Extracellular characteristics of putative cholinergic neurons in the rat laterodorsal tegmental nucleus

Brain Research, 559 (1991) 64-74 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 A DON1S 000689939116942G

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Extracellular characteristics of putative cholinergic neurons in the rat laterodorsal tegmental nucleus S.J. Grant and D.A. Highfield Department of Psychology and Program in Neuroscience, University of Delaware, Newark, DE 19716 (U.S.A.)

(Accepted 16 April 1991) Key words: Acetylcholine; Noradrenaline; Lateral-dorsal tegmental nucleus; Locus coeruleus; Anterior-ventral nucleus; Thalamus; Single unit activity; Rat

The extracellular electrophysiological properties of neurons in the laterodorsal tegmental nucleus (LDT), a major source of eholinergic afferents to the thalamus, were studied in chloral hydrate-anesthetized rats. A combination of antidromie activation from the thalamus and histological verification of recording sites was used to correlate the identity of extraeellular recordings in the rat LDT with eholinergic neurons in that region. All neurons antidromically activated by stimulation of the anteroventral thalamus were histologically verified to be within dusters of eholinergie (NADPH-d-positive) cells in the LDT or in the adjacent nucleus locus coerulens (LC). The thalamically projecting LDT neurons had a homogeneous neurophysioiogical profile consisting of long duration action potentials (mean = 2.5 ms), slow conduction velocities (mean = 0.78 m/s), and lengthy ehronaxie values (mean = 0.725 ms). The apl~aranee and axonal characteristics of these neurons resembled those of noradrenergic LC neurons, but the two populations exhibited substantially different spontaneous activity patterns and sensory responsiveness. These characteristics may be useful in the preliminary identification of putative eholinergie neurons in vivo, and thereby provide a foundation for exploring the neuropharmacology, afferent modulation, sensory responsiveness and behavioral correlates of the brainstem eholinergic system. INTRODUCTION There is great interest in the activity of brainstem cholinergic neurons because of their hypothesized relation to the classic reticular activating system z7'56'6°. Cholinergic receptors in the thalamus modulate thalamic rhythmicity and sensory responsiveness 39'56, and neurons along the mesopontine border are the major source of cholinergic innervation of the thalamus as well as other subcortical sites 24'5°'52'54. Like neighboring noradrenergic and serotonergic neurons, these brainstem cholinergic neurons are clustered in specific nuclei. The Ch6 group lies in the central gray within the laterodorsal tegmental nucleus (LDT) and the Ch5 group on the lateral edge of the brachium conjunctivum within the pedunculopontine tegmental nucleus (PPT) 42. Substantially less is known of the activity of cholinergic neurons compared to other systems with similar organization and functions, such as monoamine neurons. Neurophysiological studies of these cholinergic neurons in vivo have been hampered by the lack of extracellular criteria for their identification. The discovery of specific extracellular neurophysiological properties for other chemically defined systems, such as norpinephrine-, se-

rotonin- and dopamine-containing neurons, permitted extensive studies into the physiological, pharmacological and behavioral properties of these systems s'2°'41. Differentiation of monoamine neurons from surrounding populations was initially based on a combination of indirect criteria including location of recording sites, firing patterns, waveform, antidromic activation, and the absence of such activity after lesions with specific neurotoxic agents (e.g. 6-hydroxydopamine) l'5,tl'16,26'33,43,64This study uses a similar approach to identify putative cholinergic neurons in the brainstem. Antidromic activation from the thalamus allows a strong inference to be made about the cholinergic identity of extracellularly recorded neurons in the rat LDT. Retrograde tracing studies show that over 90% of thalamieally projecting neurons from the rat L D T and PPT are cholinergic 24's°'52'54. In particular, the anteroventral nucleus (AV) contains the densest plexus of cholinergic fibers in the thalamus, and the L D T is the major source of cholinergic afferents to the AV thalamus 24'36'52. Thus, stimulation of the A V thalamus should yield many antidromieally driven neurons in the LDT, and these neurons have a high probability of being cholinergic. Explicit histological localization of the extracellular

Correspondence: S.J. Grant, Department of Psychology, 220 Wolf Hall, University of Delaware, Newark, DE 19716, U.S.A. Fax: (1) (302) 292-3645.

65 r e c o r d i n g sites is necessary as a s e c o n d c r i t e r i o n since n e u r o n s in the adjacent locus c o e r u l e u s a n d dorsal rapile also project to t h e t h a l a m u s 5°. T h o u g h it is n o t possible with e x t r a c e l l u l a r t e c h n i q u e s to identify the specific n e u r o n r e c o r d e d , cells a n t i d r o m i c a l l y activated f r o m the t h a l a m u s are likely to b e cholinergic o n l y if t h e y lie

and a second overlying the LDT and LC (1.0-3.5 ram posterior to lambda, 0.5-1.3 mm lateral) 4s. The dura was reflected taking care to spare nearby sinuses. A co-axial bipolar stimulating electrode (Rhodes SNE-100 ) was advanced to the approximate stereotaxic depth of the AV thalamus. Multi-unit activity was recorded through the inner electrode to improve the accuracy of the placements 14. After placing the stimulating electrode the bone defect was covered with saline-soaked Gelfoam and a thin layer of bone wax.

w i t h i n clusters of n e u r o n s s t a i n i n g d e e p l y for a specific m a r k e r for Ch5 a n d Ch6 cholinergic n e u r o n s , such as nicotinamide adenine dinucleotide phosphate diaphorase ( N A D P H - d ) 6°. I n t e r p r e t a t i o n of histological d a t a is facilitated in the rat b y the spatial s e g r e g a t i o n of the Ch6 cholinergic n e u r o n s f r o m n o r a d r e n e r g i c a n d serotonergic n e u r o n s 5°. This contrasts with the cat w h e r e the interdigitation of n o r a d r e n e r g i c a n d cholinergic n e u r o n s a n d the g r e a t e r p r o p o r t i o n of n o n - c h o l i n e r g i c L D T a n d P P T n e u r o n s projecting to t h a l a m u s complicates b o t h the use of r e c o r d i n g site histology a n d a n t i d r o m i c activation from t h a l a m i c nuclei as m e a n s to identify cholinergic activity31,38,44,55,59. T h e r e f o r e , the c o m b i n a t i o n of a n t i d r o m i c activation f r o m the a n t e r o v e n t r a l t h a l a m u s a n d histological verific a t i o n o f r e c o r d i n g sites was used to infer the cholinergic i d e n t i t y of e x t r a c e l l u l a r recordings in the rat LDT. It was h y p o t h e s i z e d that these p u t a t i v e cholinergic n e u r o n s w o u l d h a v e o n e o r m o r e n e u r o p h y s i o l o g i c a l characteristics (e.g. c o n d u c t i o n velocity, w a v e f o r m , s p o n t a n e o u s activity) that w o u l d d i f f e r e n t i a t e t h e m f r o m s u r r o u n d i n g n e u r o n a l p o p u l a t i o n s . Specific e m p h a s i s was p l a c e d o n c o m p a r i s o n s with n e u r o n s in the a d j a c e n t locus coeru l e u s since cholinergic L D T n e u r o n s a n d n o r a d r e n e r i g c L C n e u r o n s have similar p r o j e c t i o n s to the t h a l a m u s , similar p o s t s y n a p t i c actions o n t h a l a m i c n e u r o n s 39, a n d s o m e m a i n t a i n that the e x t r a c e l l u l a r profile of L D T n e u r o n s r e s e m b l e s that of m o n o a m i n e n e u r o n s 32. P o r t i o n s of this d a t a have b e e n p u b l i s h e d in abstract f o r m TM. MATERIALS AND METHODS

