Electrophysiological-Pharmacological Study ...

Mesolimbic Component of the Ascending Cholinergic Pathways: Electrophysiological-Pharmacological Study Stefan M. Brudzynski, Ludmila Kadishevitz and Xiao-Wen Fu J Neurophysiol 79:1675-1686, 1998. ; You might find this additional info useful... This article cites 82 articles, 9 of which you can access for free at: http://jn.physiology.org/content/79/4/1675.full#ref-list-1 Updated information and services including high resolution figures, can be found at: http://jn.physiology.org/content/79/4/1675.full Additional material and information about Journal of Neurophysiology can be found at: http://www.the-aps.org/publications/jn

Journal of Neurophysiology publishes original articles on the function of the nervous system. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 1998 the American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our website at http://www.the-aps.org/.

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Mesolimbic Component of the Ascending Cholinergic Pathways: Electrophysiological-Pharmacological Study STEFAN M. BRUDZYNSKI, 1 LUDMILA KADISHEVITZ, 2 AND XIAO-WEN FU 2 1 Department of Psychology, Brock University, St. Catharines, Ontario L2S 3A1; and 2 Department of Clinical Neurological Sciences, London Health Sciences Centre, London, Ontario N6A 5A5, Canada

INTRODUCTION

On the basis of results of immunohistochemical studies and in situ hybridization for choline acetyltransferase mRNA, two distinctive subgroups of cholinergic perikarya (CH5 and Ch6) were distinguished in the pontomesencephalic region within the pedunculopontine and in the laterodorsal tegmental nuclei (LDT) (Armstrong et al. 1983; Butcher 1995; Kimura et al. 1981; Lauterborn et al. 1993; Mesulam et al. 1983, 1984, 1989; Vilaro et al. 1994). Although a substantial portion of the pedunculopontine cholinergic neurons has descending projections (Jones 1990; Woolf and Butcher 1989), the LDT has diverse and extensive ascending projections. The ascending projections innervate numerous

nuclei in the thalamus, hypothalamus, basal forebrain, septum, basal ganglia and medial frontal, and olfactory cortices (Cornwall et al. 1990; Fibiger 1982; Hallanger et al. 1987; Herrero et al. 1991; Jones and Beaudet 1987; Paxinos and Butcher 1985; Satoh and Fibiger 1986; Semba et al. 1988; Vincent et al. 1986; Woolf and Butcher 1986; Woolf et al. 1990). The functional role of this ascending cholinergic system remains the key for understanding sleep-waking cycle, emotional arousal, and the initiation of a number of defensive and alarm behavioral patterns. The present study is focused on the component of the ascending projections from the pontomesencephalic cholinergic cells, which are targeted at the medial limbic structures (Consolo et al. 1990; Cornwall et al. 1990; Lewis and Shute 1967; Mesulam et al. 1989). The ascending projections form a mesolimbic component of the ascending reticular activating system (Moruzzi and Magoun 1949; Shute and Lewis 1963, 1967), which plays an important role in the regulation of limbically driven behavior. The ascending cholinergic projections are associated with the activation that accompany the states of arousal, wakefulness, and paradoxical sleep, behavioral activation, emotional arousal manifested by species-typical threatening or alarm vocalizations, and autonomic symptoms (Brudzynski 1981, 1994; Imeri et al. 1995; Jones 1993; Talwar and Kumar 1994). It has been demonstrated in the past that systemic or intraventricular cholinomimetic agents with strong muscarinic properties have potent effects on the general behavior, particularly in inducing autonomic manifestations, defensive vocalization, and agonistic behavioral responses to external stimuli (Baker et al. 1960; Funke et al. 1962; Leslie 1965; Koff and Langfitt 1966). However, all these symptoms could not be obtained when lesions of various limbic, particularly medial diencephalic structures, had been made (Gellen et al. 1972; Koff and Langfitt 1966). It has been demonstrated by using direct intracerebral injection of muscarinic cholinergic agents in cats, that these behavioral symptoms were induced from an elongated, but limited, strip of medial structures, from the periaqueductal gray, through the medial hypothalamic and preoptic regions, and intralaminar thalamic nuclei to the septal nuclei (Baxter 1967, 1968; Brudzynski and Eckersdorf 1988; Brudzynski et al. 1995; Decsi 1974; Decsi and Nagy 1977; Decsi et al. 1969; Myers 1964; Varszegi and Decsi 1967). Similar responses to direct cholinergic stimulation, measured by changes in locomotor activity and by the production of 22-kHz ultrasonic alarm calls, also have been reported for the rat (Brudzynski and Bihari 1990; Brudzynski et al.

0022-3077/98 $5.00 Copyright q 1998 The American Physiological Society

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Brudzynski, Stefan M., Ludmila Kadishevitz, and Xiao-Wen Fu. Mesolimbic component of the ascending cholinergic pathways: electrophysiological-pharmacological study. J. Neurophysiol. 79: 1675–1686, 1998. The cholinergic input from the pontomesencephalic cholinergic neurons to the diencephalic and basal forebrain structures has been implicated in a number of limbically controlled overt behaviors. The cellular mechanism by which the cholinergic terminals initiate behavioral manifestations is not clear. The objective of this study was to investigate the effects of the ascending cholinergic projection from the laterodorsal tegmental nucleus (LDT) on neuronal firing in the anterior hypothalamicmedial preoptic region (AHMP), known to be involved in agonistic behavior. Experiments were performed on urethan-anesthetized rats. Iontophoretic application of carbachol (CCh) into the vicinity of single cells in the AHMP caused a dose-dependent decrease in the mean firing rate of 83% of units and an increase in 10% of units. The inhibitory effect of CCh, but not the excitatory effect, was reversed by iontophoretic pretreatment with scopolamine. The inhibition of the firing rate was repeatable for the same dose of CCh and dose dependent. Electrical stimulation of neurons in the LDT caused a comparable, current-dependent decrease in the mean firing rate of AHMP neurons that also was reversed by pretreatment of neurons in the AHMP with scopolamine. The antagonizing effects of scopolamine were reversible with time. The same units in the AHMP that inhibited their firing to stimulation of the LDT also responded with a similar inhibition to local iontophoretic CCh. Finally, the fluorescent carbocyanine dye, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide, (DiA), has been used as a retrograde axonal tracer and was injected into the recording sites immediately after the electrophysiological recordings. After 1 wk, DiA dye was found in numerous neurons in the LDT as shown by the fluorescence confocal microscopy. Results of the study suggest that LDT cholinergic neurons project and terminate in the AHMP and that their activation causes a decrease in the mean firing rate of the AHMP neurons. It is postulated that this inhibitory effect is implicated in the initiation of some of the behavioral patterns like defensive or alarm vocalization and behavioral inhibition.

