Modulation of presumed cholinergic mesopontine tegmental neurons by acetylcholine and monoamines applied iontophoretically in unanesthetized cats

Mesopontine cholinergic neurons in vivo

Pergamon PII: S0306-4522(00)00004-X

Neuroscience Vol. 96, No. 4, pp. 723–733, 2000 723 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

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MODULATION OF PRESUMED CHOLINERGIC MESOPONTINE TEGMENTAL NEURONS BY ACETYLCHOLINE AND MONOAMINES APPLIED IONTOPHORETICALLY IN UNANESTHETIZED CATS Y. KOYAMA* and K. SAKAI† INSERM U480, De´partement de Me´decine Expe´rimentale, Universite´ Claude Bernard, 69373 Lyon, France

Abstract—The mesopontine tegmentum, which contains both cholinergic and non-cholinergic neurons, plays a crucial role in behavioral state control. Using microiontophoresis in unanesthetized cats, we have examined the effect of cholinergic and monoaminergic drugs on two putative cholinergic neurons located mostly in the laterodorsal tegmental nucleus and X area (or the cholinergic part of the nucleus tegmenti pedunculopontinus, pars compacta): one (type I-S) exhibiting slow tonic discharge during both waking and paradoxical sleep, and the other (PGO-on) displaying single spike activity during waking and burst discharges in association with ponto-geniculo-occipital (PGO) waves during paradoxical sleep. We found that: (i) application of carbachol, a potent cholinergic agonist, inhibited single spike activity in both PGO-on and type I-S neurons, but had no effect on the burst activity of PGO-on neurons during paradoxical sleep; the inhibition was associated with either blockade or increased latency of antidromic responses, suggesting membrane hyperpolarization; (ii) application of glutamate, norepinephrine, epinephrine, or histamine resulted in increased tonic discharge in both PGO-on and type I-S neurons; this was state-independent and resulted in a change in the firing mode of PGO-on neurons from phasic to tonic; (iii) application of serotonin had only a weak state-dependent inhibitory effect on a few type I-S neurons; and (iv) application of dopamine had no effect on either type of neuron. The present findings suggest that cholinergic, glutamatergic and monoaminergic (especially noradrenergic, adrenergic and histaminergic) inputs have the capacity to strongly modulate the cholinergic neurons, altering both their rate and mode of discharge, such as to shape their state specific activity, and thereby contribute greatly to their role in behavioral state control. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: cholinergic neurons, carbachol, monoamines, paradoxical sleep, PGO wave, iontophoresis.

The mesopontine tegmentum, which contains both cholinergic and non-cholinergic neurons, plays a crucial role in behavioral state control. 12,34,56 In particular, mesopontine cholinergic neurons are thought to be involved in the regulation of wakefulness (W) and paradoxical sleep (PS), and in the generation of the tonic and phasic phenomena of PS, such as desynchronization of the neocortical electroencephalogram (EEG) and the occurrence of ponto-geniculo-occipital (PGO) waves. 8,11,44,47,52,56 Recent single-unit recording studies in unanesthetized cats and rats have demonstrated the existence of several populations of putative cholinergic neurons in the mesopontine tegmentum. 3,15,54 Of these, those showing slow tonic discharge during both W and PS (type I-S) and those showing single spike activity during W, but burst discharges in conjunction with PGO-waves during PS (PGO-on), have been reported, in the cat, to be most probably cholinergic. 4,46 Indeed, in addition to their exclusive localization within

cholinergic neuron cluster, both type I-S and PGO-on neurons have a broad action potential and exhibit slow repetitive firing during W, have thalamic projections with a slow conduction velocity and are inhibited by carbachol. 3,4,46,54 These features are consistent with properties shown by anatomical findings and in vivo extracellular and in vitro intracellular studies on mesopontine cholinergic neurons in the rat and guinea-pig. 13,14,19,22,23,27 Anatomical studies have shown that the mesopontine cholinergic region receives several cholinergic and monoaminergic (serotoninergic, noradrenergic, adrenergic, dopaminergic and histaminergic) afferent projections from the brainstem. 25,47,51 These anatomical findings suggest that the mesopontine cholinergic neurons are under the control of cholinergic and monoaminergic inputs. Recent in vitro studies have shown marked responses to cholinergic or monoaminergic stimulation, e.g., acetylcholine- or serotonin-induced hyperpolarization, 22,23,27,35 histamine-induced depolarization, 16 and noradrenaline-induced depolarization 35 or hyperpolarization. 35,60 However, there is little pharmacological data available in the cat, the animal in which most is known about the behavior of putative cholinergic neurons during the sleep– waking cycle. 3,4,46,54,55 Using microiontophoretic drug application in unanesthetized cats, we have therefore examined the action of cholinergic and monoaminergic drugs on two putative mesopontine cholinergic neuronal populations (type I-S and PGO-on) in the laterodorsal tegmental nucleus (LDT), X area (or the cholinergic part of the pedunculopontine tegmental nucleus, PPT), and peri-locus coeruleus alpha (peri-LCa), all of which play a critical role in behavioral state control. A preliminary report of the present work has been published in abstract form. 18