Subjects Subjects were 40 male albino rats (250-350 g) obtained from commercial suppliers (Charles River). They were housed in group cages with ad libitum access to food and water. Lighting was controlled on a 12:12 h light-dark cycle and experiments were conducted during daylight hours.

Surgery Subjects were anesthetized with either chloral hydrate (400 mg/ kg, i.p.; n = 29) or urethane (1.8 g/kg, i.p.; n = 11) at a level sufficient to abolish corneal and hindlimb withdrawal reflexes. Chloral hydrate anesthesia was supplemented throughout the experiment with periodic injections via a lateral tail vein. Core temperature was monitored with a rectal probe and maintained at 3637 °C by a heating pad. Following induction of anesthesia the rat was placed in a small animal stereotaxic frame with the head level between bregma and lambda. The skull was exposed with a midline incision and two small holes in the bone were drilled: one overlying the AV thalamus (1.3-2.0 mm caudal to bregma, 1.5 mm lateral to the midline),

Electrophysiology Glass mieroelectrodes were used to record extracellular action potentials of single neurons. Micropipettes were pulled from single barreled omega dot tubing (1.5 mm o.d.), and the tips broken under microscopic control to a diameter of 1-3/~m. The micropipettes were backfilled with 2% Pontamine sky blue in 0.5 M Na acetate yielding electrodes with impedances of 3-7 Mfl at 1 kHz. The recording electrodes were slowly advanced through the brain at a 16° caudorostral angle via a hydraulic microdrive. ExtraceUular signals were routed through a single ended high impedance probe. Amplified and filtered (200 Hz to 8 kHz) signals were displayed on oscilloscopes and an audio monitor using standard electronics. Extracellular action potentials from well-isolated (> 3:1 signal/noise ratio) single neurons were convened to standard TI'L spike pulses by a window discriminator. The standardized pulses were directed to a microcomputer for construction of interspike interval or post-stimulus time interval histograms. Continuous time interval histograms of spontaneous activity were generated by a ratemeter and displayed on a strip chart recorder. Recording locations were estimated in vivo by a series of physiological landmarks. Passage through the overlying cerebellum was marked by appearance of climbing fiber activity while a sudden drop in baseline noise signaled entrance into the fourth ventricle. Jaw stretch was used to activate neurons of the mesencephalic sensory nucleus of the trigeminal nerve (Mes V). LC single unit activity was recognized in vivo using previously published physiological criteria including long duration action potentials with a notch on the ascending limb, slow spontaneous activity (<3 Hz), and a transient biphasic burst-panse response to brief hindpaw compression11"19"33. Since the LDT lies immediately rostral and medial to the LC, and dorsal to the fiber-like activity of the medial longitudinal faseiculus (mlf), penetrations could be reliably directed into the region of the LDT after locating Mes V and the LC. Data were collected on all neurons encountered 5.0-7.0 mm below the skull surface. In order to unmask non-spontaneons neurons during the electrode penetration a suprathreshold 'hunting' stimulus (0.75 ms, 500-750/~A) was continuously delivered at 0.5 Hz during the pen7 etration. Monophasic cathodal pulses were delivered by a constantcurrent unit through the center pole of the stimulating electrode with the outer barrel serving as the anode. Evoked activity was continuously monitored on an analog storage oscilloscope triggered by the stimulus onset. The criteria used to identify antidromically evoked action potentials included constant latency of the evoked spike, following of high frequency stimulation (>250 Hz), and, for spontaneously active neurons, collision of stimulus evoked and spontaneous action potentials37. Evoked activity not meeting these criteria was considered to represent orthodromie (synaptic) responses. Threshold was defined as the current intensity that evoked antidromic responses on 50% of the trials. Latencies were determined at current intensities of 1.5 times threshold. Refractory periods were measured with twin pulse stimulation also at 1.5-times threshold. Chronaxie curves were derived by determining threshold at pulse widths varying from 0.05 to 2.0 ms 46.

Histology Dye spots were made to verify the anatomical location of each antidromicaUy activated neuron. Deposits of Pontamine sky blue were made by passing -10/~A through the microelectrode for 5 min via a constant-current unit (Fintronics). Successive penetrations

66 were separated by at least 300/~m so that individual dye spots could be unambiguously resolved on microscopic sections. Stimulation sites were also marked after the experiment by passing +20/~A for 30 s through the center electrode. At the end of each experiment the rat was rapidly killed by injecting chloral hydrate i.v. sufficient to abolish breathing. The rat was immediately perfused through the ascending aorta with saline followed by 10% formalin in 0.1 M phosphate buffer (pH 7.4). The brain was removed and cut into rostral and caudal blocks. The caudal block containing the recording sites was placed in formalin perfusate. The rostral block encompassing the stimulation site was placed into a vial containing 10% formalin plus 1% potassium ferricyanide. Both blocks were post-fixed for 12-18 h at 4 °C, then transferred to a 30% sucrose in 0.1 M phosphate buffer solution (pH 7.4) and stored at 4 °C until the blocks sank. Serial 50-/~m sections were cut on a freezing microtome. Alternate sections were stained for Nissl substance (neutral red) and either Perls (Prussian blue) iron reaction (stimulation sites) or reduced NADPH-d (recording sites) 53'6°. NADPH-d staining was used as a simple empirical histochemical marker for the Ch5 and Ch6 neurons since all neurons that stain deeply for NADPH-d activity in the LDT and PPT have been shown to contain choline acetyltransferase 6°. Neighboring noncholinergic neurons, including those in the LC and dorsal raphe and glia do not stain for NADPH-d activity. A modification of the

