and even though thepreparation and species are different, the doubt is raised ofwhether the cholinergic inputs are sufficiently powerful togenerate the in vivo effects. As already mentioned, it may bethat other neurotransmitter systems (such as the excitatoryamino acids) also contribute. In other words, the weight ofcholinergic synaptic inputs to the decrease in recruitmentthreshold and firing rate gain in the context of excitatoryneurotransmission to the oculomotor nucleus Mns remains tobe clarified.

The eye-position signals in the prepositus hypoglossi neu-rons are the result of the combined action of paramedianpontine reticular formation neurons, whose monosynaptic ex-citatory projections are glutamatergic in nature, and the facil-itative role of cholinergic terminals (Delgado-García et al.2006). The cholinergic interactions with glutamatergic trans-mission may also be present in the Mns of the oculomotornucleus. It may be hypothesized that in alert preparation theoculomotor nucleus Mns belonging to the low-threshold/low-Kgroup would receive a tonic cholinergic input, whereas thoseMns of the high-threshold/high-K group would not. Thesedifferences could be underlying the proposal that Mns recruitedearliest—those with the lowest thresholds, exerting minimalforce—have a more shallow I–F gain than that of Mns re-cruited later, exerting considerable force (Fuchs et al. 1988).

A C K N O W L E D G M E N T S

We thank the anonymous reviewers, whose commentaries greatly strength-ened this manuscript; Drs. J. L. Ribas-Salgueiro, F. Romero, M. C. Limon-Mortes, and V. Sobrino for technical assistance with Western blots andconfocal laser microscopy; and R. Churchill for editorial help.

G R A N T S

This work was supported by Spanish Ministerio de Educacion y CienciaGrant BFU 2006-08895) and Proyecto de Investigacion de Excelencia,Consejería de Innovacion Grant 2005/CVI-647.

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