Postnatal Development Enhances the Effects of Cholinergic Inputs on Recruitment Threshold and Firing Rate of Rat Oculomotor Nucleus Motoneurons

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: NEUROSCIENCE on 2010, available at: https://doi.org/10.1016/j.neuroscience.2010.09.001” POSTNATAL DEVELOPMENT ENHANCES THE EFFECTS OF CHOLINERGIC INPUTS ON RECRUITMENT THRESHOLD AND FIRING RATE OF RAT OCULOMOTOR NUCLEUS MOTONEURONS. L. CARRASCAL, M.A. LUQUE, V. SOBRINO, B. TORRES, P. NUNEZ-ABADES1 Department of Physiology and Zoology, University of Seville, Spain Running head: Cholinergic changes in oculomotor nucleus Mns with age. Number of pages: 26 Number of figures: 3 1 To whom correspondence should be addressed: Dr. PA Núñez-Abades Dept. Fisiología y Zoología Fac. Farmacia C/ Profesor González García 41012 Sevilla Spain Telephone number: 34-95-4556130 Fax number: 34-95-4233480 Email: [email protected] 2 ABSTRACT Changes in the electrophysiological and morphological characteristics of motoneurons (Mns) of the oculomotor nucleus during postnatal development have been reported, however synaptic modifications that take place concurrently with postnatal development in these Mns are yet to be elucidated. We investigated whether cholinergic inputs exert different effects on the recruitment threshold and firing rate of Mns during postnatal development. Rat oculomotor nucleus Mns were intracellularly recorded in brain slice preparations and separated in neonatal (4 - 7 postnatal days) and adult (20 - 30 postnatal days) age groups. Stimulation of the medial longitudinal fasciculus evoked a monosynaptic excitatory potential in Mns that was attenuated with atropine (1.5 µM, a muscarinic antagonist). Mns were silent at their resting membrane potential, and bath application of carbachol (10 µM, a cholinergic agonist) induced depolarization of the membrane potential and a sustained firing rate that were more pronounced in adult Mns. Pharmacological and immunohistochemical assays showed that these responses were attributable to muscarinic receptors located in the membrane of Mns. In addition, compared to control Mns, carbachol-exposed Mns exhibited a higher firing rate in response to the injection of the same amount of current, and a decrease in the current threshold required to achieve sustained firing. These latter effects were more pronounced in adult than in neonatal Mns. In conclusion, our findings suggest that cholinergic synaptic inputs are already present in neonatal Mns, and that the electrophysiological effects of such inputs on recruitment threshold and firing rate are enhanced with the postnatal development in oculomotor nucleus Mns. We propose that cholinergic input maturation could provide a greater dynamic range in adult Mns to encode the output necessary for graded muscle contraction. 3 INTRODUCTION. It is widely accepted that the different brain areas are not mature at birth, and the adult neuronal phenotype is postnatally shaped by genetic and environmental factors (Steljes et al., 1999; van Aelst and Cline, 2004; Wu et al., 2007). The oculomotor system has been extensively studied in adult preparations over the last four decades. The functional and anatomical features of the neurons within different nuclei, and the synaptic circuitry, contributing to encode eye movements have been characterized in detail (reviewed in Scudder et al., 2002; Sparks, 2002; Büttner-Ennever, 2006). However, relatively little information is available concerning postnatal changes in this system, which are required to achieve accurate eye movements in the adult. To better understand such modifications, we have investigated the physiological and anatomical changes that take place in rat oculomotor nucleus motoneurons (Mns) during the postnatal development (Carrascal et al., 2005, 2006, 2009). In short, the passive membrane properties (input resistance and membrane time constant) decline early in the maturation process (about four days after birth), subsequently exhibiting values similar to those of adult Mns. Active membrane properties (rheobase, action potential features