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S. M. BRUDZYNSKI, L. KADISHEVITZ, AND X.-W. FU

METHODS

1.5 mm from midline, and from 5.0 to 6.5 mm below the cortex surface (Paxinos and Watson 1986).

Electrical stimulation and extracellular single-unit recordings Electrical stimulation was delivered by stainless steel concentric bipolar electrodes (NE-100, Rhodes Medical Instruments, Woodland Hills, CA) with tip separation of 0.5 mm. The monophasic square-wave pulses (1-ms width, 100–800 mA, 1 Hz) were generated by a Grass S44 stimulator coupled to a Grass stimulation isolation unit (SIU-5). Six-barrel pipettes for recording and iontophoresis were pulled from F. Haer capillary glass tubing (1.0 mm OD) with a Narishige vertical puller (Model PE-2). The central barrel was filled with 2 M NaCl solution and served as a recording electrode. The electrode impedance ranged from 3 to 7 MV, as measured by microelectrode tester (model BL-100, Winston Electronics, San Francisco, CA) using 135 Hz AC test current of 5 1 10 010 A. One of the side barrels was filled with 2% pontamine sky blue (Chicago sky blue, Sigma Chemical, St. Louis, MO) in 0.5 M sodium acetate for marking of the recording site. Recorded signals were connected to the probe headstage of the Axoprobe-1 DC amplifier (Axon Instruments, Forest City, CA) and then coupled to a signal processing unit for high-pass filtering and further amplification (Intronix, Rexdale, ON). The amplified signal was fed into a window discriminator (Frederick Haer, Brunswick, MA) to isolate action potentials. Standard pulses corresponding to the individual action potentials were multiplexed with stimulus triggers and event markers and fed to an AST 286 personal computer equipped with a Data-Translation DT2801A A/D conversion card. The multiplexed signal was sampled and decoded at 1 kHz by a PCIPEE program (CY Electronics, London, ON). The data were displayed continuously in real time as running time histogram or poststimulus time histogram during sampling and saved for analysis. Peristimulus time histograms were compiled from the stored data files off-line. Significant changes in activity of neurons after stimulation were quantified by comparing the height of each 1-ms poststimulus bin of the peristimulus time histogram with the average height of the bin for 100 ms before the stimulus. These data also were recalculated for the mean firing rate/s and compared for three arbitrary chosen, 100-ms bouts of time: 100 ms before the stimulus, 100 ms immediately after the stimulus, and 101–200 ms after the stimulus. Significant changes in the activity of neurons after iontophoretic ejection were quantified by comparing the mean firing rate for 20-s ejection bout with the mean firing rate/20 s immediately before and after the ejection.

Animals and surgery

Microiontophoresis

Seventy-four male, Wistar rats, weighing 230–350 g were used in the study. Animals were anesthetized with an initial urethan dose of 1.15 g/kg ip and a supplementary dose of 0.08 g/kg was added after a couple of hours if necessary. The total dose of urethan was within the range of doses that do not suppress spontaneous activity of neurons in explored areas (Cross and Dyer 1971; Dyball and McPhail 1974). Rats were placed in a Kopf stereotaxic apparatus in the Faraday enclosure and their body temperature maintained at 37.5 { 0.37C as measured by the rectal probe. The skull was opened, the dura reflected, and the exposed brain surface was covered with warmed 4% agar in Ringer solution after the electrode penetration. The recording of single-unit activity was performed from the AHMP between stereotaxic frontal planes 7.0 and 8.5 mm anterior from the interaural zero plane, between sagittal planes 0.1 and 1.3 mm lateral from midline, and from 7 to 9.5 mm below the surface of the cortex according to the stereotaxic atlas by Paxinos and Watson (1986). Electrodes for electrical stimulation were placed stereotaxically in the LDT and adjoining structures, between frontal planes 0.9 and 00.4 mm, between sagittal planes 0.1 and

Side barrels of the multibarrel pipette were filled with 0.5 M centrifuged solutions of carbachol (CCh, carbamylcholine chloride), sodium glutamate, or scopolamine hydrochloride, saline and 2% pontamine sky blue (all compounds from Sigma Chemical). All pharmacological agents were dissolved in distilled water and retained with 1 nA current. The current was generated by a Dagan 6400 Micro-iontophoresis current generator (Dagan, Minneapolis, MN). For the technical description of the generator, see Stone (1985). The drugs were ejected using °120 nA current (usually 20–60 nA), positive for CCh, scopolamine, or saline and negative for glutamate, applied for 20–30 s for a single agent and °70 s for double ejection. Periods of maintaining the retention current between ejections were controlled in time to avoid build-up of solute at the tip of the pipette and to allow a quantitative assessment of the successive ejection-induced changes in the firing rate (Bradshaw et al. 1973). The current effect was compensated automatically by an additional barrel filled with 2 M NaCl. For further details of the recording and iontophoresis, see Brudzynski et al. (1991). To antagonize the effects of CCh, scopolamine was ejected

