Relation of pontine choline acetyltransferase immunoreactive neurons with cells which increase discharge during REM sleep

&ajn Research Bulletin, Vol. 18, pp.447455.

CJPergamon Journals

Ltd.,

1987.Printedin the U.S.A.

0361~9230/87$3.00 + .OO

Relation of Pontine Choline Acetyltransferase Immunoreactive Neurons With Cells Which Increase Discharge During REM Sleep’ PRIYATTAM J. SHIROMANI,* DAVID M. ARMSTRONG,+ GORDON BRUCE,5 LOUIS B. HERSH,8 PHILIP M. GROVES? AND J. CHRISTIAN GILLIN*

*VA Medical Center, San Diego, and Department of Psychiatry, University of California San Diego, CA 92093 tDepartment of Psychiatry and SNeurosciences, University of California San Diego, CA 92093 8Department of Biochemistry, The University of Texas Health Science Center at Dallas Dallas, TX 75235 Received

29 August 1986

SHIROMANI, P. J., D. M. ARMSTRONG, G. BRUCE, L. B. HERSH, P. M. GROVES AND J. C. GILLIN. Relation of gontine choline acetyltransferase immunoreactive neurons with cells which increase discharge during REM sleep. BRAIN RES BULL 18(3) 447-455, 1987.-The purpose of this study was to determine whether neurons in the medial pontine reticular formation with high discharge rates during REM sleep could be localized in regions of the brainstem having neurons displaying choline acetyltransferase immunoreactivity. Six cats were implanted with sleep recording electrodes and microwires to record extracellular potentials of neurons in the pontine reticular formation. Single-units with a S:N ratio greater than 2: 1 were recorded for at least two REM sleep cycles. A total of 49 units was recorded from the pontine reticular formation at medial-lateral planes ranging from 0.8 to 3.7 mm. The greatest proportion of the units (28.6%) showed highest discharge during active waking and phasic REM sleep compared to quiet waking, non-REM sleep, transition into REM sleep or quiet REM sleep periods. A percentage (20.4%) of the cells had high discharge associated with phasic REM sleep periods while 8.2% of the cells showed a progressive increase in discharge from waking to REM sleep. Subsequent examination of the distribution of choline acetyltransferase immunoreactive cells in the PRF revealed that cells showing high discharge during REM sleep were not localized near presumed cholinergic neurons. Indeed, we did not find any ChAT immunoreactive somata in the medial PRF, an area which has traditionally been implicated in the generation of REM sleep. These results suggest that while increased discharge of PRF cells may be instrumental to REM sleep generation, these cells are not cholinergic. Cats

REM sleep

Choline acetyltransferase

immunoreactivity

Pontine reticular formation

majority of medial PRF neurons subserve both REM sleep and motor functions [47]. Nevertheless, Sakai [36] has found some cells in the medial PRF which progressively increase discharge from waking to REM sleep. While most medial PRF neurons increase discharge during waking and REM sleep, there are cells in the; locus coeruleus (LC) and dorsal raphe nucleus (DRN) that decrease or cease fling only during REM sleep [9, 10,281. The divergent firing profiles of LC, DRN and medial PRF neurons form the basis of the reciprocal-interaction hypothesis of REM sleep generation [ 17, 18,271. This theory postulates that decreased activity of noradrenergic LC and serotonergic DRN neurons permits activation of medial PRF

TRANSECTION [22, 49, SO]and lesion [21,22] studies have shown that the medial pontine reticular formation (PRF) plays an important role in the generation of rapid eye movement sleep (REMS). This region corresponds to nucleus pontis oralis and caudalis in the rat brain, while in the cat, using Berman’s [7] terminology, the medial PRF corresponds to the regions designated as the lateral, paralemniscal and gigantocellular tegmental fields between the abducens and mesencephalic trigeminal nuclei. Single-unit recording studies have shown that most medial PRF neurons increase discharge during REM sleep compared to non-REM sleep [17,27,45,46,48,53,54]. However, many of these neurons also show high discharge during waking, suggesting that a

‘Supported by The American Narcolepsy Association (P.J.S.), VAMC research (J.C.G.), NIMH-38738 (J.C.G.), NIH AG-05344 (D.M.A.), NIDA DA 00079 (P.M.G.) and NIH AG-05893 (L.B.H.).