and the relationship between injected current and firing rate) are characteristically mature about two-three weeks after birth (Carrascal et al., 2005, 2006; Nieto-Gonzalez et al., 2007). Early in the maturation process (about ten days after birth) the membrane surface area increases twofold, mainly due to the development of dendrites, reaching a similar area to that in adult Mns. Furthermore, the dendritic architecture is largely reorganized with age (Carrascal et al., 2009). These results have been interpreted in the context of the maturation of the synaptic circuitry of the oculomotor nucleus. To obtain insights into synaptic modifications accompanying postnatal development, the present study examines electrophysiological changes that take place in response to the activation of cholinergic receptors in developing oculomotor nucleus Mns. 4 The neurotransmitter acetylcholine exerts a profound influence over neuronal excitability (Klink and Alonso, 1997a,b; Chamberlin et al., 2002; Fernández de Sevilla et al., 2006). To this end, the physiology of cholinoceptive adult neurons has been studied using in vitro brain slice preparations accompanied by bath applications of agonists and antagonists of cholinergic receptors. Cholinergic agonists (carbachol, muscarine and DMPP) produce depolarization of the cell membrane potential, which usually leads to an increase in the firing rate, as reported for Mns of the hypoglossal nucleus and spinal cord (Lape and Nistri, 2000; Alaburda et al., 2002; Hornby et al., 2002; Chevallier et al., 2006; Miles et al., 2007) and neurons of different nuclei (Klink and Alonso, 1997a,b; Nuñez et al., 1997; Yajeya et al., 1997, 1999; McQuiston and Madison, 1999; Navarro-López et al., 2004). These drugs can also cause hyperpolarizations and biphasic responses (rapid hyperpolarizing response followed by a slower depolarizing one) in other neuronal populations (Good et al., 2007; Ye et al., 2009). It has recently been demonstrated in oculomotor nucleus Mns of young adult rats that the activation of cholinergic receptors by carbachol has two effects. First, membrane potential depolarization is produced along with sustained neuronal firing – which is not silenced upon return of the membrane potential to precarbachol values via DC current injection. Second, a higher firing rate than control is produced in response to depolarizing current steps of the same amplitude (Nieto-Gonzalez et al., 2009). These responses are of a postsynaptic nature, i.e. attributable to the activation of cholinergic receptors located in the membrane of the Mns. Therefore, it has been concluded that cholinergic synaptic inputs play an important role in determining the recruitment threshold and firing rate of oculomotor nucleus Mns (Nieto-Gonzalez et al., 2009). In view of these findings, we sought to determine whether cholinergic synaptic inputs exert different effects on the recruitment threshold and firing rate of these Mns as a function of postnatal age. 5 MATERIALS AND METHODS Surgery and solutions. Experiments were carried out on Wistar rats. All studies were performed in accordance with the European Community Directive 2003/65, as well as with the Spanish Royal Decree 120/2005 and University of Seville regulations on the care of laboratory animals. Rats were anesthetized with sodium pentobarbital (50 mg kg-1) and quickly decapitated. The methods used to obtain the slices, recordings, and analysis are described in detail elsewhere (Carrascal et al. 2006; Nieto-Gonzalez et al., 2007, 2009). In brief, brain slices (thickness 300 µm) including the oculomotor nucleus were incubated in a chamber containing cold sucrose-artificial cerebrospinal fluid (ACSF) for 35-45 minutes, and then transferred to a second chamber containing ACSF maintained at a constant temperature of 33 ºC. Single slices were transferred to the recording chamber and superfused at 2 mL/min (Harvard, MPII) with ACSF bubbled with 95% O2 - 5% CO2 (pH 7.4; 33 ºC). The composition of the ACSF was as follows (data are in mM): 126 NaCl, 2 KCl, 1.25 Na2HPO4, 26 NaHCO3, 10 glucose, 2 MgSO4, and 