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1990). Functional mapping of the decrease in locomotor activity and vocalizational responses in the rat brain delineated a similar brain system to that in the cat brain (Brudzynski 1994; Brudzynski et al. 1989). Moreover, results of the mapping studies in these two species outlined a system strikingly similar to the pattern of projection of the ascending pontomesencephalic cholinergic neurons (Satoh and Fibiger 1986; Woolf at al. 1990). It has been postulated, therefore, that cholinergically induced behavioral responses are triggered by cholinergic terminals originating from the pontomesencephalic cholinergic cells (Brudzynski and Barnabi 1996). It has been shown in recent experiments on rats, that stimulation of neurons of the LDT with L-glutamate induced comparable 22-kHz alarm calls to those induced by cholinergic stimulation of mediobasal diencephalic structures. Moreover, L-glutamate stimulation of the LDT was ineffective, or less effective, when the terminal fields in the anterior hypothalamic-medial preoptic region (AHMP) were pretreated with scopolamine, a muscarinic antagonist (Brudzynski and Barnabi 1996). The cellular mechanism by which the ascending cholinergic inputs initiate behavioral manifestations is not clear. It has been found in the acute rat preparation that a predominantly muscarinic agent—carbachol (CCh) —caused a decrease in the firing rate of spontaneously active neurons in the AHMP (Brudzynski et al. 1991). The question arises whether or not these neuronal responses can be attributed to the effects of the ascending cholinergic projection from the pontomesencephalic cell groups. The goal of the present study is to test the hypothesis that the ascending cholinergic fibers originating from the pontomesencephalic cells have a predominantly inhibitory influence on the firing rate of neurons in the rat mediobasal diencephalic regions from which behavioral responses to cholinergic stimulation have been obtained. The AHMP region has been chosen for the study because the prominent behavioral manifestations with the decrease in locomotor activity (Brudzynski et al. 1989) and high magnitude of alarm vocalizations (Brudzynski 1994) have been obtained from that region.

MESOLIMBIC CHOLINERGIC PATHWAYS

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Confocal microscopy The DiA-injected preparations were analyzed under Bio-Rad MRC-600 confocal laser scanning microscope equipped with krypFIG . 1. Responses of anterior hypothalamic-medial preoptic neurons (AHMP) to inotophoretically applied carbachol at 20–60 nA of the ejecting current. A: percentage of inhibitory, excitatory responses, and unresponding neurons to carbachol. B: averaged neuronal activity of the same neurons expressed in the mean firing rate per second during 20-s ejection of carbachol (CCh) in comparison with 20-s control period before and after application of carbachol (Con). Data are shown for inhibitory results (left), excitatory results (middle), and no responding units (right 3 bars). Vertical bars represent {SE. **t[82] Å 5.5, P õ 0.0001 in comaprison to the initial baseline firing rate (Con). There was no significant difference between baseline firing rates of cells that showed inhibition vs. excitation (t-test, P ú 0.5).

from an adjoining barrel (1–5 nA) just before the ejection of CCh in such a way that both ejection periods partially overlapped. In some cells, no ejecting current was applied and scopolamine was allowed to passively diffuse from the tip of the pipette during the concurrent ejection of CCh. Effects obtained by ejection of 1–5 nA of scopolamine or by its passive diffusion were basically the same. Passive diffusion appeared to sufficiently antagonize the effects of CCh and provided evidence that effects of scopolamine were not caused by a potential local anesthetic action of this drug (Curtis and Phillis 1960).

Fluorescent tracer injections To study neuronal connections of the recording site, the group of a long-alkyl chain, carbocyanine and aminostyryl dyes have been selected (Balice-Gordon et al. 1993; Honing and Hume 1986, 1989). This group of dyes was chosen because they can be injected postmortem in fixed tissues (Godement et al. 1987; Mufson et al. 1990) in the same animals that were used for electrophysiological recordings in nonsurvival experiments. The dyes do not transfer between intact membranes and have been used as retrograde tracers (Godement et al. 1987; Honig and Hume 1989). These lipophilic dyes can bind both in vivo and in vitro to lipid components of the plasma membrane, diffuse in the plane of the membrane, resulting in uniform staining of the entire cell plasma membrane (Godement et al. 1987). After electrophysiological recording, the lipophilic

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FIG . 2. Examples of inhibitory responses of 2 anterior hypothalamicmedial preoptic neurons (1 in A and the other in B) to iontophoretic application of CCh for 20 s with the increasing ejecting current. A: running time histogram showing inhibitory responses to the increasing doses of carbachol at the ejecting current of 20–60 nA. Ejection time for carbachol at 60 nA was prolonged to 30 s. B: inhibitory responses to carbachol were repeatable for the same ejecting current (20 nA) but potentiated by an increase in the ejecting current (40 nA). Same unit responded with an increase in firing rate to ejection of glutamate (GLU) at 20 nA.

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dialkylaminostyryl probe, DiA [4-(4-(dihexadecylamino)styryl)N-methylpyridinium iodide, D-3883, Molecular Probes, Eugene OR], has been used as a retrograde axonal tracer. DiA is bright fluorescent green (Snider et al. 1992), diffuses more rapidly in membranes, and stains membranes more uniformly than other carbocyanine dyes. Injection solution of DiA was prepared by dissolving 2.5 mg of DiA in 0.9 ml of 100% ethanol with 0.1 ml of dimethylsulfoxide and subsequently filtering through 5-mm pore filter (Millipore, Bedford, MA). After the electrophysiological recordings were finished, 0.1 ml of DiA was injected into the recording site in the AHMP via a glass pipette (50- to 60-mm tip diam) connected with a Hamilton 1-ml syringe. Seven animals with a body weight not exceeding 300 g were injected. After 6 h survival time, the rats were anesthetized deeply with urethan and perfused transcardially with 0.9% NaCl solution followed by 10% formaldehyde in 0.1 M phosphate buffer saline (PBS). The brains were removed, stored in fixative for 48 h at 277C and subsequently immersed in 10, 20, and 30% sucrose in PBS for 24 h in each concentration. After 1 wk of storage in a black box at room temperature, the brains were sectioned for 50-mm horizontal preparations on a cryostat microtome (Leitz, Canada), transferred into PBS solution, mounted immediately on glass slides, coverslipped using DPX mountant (BDH Chemicals, Toronto, ON) and stored at 0157C for confocal microscopy analysis.