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AWOWNon

REM

REM Tnn

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Non REM

REM Tra”

Sleep-Wake

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States

FIG. 1. Sleep-state related discharge rates of 49 PRF units. Unit discharge was monitored during waking with movement (W), quiet waking without movement (QW), non-REM sleep without PGO waves (Non-REM), the one minute period before REM sleep onset when PGO waves were present (REM Tran), REM sleep periods without rapid-eye movements or PGO waves (QR), and REM sleep periods with abundant rapid-eye movements and PGO waves (Phasic REMS). The majority of units (Group A) showed highest discharge during both active waking and phasic REM sleep. There were units which showed an increase in discharge during REM sleep compared to the other states (Group D).

neurons, which is instrumental to REM sleep generation. Anatomical and pharmacological studies support the presumed neurochemical interactions of LC and DRN neurons as postulated by the reciprocal-interaction theory [9, 10, 14,281. However, the neurotransmitter identity of the medial PRF neurons is not clear. Pha~acologica~ studies have suggested that medial PRF neurons involved in REM sleep control may be cholinergic or at least cholinoceptive. For example, microinfusion of cholinergic agonists, such as neosti~~ne, carbachol, or bethanechol into the medial PRF readily elicits REM sleep [l, 4-6, 15, 19, 23, 31, 41-44, 511, and some medial PRF neurons increase discharge before and in conjunction with carbacho1-induced REM sleep [#I. However, these studies only indicate that medial PRF neurons are sensitive to acetylcholine and are therefore, likely to be cholinoceptive. Kimura et al. [24] have suggested that some PRF neurons are cholinergic. The relationship between these cells and neurons demonstrating high discharge rates during REM sleep, however, is not known. Such evidence would provide much stronger support for a link between cholinergic transmission and REM sleep. Therefore, in this study we used the extracellular recording method in freely behaving cats to de-

termine whether cells with high discharge during REM sleep are localized in regions of the PRF which also contain neurons immunoreactive for the cholinergic biosynthetic enzyme, choline acetyltransferase. METHOD

Six adult cats were implanted under halothane anesthesia with a standard array of sleep electrodes to record EEG, EOG, EMG and tripolar depth electrodes in the lateral geniculate nucleus to record ponto-geniculate-occipital (PGO) waves. One or two barrel microdrives were targeted into both left and right sides of the pontine tegmentum at 30” or 40” from the frontal plane. The construction of the microdrives was as previously described [ 161. Briefly, each microdrive consisted of one or two 38 mm length, 24 gauge, thin-wall stainfess steel tubes spaced 1 mm apart supported by guide cannulae mounted on the skull. A screw-nut assembly permitted vertical movement of the inner cannula. Each barrel of the microdrive housed four to seven microwires (32 ,u) which extended 6 mm beyond the cannula tip. At least two weeks were allowed for recovery from surgery. During each experimental session, the microdrives

ChAT CELLS IN THE PRF were advanced until a stable single neuron recording with a signahnoise ratio greater than 2:l was obtained. The polygraph was then switched on and sleep and unit recordings were obtained until the end of at least two REM sleep cycles. Subsequently, the discharge rate of the unit was monitored during a behavioral test session. During this session various portions of the cat’s face and body were massaged and stroked to determine if the unit was related to sensory stimulation. Unit discharge was also monitored during walking, eating, drinking or grooming to determine the relationship of unit discharge with motor acts. Single neuron action potentials were detected by passing the microwire signals through ~erential amplifiers, filters and an adjustable window discriminator. The triggered output from the window discriminator was then displayed on the polygraph and also fed into an Apple-Isaac computer system which provided counts every 10 seconds. After completing the full dorso-ventral excursion of the microdrive, microwire positions were determined by making anodal lesions (150 PA, 10 set) through each recorded microwire. All lesions were made while the animals were deeply anesthetized with Nembutal. Subsequently, the animals were perfused through the heart with a phosphate buffered 4% formaIdehyde solution and the brains quickly removed and placed in the buffered formaldehyde solution for later histological analysis. Unit Data Analysis