2 CaCl2. For the sucrose-ACSF solution, the 126 NaCl was substituted by 240 sucrose. The following drugs (all from Sigma-Aldrich) were used in this study: carbamylcholine chloride (carbachol, 10 µM), atropine sulfate (1.5 µM), pirenzepine dihydrocloride (2 µM), and tetrodotoxin (TTX, 1 µM). The time taken to completely exchange the recording chamber was about 50 s, and bath application of drugs was always for >2 min. Drugs were usually applied to only one Mn per slice; if it was necessary to apply more than one drug, the slice was washed with ACSF solution for >20 min (Nunez-Abades et al., 2000; Nieto-Gonzalez et al., 2009). Electrophysiological recordings and data analysis. All recorded neurons were identified as Mns by their antidromic activation from the root of the third nerve and by the collision test (for details see Carrascal et al., 2006). The micropipettes used for recordings 6 were filled with a 3 M KCl (40-70 M) solution. Monosynaptic potentials were elicited by stimulating the region of the medial longitudinal fasciculus close to the boundaries of the oculomotor nucleus. Metallic bipolar microelectrodes (tips ~300 µm apart) were constructed from 25 µm diameter stainless steel wire insulated in glass micropipettes. Cathodal pulses of 150 µs were used, and the current adjusted at subthreshold and increased to suprathreshold values. Recordings were stored on videotape (Neuro-Corder, Neurodata Instruments, PA, USA), and subsequently played back and acquired with a PCI-6070E card (National Instruments, Austin, TX, USA) for off-line analysis. All Mns included for analysis showed a stable resting membrane potential of -55 mV or more-negative, an action potential amplitude larger than 60 mV, and fired repetitively in response to suprathreshold depolarizing current steps of one second. In addition, negative current pulses (0.1 nA, 500 ms, 1 Hz) were used to determine the membrane input resistance of each Mn. With the exception of the exposure of Mns to TTX, the effects of the pharmacological manipulations were reversible. The data for any given Mn in response to the different drugs were accepted only if the membrane potential and spike amplitude returned to the control value (measured when the Mn was impaled) after a wash-out period. We focused our analysis on Mns (n = 36) showing a phasic-tonic discharge in response to suprathreshold current steps (Carrascal et al., 2005; Nieto-Gonzalez et al., 2007). Two separate postnatal age groups of Mns were used: neonatal and adult. The former were obtained in rats of 4-7 postnatal days and the second from young adult rats (20-30 postnatal days) of both sexes. These age groups were selected taking into account the fact that the passive membrane properties of Mns are already established by postnatal day 4 (Carrascal et al., 2006), but the dendritic field is immature (Carrascal et al., 2009). Mns from both age groups were included for analysis when their input resistance was 40 - 80 MΩ. To investigate 7 the effects of the activation of cholinergic receptors we used carbachol at 10 µM. The effects of carbachol on the membrane potential and firing frequency for each Mn were quantified before exposure (control condition), during perfusion, and after wash-out with ACSF. The effects on membrane potential were measured as the difference between resting membrane potential and spike threshold at a steady tonic rate. To assess whether the membrane potentials during DC current injection were correct, we routinely monitored the bridgebalance over the course of the experimental session (for further details see Nunez-Abades et al., 1993). To determine whether carbachol responses were only attributable to the activation of postsynaptic cholinergic receptors, TTX (1 µM) was added to the extracellular solution to block voltage-gated Na+ channels and, thereby, to inhibit spike generation and synaptic transmission. The repetitive-firing frequency was evoked by depolarizing current steps (1 s, 0.5 Hz) with 0.1 nA increments. The steady-state firing frequency was the average of the instantaneous frequencies during the last 