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ton/argon mixed gas laser (Baker and Reese 1993). The filter block, which allows detection of a green-emitting probe was used. The confocal imaging was made with a 110, 140, and 160 Nikon oil immersion Fluotar lens. The gain setting varied from 3 to 8 depending on the contrast of the image. Sequential optical sections were collected as a function of the depth of the tissue by automatically indexing the position of the specimen stage along the z axis and recording the image at each step. Each optical section was the synthesis of 15–17 scans, which were processed digitally by Kalman filtering to optimize the signal-to-noise ratio. The resulting optical images appeared as white neurons on a black background. Occasionally, for better visualization of the dendritic arborization, pseudocolor was used and the images were merged using all-pixel merge mode, to indicate the site of maximal luminescence. To separate the labeled and unlabeled regions of the preparation, line scanning through the labeled neuron and unlabeled surrounding background areas were performed and curves of maximum pixel intensity were obtained. Prints of selected images were obtained from a color video printer (Sony, model UP-5000).

Histological verification and statistics Recording sites were marked by expelling pontamine sky blue by a cathodal current of 5–8 mA for 10–20 min (Lodge et al. 1974)

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FIG . 5. Peristimulus histograms showing 3 types of neuronal responses of the AHMP neurons to single pulse (2 ms) stimulation (S) of the laterodorsal tegmental nucleus. A: inhibitory response to 400-mA stimulus compiled from 123 superimposed sweeps. B: excitatory response to 900-mA stimulus compiled from 151 superimposed sweeps. C: excitatory response followed by an inhibition to 800-mA stimulus compiled from 95 superimposed sweeps.

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FIG . 3. Effects of iontophoretically applied scopolamine (SCO) on the neuronal responses in the anterior hypothalamic-medial preoptic region (AHMP) induced by ejection of CCh. A: running time histogram showing mean firing rate of an AHMP neuron. Unit showed decrease in the mean firing rate to ejection of CCh (20 nA) for 25 s. Inhibitory response was reversed by pretreatment with scopolamine (SCO) ejected at 1 nA for 25 s. This antagonism was repeatable for the same cell. B: averaged neuronal activity of 8 neurons expressed in mean firing rate ({SE) during ejection of CCh (20–40 nA) compared with the control activity 20 s before and after application of carbachol (Con, left 3 bars). Inhibitory response to carbachol was reversed by pretreatment with scopolamine at 1–2 nA (SCO / CCh, right 3 bars). **t[7] Å 6.5, P õ 0.0001 in comparison with the activity before carbachol (Con) and t[7] Å 8.2, P õ 0.0001 in comparison to the activity during carbachol with scopolamine pretreatment (SCO / CCh, right).

FIG . 4. Location of the recording site with ejecting pipettes for CCh and SCO in the anterior hypothalamic medial preoptic region (AHMP), and the stimulating electrode in the laterodorsal tegmental nucleus (LDT) shown on the parasagittal section of the rat brain. Scale is in mm from the frontal, interaural zero plane, according to the Paxinos and Watson (1986) stereotaxic atlas. BN, bed nucleus of stria terminalis; cc, corpus callosum; CE, cerebellum; HI, hippocampal formation; HY, medial and posterior hypothalamus; NA, nucleus accumbens; OB, olfactory bulb; PAG, periqueductal grey matter; RF, reticular formation; SE, septal nuclei; TH, thalamic nuclei.


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by a local concurrent application of scopolamine in the AHMP. Some of the neurons were tested for both, iontophoretic CCh and stimulation of the LDT and for the effects of scopolamine on both of these responses. These results would demonstrate electrophysiologically that activation of the LDT may produce comparable, muscarinic neuronal responses in the AHMP to those induced by CCh in the same structure. Finally, the last step included the injection of DiA into the recording site to demonstrate retrogradely labeled cells within the region of the LDT. RESULTS

Effects of CCh on firing rate of AHMP neurons

FIG . 6. Responses of AHMP neurons to single pulse (2 ms) stimulation of the laterodorsal tegmental nucleus. A: percentage of inhibitory (Inhib.), excitatory responses (Excit.), and unresponding units (No Resp.) to stimulation. B: averaged neuronal activity of neurons that showed inhibitory responses (n Å 52), expressed in the mean firing rate per second during 100ms bout immediately after the stimulus (S) and compared with 100-ms control period before the stimulus (Before Stim.) and the following 100 ms, i.e., from 101–200 ms poststimulus (After Stim.). Vertical lines represents SE. **t[51] Å 8.14, P õ 0.0001.

and later were observed as an intense blue dot on the histological preparations. Stimulation sites were marked with an electrolytic iron deposition by passing 10 mA anodal current through the stimulating electrode for 1 min. The animal then was killed with an overdose of urethan and transcardially perfused with 0.9% NaCl solution followed by 10% formaldehyde in PBS containing 2% potassium ferricyanide. The ferricyanide reacted in the iron deposit in the tissue to form a Prussian blue spot that marked the tip of the stimulating electrode. The sites of recording and stimulation were marked on composite diagrams using a drawing attachment of the stereo-zoom microscope (SZH, Olympus Optical, Japan). Results were analyzed using Student’s t-test for paired comparisons.

Experimental design The study was conducted in three steps. The first step was focused on the effects of iontophoretically ejected CCh on the firing rate of spontaneously active AHMP neurons and the antagonizing effects of scopolamine on CCh-induced responses. The results would demonstrate predominance of cholinergically induced inhibitory neuronal responses in the AHMP mediated by muscarinic receptors. Second, responses of AHMP neurons to electrical stimulation of the LDT and its vicinity were recorded and antagonized

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FIG . 7. Responses of the AHMP neurons showing inhibition to increasing intensities of the stimulating current applied to the electrode in the laterodorsal tegmental nucleus. A: three peristimulus time histograms showing increasing inhibition to stimulation (S) with 200-, 400-, and 600-mA current, respectively. Histograms are compiled from 44 to 64 superimposed sweeps. B: relationship between the average inhibition time in ms caused by stimulation of the laterodorsal tegmental nucleus and the intensity of the stimulating current in mA. Vertical lines represent SE.