*Present address: Department of Physiology, Fukushima Medical University, 1 Hikari-ga-oka, Fukushima 960-1295, Japan. †To whom correspondence should be addressed. Abbreviations: CL, nucleus centralis lateralis; CM, nucleus centrum medianum; CV, coefficient of variation; DA, dopamine; E, epinephrine; EEG, electroencephalogram; Glu, glutamate; HA, histamine; 5-HT, serotonin; INTH, interspike interval histogram; LDT, laterodorsal tegmental nucleus; LGNd, dorsal lateral geniculate nucleus; LTS, low-threshold calcium spike; NE, norepinephrine; 8-OH-DPAT, (^)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthaline hydrobromide; periLCa, peri-locus coeruleus alpha; PGO, ponto-geniculo-occipital; PPSH, peri-PGO spike histogram; PPT, pedunculopontine tegmental nucleus; PS, paradoxical sleep; PSTH, post-stimulus time histogram; SWS, slow wave sleep; W, wakefulness; X area, cholinergic portion of the nucleus tegmenti pedunculopontinus, pars compacta. 723

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Y. Koyama and K. Sakai EXPERIMENTAL PROCEDURES

Four adult cats were used for these painless chronic stereotaxic recording experiments. Electrodes were implanted under sodium pentobarbital anesthesia (25 mg/kg, i.v.) in order to record the neocortical and dorsal lateral geniculate (LGNd) EEG, eye movement (electro-oculogram), and neck muscle activity (electromyogram). In addition, an H-shaped plastic plate (42 mm wide, 33 mm long, 8 mm thick) was fixed stereotaxically to the skull using dental acrylic cement, so that the cranium could be painlessly returned to the same stereotaxic position using a semi-chronic head holder (Narishige, SA-8). After opening the skull, wells were fixed in place over the cerebellum and cerebral cortex for subsequent microelectrode penetration. For antidromic identification of the axonal trajectories of neurons, bipolar stimulatory electrodes, consisting of two stainless steel wires (200 mm diameter, bare for 0.5 mm at the tip and spaced 1.0 mm apart) were implanted into the nucleus centrum medianum (CM), the nucleus centralis lateralis (CL), the lateral posterior hypothalamus, and the nucleus reticularis magnocellularis of the medulla. Stimulation was delivered by means of 1-Hz square pulses (0.1–0.5 ms, 0.05– 2.5 mA) that were sub-threshold for movement. The criteria used to identify antidromically-evoked action potentials included the fixed latency of the spike, the ability to follow high-frequency stimulation (.200 Hz), and, for spontaneously active neurons, the collision of stimulus-induced and spontaneous action potentials. Conduction velocity was estimated on the basis of: (i) a straight line distance between the recording and stimulating sites; (ii) the shortest antidromic latency when the antidromic latency was decreased step-wise with increasing intensity of stimulation; and (iii) the fastest conduction velocity when the same neuron was activated antidromically from different structures. After surgery, the animals were treated daily with antibiotics, then, after recovery, they were habituated to the head-restrained position: they were comfortably placed on a towel laid within a stainless steel basin, put in the heavy base of stereotaxic instrument (Narishige, SN-3). The head was painlessly restrained with a semi-chronic head holder and large body movements were prevented by a wooden covering inserted between the basin and the stereotaxic frame. In the first day of habituation, the cats soon became calm and could stay for four to five consecutive hours without showing any signs of discomfort. They displayed normal sleep–waking cycles during all experiments which could start from the second day of habituation. Single action potentials were recorded extracellulary via a 32-mm Formvar-coated stainless steel wire, glued to an eight-barrel glass micropipette, with the tip of the recording electrode protruding less than 5 mm. Using this assembly, high-quality extracellular recordings were obtained over several sleep–waking cycles. Using a digital microdrive, the electrode was inserted through a 0.8-mm-diameter guide tube, positioned vertically, or at 468, 528 or 568 from the horizontal. The amplified and filtered (100 Hz to 10 kHz) signals were displayed on oscilloscopes. Sixty-four or 128 action potentials were averaged to determine spike shape using a digital memory oscilloscope equipped with a processor for waveform averaging. Stable and wellisolated extracellular action potentials were converted to standard pulses using a window discriminator. Both polygraphic and standard spike pulses were stored on a microcomputer using a CED 1401 data processor. Unitary activity was analysed to obtain the change in discharge rate, interspike interval histogram (INTH), peri-stimulus time histogram (PSTH), and coefficient of variation (CV) using Spike 2 software. The effect of iontophoretically-applied drugs was determined by a paired two-tailed t-test; differences between the mean pre-drug application (baseline) firing rate and that during drug application were evaluated with 1 s bins. A significance level of 0.05 was adopted. Other statistical analyses used were one-way analysis of variance (ANOVA) with repeated measures and cluster analysis (kmeans method). For the description of mesopontine tegmental structures, we used the nomenclature defined by Sakai, 43,47 in the cat, such as the LDT, locus coeruleus (LC), locus coeruleus alpha (LCa), peri-LCa) and X area (equivalent to the cholinergic component of the nucleus tegmenti pedunculopontinus, pars compacta (PPT) of Nauta 36). In the cat, the peri-LCa is located just ventromedial to the LCa containing a high density of noradrenergic neurons and distinct from the LDT and PPT by its location and cyto- and chemoarchitecture, as well as by its efferent and afferent connections. 43,47 The iontophoretically-applied drugs were carbamylcholine chloride (carbachol) (0.2 M, pH 4.0), norepinephrine hydrochloride (NE)