500 ms of the current step. For each Mn, the relationship between the steady-state firing frequency and injected current amplitude was represented (I-F plot). We defined current threshold as the intensity of stimulating current capable of eliciting maintained repetitive firing at 20 spikes s-1 (Nieto-Gonzalez et al., 2007, 2009). All statistical analyses were carried out on the raw data. Significant differences between the Mns before (control) and during bath application of carbachol were determined by using a one-way ANOVA. This test was also used to determine differences between neonatal and adult age groups in response to exposure to drugs. The significance level was established at P< 0.05. All data are reported as mean ± standard error. 8 Immunohistochemistry. Immunohistochemical experiments (n = 5) were carried out to demonstrate the presence of muscarinic receptors (M1) in the oculomotor nucleus (for details see Nieto-Gonzalez et al., 2009). Transverse sections of 50 m thickness were processed to reveal M1 by confocal laser microscopy. Free-floating sections were incubated for 1 h at room temperature in 3% normal goat serum and 0.15% Triton X-100 in PBS, and then in the diluted primary antibody (rabbit anti-M1, Millipore; 1:100) overnight at 4 ºC. Finally, sections were incubated with secondary antibody labeled with Alexa 488 goat antirabbit IgG (Invitrogen; 1:200) overnight at 4 ºC. RESULTS All recorded Mns showed stable resting membrane potentials, which were not significantly different between neonatal (-60.6 ± 2.8 mV) and adult (-62.5 ± 3.3 mV ) Mns, and required depolarizing current injection to cause repetitive firing. The input resistance was also similar for both age groups (neonatal Mns = 61.3 ± 3.7 MΩ and adult Mns = 59.7 ± 4.1 MΩ). Exposure of slices to carbachol (10 M), an agonist of cholinergic (muscarinic and nicotinic) receptors, produced membrane potential depolarization and sustained firing in all neonatal (n = 12) and adult (n = 12) Mns tested. As shown by the examples in Fig. 1A,B, the response of Mns was a slow depolarization of membrane potential that resulted in the triggering of action potentials. Once this repetitive firing commenced, the membrane potential continued depolarizing until it reached a stable value; the instantaneous firing frequency also gradually increased, reaching a sustained repetitive firing (11 mV and 13 spikes s-1 for the representative neonatal Mn showed in Fig. 1B). The mean effects of carbachol on membrane potential and firing rate in all recorded neonatal and adult Mns are plotted in Fig. 1C,D. The carbachol evoked a membrane potential depolarization that increased with the postnatal age (Fig. 1C). The mean of the depolarization for the neonatal group was 8.0 ± 0.9 mV, while for 15 excitability and repetitive discharge with age (Carrascal et al., 2005, 2006). If we could assume that cholinergic synaptic inputs would exert the same influence on neonatal and adult Mns, the rise in excitability and firing rate of the oculomotor nucleus Mns with age support the fact that the effects of the carbachol would be more pronounced in adult Mns. The results reported in the present work could also be interpreted in the context of cholinergic synaptic proliferation. Indeed, a direct link has been established in spinal Mns between an increase in cholinergic synaptic inputs with postnatal development and the maturation of the motor system that permits weight bearing and locomotion (Wilson et al., 2004). In oculomotor nucleus Mns, two phases (1-10 and 11-30 postnatal days) have been distinguished in the changes of somatodendritic morphometry during postnatal maturation (Carrascal et al., 2009). During the first phase, dendritic complexity and membrane somatodendritic surface area increases, while during the second phase, dendritic elongation takes place –with a simplification of dendritic complexity– and membrane surface area continues to increase slowly. The increase in somatodendritic surface area with development, particularly from 4 days after birth, and the reorganization of the dendritic architecture are