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A total of 100 spontaneously active neurons (100%) were studied in the AHMP. Neurons with a stable firing rate for at least 40–60 s were selected for further study. The average firing rate of units was 2.01 { 0.22 Hz (mean { SE). In response to iontophoretically applied CCh (20- to 60-nA ejecting current), the vast majority of these cells (83%) responded with a decrease in the mean firing rate and only 10% of cells showed increase in the mean firing rate (Fig. 1A). On average, the mean firing rate of 2.48 { 0.15 Hz was significantly reduced to 0.9 { 0.09 Hz during ejection

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of CCh at 20- to 40-nA level (63.7% reduction, t[82] Å 5.5, P õ 0.0001, Fig. 1B). Firing rate returned to the preejection level almost instantly with termination of the ejecting current for each neuron (Fig. 1B). Ten neurons increased their firing rate from an average of 1.53 { 0.41 Hz to 2.79 { 0.57 Hz during ejection of the same doses of CCh. The increase, however, did not reach the significance level (t[12] Å 2.0, n.s.). The inhibitory responses to CCh were dose dependent (Fig. 2, A and B) and repeatable for the same current (Fig. 2B, left). The same cells responded invariably with an increase in the mean firing rate to ejection of 10–20 nA of glutamate as tested for n Å 65 units (Fig. 2B, right). Effects of scopolamine on CCh-induced effects Ejection of scopolamine (1–5 nA) before or during the application of CCh attenuated or reversed the CCh-induced inhibitory effects (Fig. 3, A and B). The antagonizing effects of scopolamine were repeatable on the same cells (Fig. 3 A). As tested for eight cells, an average decrease in the mean firing rate by 72% (t[7] Å 6.5, P õ 0.0001) was reversed by scopolamine to a nonsignificant value of 25% below the preejection control level (t[7] Å 3.48, n.s., Fig. 3B). The increase in firing rate to CCh was not sensitive to preceding or concurrent ejection of scopolamine for the range of the ejecting current of 1–5 nA. Responses of AHMP neurons to stimulation of LDT The experimental set-up is illustrated in Fig. 4. The mean firing rate of AHMP neurons was recorded before and immediately after a 1-ms electrical stimulus was delivered to the LDT. A total of 255 spontaneously active neurons (100%) were tested in the AHMP with the mean firing rate of 1.01 { 0.09 (for n Å 106, which includes all responding cells and 37

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FIG . 9. Example units in the AHMP region showing reversal of the the inhibitoty, but not excitatory, responses caused by stimulation (S) of the laterodorsal tegmental nucleus by iontophoretic application of scopolamine (SCO). Stimulation of the laterodorsal tegmental nucleus caused inhibition of firing rate of the AHMP unit (A). Application of scopolamine ( /SCO) into the vicinity of the recorded neuron abolished the inhibition (B). Inhibitory response returned 45 min later in the same neuron (C). Increase in firing rate of the AHMP unit to stimulation (D) was not reversed by pretreatment with scopolamine ( /SCO, E) and remained unchanged 45 min later (F). A–C, 125 superimposed sweeps; D–F, 116 superimposed sweeps.

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FIG . 8. Antagonism by scopolamine of the inhibitory responses of the AHMP neurons to stimulation of the laterodorsal tegmental nucleus. A: averaged neuronal activity of 6 neurons that showed inhibitory responses to stimulation of the laterodorsal tegmental nucleus. Activity is expressed as a mean firing rate/s during 100-ms bout immediately after the stimulus (S) and compared with 100-ms control period before the stimulus (Before Stim.), and the following 100 ms, i.e., from 101–200 ms poststimulus (After Stim.). B: average neuronal activity of the same 6 neurons during concurrent application of scopolamine (S / SCO) into the vicinity of the recorded neuron in the AHMP region. Vertical lines represent SE. *t[5] Å 2,86, P õ 0.03 in comparison with the activity before stimulation (Before Stim.) and t[5] Å 2.72, P õ 0.04 in comparison with the activity during stimulation with scopolamine pretreatment (S / CCh, right).

not responding cells). Responses of the units were classified according to their primary response. Examples of an inhibitory and two excitatory responses are shown in Fig. 5, A– C. Although two types of excitatory responses could be distinguished, excitation (Fig. 5B) and excitation followed by inhibition (Fig. 5C), both types showed primary excitation and were counted jointly. Thus three types of responses were distinguished: inhibition, excitation, and no change in the mean firing rate. Sixty-nine units (27%) responded to stimulation of LDT (Fig. 6A). The majority of the responding neurons (75%) were inhibited by LDT stimulation, i.e., three times more cells responded with inhibition than with excitation. Moreover, the latency period (from the time of the stimulus occurrence to the first significantly changed bin) was different for the excitatory and inhibitory effects. Average latency for the total inhibition of firing was 3.2 { 0.5 ms, whereas the average latency for the significant excitatory effects was 11.8 { 1.8 ms (see Fig. 5, A and B). Most


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of the neurons inhibited by stimulation of the LDT had a lower firing rate than cells responding with excitation, however, there was no significant difference between baseline firing of cells showing inhibitory versus excitatory responses (t-test, P ú 0.08). On average, the value of firing rate per second, calculated for the 100-ms bouts of time, significantly decreased from 0.77 { 0.1 Hz before the stimulus to 0.36 { 0.05 Hz immediately after the stimulus (t[51] Å 8.14, P õ 0.0001, Fig. 6B) and returned the a similar level in the following 100-ms bout after the stimulus. The average time of the total inhibitory period induced by stimulation of LDT was 26.1 { 1.4 ms (maximum 57 ms). The inhibition time was dependent on the stimulating current and increased proportionally to the intensity of the stimulus in the LDT (Fig. 7, A and B). Effects of scopolamine on effects induced by LDT stimulation Iontophoretic application of scopolamine into the AHMP during the period of stimulation of the LDT (see Fig. 4) reversed the effects of stimulation. The mean firing rate per second, calculated for 100-ms bouts of time, showed a significant decrease immediately after the stimulus, as compared with 100-ms bout before the stimulus (Fig. 8A). The average value of the firing rate significantly decreased from 0.48 { 0.2 Hz to 0.23 { 0.09 Hz as a result of the stimulation of the LDT (t[5] Å 2.86, P õ 0.03) and returned to the prestimulus level in the following 100-ms bout (Fig. 8 A). However, similar LDT stimulation had no effect on the firing of the same neurons when scopolamine was ejected into the vicinity of the recorded neuron (Fig. 8B). Effects of scopolamine were reversible as tested in four neurons (Fig. 9, A–C). Stimulation of the LDT caused significant inhibition of the firing rate of a AHMP neuron before the drug application (Fig. 9A). This inhibitory effect was reversed when scopolamine was ejected concurrently into the vicinity of the recorded neuron in the AHMP (Fig. 9B, /SCO). The