Fig. 1. Drawings of sagittal sections showing the localization of the four (type I-S, PGO-on, type I-R and phasic) types of neurons recorded in the cholinergic region of the mesopontine tegmentum.7G, genu of the facial nerve; 7N, facial nerve; BC, brachium conjunctivum; FTC, central tegmental field; IC, inferior colliculus; mV, mesencephalic root of the trigeminal nerve; PAG, periaqueductal gray; Poo and Poc, nucleus reticularis pontis oralis and caudalis, respectively; R, red nucleus.

(0.1–0.2 M, pH 4.0), epinephrine hydrochloride (E) (0.2 M, pH 4.0), serotonin hydrochloride (5-HT) (0.04 or 0.2 M, pH 4.0), histamine dihydrochloride (HA) (0.2 M, pH 4.4), dopamine hydrochloride (DA) (0.2 M, pH 4.0), sodium glutamate (Glu) (0.1 M, pH 8.0) and sodium chloride (NaCl) (0.2 M, pH 4.0). All were obtained from Sigma. All compounds were dissolved in distilled water; 0.1% ascorbate was added to the catecholamine and serotonin solutions. The central barrel was filled with NaCl (1 M) and used for current balancing or as the reference electrode. The tip diameter of the eight-barrel micropipette was 7–10 mm and the impedance of each barrel 50– 300 MV, depending upon the drug used. A retaining current of 2– 10 nA was passed through each electrode. As the same effect was seen using the current balancing and unbalancing modes, the latter was used in most cases. After the experiments, several recording sites were coagulated by passing a 20 mA cathodal current for 10 s and the localization of the tip and microelectrode tracts determined histologically using Prussian Blue. The localization of cholinergic and noradrenergic neurons was determined immunohistochemically by the presence of choline acetyltransferase or tyrosine hydroxylase, as previously described. 3 All experiments followed EEC guidelines (86/609/EEC) and all efforts were made to minimize the number of animals used and their suffering. RESULTS

Cell identification and localization Extracellular action potentials were recorded from 307 neurons during the sleep–waking cycles. Of these, 214 were from the cholinergic part of the mesopontine tegmentum (Fig. 1). Sixty-eight of the 214 (32%) were identified as “type I-S” neurons and 27 (13%) as “PGO-on” neurons. The localization of type I-S, PGO-on, type I-R, and phasic

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Table 1. Summary of spike duration and conduction velocity for four different types of neuron (type I-S, PGO-on, type I-R and phasic) in the mesopontine tegmentum Cell type

Fig. 2. Averaged spike shape of four (type I-S, PGO-on, type I-R and phasic) populations of mesopontine tegmental neurons showing their respective broad (type I-S and PGO-on) and short (type I-R and phasic) action potentials. Sixty-four spikes were averaged.

neurons is illustrated in Fig. 1. Type I-S neurons were mainly located in, or near, the LDT and X area, while PGO-on neurons were mostly recorded in the X area and in the rostral part of the peri-LCa. As previously described, 3,4 the type I-S neurons displayed slow (,10 Hz) tonic discharge during waking (W), a reduction in discharge rate during slow-wave sleep (SWS), and an increased discharge rate during paradoxical sleep (PS) compared with during SWS. Type I-S neurons could be distinguished from Type I-R neurons which had faster (.10 Hz) tonic discharging during waking and showed a similar behavioral state-related discharge profile (n ˆ 29, 14%). The mean values (^S.D.) for the spontaneous firing rates (and range) of type I-S and type I-R neurons during W were, respectively, 4.0 ^ 2.0 Hz (0.9–8.7 Hz) and 21.6 ^ 9.2 Hz (11.0–41.2 Hz), the difference being distinct and highly statistically significant (F ˆ 216.4, P , 0.0001). PGO-on (or PGO-on burst) neurons are defined in the present account by their short burst activity, which preceded by 5–25 ms the onset of every “primary” PGO wave recorded in the ipsilateral LGNd. 29,42,45 Each burst usually consisted of three to seven spikes, but sometimes consisted of single or double spikes, as previously described. 46,55 It is also important to note that the vast majority of PGO-on neurons show slow tonic firing during attentive wakefulness. 45,46 PGO-on neurons therefore display two basic patterns of activity, i.e. burst firing and single spike activity. Other PGO-unrelated neurons, displaying phasic discharge patterns during W and/or PS, will be described as “phasic” neurons (n ˆ 45; 21%). The remaining 45 neurons either exhibited tonic discharge selective (PS-on; n ˆ 23; 11%), or highly selective (tonic type II, n ˆ 16; 7%) for the periods of PS, or showed PS-selective cessation of discharge (PS-off, n ˆ 6; 3%). In addition to their firing rate and pattern, both type I-S and PGO-on neurons were readily distinguishable from type I-R and phasic neurons by their broad action potential (Fig. 2). Although the extracellular waveform of the action potential is dependent on several factors, the broad action potential of both type I-S and PGO-on neurons was readily recognizable on the oscilloscope at the time of recording. When averaged and measured from onset to the zero crossing of the negative component (D1) or to the peak of the large positive