probably related to changes in synaptic inputs. In other words, it is plausible that these anatomical changes support the production and / or refinement of synaptic connections with development as reported in other neural systems (Nunez-Abades et al., 1994; Nunez-Abades and Cameron, 1995; Vincent et al., 2004; Wilson et al., 2004). Changes in trophic factors and / or neurotransmission with age could also support the increase in recruitment threshold and firing rate because they modify the kinetics of the conductances underlying such processes (Gonzalez and Collins, 1997; Kernell, 1999; Alaburda et al., 2002). With regards to the inputs, the activity of Mns located in the oculomotor nuclei is essentially triggered by vestibular and visual sensory signals (Büttner- Ennever and Horn, 1997). In rats, the 16 vestibular circuitry is functional about one week after birth (Curthoys, 1981; Rusch et al., 1998). Since the eyelids open at about postnatal day 12, visual signals could play a role later. Furthermore, eye movements evoked by visual and vestibular stimuli are performed from about postnatal day 21 (Lannou et al., 1980; Faulstich et al., 2004). We have demonstrated that acetylcholine plays an excitatory role in Mns of the oculomotor nucleus early in development. Nevertheless, the visual synaptic inputs could contribute to reinforce the cholinergic excitatory effects with postnatal maturation. In short, the proposal of proliferation-refinement of cholinergic synapses in oculomotor nucleus Mns could explain the strengthening of the electrophysiological effects with postnatal development. The functional implications of the present findings can be related to the co-variation between threshold and firing frequency gain observed in in vivo preparations, which are primarily dependent on synaptic inputs to the Mns (Nieto-Gonzalez et al., 2007, 2009). Indeed, the recruitment threshold and the slopes of the I-F plots in ocular Mns are modulated by cholinergic synaptic inputs. The present work increases our understanding of these processes by showing that postnatal development strengthens the efficacy of cholinergic synaptic modulation on the activity of the Mns. This latter finding could support the fact that the threshold and firing frequency could be dynamically modulated by acetylcholine across a greater range with age. Under such circumstances the level of tonic excitatory cholinergic inputs could contribute to determine the threshold and firing rate required to generate the output necessary for graded muscle contraction. 17 ACKNOWLEDGMENTS We thank Dr. José Luis Nieto Gonzalez, whose study of the effects of carbachol on adult oculomotor nucleus Mns is one of the bases of the present work, and Dr. Luis Herrero for their comments on the manuscript. This work was supported by the Spanish Ministerio de Innovacion y Ciencia (Grant BFU 2009-07867) and by Andalusian government grants (Proyecto de Investigacion de Excelencia: P08-CVI-039 and P09-CVI-4617). 18 LEGENDS Figure 1. Effect of carbachol (10 M) on the membrane potential and firing frequency in developing motoneurons (Mns) from the rat oculomotor nucleus. A. Responses evoked by bath application of carbachol in a representative neonatal Mn (5 days) to illustrate the time course of the membrane potential depolarization and firing. B. Details of the intervals (1, 2, and 3) indicated in A. Note that Mn was silent at rest; carbachol initially evoked a slow depolarization that triggered action potentials (1), and then sustained firing (2). Finally, the Mn still discharged, although at a lower frequency, when repolarized to pre-carbachol values (3). C, D, E. Histograms showing mean values and standard errors of membrane potential depolarization and firing frequency produced by bath application of carbachol in neonatal and adult age groups. Note that histograms in D and E correspond to the firing frequency measured when the carbachol-exposed Mns were depolarized and exhibited a sustained discharge (as shown in period 2 of panel B), and when repolarized to values prior to drug treatment (as shown in period 3 of