Unit activity was examined during the following six conditions: active waking, quiet waking, non-REM sleep, one minute period before REM sleep, tonic REM sleep periods with no eye movements or PGO activity, and phasic REM sleep with considerable eye movements and PGO activity. Mean neuronal tiring rate during each condition was determined by taking the average of six 10 second epochs distributed evenly throughout the sleep recording period. Unit discharge during the active waking condition was assessed during portions of the behavioral test session in order to obtain peak firing rates related to motor or sensory events. Units were placed into one of six groups on the basis of well defined strict criteria. In group A unit discharge had to be 50% higher in active waking and phasic REM sleep compared to the other states. In group B unit discharge during active waking had to be 50% higher compared to the other states while in group C, unit discharge during phasic REM sleep had to be 50% higher compared to the other states. Units with a profile suggestive of a progressive increase in discharge from active waking to phasic REM sleep were assigned to group D while group E units had to show a progressive decrease in discharge from waking to phasic REM sleep. Group F was comprised of units which did not meet the requirements of the other groups. In each group a repeated measures ANOVA with Neuman-Keuls post-hoc test was used to analyze the data. Choline Acetyitransferase

Staining Procedure

Details of the production and characterization of the anti-ChAT antiserum have been recently described [8]. In brief, 150 fig of electrophoretically pure human placental ChAT was emulsified in complete Freund’s adjuvant and injected directly into the popliteal lymph nodes of New Zealand white rabbits. After four weeks a booster injection of 150 pg of the protein was given in the thick part of the skin above the scapula and in the neck region, followed by a

449

second boost after an additional 21 days. Seven days after the last injection, the rabbits were bled, and the immunoglobulin fraction was purified on a DEAE cellulose column. Anti-ChAT antiserum was adsorbed against the purified enzyme as follows: highly purified human placental ChAT (30 pg; >80% pure) was subjected to SDS-PAGE, and electrophoreticahy transferred to nitrocellulose paper. Strips of nitrocellulose containing ChAT or BSA (the former allocated by its co-migration with BSA and reaction with antiChAT antisera) were cut out and incubated with the anti-ChAT antiserum overnight at 4°C. The efftcacy of adsorption was confirmed by demons~ti~ the inability of this antiserum to visualiie ChAT in a Western blot procedure compared to untreated antiserum or to antiserum adsorbed against BSA. The polyclonal anti-human placental ChAT antibody was shown to react mono-specifically with ChAT. In addition, our recent immunohistochemical studies demonstrated that within the rat the distribution and general cytological features of cholinergic neurons labeled with this polyclonal antibody [31 were identical to those of cholinergic neurons immonolabelled with monoclonal anti-ChAT antibodies [2]. Further specificity of this polyclonal antibody was confirmed by the absence of immunolabelled profiles in tissue sections incubated with non-immune serum or when the antiserum was previously adsorbed with microgram concentrations of pure ChAT protein. In this study the following i~un~ytochemic~ labelling procedure was used. Brains of perfused cats were sectioned on a Vibratome (4 cats, sag&al sections 50 pm thick) or a freezing microtome (2 cats, sagittal sections 30 pm thick) and transferred to spot-test plates containing 0.1 M PO, buffer in preparation for the avidin-biotin immunocytochemical labelling procedure. This procedure consisted of the following series of steps: (a) 48 hour incubation with 1~500dilution of primary antiserum to ChAT or control serum. Dilutions of the primary antiserum were prepared with 1% goat serum and 0.25% T&on X-100 in 0.1 M T&-buffer; (b) One hour incubation with biotinylated goat anti-rabbit IgG (Vector) dihtted I:200 with 1%goat serum in Tris-saiine; (c) One hour incubation with ABC complex (Vectors diluted 1:lOOwith 1% goat serum in Tris-saline. Following each incubation the sections were rinsed with 1% goat semm in Tris-saline. The sections were then treated for 6 min with 0.05% solution of 3,3’-diaminobenzidine, 0.01% hydrogen peroxide and 0.04% nickel chloride to yield a dark reaction product. Subsequently, the sections were rinsed in phosphate buffer. Following the diaminobenzidine reaction the sections were mounted on glass slides and examined and photographed with a Zeiss microscope. The figures were prepared by drawing the outline of the tissue sections with an overhead projector and then plotting with a drawing tube attached to the Zeiss microscope the location of immunoreactive neurons onto these outlines. ~istol~ic~ verification of microwire placements were made by examitiing adjacent sections stained with cresyl violet and consulting Berman’s atlas [7]. RESULTS Single Unit Data

Forty-nine single units were recorded. The units were placed into one of six groups depending upon the sleep state related pattern of discharge. Figure 1 summarizes the discharge rates of units in each group. The greatest proportion of units (Group A, 28.6%) showed significantly higher dis-