Spike duration (ms)

Conduction velocity (m/s)

Mean ^ S.D. (range)

Mean ^ S.D. (range)

D1

D2

Type I-S (n ˆ 68)

0.65 ^ 0.12 (0.48–1.05)

1.18 ^ 0.18 (0.90–1.75)

1.78 ^ 0.97 (12 of 35) (0.46–3.30)

PGO-on (n ˆ 27)

0.70 ^ 0.10 (0.54–0.88)

1.14 ^ 0.13 (0.90–1.35)

1.88 ^ 1.24 (7 of 18) (0.71–3.98)

Type I-R (n ˆ 29)

0.43 ^ 0.11 (0.26–0.56)

0.60 ^ 0.14 (0.38–0.84)

6.59 ^ 1.11 (3 of 14) (5.38–7.57)

Phasic (n ˆ 45)

0.36 ^ 0.12 (0.25–0.54)

0.51 ^ 0.24 (0.33–0.80)

7.77 ^ 5.19 (18 of 38) (1.18–18.40)

D1 and D2 indicate the duration of the averaged action potential measured from the onset to either the zero crossing of the negative component (D1) or to the peak of the large positive component (D2). Differences in both D1 and D2 for the four types of neuron were highly significant (d.f. ˆ 3,158; D1, F ˆ 83,35, P , 0.0001; D2, F ˆ 141,71, P , 0.0001). D2 for type I-S and PGO-on neurons were clearly distinct from those for type I-R and phasic neurons as shown by their ranges. No significant difference was found in spike duration between type I-S and PGO-on neurons and between type I-R and phasic neurons (P . 0.05). Cluster analysis (k-means method) using D1 and D2 as parameters also revealed that the tested neurons can be divided into two groups: one (Type I-S and PGO-on) with a long duration action potential and the other (Type I-R and Phasic) with a short duration action potential.

component (D2), the difference in spike duration between the two populations of neurons (type I-S 1 PGO-on cf type I-R 1 phasic) was distinct and highly statistically significant, while no significant difference was seen in spike duration between type I-S and PGO-on neurons and between phasic and type I-R neurons (Table 1). Twelve out of the 35 type I-S neurons tested responded antidromically to stimulation of the intralaminar thalamic nuclei (CM or CL) (n ˆ 9) and/or posterior hypothalamus (n ˆ 3) with a mean conduction velocity of 1.78 ^ 0.97 ms. Seven out of 18 PGO-on neurons were antidromically driven by stimulation of the intralaminar thalamic nuclei (n ˆ 6) or LGNd (n ˆ 1) with a mean conduction velocity of 1.88 ^ 1.24 ms. These two values were not statistically different (F ˆ 0.036, P ˆ 0.851), and are consistent with the results of previous studies in freely moving cats. 3 Sixty-four type I-S and 24 PGO-on neurons were sufficiently stable to allow study of the effects of the repeated application of cholinergic and/or monoaminergic drugs during the sleep–waking cycles. Firing pattern and pharmacological features of type I-S neurons Although type I-S neurons showed tonic discharge during both W and PS, the same individual neurons displayed different patterns of tonic discharge during these two states. Figure 3 shows the time array of spike discharge and INTHs for a type I-S neuron during W and PS. During W, the INTH showed a narrow peak while, during PS, the peak was broad, indicating increased fluctuation during PS compared with during W. As the CV can be used as an index of discharge variability, 38 we compared the CVs for type I-S

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Y. Koyama and K. Sakai

Fig. 3. Raster displays (1) and INTHs (2) of a type I-S neuron during W and PS. In A1 and B1 the recording starts from the bottom left. Note the increased variability during PS compared with during W. LGN, EEG recordings from the dorsal lateral geniculate nucleus.

neurons (n ˆ 21) during W and PS. As expected, the mean CV during W (0.20 ^ 0.09) was significantly less than that during PS (0.50 ^ 0.19) (F ˆ 30.0, P , 0.0001), reflecting the irregular discharge of these neurons during PS. As shown in Fig. 3, type I-S neurons did not exhibit either burst firing or any obvious time-locked discharge in relation to PGO waves. However, when peri-PGO spike histograms (PPSH) were constructed, the neuronal discharges of every type I-S neuron examined (n ˆ 29) showed a weak, but clear, correlation with PGO waves. Indeed, as shown in Fig. 4B, the PPSHs in this cell group showed increased activity which preceded the onset of LGNd PGO waves by 10–20 ms, this correlation being very similar to that for PGO-on neurons (Fig. 4A). These observations suggest that type I-S neurons receive PGO-related phasic excitatory inputs during PS, but receive fewer steady tonic excitatory inputs during PS than during W. The effects of cholinergic and monoaminergic drugs on type I-S neurons are shown in Figs 5 and 6. One of the most striking features was that carbachol induced complete