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inhibitory effects of the LDT reappeared in the same neuron 45 min after application of scopolamine (Fig. 9C). Ejection of scopolamine into the vicinity of neurons that responded with an excitation to the LDT stimulation had no effect on the response (Fig. 9, D–F). Finally, responses of two neurons were studied both to iontophoretic CCh and to stimulation of the LDT (Fig. 10). Stimulation of LDT, which caused inhibition of the firing rate in the AHMP neurons (Fig. 10A), was ineffective when scopolamine was ejected into the vicinity of the recorded neuron. The same neuron was tested for its responses to ejection of CCh (Fig. 10B). Ejection of CCh caused a current (dose)-dependent decrease in the mean firing rate of this neuron (40–120 nA CCh). Application of scopolamine (in this case by passive diffusion from the pipette tip) partially antagonized the effects of CCh (Fig. 10B, SCO / CCh). The same neuron responded with the usual excitatory response to iontophoretic glutamate (Fig. 10B, GLU). Localization of recording and stimulation sites and their anatomic connection Localization of all neurons tested with CCh and studied in response to the stimulation of the LDT is illustrated in Fig. 11, on the right, and the left side of the brain, respectively. The majority of cells that showed inhibitory responses occupied the region of the medial AHMP and, partially, the lateral portion of the paraventricular nucleus. Neurons with excitatory responses are more scattered but show a tendency to be located in the bed nucleus of stria terminalis, perifornical region, and in the vicinity of the retrochiasmatic region. Localization of the stimulation sites in the tegmentum is shown in Fig. 12. All stimulation sites that caused inhibition of the firing rate of AHMP neurons were located in or at the border of the LDT. Locations of the stimulation electrode that yielded an increase in the firing rate or no change are scattered further around from the LDT. The excitatory ef-

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FIG . 10. Responses of the same AHMP neuron to stimulation of the laterodorsal tegmental nucleus or to carbachol, and effects of scopolamine on these responses. A: stimulation (S) of the laterodorsal tegmental nucleus caused inhibition of the firing rate (top). Inhibition was reversed by iontophoretic pretreatment of the neuron with scopolamine ( /SCOP, bottom). Iontophoretic application of carbachol (CCh) into the vicinity of the same neuron caused a dose-dependent (40–120 nA) decrease in the mean firing rate as shown on the running time histogram (B, left). Responses to iontophoretic carbachol (80–120 nA) were reversed or attenuated by concurrent application of scopolamine (SCO / CCh in B). Same unit responded with an increase in firing rate after iontophoretic application of glutamate (GLU, 30 nA, right). Ejection time for carbachol was 30 s and for scopolamine / carbachol 70 s. ●, application of scopolamine; retaining current was stopped and the scopolamine was allowed to passively diffuse from the pipette without any ejecting current.

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acetyltransferase (Dencev et al. 1996). These observations will be further studied and published separately. DISCUSSION

Inhibitory responses of medial AHMP neurons to cholinergic stimulation

fects were obtained primarily from the periaqueductal gray and the vicinity of the dorsal tegmental nucleus. To demonstrate that LDT neurons project to the AHMP, to the areas where the recorded neurons were localized, DiA was injected into the medial portion of AHMP in seven animals. A summary diagram of all injection sites for the DiA in the AHMP and an example of LDT staining are shown in Fig. 13, A and B. One week after the DiA injection, a dense group of labeled cells was found within the LDT in all of the animals (Fig. 13B). DiA was found uniformly distributed on the cell body and dendrites and in the cell in cytoplasmic granules but not within the cell nucleus. The vast majority of neurons was stained reliably by DiA. Our latest preliminary results indicate that, after injection of fluorogold into the AHMP, many, but not all of the stained LDT cells are double labeled with the antibody against choline

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Inhibitory responses of AHMP neurons to LDT stimulation Inhibitory neuronal responses in the AHMP to stimulation of the LDT were obtained only from electrodes localized almost precisely within the LDT nucleus. Furthermore, after injection of the fluorescent lipophilic carbocyanine dye, DiA, into the recording sites immediately after an electrophysiological experiment, DiA was found in numerous neurons in the LDT, 1 wk later. Inhibitory responses of the AHMP neurons to stimulation of the mesencephalic tegmentum have been reported. Highfrequency electrical stimulation of the mesencephalic reticu-

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FIG . 11. Localization of all the recorded units in the AHMP region shown on the frontal section of the rat brain 7.2 to 8.2 mm from the zero, interaural plane. All cells localized on the right side of the brain were tested with carbachol, and all the cells localized on the left side of the brain were tested for effects of stimulation of the laterodorsal tegmental nucleus. ●, inhibitory responses; m, excitatory responses; s, no response. Neurons showing no response were marked for carbachol only (n Å 6, right side of the brain), whereas neurons with no responses to electrical stimulation of the laterodorsal tegmental nucleus (n Å 186) were omitted on the left side of the brain for clarity. AA, anterior amygdalar area; AM, amygdalar complex; CP, caudate putamen; DB, diagonal band (horizontal limb); EN, endopiriform nucleus; fx, fornix; GP, globus pallidus; ic, internal capsule; LH, lateral hypothalamic area; LP, lateral preoptic area; mt, mammillothalamic tract; ox, optic chiasm; PA, paraventricular hypothalamic nucleus; RC, retrochiasmatic area; RE, reuniens thalamic nucleus; RT, reticular thalamic nucleus; sm, stria medullaris; SP, subpallidal area; VP, ventral pallidum; ZI, zona incerta.