panel B), respectively. Asterisks indicate significant differences between age groups. Figure 2. The cholinergic response in the rat oculomotor nucleus Mns acts via muscarinic receptors. A. Superimposed recordings of EPSPs showing graded responses as a function of current stimulus strength. A suprathreshold stimulus is seen to evoke an action potential. B. Superimposed recordings showing temporal summation of EPSPs with paired stimuli that could trigger an action potential. C. Superimposed EPSP recordings under control conditions and following exposure to the muscarinic antagonist atropine (1.5 µM) in neonatal (6-day-old) Mn. Note the decrease in EPSP amplitude, even though stimulus current strength was maintained. D. Recording from a neonatal (5-day-old) Mn showing that pirenzepine (2 µM; a M1 muscarinic antagonist) blocks carbachol-dependent firing. The framed area includes 19 an expanded recording at the time indicated by the star symbol. E. Time course of the inhibition of carbachol-dependent firing by pirenzepine in neonatal (5-day-old) and adult (25- day-old) Mns. Dots correspond to instantaneous frequency sampled at a fixed rate. F. Confocal photomicrograph showing immunopositive cells for muscarinic M1 receptors located inside the boundaries of the oculomotor nucleus (OCM) in a brain slice from a neonatal (6-day-old) rat. The inset illustrates a higher magnification of the cells indicated by arrowheads. Figure 3. Response to depolarizing current steps in control and carbachol conditions in oculomotor nucleus Mns. A, B. Firing discharge evoked by same-amplitude current steps in neonatal (4-day-old) and adult (22-day-old) Mns under control conditions and then exposed to carbachol (10M). C, D. Current-firing frequency (I-F) relationship in a representative Mn of each age group under control conditions (filled circles) and then exposed to carbachol (empty circles). Note that carbachol decreased the recruitment threshold of both Mns. E. Histogram showing the mean firing frequency evoked by 0.4 nA current steps in control and carbacholexposed Mns of neonatal and adult rats. F. Histogram showing the mean current threshold (nA) in control and carbachol-exposed Mns of neonatal and adult rats. Asterisks indicate significant differences between both conditions for each age group. 20 REFERENCES Alaburda A, Perrier JF, Hounsgaard J (2002) An M-like outward current regulates the excitability of spinal motoneurones in the adult turtle. J Physiol 540:875–881. Bayliss DA, Viana F, Bellingham MC, Berger AJ (1994) Characteristics and postnatal development of a hyperpolarization-activated inward current in rat hypoglossal motoneurons in vitro. J Neurophysiol 71:119–128. Brown BS, Yu SP (2000) Modulation and genetic identification of the M channel. Prog Biophys Mol Biol 73:135–166. Büttner-Ennever JA, Horn AK (1997) Anatomical substrates of oculomotor control. Curr Opin Neurobiol 7:872–879. Büttner-Ennever JA (2006) The extraocular motor nuclei: organization and functional neuroanatomy. Prog Brain Res 151:95–125. Cantrell AR, Catterall WA (2001) Neuromodulation of Na+ channels: an unexpected form of cellular plasticity. Nat Rev Neurosci 2:397–407. Carrascal L, Nieto-Gonzalez JL, Cameron WE, Torres B, Nunez-Abades PA (2005) Changes during the postnatal development in physiological and anatomical characteristics of rat motoneurons studied in vitro. Brain Res Rev 49:377–387. 21 Carrascal L, Nieto-Gonzalez JL, Nunez-Abades P, Torres B (2006) Temporal sequence of changes in electrophysiological properties of oculomotor motoneurons during postnatal development. Neuroscience 140:1223–1237. Carrascal L, Nieto-Gonzalez JL, Torres B, Nunez-Abades P (2009) Changes in somatodendritic morphometry of rat oculomotor nucleus motoneurons during postnatal development. J Comp Neurol 514:189–202. Chamberlin NL, Bocchiaro CM, Greene RW, Feldman JL (2002) Nicotinic excitation of rat hypoglossal motoneurons. Neuroscience 115:861–870. Chevallier S, Nagy F, Cabelguen JM (2006) Cholinergic control of excitability of spinal motoneurones in the salamander. J Physiol 570:525–540. Curthoys IS (1981) The organization of the horizontal semicircular duct, ampulla and utricle in the rat and guinea pig. Acta Otolaryngol 92:323–330. Faulstich BM, Onori KA, du Lac S (2004) Comparison of plasticity and development of mouse optokinetic and vestibulo-ocular reflexes suggests differential gain control mechanisms. Vision Res 44:3419–3427. Fernández de Sevilla D, Nuñez A, Borde M, Malinow R, Buño W (2008) Cholinergicmediated IP3-receptor activation induces long-lasting synaptic enhancement in CA1 pyramidal neurons. J Neurosci 28:1469–1478. 