Fig. 4. Analysis of PGO-related discharges of a PGO-on and a type I-S neuron during PS. The upper trace show the peri-PGO spike histogram (2-ms bins), while the lower shows the averaged PGO waves from the analysed epoch.

Fig. 5. Effect of iontophoretically-applied drugs on three type I-S neurons during waking. 5-HT applied during attentive wakefulness had no effect, while application of carbachol resulted in complete long-lasting suppression of tonic discharges. In contrast, Glu, NE, HA and E all caused an increased tonic discharge. The current intensity (nA) is shown under the drug name.

Fig. 6. Effect of iontophoretically-applied drugs on two type I-S neurons during waking. Note the inhibitory effect of 5-HT (60 nA) seen during slow-wave sleep with high-voltage delta waves, but absent during paradoxical sleep (B) and waking (A), both characterized by EEG desynchronization. Also note the respective excitatory and inhibitory effects of HA and carbachol (Carb). The current intensity (nA) is indicated under the drug name.

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Fig. 7. Effect of iontophoretically-applied Glu and serotonin (5-HT) on a dorsal raphe (RD) PS-off neuron during waking, slow-wave sleep, and the transition to paradoxical sleep (indicated by arrows). Note the pronounced inhibitory action of 5-HT on the presumed serotoninergic dorsal raphe neuron. Table 2. Responses of type I-S (A) or PGO-on (B) neurons to microiontophoretically-applied drugs A

Type I-S Carb

Glu

NE

E

HA

5-HT

DA

0 53 0 53

37 0 1 38

41 0 1 42

15 0 0 15

30 0 0 30

0 10 29 39

0 0 5 5

Excitation Inhibition No effect Total

B

PGO-on Carb

Glu

NE

E

HA

5-HT

DA

0 22 0 22

21 0 2 23

16 0 2 18

4 0 1 5

9 0 1 10

0 0 14 14

0 0 2 2

Excitation Inhibition No effect Total Carb, carbachol.

suppression of spontaneous and evoked discharges in this cell group. When applied during W, carbachol (,20 nA) induced complete inhibition of the tonic spontaneous discharge within a few seconds of onset of current application, with the unit remaining silent for several minutes (Fig. 5). Carbachol inhibition was state-independent and seen in every type I-S neuron tested (n ˆ 53). In sharp contrast, Glu, NE, E and HA had a strong excitatory action on type I-S neurons. Application of these drugs (20–60 nA) resulted in a marked increase in tonic discharge rate in almost all type I-S neurons examined (Fig. 5, Table 2A). The excitatory action of Glu and monoaminergic drugs was state-independent and could overcome the inhibitory action of carbachol. In contrast to the Glu response, which was rapid in onset (,2 s) and of short duration (typically restricted to the application period), the excitatory responses to NE, E and HA were slow in onset

Fig. 8. Effect of iontophoretically-applied drugs on a PGO-on neuron during slow-wave sleep with PGO waves. Application of Glu (A) or NE (B) resulted in increased tonic discharge, while serotonin (5-HT) had no effect on Glu-evoked or spontaneous tonic firing (A), or spontaneous burst firing (B), of the PGO-on neuron. Note the complete carbachol-induced inhibition of the tonic, but not phasic, discharges of the PGO-on neurons. Stim indicates the period of electrical stimulation in the antidromic invasion experiment.

(.3–5 s) and of long duration (up to several minutes) (Fig. 5). However, application of 5-HT (40–90 nA) had no effect on this cell type either during W or PS, at which times the cells were most active (Fig. 5A). In contrast, regardless of the drug concentration used (0.04 or 0.2 M), in 10 out of 64 type I-S neurons, a slight inhibitory effect was seen during behavioral states in which the cells were less active, such as SWS or quiet W (Fig. 6). The action of 5-HT therefore appears to be minor and state-dependent. DA had no significant effect on the five type I-S neurons tested (60–80 nA). Since in vitro intracellular studies of the action of 5-HT, both in the rat and guinea-pig, have shown that 5-HT has an exclusively inhibitory action on mesopontine cholinergic neurons by causing pronounced membrane hyperpolarization, 23,27,35 and our in vivo extracellular study in the rat also demonstrated the same inhibitory action of 5-HT on putative cholinergic neurons in the LDT, 17 we examined whether the minor 5-HT effect seen in the present study could result from technical problems. We performed extracellular recording from the dorsal raphe nucleus and examined the effect of 5-HT on 25 putative serotoninergic neurons, characterized by long spike duration, slow tonic firing during W, reduction in discharge rate during SWS, and cessation of discharge during PS (PS-off neurons). 32 In agreement with previous reports, 1 application of 5-HT (20–60 nA; 0.04 M) resulted in marked inhibition of the spontaneous discharge of these dorsal raphe neurons (Fig. 7), indicating that the technique was indeed valid.