method of Scherer-Singler et al. 53 was used for NADPH-d staining. Alternate serial sections through the LDT were collected into ice-cold 0.1 M phosphate buffer (pH 7.4). Free floating sections were incubated immediately in a solution containing 15 mM sodium malate, 1 mM NADPH, 20 mM MgCI and 0.2 M nitro blue tetrazolium in 0.1 M Tris-HCl (pH 8.0) for 20-40 min at 37 °C in the dark. Following incubation, the sections were rinsed in 0.1 M Tris buffer, mounted, allowed to dry overnight, then dehydrated in alcohol, cleared in xylene, and cover slips were attached. The serial order of the NADPH-d sections was determined by comparisons with the corresponding Nissl sections. The locations of the recording and stimulation sites were reconstructed from the histological sections using the dye spots as reference points. A projected image of each histological section containing a spot was traced onto graph paper along with the outlines of prominent nuclear structures and the position of any NADPHd-positive neurons. The reconstructed recording sites were collapsed across all animals and plotted on tracings derived from a standard stereotaxic atlas 45.

Data analysis Single unit activity was classified according to their histological location and electrophysiologieal responses. The LDT was defined as co-extensive with the cluster of NADPH-d-stained neurons located lateral to the dorsal tegmental nucleus of Gudden, medial to

Fig. 1. Anatomical verification of representative recording (A,B) and stimulation (C,D) sites of antidromically driven LDT neurons. Adjacent Nissl (A) and NADPH-d (B) sections contain a dye spot marking a recording site (arrow) in proximity of cholinergic neurons. The neuron recorded at this site was antidromically driven by a stimulating electrode whose tip (arrowhead) was in the dorsal anterior-ventral thalamus shown in adjacent Nissl (C) and acetylcholinesterase (D) stained sections. DTg, dorsal tegmental nucleus of Gudden, V, ventricle, sm, stria medullaris, Rt, thalamic reticular nucleus. Calibration bar : A,B, 100 gm; C,D, 250/am.

67 the LC and Mes V and dorsal to the fibers of the mlf (Fig. 1). Thus, the adjacent NADPH-d-POor parvicellular Barrington's nucleus and the NADPH-d neurons in the parabrachial region lateral to Mes V were not considered part of the LDT. The LC, known to be a homogeneons duster of noradrenergic neurons, was easily recognized in Nissl sections as a compact collection of hyperehromic cells 2:'2s. LC neurons had to be located within the borders of the LC and conform to the standard physiological criteria described above. Most electrophysiological measurements were made from photographs of oscilloscope traces. Action potential duration was measured from the initial voltage deflection to the second zero (baseline) crossing. Latency was measured from stimulus onset to the initial action potential deflection. Conduction velocity was estimated using distances derived from published figures of the thalamic trajectory of LC and LDT axons 2s'52. Chronaxie values were derived by graphically estimating the stimulus duration at twice the rheobase (plateau) stimulus current 46. The interval at which the neuron failed to follow twin pulse stimulation at 1.5-times threshold intensity was considered the relative refractory period. Spontaneous firing rates were derived from interspike interval histograms or ratemeter generated continuous time histograms. Histograms were collected during a 5-15 min period in the absence of any stimulation. In the statistical analysis non-spontaneous neurons were assigned a firing rate of zero and included in the calculation of the average population firing rate. Data were analyzed by

parametric statistical tests (t-tests, ANOVA with post hoc comparisons) using a microcomputer program (SysStat).

RESULTS

Histological location Out of a total of 248 cells recorded from 40 rats, all neurons activated by antidromic stimulation were restricted to recording sites in either the LDT or LC. All recording sites in the LDT yielding antidromic activity were in proximity to neurons staining deeply for NADPH-d (Fig. 1); all other antidromic neurons were located in the rostral and middle portions of the LC (Fig. 2). All cells provisionally identified as noradrenergic by established physiological criteria were histologically located within the borders of the LC. The remaining nonantidromically activated cells were found within a region encompassing the LDT, Barrington's nucleus, dorsal tegmental nucleus of Gudden and the reticular formation immediately ventral to the mlf (Fig. 2).

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Fig. 2. Reconstruction of recording sites. Sections are redrawn from the atlas of Paxinos and Watson. Borders of the LDT were based on the distribution of NADPH-d neurons. Note that antidromically activated neurons were confined to either the LC (LC-AD) or the LDT (LDT-AD) whereas neurons not antidromically activated (LDT-ND) were also found beyond the LDT. For clarity only antidromically activated locus coeruleus neurons (LC-AD) were plotted, sc, sub coeruleus.

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Fig. 3. Antidromic activation of two LDT neuroas. A: collision test. In the top trace, a stimulation pulse (triangle) triggered by a spontaneous action potential (left solid circle) after a critical interval eficited a constant latency action potential (right solid circle). In the bottom trace a slight decrease in the interval blocked the evoked action potential. B: high frequency follo~ng. In the top trace dual pulse stimulation (triangles) at 300 Hz elicits two constant latency action potentials (solid circles). In the bottom trace a small decrease in the interpulse interval fails to elicit the second action potential. All figures are 5 overlapping traces.

68 Effective stimulation sites were located predominantly in the anterior-ventral thalamic nucleus, but since stimulation currents could spread into adjacent anterior nuclei (e.g. anterior-medial, anterior-dorsal and lateral-dorsal nuclei, effective stimulation sites will be collectively called the anterior thalamus. A typical stimulation site in the anterior-ventral thalamus is shown in Fig. 1. No antidromically driven neurons were recorded when the stimulating electrodes were located outside the thalamus, nor were antidromic L D T neurons recorded when the stimulation sites were in the reticular or ventrolateral thalamic nuclei. Stimulation electrodes were histologically located in the anteroventral (29/40), reticular (5/40), ventrolateral (1/40), and central lateral/medial dorsal (2/40) thalamic nuclei. In 3 rats the stimulation electrodes were located outside the thalamus, in the hippocampus (2/40) and the bed nucleus of the stria terminalis (1/40).