22 Fernández de Sevilla D, Rodrigo-Angulo M, Nuñez A, Buño W (2006) Cholinergic modulation of synaptic transmission and postsynaptic excitability in the rat gracilis dorsal column nucleus. J Neurosci 26:4015–4025. Fisahn A, Yamada M, Duttaroy A, Gan JW, Deng CX, McBain CJ, Wess J (2002) Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents. Neuron 33:615–624. Franceschetti S, Taverna S, Sancini G, Panzica F, Lombardi R, Avanzini G (2000) Protein kinase C-dependent modulation of Na+ currents increases the excitability of rat neocortical pyramidal neurones. J Physiol 2:291–304. Gonzalez M, Collins WF 3rd (1997) Modulation of motoneuron excitability by brain-derived neurotrophic factor. J Neurophysiol 77:502–506. Good CH, Bay KD, Buchanan R, Skinner RD, Garcia-Rill E (2007) Muscarinic and nicotinic responses in the developing pedunculopontine nucleus (PPN). Brain Res 1129:147–155. Honda E, Ono K, Toyono T, Kawano H, Masuko S, Inenaga K (2003) Activation of muscarinic receptors in rat subfornical organ neurones. J Neuroendocrinol 15:770–777. Hornby TG, McDonagh JC, Reinking RM, Stuart DG (2002) Motoneurons: a preferred firing range across vertebrate species? Muscle Nerve 25:632–648. 23 Kernell D (1999) Repetitive impulse firing in motoneurons: facts and perspectives. Prog Brain Res 123:31–37. Klink R, Alonso A (1997a) Muscarinic modulation of the oscillatory and repetitive firing properties of entorhinal cortex layer II neurons. J Neurophysiol 77:1813–1828. Klink R, Alonso A (1997b) Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons. J Neurophysiol 77:1829–1843. Krnjevic K (1993) Central cholinergic mechanisms and function. Prog Brain Res 98:285–292. Lannou J, Precht W, Cazin L (1980) Development of optokinetic responses in vestibular nuclear neurons in the young rat. Brain Res 202:217–222. Lape R, Nistri A (2000) Current and voltage clamp studies of the spike medium afterhyperpolarization of hypoglossal motoneurons in a rat brain stem slice preparation. J Neurophysiol 83:2987–2995. Marchetti C, Pagnotta S, Donato R, Nistri A (2002) Inhibition of spinal or hypoglossal motoneurons of the newborn rat by glycine or GABA. Eur J Neurosci 15:975–983. McQuiston AR, Madison DV (1999) Muscarinic receptor activity has multiple effects on the resting membrane potentials of CA1 hippocampal interneurons. J Neurosci 19:5693–5702. 24 Miles GB, Hartley R, Todd AJ, Brownstone RM (2007) Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion. Proc Natl Acad Sci USA 104:2448–2453. Navarro-López Jde D, Alvarado JC, Marquez-Ruiz J, Escudero M, Delgado-García JM, Yajeya J (2004) A cholinergic synaptically triggered event participates in the generation of persistent activity necessary for eye fixation. J Neurosci 24:5109–5118. Nieto-Gonzalez JL, Carrascal L, Nunez-Abades PA, Torres B (2007) Phasic and tonic firing properties in rat oculomotor nucleus motoneurons, studied in vitro. Eur J Neurosci 25:2682– 2696. Nieto-Gonzalez JL, Carrascal L, Nunez-Abades PA, Torres B (2009) Muscarinic modulation of recruitment threshold and firing rate in rat oculomotor nucleus motoneurons. J Neurophysiol 101:100–111. Nunez-Abades PA, Cameron WE (1995) Morphology of developing rat genioglossal motoneurons studied in vitro: relative change in diameter and surface area of somata and dendrites. J Comp Neurol 353:129–142. Nunez-Abades PA, He F, Barrionuevo G, Cameron WE (1994) Morphology of developing rat genioglossal motoneurons studied in vitro: Change in length, branching pattern, and spatial distribution of dendrites. J Comp Neurol 339:401–421.