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Fig. 9. Effect of iontophoretically-applied histamine and carbachol (Carb) on a PGO-on neuron during the sleep–waking cycle. Periods indicated by bars 1 and 2 in A are expanded in B1 and B2, respectively. Application of histamine (10 nA) resulted in a marked increase in tonic discharge and the subsequent suppression of the phasic burst discharges in conjunction with PGO waves during sleep (A, B2). In sharp contrast, carbachol (40 nA) induced complete longlasting suppression of the tonic, but not phasic, discharges of the PGO-on neuron (A, B1). Note that the traces of neural discharges (B1, B2) begin from the bottom left.

Pharmacological features of PGO-on neurons The pharmacological properties of PGO-on neurons were very similar to those described above for type I-S neurons. All PGO-on neurons were inhibited by carbachol and stimulated by Glu, HA, NE and E, but did not respond significantly to either 5-HT or DA (Table 2B). Figures 8 and 9 show examples of these responses. Carbachol (10 nA), applied alone or in combination with Glu (10 nA), completely suppressed both spontaneous or evoked tonic repetitive firing, but did not inhibit the phasic burst discharges seen in conjunction with PGO waves (Figs 8A, 9A). Application of Glu (10 nA) caused short latency excitation of PGO-on neurons (Fig. 8A), while application of NE (40 nA) (Fig. 8B) or HA (10 nA) (Fig. 9A) resulted in similar slow prolonged excitation of PGO-on neurons to that seen for type I-S neurons. The effect of E (40–60 nA, 4/5) was the same as that for NE and HA (Table 2B). Figure 9 illustrates changes in the firing mode of a PGO-on neuron. Application of HA during SWS accompanying PGO waves resulted in increased tonic repetitive firing of the PGOon neuron (see Fig. 9A, first HA application, and B1, lower three traces). The tonic discharges were rapidly inhibited by carbachol and replaced by a phasic mode of firing (see Fig. 9A, after carbachol application; and B1, upper three

traces). Conversely, application of HA during a PS episode caused increased single spike activity and suppression of phasic burst discharges (see Fig. 9A, last HA application, and B2). The same shift in firing mode from burst firing to tonic single spike activity was seen with Glu, NE and E. In two PGO-on neurons, carbachol completely inhibited tonic spontaneous firing and was able to modulate the firing mode even during W, inducing a shift from tonic single spike activity (Fig. 10A) to burst firing in response to moving visual stimuli (Fig. 10B). The intraburst intervals varied from 2 to 6 ms (Fig. 10B-2, 3), these values being very similar to those seen for high-frequency burst discharges in conjunction with PGO waves during PS. Finally, it is worth mentioning that the application of carbachol had no inhibitory effect on 16 type I-R and 27 phasic neurons tested, but, in contrast, resulted in excitation in six out of 16 type I-R neurons. NE, 5-HT, or HA had no significant effect on these presumed non-cholinergic neurons (data not shown). Antidromic latency prolongation The antidromic latency technique has been used to investigate neuronal excitability. 26 Using this technique, we examined whether inhibition of the tonic discharge of type I-S and

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Fig. 10. Raster displays (A1, B1) and INTHs (A3, B3) of a PGO-on neuron during waking before (Control), and after, carbachol application. Note the shift in firing mode from tonic single spike activity to high-frequency burst activity in response to moving visual stimuli after carbachol application. All unit firings in B were responses to moving visual stimuli.

PGO-on neurons was accompanied by any change in neuronal excitability. Carbachol inhibition of both type I-S and PGOon neurons appeared to be associated with hyperpolarization of the soma membrane, since it induced either an increase in antidromic latency or abolition of antidromic responses in all type I-S (n ˆ 4) and PGO-on (n ˆ 3) neurons tested. Figures 11 and 12 show examples of antidromic latency experiments on one type I-S and one PGO-on neuron, respectively. With the type I-S neuron, stimulation of the centrum medium of the thalamus (CM) evoked antidromic responses, as shown by the fixed latency and collision with a spontaneous spike (Fig. 11 C1). Shortly after onset of carbachol application (10 nA for 30 s), the latencies increased by about 0.1 ms (Fig. 11A, B), to be followed by the almost complete abolition of antidromic responses for about 30 s (Fig. 11A, C2). The antidromic response returned to the baseline level within 1 or 2 min after cessation of current application (Fig. 11A, upper third, and C3). In the PGO-on neuron, carbachol application resulted in a marked increase (.0.2 ms) in antidromic latency, with no accompanying marked response failure (Fig. 12). For both types of neuron, the increased latency varied from 0.08 to 0.4 ms and was not correlated with the initial latency. The reduction in response to stimulus varied from 25 to 95%, compared with control, over the first minute after onset of current application. 5-HT application (up to 80 nA) had no effect on antidromic latency variability (n ˆ 2).