Our results have indicated that iontophoretic application of CCh into the vicinity of single cells in the AHMP caused a dose-dependent decrease in the mean firing rate of the majority of units. The inhibitory effect of CCh, but not the excitatory effect, was reversed by iontophoretic pretreatment with scopolamine. Electrical stimulation of the LDT neurons caused a comparable current-dependent decrease in the mean firing rate of AHMP neurons. Effects of LDT stimulation were reversed by iontophoretic pretreatment of the recorded neurons in the AHMP with scopolamine. The same AHMP neurons that were inhibited by LDT stimulation also responded to CCh with the decrease in firing rate. Numerous studies reported both the excitatory and inhibitory effects of cholinergic agents on the firing rate of AHMP neurons (Bloom et al. 1963; Hsieh and Pan 1990; Jell 1973; Kow and Pfaff 1985; Oomura et al. 1969). It seems, however, that the most medial neurons, which are not necessarily embedded within any particular nucleus and are not projecting to the neurohypophysis, show predominantly inhibitory responses to iontophoretic ejection of CCh or acetylcholine (Brudzynski et al. 1991; Hsieh and Pan 1990; Moss et al. 1972). More caudally, neurons of the ventromedial hypothalamic nucleus showed a comparable number of excited or inhibited neurons to acetylcholine (Kow and Pfaff 1985). On the other hand, cells of the paraventricular nucleus that project to the neurohypophysis or neurons in the supraoptic nucleus or arcuate nucleus predominantly increased their mean firing rate after application of acetylcholine or CCh (Dreifuss and Kelly 1970; Gribkoff et al. 1988; Lin et al. 1993; Moss et al. 1972). Excitatory responses to acetylcholine or CCh also were obtained from the forebrain cholinergic neurons projecting to the neocortex (Lamour et al. 1986; Levine et al. 1986). All of these results suggest that there is a subpopulation of medially located, cholinoceptive neurons in AHMP that respond with a decrease in firing rate to acetylcholine. Medial localization of these neurons is consistent with results of functional mapping of behavioral responses in the rat and cat brain (Brudzynski 1994; Brudzynski et al. 1995). Further functional and anatomic evidence suggest that these inhibitory effects are caused by cholinergic input from the LDT nucleus.


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lar formation in the central tegmental field resulted in the inhibition of a majority of the preoptic neurons (Mallick et al. 1986; Mohan-Kumar et al. 1984, 1985). Inhibition of firing in the subpopulation of the basal forebrain neurons in response to a short train stimulation of the medial pontomesencephalic region also has been reported recently (Semba et al. 1995). Cholinergic input to the AHMP, presumably from the LDT, has been implicated in the sleep-wakefulness cycle (Imeri et al. 1995, 1996; Jones 1993; Mallik and Joseph 1997). Low-amplitude stimulation of the LDT induced significant increase in total rapid eye movement (REM) and in the duration of REM sleep (Thakkar et al. 1996). Similar stimulation of neighboring areas in the mesencephalic reticular formation during non-REM sleep evoked long-lasting suppression of discharges in sleep-related neurons in the magnocellular preoptic nucleus (Szymusiak 1995; Szymusiak and McGinty 1989). The inhibitory effects of the ascending cholinergic fibers are not unique for the mediobasal forebrain. Cholinergic inhibitory input, which modulates the thalamo-cortical relay transmission, has been well documented (Ben-Ari et al. 1976; Dingledine and Kelly 1977; Purpura et al. 1966; Steriade and Buzsaki 1990). Although inhibitory influences in the thalamus are associated with the regulation of the electrocortical activity, the mesolimbic component seems to be involved in the regulation of limbic activity in aversive and agonistic behavioral situations. Inhibition of firing rate of AHMP neurons in dangerous or agonistic behavioral situations The results suggest that activity of the LDT neurons would cause a release of acetylcholine in the medial structures, including AHMP, and would cause cellular inhibition. This should be accompanied by a change in behavior or initiation of behavior, particularly associated with a dangerous or ago-

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nistic situation, as indicated by pharmacological-behavioral studies (Brudzynski et al. 1993). Indeed, a number of studies provided evidence for a behavior-dependent decrease in the firing rate of AHMP or preoptic neurons (Adams 1968; Mink et al. 1983; Naka and Kido 1967). Adams (1968) reported that about one-third of single cells, recorded from the hypothalamus and midline thalamus of freely behaving cats, inhibited their firing rate during various manipulations. The decrease in firing rate was not specific to any particular stimulus; however, certain manipulations such as an affective defense in response to a second attacking cat, lifting and dropping the cat, or opening the cage were more effective than other stimuli. Interestingly, such an inhibition of the firing rate was not recorded from the midbrain where none of the cells were exclusively inhibited in different behavioral situations (Adams 1968). Similar results have been reported in other studies on cats. A certain class of hypothalamic neurons (‘‘medium units’’) stopped firing as soon as the cat was presented with a mouse or during a defensive response (‘‘rage’’) with accompanying growling vocalization (Naka and Kido 1967). Significance of the cholinergic mesolimbic projection Inhibition of neuronal firing by the ascending pontomesencephalic cholinergic projection may mean that the pontomesencephalic system can attenuate or block input from other limbic and extralimbic structures to the medial diencephalic and forebrain regions. This inhibition in the medial forebrain and diencephalon can stop the ongoing behavior or facilitate defensive responses associated with behavioral inhibition. There are experimental data to support this conclusion. High-frequency stimulation of the medial mesencephalic tegmentum, including LDT, caused inhibition of the firing rate of the ventromedial hypothalamic neurons, which were driven by amygdaloid stimulation (Tsubokawa and

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FIG . 12. Composite diagram of stimulation sites in the pontomesencephalic tegmentum shown on frontal sections of the rat brain /1.0 to 00.3 mm from the interaural 0 plane. Depending on the result of the stimulation, the sites are marked with the following responses: ●, inhibitory; n, excitatory; s, no response. Some of the sites with inhibitory results are superimposed. All the stimulation sites were ipsilateral to the recorded neuron in the AHMP area. AQ, aqueduct; CI, colliculus inferior; cp, superior cerebellar peduncle; CU, cunneiform nucleus; DR, dorsal raphe nucleus; DT, dorsal tegmental nuclei; ml, medial lemniscus; MR, medial raphe nucleus; MT, motor trigeminal nucleus; PB, parabrachial nucleus; PPN, pedunculopontine nucleus; PR, raphe pontis nucleus; py, pyramidal tract.