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Antidromic activation Out of the total population, 60 cells (24%) exhibited constant latency spikes following stimulation of the anterior thalamus, and 44 (17%) satisfied at least one other criterion for antidromic activation. Of all L D T neurons, 16% (20/126) were antidromically driven, while 19% (24/ 122) of all LC neurons were antidromically driven. Recordings of 16 neurons were not sufficiently stable to permit testing for additional criteria, with the LC neurons constituting most of this population (13/16). Since the antidromic status of these cells is ambiguous, they were excluded from further analysis. The antidromically driven L D T and LC neurons will be hereafter referred to as LDT-AD and LC-AD, respectively. The neurons that were not antidromically driven are designated as LDT-ND and LC-ND. Fig. 3 shows the responses of two representative LDT=AD neurons meeting the criteria for antidromic activation. Collision testing was precluded for many LDT-AD neurons due to absence of spontaneous activity. Therefore, non-spontaneous LDT neurons that exhibited constant latency and followed high frequency stimulation (>250 Hz) were considered antidromic 37. These criteria were considered reasonable as we have previously found that the orthodromic activation of LDT neurons produced by single pulse stimulation of the medial prefrontal cortex fails to exhibit constant latency or follow stimulation frequencies greater than 50 Hz (Grant and Highfield, unpublished results). In addition, all LC-AD neurons that were spontaneously active and satisfied the collision criterion also exhibited high frequency following in excess of 250 Hz. As seen in Fig. 4A, the antidromic response latency for L D T neurons ranged from 6 to 30 ms with an average latency of 16.5 - 3.7 (S.E.M.) ms. By contrast, LC neurons had a longer average latency (25.2 -+ 2.3 ms), and a slightly wider, but overlapping range (6-52 ms). These latency differences were statistically significant (t = 2.87, df = 42, P < 0.01) (Fig. 4B), but the calculated conduction velocities for L D T (0.78 --- 0.09 m/s) and LC (0.62 - 0.08 s) neurons were not (t = 1.29, df = 42, P < 0.075). This was probably because the shorter latency

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Fig. 4. Distribution of antidromic latencies for LDT and LC neurons. The distributions have overlapping ranges (A), but LC neurons had a significantly longer mean latency due to a few neurons with especially long latencies (** P < 0.01, t.test). Ordinate labels indicate the range of the histogram bins (B). There was no significant difference between LC and LDT neurons in calculated conduction velocities because of differences in axonal pathway lengths (see text).

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Fig, 5. Representative waveforms for antidromically activated LC (A) and LDT (B) neurons in filtered recordings (200-8 kHz). The major distinguishing feature is the prominent notch (IS-SD break)

on the ascending limb of the LC action potential.

69 of L D T neurons was offset by its more rostral location. No significant differences were found between L D T - A D and L C - A D neurons with respect to stimulation threshold (LDT, 489 -+ 88 # A ; LC neurons, 476 __ 57/zA), relative refractory period (LDT, 2.1 +- 0.2 ms; LC, 2.2 -- 0.2 ms), or chronaxie (LDT, 0.725 -+ 0.275 ms; LC, 0.470 -+ 0.20 ms). The conduction velocity, refractory period, and antidromic stimulation thresholds of L C neurons are within the range of previous descriptions for this population 11'13'43.

single unit activity. This notch is usually interpreted as marking the spread of the action potential from the axon hillock/initial segment to the soma-dendritic compartment (IS-SD break). Most of the non-driven L D T cells also had initially positive biphasic waveforms. Only a few non-driven neurons (12/126) were initially negative, and they were all located ventral to the mlf at the border of the reticular formation. Although there was substantial overlap in the range of values, there were reliable quantitative differences among the action potential duration widths of 3 populations (Fig. 6). L D T - A D neurons (2.5 - 0.2 ms, n = 14) had shorter duration action potentials than L C - A D neurons (3.1 - 0.4 ms, n = 13), but both were longer than L D T - N D neurons (1.6 - 0.1 ms, n = 82). All of these differences were found to be significant by A N O V A (F = 40.2, df = 2, 106, P < 0.001) and post-hoc comparisons (Newman-Keuls, P <

Waveforms In filtered recordings, L D T - A D single unit activity consisted of stable, large amplitude (0.5-1.6 mV), initially positive biphasic or triphasic waveforms. These waveforms were similar but not identical with L C neurons (Fig. 5). Both L C and L D T waveforms exhibited prominent negative-going afterpotentials, but L D T - A D neurons lacked the notch on the ascending limb of the action potential that is a conspicuous component of L C

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Fig. 6. A: distribution of action potential widths for antidromically activated LDT (LDT-AD) and LC neurons compared to non-antidromically activated LDT (LDT-ND) neurons. Ordinate labels indicate the midpoint of the histogram bins. B: although there was overlap in the ranges, LC action potentials were significantly longer than LDT neurons, while other neurons were significantly shorter (**P < 0.01, Newman-Keuls). Note however, the wider distribution of non-antidromically activated LDT neurons and the presence of neurons with long duration action potentials in that population.

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Fig. 7. Distribution of spontaneous firing rates of antidromically activated LDT (LDT-AD) and LC neurons compared to non-antidromically activated LDT (LDT-ND) neurons. Ordinate labels indicate the upper limit of the histogram bin width. Note the high proportion of non-spontaneous LDT-AD neurons (0 Hz). In contrast to the narrow distribution of LDT-AD and LC activity, other LDT neurons have a wider range containing both relatively slow (<5 Hz) and fast firing (>10 Hz) populations. B: significant differenees were present between the mean firing rates of all 3 populations (** P < 0.01, * P < 0.05 Newman-Keuls).

70

Spontaneous activity As seen in Fig. 7, L D T - A D neurons exhibited less spontaneous activity than surrounding neurons. LDT-AD neurons had an average firing rate (0.57 --+ 0.31 Hz) that was considerably slower than both LC neurons (1.69 - 0.26 Hz) and non-driven neurons in the L D T region (9.8 --- 1.37 Hz) (F = 7.18, df = 3, 98, P < 0.01; Newman-Keuls, P < 0.05 for all comparisons). Most notably, 75% of LDT-AD neurons were either not spontaneously active or had intermittent activity lower than 0.1 Hz (n = 13/17). Nearly all LC neurons and LDT-ND neurons were spontaneously active. Consistent with previous reports most LC neurons exhibited regular spontaneous activity between 0.2 and 4 Hz, and only 10% (2/19) were not spontaneously active 16'19'33. By contrast, LDT-ND neurons exhibited a wider distribution of spontaneous rates. About half (30/65) had moderate firing rates in the range of L D T - A D neurons (<5 Hz), while the remainder had spontaneous rates greater than 10 Hz (25/65). The slower neurons were located dorsal to the mlf while the cells with the faster firing rates tended to lie ventral to the mlf bordering the reticular formation. No differences were noted between chloral hydrate and urethane anesthesia with respect to the range of spontaneous activity or incidence of nonspontaneous cells.