DISCUSSION

Type I-S and PGO-on neurons as putative cholinergic neurons Based on intrinsic electrophysiological characteristics, three types of neurons (I, II and III) was described in the cholinergic region of the LDT and pedunculopontine (PPT) (area X of the present report) tegmental nucleus of the guineapig and rat brainstem slices. Type I neurons showed a highfrequency burst discharge due to a low-threshold calcium spike (LTS) and were reported to be non-cholinergic in the PPT of the adult guinea-pig and rat, 14,22 but mainly cholinergic in the LDT of the neonatal rat. 28,60 Both type II and type III neurons contained NADPH-diaphorase and were therefore defined as cholinergic. Both showed a broad action potential, long-lasting post-spike afterhyperpolarization, and a transient outward conductance (A-current). Type III neurons were characterized by the presence of both A-current and LTS. 13,22,23,35,58 The intrinsic electrophysiological properties of these identified type II and type III cholinergic neurons were very reminiscent, respectively, of the discharge properties of type I-S and PGO-on neurons. In vitro studies further demonstrated that mesopontine cholinergic neurons showed hyperpolarization in response to the application of muscarinic agonists, due to activation of an inwardly rectifying potassium current. 23,28,35 In line with previous anatomical data, 7,40 both type I-S and PGO-on neurons send axons to the

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Fig. 11. Change in the antidromic responses of a type I-S neuron to stimulation of the nucleus CM, before (Control), during, and after, iontophoretic application of carbachol (10 nA). (A) Raster display of post-stimulus spike responses, (B) post-stimulus time histograms (PSTH) and (C) photomicrographs showing antidromic responses before (1), during (2), and after (3), carbachol application.

intralaminar thalamic complex. 3,46,54,55 The slow conduction velocity of type I-S and PGO-on neurons is also consistent with anatomical data showing that cholinergic neurons contain small diameter unmyelinated, or finely myelinated, axons. 21,24 In addition, afferent projections to the cat LGNd from the cholinergic mesopontine tegmentum originated almost exclusively from cholinergic neurons, 5 while all mesopontine tegmental neurons that were antidromically driven by stimulation of the LGNd were PGO-on, and not type I-S, neurons. 45,46 Moreover, thalamic PGO waves was shown to result from nicotinic cholinergic depolarization of relay cells. 9,41 These observations therefore strongly suggest that type I-S and PGO-on neurons are cholinergic. We cannot, however, rule out the possibility of cholinergic property of type I-R neurons, which were found in a previous study to represent a great part of mesopontine neurons recorded from cholinergic nuclei. 54 Further intracellular or juxtacellular recordings and single cell labeling with biocytin followed by immunohistochemical analysis are required to determine the exact neurochemical identity of the mesopontine neurons in conscious cats. Cholinergic and monoaminergic modulation and implications in behavioral state control It has been suggested that the high-frequency burst discharges of PGO-on neurons result from a low-threshold Ca 21 conductance 22,46,55 that is essentially inactive at the membrane resting potential, but is activated by membrane hyperpolarization. 53 In support of this theory, carbachol application resulted in complete suppression of tonic discharges in

Fig. 12. Change in the antidromic responses of a PGO-on neuron to stimulation of the CL of the thalamus, before (Control), during, and after, iontophoretic application of carbachol (40 nA). (A) Raster display of poststimulus spike responses, (B) post-stimulus time histograms (PSTH) and (C) photomicrographs showing antidromic responses before (1), during (2), and after (3), carbachol application. Note the marked increase in antidromic latency in the absence of any accompanying marked response failure.

all PGO-on neurons examined, but had no effect on their burst discharges associated with PGO waves. We also found that in vivo carbachol inhibition was associated with blockade of the antidromic response or an increase in antidromic latency. As the magnitude of the antidromic latency increase did not correlate with the initial latency, this appeared to result from soma membrane, rather than axonal, hyperpolarization. The persistence of burst firing in PS during carbachol inhibition and the occurrence of carbachol-induced burst discharges during W are also consistent with the hypothesis that the basic prerequisite for bursting of PGO-on neurons is hyperpolarization of the neuronal membrane. In a similar fashion to thalamocortical neurons, 31,54,57 PGO-on neurons therefore appear to change their firing pattern from single to burst activity in association with membrane hyperpolarization, as a result of either inhibition or disfacilitation. Similar mechanisms may be involved in the neuronal dynamics of type I-S neurons during the sleep–waking cycle, since their discharge variability was much higher during PS than during W. It is important to note that putative central monoaminergic (noradrenergic, serotoninergic and histaminergic) neurons are mainly active during W and are silent during both PS and SWS accompanying PGO waves. 10,56 The decreased tonic excitatory inputs seen during PS might be ascribed to disfacilitation due to cessation of discharge of PS-off neurons. The present findings on the tonic excitatory actions of NE, E, and HA favor this hypothesis. Our findings on the excitatory action of HA on putative cholinergic PGO-on and type I-S neurons are in general agreement with those from previous in vitro studies in the