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The study was supported by the grant from the Natural Sciences and Engineering Research Council of Canada to Dr. Stefan M. Brudzynski. Address for reprint requests: S. M. Brudzynski, Dept. of Psychology, Brock University, 500 Glenridge Ave., St. Catharines, Ontario, L2S 3A1 Canada. Received 7 May 1997; accepted in final form 18 December 1997. REFERENCES

FIG . 13. Labeling of the laterodorsal tegmental nucleus with the fluorescent dye, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide, (DiA), after its injection into the recording site in the AHMP region. A: summary diagram of the maximal extent of the DiA spread from 7 DiA injections in the AHMP region is shown on the left side of the rat brain sections between the frontal stereotaxic planes 7.2–8.2 mm anterior to the interaural line according to the Paxinos and Watson (1986) atlas. An exemplary injection site of the DiA (0.1 ml) into the anterior hypothalamic area (AH) is shown on the right side of the brain ( r ), with the resulting staining in B. B: dense group of labeled cells in the laterodorsal tegmental nucleus on the horizontal cross-section of the brain, 1 wk after the injection. Scale bar Å 50 mm. AH, anterior hypothalamic area;

Sutin 1963). For comparison, the same stimulation could facilitate or had no effect on the firing of neurons driven by stimulation of the septum. Although, acetylcholine is not the only transmitter released by tegmental stimulation, it seems likely that this inhibitory effect is due to pontomesencephalic cholinergic input and release of acetylcholine. Further evidence for cholinergic inhibition of the input from other structures was reported for medial zona incerta. The firing rate

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ADAMS, D. B. The activity of single cells in the midbrain and hypothalamus of the cat during affective defense behavior. Arch. Ital. Biol. 106: 243– 269, 1968. ARMSTRONG, D. M., SAPER, C. B., LEVEY, A. I., WAINER, B. H., AND TERRY, R. D. Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase. J. Comp. Neurol. 216: 53–68, 1983. BAKER, W. W., HOSKO, M. J., RUTT, W. J., AND MC GRATH, J. R. Tremorine-induced rage and its antagonism by atropine. Proc. Soc. Exp. Biol. Med. 104: 214–217, 1960. BAKER, G. AND REESE, B. Using confocal laser scanning microscopy to investigate the organization and development of neuronal projections labelled with DiI. Methods Cell Biol. 38: 325–344, 1993. BALICE-GORDON, R. J., CHUA, C. K., NELSON, C. C., AND LICHTMAN, J. W. Gradual loss of synaptic cartels procedes axon withdrawal at developing neuromuscular junctions. Neuron 11: 801–815, 1993. BAXTER, B. L. Comparison of the same behavioral effects of electrical or chemical stimulation applied at the same brain loci. Exp. Neurol. 19: 412–432, 1967. BAXTER, B. L. Elicitation of emotional behavior by electrical or chemical stimulation applied at the same loci in the cat mesencephalon. Exp. Neurol. 21: 1–10, 1968. BEN-ARI, Y., DINGLEDINE, R., KANAZAWA, T., AND KELLY, J. S. Inhibitory effects of acetylcholine on neurons in the feline nucleus reticularis thalami. J. Physiol. (Lond.) 261: 647–671, 1976. BLOOM, F. E., OLIVER, A. P., AND SALMOIRAGHI, G. C. The responsiveness of individual hypothalamic neurons to microelectrophoretically administered endogenous amines. Int. J. Neuropharmacol. 2: 181–193, 1963. BRADSHAW, C. M., ROBERTS, M.H.T., AND SZABADI, E. Kinetics of the release of noradrenaline from micropipettes: interaction between ejecting and retaining currents. Br. J. Pharmacol. 49: 667–677, 1973. BRUDZYNSKI, S. M. Carbachol-induced agonistic behavior in cats: aggressive or defensive response? Acta Neurobiol. Exp. 41: 15–32, 1981. BRUDZYNSKI, S. M. Ultrasonic vocalization induced by intracerebral carbachol in rats: localization and dose-response study. Behav. Brain Res. 63: 133–143, 1994. BRUDZYNSKI, S. M. AND BARNABI, F. Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat. Behav. Brain Res. 80: 145–152, 1996. BRUDZYNSKI, S. M. AND BIHARI, F. Ultrasonic vocalization in rats produced

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of all the neurons in the zona incerta, which were excited by stimulation of the ventromedial hypothalamic nucleus, were inhibited by acetylcholine (Eaton and Moss 1989). In summary, the results of the present study suggest that the LDT cholinergic neurons project and terminate on neurons in the mediobasal regions of the forebrain with their activation causing a decrease in the mean firing rate of these neurons. This projection can be attributed to the cholinergic component of the ascending reticular activating system (Satoh and Fibiger 1986; Shute and Lewis 1963, 1967; Vincent et al. 1986; Woolf et al. 1990) . On the basis of the present results and previous behavioral studies, it is postulated that the inhibitory influence of the ascending cholinergic fibers in the mediobasal forebrain and diencephalon may play a role in the initiation of some of the behavioral patterns. Thus the inhibition of neuronal firing represent an electrophysiological correlate accompanying some agonistic behavioral manifestations, defensive or alarm vocalization and behavioral inhibition manifested by a decrease in locomotor activity.


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