Sensory responses LC neurons produced uniform biphasic burst-pause responses to brief hindpaw compression, consistent with previous reports 7'33. In contrast, L D T - A D neurons exhibited heterogeneous responses to hindpaw compression; 47% of the neurons increased, 41% decreased and 12% did not change their rate of activity. LDT-ND neurons exhibited a similar range of responses. In addition, both phasic and tonic response patterns were observed in both populations.

DISCUSSION Neurons in the medial mesopontine region an.tidromically activated from the anterior ~ u s could be separated into two populations based on the location of their recording sites. One population was found in the lateral dorsal tegmental nucleus (LDT-AD) and the second group was located in the locus coenfleus (LC-AD). The characteristics of L D T - A D neuronal activity will first be discussed in relation to surrounding neuronal populations especially the noradrenergie neurons of the LC. The relation of L D T - A D activity to Ch6 eholinergic neurons will then be examined.

Characteristics of LDT neurons and comparison to surrounding populations The low conduction velocity of LDT-AD neurons is consistent with anatomical descriptions of small diameter, unmyelinated axons ascending from L D T neurons and the presence of fine cholinergic terminal arborizations in the thalamus 36'5°'52. Others have reported that rat L D T neurons have low (<1 m/s) conduction velocities, but cat L D T neurons appear to have faster conduction velocities (1-8 m/s) 32"38'55. In particular, the very fast conducting (>4 m/s) subpopulation observed in the cat was not detected in this study 38-s5. The cat may therefore contain additional cell types not present in the rat. This also could reflect a general species trend as the conduction velocity of LC neurons in both cat and primate is higher than in rat ~3. There was a marked similarity in action potential waveform and axonal properties of LDT-AD and LC neurons (both AD and ND). Both types of neurons had long duration initially positive waveforms, but L D T - A D neurons consistently lacked the prominent notch on the ascending limb of the action potential seen in LC neurons. The initially positive long duration action potentials of L D T - A D and LC neurons were distinct from the waveforms of surrounding LDT-ND neurons, which were nearly a full millisecond shorter and occasionally had initially negative-going waveforms. Although extracellular waveshape is dependent on several factors (e.g. position of the electrode, electronic filtering), others have reported in both the rat and the cat that neurons in the L D T have long duration extracellular action potentials 32" 38. There was no significant difference between LDT-AD and LC-AD neurons with respect to other measures of axonal properties, e.g. conduction velocity, refractory period, chronaxie and stimulation threshold. Similarities in the extracellular properties of L D T and LC neurons have been noted by others in both rat and cat 32'3s. However, the intercalation of noradrenergic and cholinergic neurons across both the LC and L D T in the cat confounds inferences regarding neurotransmitter content based on histological position 31'59. Similarities between L D T and LC neurons have also been noted in intracellular recordings in vitro with respect to action potential and afterhyperpolarizati0n duration and intrinsic conductances 35. The most striking difference between L D T - A D cells and surrounding neurons was their respective firing patterns. Few L D T - A D neurons were spontaneously active, and many of those with spontaneous activity had highly erratic firing patterns. If the hunting stimulus had not unmasked the non-spontaneous cells, the L D T region would have appeared electrophysiologicaUy silent up through the M L R and entry into the reticular formation.

71 On the other hand, LC and raphe neurons had a characteristic slow and regular spontaneous firing rate whereas many non-antidromically driven LDT and more ventral reticular formation neurons had relatively fast firing rates. The absence of spontaneous activity of LDT-AD neurons here contrasts with the slow, tonic spontaneous firing attributed to LDT neurons in a previous study 32. Since that report did not provide a quantitative distribution of firing rates, it is possible that this discrepancy simply is due to their emphasis on the spontaneously active population. Non-spontaneous cells were observed in that study and the range of firing rates was the same as seen here. It is also possible that the continuous presentation of a 'hunting' stimulus in this study could have uncovered a greater number of non-spontaneous cells. It is unlikely that presence of the 'hunting' stimulus influenced the spontaneous firing rate of the cells as there was no difference in spontaneous fuing in the absence of stimulation. In particular, non-spontaneous cells never began to exhibit spontaneous activity when the 'hunting' stimulus was discontinued. Finally, LDT cells projecting to other forebrain sites may be more spontaneously active than cells projecting to the anterior thalamus. A more extensive study will be required to resolve this issue. It is probable that the spontaneous activity of LDT neurons is influenced by anesthesia. In unanesthetized cats the activity of thalamically projecting LDT neurons varied according to behavioral state and consisted of both slow (<2 Hz) and fast (>10 Hz) subpopulations 38' 55. Since the relation of LDT activity to behavioral state has important functional implications, it will be necessary to examine LDT activity in unanesthetized rats to determine if there are significant species differences. On the other hand, it is unlikely that LDT-AD activity was differentially influenced by the specific drug used as the anesthetic agent. In these experiments chloral hydrate and urethane anesthesia did not produce systematic differences in firing rate or other parameters of either LDT-AD or LC neurons. Precise specification of how individual anesthetics influence the activity of LDT-AD neurons will require both examination of a wider range of anesthetic agents and use of unanesthetized subjects. The diversity of response patterns to a sensory stimulus was the only heterogeneous characteristic of LDT-AD neurons. In this regard LDT-AD neurons are unlike LC neurons that have characteristic biphasic (burst-pause) response 7,33. Rather LDT-AD neurons resemble neurons in the surrounding reticular formation and periaqueductal gray that also exhibit heterogeneous response patterns to sensory stimuli 51. The varying responses of LDT-AD neurons may reflect the wider di-

versity of afferents received by these neurons compared to LC neurons, but the exact sources underlying this response diversity are unknown 2'52. This diversity also may indicate functional heterogeneity within the LDT. Are L D T - A D neurons cholinergic? The use of extracellular recording in this study precludes positive identification of the individual cell that gives rise to the recorded activity, but several lines of evidence support the hypothesis that LDT-AD neurons are cholinergic. Antidromic activation provides the most compelling evidence since anatomical studies have demonstrated that virtually all (>90%) of the projection from the LDT to the anterior thalamus originates from cholinergic neurons 24'5°'52'54. It is unlikely that antidromic activation was due to fibers of passage terminating in nonthalamic sites since the anterior thalamus contains only terminal ramifications of cholinergic neurons 24'36'52. Furthermore, the larger somas of the cholinergic neurons would favor their recording relative to the more parvicellular non-cholinergic population 24'5°'57. Thus, it is highly probable that LDT-AD cells are cholinergic. Other evidence reinforces the putative cholinergic status of LDT-AD cells. First is the location of all LDT-AD sites within clusters of cholinergic neurons, as visualized by NADPH-d histochemistry. Second, the homogeneous electrophysiological properties of LDT-AD neurons are consistent with a single underlying neuronal population. This is analogous to aminergic neurons that are homogeneous with respect to waveform, spontaneous activity, conduction velocity and other electrophysiological characteristics. Third, the long duration extracellular waveforms of LDT-AD neurons described here are compatible with the findings of in vitro studies where LDT neurons confirmed as cholinergic via intracellular dye marking generated long duration action potentials and afterpotentials35,63.