Mesopontine cholinergic neurons in vivo

adult guinea-pig 16,35 and a recent in vivo study in the unanesthetized head-restrained rat. 20 Our observations are also in good agreement with recent findings showing that microinjection or microdialysis application of HA, as well as NE or E, to the feline cholinergic mesopontine tegmentum results in a marked increase in EEG and behavioral arousal.2,25 Although both depolarization and hyperpolarization by NE have been reported in adult guinea-pig brain slices, 35 in the neonatal rat, NE exclusively hyperpolarizes identified cholinergic LDT neurons via a2-adrenoceptors. 60 In addition, in both anesthetized and unanesthetized rats, the vast majority of presumed cholinergic LDT neurons are reported to be inhibited by iontophoretically applied NE. 17,20 The reasons for the discrepancy between these and present findings are not known. Species difference might be one explanation, as shown, for example, by 30 in response to application of acetylcholine; in the cat LGNd neurons, this results in rapid nicotinic depolarization, while, in the rat and guinea-pig LGNd, it produces either slow muscarinic depolarization or muscarinic hyperpolarization. Marked differences in the regional distribution of brain a1-adrenoceptors, which mediate depolarization, 37 have also been reported in the brain of different species of animals. 39 Moreover, recent in vitro experiments on guinea-pig brain slices have shown that NE depolarizes almost all identified basal forebrain cholinergic neurons and that this is mainly mediated by a1-adrenoceptors. 6 A second explanation for this discrepancy is that NE application results in the release of excitatory amino acids from axon terminals, thereby overcoming the direct inhibitory action of NE on cholinergic neurons. Glu is reported to excite mesopontine cholinergic neurons, 49,50 and possibly glutamatergic medullary neurons appear to send tonic excitatory inputs to the cholinergic mesopontine tegmentum. 4,46,54 A direct and potent inhibitory action of 5-HT (via 5-HT1A receptors) on mesopontine cholinergic neurons has been demonstrated by in vitro intracellular studies. 23,27,35 An inhibitory action of 5-HT has also been demonstrated in presumed cholinergic LDT neurons in vivo in the anesthetized rat. 17 In the present report, however, we found it to have only a weak state-dependent inhibitory effect on a few type I-S neurons, suggesting a minor, if any, inhibitory action of 5-HT on the cat mesopontine cholinergic neurons. Our observations are in general agreement with those of Thakkar et al. showing that (^)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthaline hydrobromide (8-OH-DPAT), a 5-HT1A agonist, had no effect on “Wake/REM-on” units, which might be equivalent to our type I-S. 59 We have previously reported that applied 5-HT

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also had no effect on mesopontine PS-on neurons which exhibit no discharge during W, but exhibit a tonic discharge just prior to and throughout PS (‘selective’ PS-on neurons). 48 It should be mentioned, however, that an inhibitory effect of 8-OH-DPAT on ‘non-selective’ PS-on neurons has recently been reported in the cat. 59 A recent in vivo microdialysis experiment in the rat thalamus showed that acetylcholine release in the thalamus, to which the mesopontine cholinergic neurons send axons, is higher during W than during SWS, and is essentially the same as during PS, 61 suggesting a minor, if any, inhibitory action of 5-HT and NE even on the rat mesopontine cholinergic neurons. Recently, Morilak and Ciaranello demonstrated 5-HT2 receptors to be abundant on mesopontine cholinergic neurons; 33 such receptors are known to mediate depolarization in many cell types. 37 The minor action of 5-HT found in the present study might be due to its opposing actions at postsynaptic sites. Further work is needed to determine whether iontophoretically-applied 5-HT may act on both 5HT1A and 5-HT2 receptors. Although our in vivo data mostly corroborate previous in vitro data, it does not exclude the possibility that iontophoretic application would have indirect effects on the recording neurons mediating through the immediate neurons. Drug application under conditions of synaptic uncoupling (e.g., with tetrodotoxin or low calcium) would be required to solve this problem. In conclusion, we have found that carbachol causes inhibition of single spike activity in both type I-S and PGO-on neurons, probably via soma membrane hyperpolarization, but has no effect on the burst activity of PGO-on neurons during PS. In contrast, Glu, NE, E, and HA all induce a marked increase in tonic discharge rate of both type I-S and PGO-on neurons and change the firing mode of PGO-on neurons from phasic to tonic. 5-HT had a slight state-dependent inhibitory effect, and DA had no effects. Thus, our data show that cholinergic, glutamatergic, noradrenergic, adrenergic and histaminergic inputs have the capacity to strongly modulate the putative cholinergic neurons, altering both the rate and mode of discharge, such as to shape their state specific activity during the sleep–waking cycles. The present findings have considerable implications for the understanding of the control and modulation of the mesopontine cholinergic neurons and of the function of these neurons in behavioral state control.

Acknowledgement—This work was supported by INSERM U480, France.

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