L = 2.5

L = 2.9 I

-I

L = 1.9

L = 2.3

AGroup& oGroup B,

l C,

*E

oD,

) F

FIG. 2. Dist~bution of choline acetyltransferase cont~ning cells (shown by stipling) in relationship with the recorded cells. The symbols used to represent each group are identical to those used in Fig. 1. Note that two recorded units which showed a decreased discharge rate from waking to REM sleep were not included in this figure because accurate histological placement could not be made. The figures were derived from camera htcida drawings of sag&al sections of the cat brainstem. The medial-lateral positions are based on Berman’s stereotaxic atlas of the cat brainstem. Note that at L=3.7 recorded units were localized near ChAT cells of the motor trigeminal nucleus. These units, however, did not show a high discharge associated with REM sleep. Abbreviations: 3=occuiomotor nucleus, 4=trochlear nucleus, 4N=trochlear nerve, 6=abducens nucleus, 7=facial nucleus, 7G=genu of the facial nerve, 7N=facial nerve, nucleus dorsal division, SMD=motor trigeminal SM=motor trigeminal nucleus, 5ME=mesencephahc trigeminal nucleus, SMET=mesencephalic trigeminal tract, 6=abducens nucleus, BC=brachium conjunctivum, CNF=cuneiform nucleus, FTC=central tegmental field, tegmental field. LC=locus ~G=~gantoce~ul~ tegmental field, FTP=paralemniscal coerufeus, LDT=lateral dorsal tegmental cholinergic group, PGM=pontine gray medial division, PGR=pontine gray rostroventral division, PGL=pontine gray lateral division, SA=stria olive medial division, acustica, SOL=superior olive lateral division, SOM=superior TB=trapezoid body, TRC=tegmental reticular nucfeus central division, TRP=tegmental reticular nucleus pericentral division, VLD=lateral vestibular nucleus dorsal division, VLV=lateral vestibular nucleus ventral division.

FACING PAGE FIG. 3. Distribution of choline acetyltransferase containing cells in the medial pontine reticular formation (sagittal sections at lateral= 1.6). The top panel (A) depicts sections stained with cresyl violet and the inset (A’) shows close-ups of giant cells in the gigantocellular tegmental field. Panel B depicts an adjacent section with choline acetyhransferase containing cells in the lateral dorsal tegmental nucleus (LTD), the pedunculo-pontine group (PPG) and nucleus of the sixth nerve 161. Note the lack of ChAT iabelling in the FTG. Inserts C and D depict close-ups of ChAT immunoreactive cell bodies in the PPG and the sixth nerve, respectively. Abbrevia~ons: lV=fourth ventricle, 6=abducens nucleus, 7g=genu of the facial nerve, ~G=gigant~ellular tegmentd field, LDT=lateral dorsal tegmental cholinergic group. PPG=pedunculo pontine cholinergic group.

ChAT CELLS IN THE PRF

FIG. 3.

451

FIG. 4. The relation of ChAT immunoreactive neurons with microwire placements in the pontine reticular formation. Panel A depicts a sagittal section stained with cresyl violet. The arrow depicts microwire tract. Panel B depicts an adjacent sagittal section containing immunoreactive ChAT cell bodies and processes. Note that there are no ChAT cells in the immediate vicinity of the microwire track. Insert B’ is a close-up of the tip of the microwire tract. The staining at the top represents peroxidanr reactivity in the coagulated blood. Abbreviations are same as in Figs. 2 and 3.