It remains to be determined whether other brainstem cholinergic neurons in the rat conform to the electrophysiological profile described here. Since nearly all LDT-AD neurons were silent, it is possible that additional non-spontaneous cholinergic populations exist in the LDT that have different electrophysiological characteristics. These covert populations might be unmasked by stimulation of non-thalamic LDT target areas, such as septum, basal forebrain, hypothalamus, and medullary reticular formation. Data are not presently available on the activity of thalamically projecting, hence cholinergic, PPT neurons in the rat, but no difference was reported between thalamically projecting neurons from the LDT and the PPT in the extracellular recordings in unanesthetized cats or in intracellular recordings from the LDT and PPT in guinea pig brain slices 35'aa'55.

72 The transmitter content of the non-antidromically activated LDT neurons cannot be established from these data, but it is likely that this population includes both cholinergic and non-cholinergic neurons. Unlike the LC that is composed entirely of noradrenergic neurons, not all LDT neurons are cholinergic 9'1°'24'3°'5°'52"57. Some non-antidromically activated neurons resembled LDT-AD neurons with respect to waveform and firing pattern, and thus could be cholinergic neurons which project to different target areas. Cells with other neurophysiological characteristics may derive from non-cholinergic populations. For example, cells firing in intermittent bursts were occasionally seen in this study, although none of the LDT-AD neurons exhibited such activity. In vitro intracellular studies have described neurons in the LDT that have a calcium-dependent bursting firing pattern, and intracellular dye labeling suggests these cells are not cholinergi c35'63. Recently populations of putative glutamatergic and GABAergic neurons have been described in the LDT 9'1°'3°. This raises the possibility that non-AD cells, such as the bursting neurons, may comprise a separate neurotransmitter population. The uniform characteristics noted in this study contrast with the wide range of electrophysiological characteristics attributed to putative cholinergic neurons in the basal forebrain 3A7,34'47,as'58. The greater electrophysiological heterogeneity in the basal forebrain neurons could reflect sampling from several different cell populations rather than a lack of a distinctive electrophysiological signature for cholinergic neurons. Identification of putative basal forebrain cholinergic neurons by antidromic activation from cortex or hippocampus may have been confounded by the significant proportion of non-cholinergic neurons contributing to those projections 4'12'15. It is worth noting that some investigators report that in both rats and primates many cortically projecting basal forebrain neurons, like LDT-AD neurons, were not

spontaneously active3A7'47. The identification of monoamine neurons was initially based on convergent, indirect evidence similar to that in this study. The extracellular identification of putative cholinergic neurons would be even more compelling, if the pharmacological techniques available for manipulating monoamine neurons were available for cholinergie neurons. For example, if animals could be treated with an agent that would selectively lesion cholinergic neurons, it should abolish single unit activity with the electrophysiological signature of cholinergic neurons proposed here. This parallels the use of selective neurotoxicants (e.g. 6-hydroxydopamine) to abolish presumed cateeholamine activity 5. Unfortunately, no sufficiently selective cholinergic neurotoxicant is presently

available, but the high interest in developing such an agent is promising 23"4°'49'61"62. Differential pharmacological sensitivity may provide another criterion for identifying putative cholinergic neurons. Monoamine neurons contain impulse-modulating autoreceptors, i.e. they are inhibited by their own transmitter and activated by antagonists at that receptor 5'6'23. This would allow the response to drugs acting at such autoreceptors to be used as an identification criterion 6" 19,29. Brainstem cholinergic neurons are hyperpolarized by acetylcholine in vitro suggesting that they possess impulse modulating autoreceptors 35. Preliminary findings in this laboratory indicate that local (iontophoretic) administration of acetylcholine in vivo decreases the activity of spontaneously active LDT neurons, whereas systemic administration of atropine (but not its quaternary derivative) increases the activity of LDT neurons (Grant, unpublished observations). On the other hand, acetylcholine activates noradrenergic and reticular formation neurons 22, and LDT neurons are insensitive to clonidine, an agonist at noradrenergic autoreceptors (Grant, unpublished observations). These differential pharmacological effects need to be systematically investigated to establish their utility for the identification of cholinergic neurons. In summary, this study examined the electrophysiological characteristics and anatomical distribution of LDT neurons antidromically activated from the anterior thalamus. Thalamically projecting neurons in the LDT were found to have a common set of electrophysiological characteristics. These neurons were considered to be putatively cholinergic based on previous anatomical demonstrations that virtually all thalamically projecting LDT neurons are cholinergic and the localization of recording sites in proximity to clusters of cholinergic neurons. Although their long duration, initially positive action potentials and slow conduction velocities resembles monoamine neurons, these putatively cholinergic neurons can be distinguished from adjacent noradrenergic neurons by their general lack of spontaneous activity and heterogeneous response to sensory stimuli. This study provides a foundation for exploring the functional correlates of brainstem cholinergic neurons by describing a set of characteristics that can be used in vivo to aid in their preliminary identification.

Acknowledgements. Drs. K. Campbell, G. Christoph, and T. Scott provided numerous helpful comments during the preparation of this manuscript. This research was supported by grants to S.G. from N.I.M.H. (MH45610), University of Delaware Research Foundation, Delaware Research Partnersehip and ICI Pharmaceuticals.

73 REFERENCES 1 Aghajanian, G., Foote, W. and Sheard, M., Action of psychotogenic drugs on single midbrain raphe neurons, J. Pharm. Exp. Ther., 171 (1970) 178-187. 2 Aston-Jones, G., Ennis, M., Pierobone, V., Nickell, W. and Shipley, M., The brain nucleus locus cocruleus: restricted afferent control of a broad efferent network, Science, 234 (1988) 734-738. 3 Aston-Jones, G., Shaver, R. and Dinan, T., Cortically projecting nucleus basalis neurons in rat are physiologically heterogeneous, Neurosci. Lett., 46 (1984) 19-24. 4 Brashear, H., Zaborszky, L. and Heimer, L., Distribution of gabaergic and cholinergic neurons in the rat diagonal band, Neuroscience, 17 (1986) 439--451. 5 Bunney, B., Waiters, J., Roth, R. and Aghajanian, G., Dopaminergie neurons: effects of antipsychotic drugs and amphetamine on single cell activity, J. Pharmac. Exp. Ther., 180 (1973) 560571. 6 Cedarbaum, J. and Aghajanian, G., Noradrenergic neurons of the locus coeruleus: inhibition by epinephrine and activation by the alpha-antagonist piperoxan, Brain Research, 112 (1976) 413--419. 7 Cedarbaum, J. and Aghajanian, G., Activation of locus coeruleus neurons by peripheral stimuli: modulation by a collateral inhibitory mechanism, Life Sci., 23 (1978) 1383-1392. 8 Chiodo, L., Dopamine containing neurons in the mammalian central nervous system: electrophysiology and pharmacology, Neurosci. Biobehav. Rev., 12 (1988) 49-91. 9 Clements, J.R. and Grant, S.J., Glutamate-like immunoreactivity in neurons of the laterodorsai tegmental and pedunculopontine nuclei, Neurosci. Lett., 120 (1990) 70-73. 10 Clements, J.R., Toth, D.D., Highfield, D.A. and Grant, S.J., Glutamate immunoreactivity is present within cholinergic neurons of the laterodorsai tegmentai and pedunculopontine nuclei. In I. Hanin, C. Napier and P. Kaiivas (Eds.), Basal Forebrain: Anatomy to Function, Raven, New York, in press. 11 Faiers, A. and Mogenson, G., Electrophysiologicai identification of neurons in the locus coeruleus,.Exp. Neurol., 53 (1976) 254-266. 12 Fisher, R., Buchwaid, N., Hull, C. and Levine, M., GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons, J. Comp. Neurol., 272 (1988) 489-502. 13 Foote, S., Bloom, E and Aston-Jones, G., Nucleus locus coeruleus: new evidence of anatomical and physiological specificity, Physiol. Rev., 63 (1983) 844-900. 14 Foster, K., Orona, E., Lambert, R. and Gabriel, M., Early and later acquisition of discriminative neuronal activity during differential conditioning in rabbits: specificity within the laminae of cingulate cortex and the anteroventrai thalamus, J. Comp. Physiol. Psychol., 94 (1980) 1069-1086. 15 Freund, T. and Antal, M., GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus, Nature, 336 (1988) 170-173. 16 Graham, A., Effects of amphetamine on single cell activity in a catecholamine nucleus, the locus coeruleus, Nature, 234 (1971) 100-102. 17 Grant, S. and Aston-Jones, G., Discharge properties of cortically projecting nucleus basalis neurons in behaving animals, Soc. Neurosci. Abstr., 12 (1986) 572. 18 Grant, S., Highfield, D. and Piser, T., Electrophysiology of putative Ch6 cholinergic neurons in the iaterodorsal tegmental nucleus, Soc. Neurosci. Abstr., 14 (1988) 367. 19 Grant, S., Huang, Y. and Redmond Jr., D.E., Benzodiazepines attenuate single unit activity in the locus coeruleus, Life Sci., 27 (1980) 2231-2236. 20 Grant, S. and Redmond, D., The neuroanatomy and pharmacology of the nucleus locus coeruleus. In H. Lal and S. Fielding

(Eds.), Psychopharmacology of Clonidine, Liss, New York, 1981, pp. 5-27. 21 Grzanna, R. and Molliver, M., The locus coeruleus in the rat: an immunohistochemical delineation, Neuroscience, 5 (1980) 21--40. 22 Guyenet, P. and Aghajanian, G., ACh, substance-P and metenkephaiin in the locus coeruleus: pharmacological evidence for independent sites of action, Eur. J. Pharmacol., 53 (1979) 319328. 23 Haigler, H. and Aghajanian, G., Lysergic acid diethylamidc and serotonin: a comparison of effects on serotonergic neurons and neurons containing a serotonergic input, J. Pharm. Exp. Ther., 188 (1974) 688-699. 24 Hailenger, A., Levey, A., Lee, H., Rye, D. and Wainer, B., The origins of cholinergic and other subcortical afferents to the thalamus in the rat, J. Comp. Neurol., 262 (1987) 105-124. 25 Hanin, I., Fisher, A., Hortnagl, H., Leventer, S., Potter, P. and Waish, T., Ethylcholine aziridinium (AF64A; ECMA) and other potential cholinergic neuron-specific neurotoxins. In H. Meltzer (Eds.), Psychopharmacology: The Third Generation of Progress, Raven, New York, 1987, pp. 341-349. 26 Haselton, J. and Guyenet, E, Electrophysiological characterization of putative C1 adrenergic neurons in the rat, Neuroscience, 30 (1989) 199-214. 27 Hobson, J., Lydic, R. and Baghdoyan, H., Evolving concepts of sleep cycle generation: from brain centers to neuronal populations, Behav. Brain Sci., 9 (1986) 371-448. 28 HSkfelt, T., Martensson, R., BjSrldund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine-hydroxylase immunoreactive neurons in the rat brain. In A. Bjtirklund and T. HSkfelt (Eds.), Handbook of Chemical Neuroanatomy: Classical Transmitters and Transmitter Receptors in the CNS, Part I, Elsevier, Amsterdam, 1984, pp. 277-379. 29 Jacobs, B., Central monoamine neurons: single unit studies in behaving animals. In H. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven, New York, 1987, pp. 159-170. 30 Jones, B., Relationship of GABA-ergic to cholinergie neurons within the laterodorsal and pedunculopontine tegmental nucleus, Soc. Neurosci. Abstr., 16 (1990) 300. 31 Jones, B. and Beaudet, A., Distribution of acetylcholine and catccholamine neurons in the cat brainstcm: a choline acetyitransferase and tyrosine hydroxylase immunohistochemical study, J. Comp. Neurol., 261 (1987) 15-32. 32 Kayama, Y. and Ogawa, T., Electrophysiology of ascending, possibly cholinergic neurons in the rat laterodorsai tegmentai nucleus: comparison with monoamine neurons, Neurosci. Lett., 77 (1987) 277-282. 33 Korf, J., Bunney, B. and Aghajanian, G., Noradrenergic neurons: morphine inhibition of spontaneous activity, Eur. J. Pharmacol., 25 (1974) 165-169. 34 Lamour, Y., Dutar, P., Rascol, O. and Jobert, A., Basal forebrain neurons projecting to the rat frontopartial cortex: electrophysiological and pharmacological properties, Brain Research, 362 (1986) 122-131. 35 Leonard, C. and Llinas, R., In vitro study of peduneulopontine neurons and ACh actions. In M. Steriade and D. Biesold (Eds.), Brain Cholinergic Systems, Oxford University Press, New York, 1990, in press. 36 Levey, A., Hallanger, A. and Wainer, B., Choline acetyltransferase immunoreactivity in the rat thalamus, J. Comp. NeuroL, 257 (1987) 317-332. 37 Lipski, J., Antidromic activation of neurones as an analytic tool in the study of the central nervous system, J. Neurosci. Meth., 4 (1981) 1-32. 38 Mansari, M.E., Sakai, K. and Jouvet, M., Unitary characteristics of presumptive cholinergic tegmental neurons during the sleep waking cycle in freely moving cats, Exp. Brain Res., 76 (1989) 519-529. 39 McCormick, D., Cholinergic and noradrenergic modulation of

74

40

41 42

43 44

45 46

47

48

49

50

51

52

thalamocortical processing, Trends Neurosci., 12 (1989) 315321. McGurk, S., Hartgraves, S., Kelley, P., Gordon, M. and Butcher, L., Is ethylcholine mustard aziridinium ion a specific cholinergic neurotoxin, Neuroscience, 22 (1987) 215-224. Meltzer, H. (Ed.), Psychopharmacology: The Third Generation of Progress, Raven, New York, 1987. Mesulam, M., Mufson, E., Wainer, B. and Levey, A., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 11851201. Nakamura, S., Some electrophysiological properties of neurons in the rat locus coeruleus, J. Physiol., 267 (1977) 641--658. Pare, D., Smith, Y., Parent, A. and Steriade, M., Projections of brainstem core cholinergic and non-ehofinergic neurons of eat to intralaminar and reticular thalamic nuclei, Neuroscience, 25 (1988) 68-86. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic, New York. Ranck, J., Extracellular stimulation. In M. Patterson and R. Kesner (Eds.), Electrical Stimulation Research Techniques, Academic, New York, 1981, pp. 1-36. Reiner, P., Semba, K., Fibiger, H. and McGeer, E., Physiological evidence for subpopulations of cortically projecting basal forebrain neurons in the anesthetized rat, Neuroscience, 20 (1987) 629--636. Richardson, R. and DeLong, M., Nucleus basalis of Meynert neuronal activity during a delayed response task in monkey, Brain Research, 399 (1986) 364--368. Robbins, T., Everitt, B., Ryan, C., Marston, H., Jones, G. and Page, K., Comparative effects of quisqualie and ibotenic acid induced lesions of the substantia innominata and globus pallidus on the acquisition of a conditional visual discrimination: differential effects on cholinergic mechanisms, Neuroscience, 28 (1989) 337-352. Rye, D., Saper, C., Lee, H. and Wainer, B., Peduneulopontine tegmental nucleus of the rat: cytoarehitecture, cytoehemistry, and some extrapyramidal connections of the mesopontine tegmentum, J. Comp. Neurol., 259 (1987) 483-528. Sanders, K., Klein, C., Mayer, T., Heym, C. and Handwerker, H., Differential effects of noxious and non-noxious input on neurons according to location in ventral periaqueductal grey or dorsal raphe nucleus, Brain Research, 186 (1980) 83-97. Satoh, K. and Fibiger, H., Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections, J.

Comp. Neurol., 253 (1986) 277-302. 53 Scherer-Singler, U., Vincent, S., Kimura, H. and McGeer, E., Demonstration of a unique population of neurons with NADPH diaphorase histochemistry, J. Neurosci. Meth., 9 (1983) 229234. 54 Sofroniew, M., Priestly, J., Consolazione, A., Eckenstein, E and Cuello, A., Cholinergic projections from the midbrain and pons to the thalamus in the rat, identified by combined retrograde tracing and choline acetyltransferase immunohistochemistry, Brain Research, 329 (1985) 213-223. 55 Steriade, M., Datta, S., Oakson, G. and Dossi, R.C., Neuronal activities in brain-stem cholinergic nuclei related to tonic activation processes in the thalamocortical systems, J. Neurosci., 10 (1990) 2541-2559. 56 Steriade, M. and McCarley, R.W., Brainstera Control of Wakefulness and Sleep, Plenum, New York, 499 pp. 57 Sutin, E. and Jacobowitz, D., Immunocytoehemical localization of peptides and other neurochemicals in the rat laterodorsal tegmental nucleus and adjacent area, J. Comp. Neurol., 270 (1988) 243-270. 58 Szymusiak, R. and McGinty, D., Sleep related neuronal discharge in the basal forebrain of cats, Brain Research, 370 (1986) 82-92. 59 Vincent, S. and Reiner, P., The immunohistoehemical localization of choline acetyltrans/erase in the cat brain, Brain Res. Bull., 18 (1987) 371-415. 60 Vincent, S., Satoh, K., Armstrong, D. and Fibiger, H., NADPH-diaphorase: a selective histochemical marker for the cholinergic neurons of the pontine reticular formation, Neurosci. Lett., 43 (1983) 31-36. 61 Webster, H. and Jones, B., Neurotoxic lesions of pontomesencephalic tegmentum-cholinergic cell area in the cat: I. Effects upon the eholinergic innervation of the brain, Brain Research, 451 (1988) 13-32. 62 Wenk, G., Cribbs, B. and McCall, L., Nucleus basalis magnocellularis: optimal coordinates for selective reduction of choline acetyltransferase in frontal neocortex by ibotenic acid injections, Exp. Brain Res., 56 (1984) 335-340. 63 Wilcox, K., Grant, S,, Burkhardt, B. and Christoph, G., Electrophysiological properties of lateral dorsal tegmental neurons in vitro, Brain Res. Bull., 22 (1989) 557-560. 64 Yim, C. and Mogenson, G., Electrophysiological studies of neurons in the ventral tegmental area of Tsai, Brain Research, 181 (1980) 301-313.