ChAT CELLS IN THE PRF

charge during both active waking and phasic REM sleep compared to the other states, F(5,65)=10.04, p~O.05. This pattern of discharge is typical of most medial PRF neurons [45, 46, 48, 53, 541. A percentage (14.3%) of units (Group B) showed signi& cantly higher discharge during active waking compared to the other states (Group B), F(5,30)=8.30, p-=0.05. All of these neurons discharged in association with gross motor events such as walking or head turn. We did not encounter any cells discharging only in response to specific sensory stimulation. We note, however, that often it was difficult to separate sensory from motor related discharge because when the cat was stroked it would invariably move its head and forelimbs. A percentage (20.4%) of cells (Group C) showed significantly increased firing during phasic REM sleep compared to the other states, F(5,45)=26.27,~<0.05. The activity of some of these units was related to the occurrence of eye movements. However, the intensity of firing was much less during waking than during phasic REM sleep. A percentage (12.2%) of cells (Group E) showed a gradual decrease in fling from waking to REM sleep. These units displayed a regular discharge rate (about one to two spikes per second) during waking and non-REM sleep. A percentage (8.2%) of units (Group D) showed a progressive increase in discharge from waking to REM sleep. All of these units had lowest discharge during waking and, in fact, two units ceased discharging in association with gross motor acts. During REM sleep this group of cells displayed a tonic tiring pattern with sharp increases in discharge occurring in phasic REM sleep. The remaining group (Group F) contained cells which showed no clearly evident state-related discharge pattern (16.3%). tmmunohistochemistty Figure 2 summarizes the distribution of ChAT immunoreactive neurons in relation to the recorded units. A major cluster of ChAT containing cells was located in the anterior-posterior plane contiguous with the brachium conjunctivum. This group represents the pedunculo-pontine group (PPG). A second group of smaller, more closely packed, cells were located below the floor of the fourth ventricle just dorsal to the locus coeruleus. In the coronal plane this group of cells lies medial to the locus coeruleus. This group has been noted by several investigators [2,24,29] and is called the lateral dorsal tegmental (LDT) group. The medial PRF between the locus coeruleus and the nucleus of the sixth nerve did not contain any cholinergic neuronal somata (see Fig. 3). However, cholinergic cells were noted in the nuclei of the 3,4,5,6, and 7 cranial nerves. The most densely packed cells were noted in the nucleus of the seventh nerve. A few lightly stained scattered cells were located in the dorsal and lateral divisions of the vestibular nucleus. Only two units were found to be localized near ChAT containing cells in the motor nucleus of the fifth nerve. One of these units fired most frequently during waking while the other unit was assigned to the miscellaneous group. Figure 4 depicts photomicrographs of ChAT immunoreactive neurons in the PPG and LDT and the relation of ChAT cells with a microwire used to record neuronal activity. DISCUSSION

We found a major collection of ChAT immunoreactive neurons in the PPG and LDT groups. This is the largest

453 collection of cholinergic cells in the pontine reticular formation [2,24,29] and it has been suggested that ceils from these two groups form the ascending dorsal cholinergic pathway which innervates the thalamus, hippocampus, hypothalamus and cingulate cortex [29, 37-39, 52, 55, 571. The PPG and LDT may also comprise the ascending reticular activating system described by Moruzzi and Magoun [32]. ChAT containing cells were also found in the nuclei of the 3,4,5,6, and 7 cranial nerves. The distribution of ChAT neurons in this study is similar to the findings by other investigators in the cat [24],rat [2,29] and non-human primate [33,40]. Thus, it is clear that our immunohistochemical procedures were able to delineate cholinergic cell populations described by other investigators. We did not fmd any ChAT immunoreactive neurons in the medial PRF, an area which has traditionally been implicated in the generation of REM sleep [223. Electrolytic lesions in the medial PRF have been shown to totally eliminate REM sleep [13, 21, 221 and it has been suggested that the medial PRF contains cells which increase their discharge rate to trigger REM sleep [17, 18, 271. On the basis of data from pharmacological studies it has been suggested that the medial PRF cells instrumental to REM sleep are cholinergic or at least cholinoceptive [18,20]. In the present study we found that medial PRF cells showed a heterogenous sleep-state related firing pattern. The largest number of the neurons (28.6%) demonstrated highest firing rates during active waking and phasic REM sleep. This pattern of discharge has been found to be typical of most PRF cells [45, 46, 48, 53, 541. We also noted two groups of cells with high discharge during active waking and phasic REM sleep, respectively. These findings are consistent with other reports [45, 46, 48, 53, 541. We found some cells (8%) that showed a progressive increase in tiring from waking to REM sleep. The REM sleep-related discharge pattern of this group suggests that this cell type may be involved in REM sleep generation. The distribution and discharge pattern of this cell type is similar to that reported by Sakai [36]. Indeed, Sakai [36] also noted that only a small percentage of cells in the medial PRF showed a progressive increase from waking to REM sleep. We used immunohistochemical methods to determine whether cells with high discharge during REM sleep were localized to areas with ChAT immunoreactive neurons. Our results showed that these cells were not localized within or near ChAT containing somata. Indeed, we did not find any ChAT immunoreactive neurons in the medial PRF, thus suggesting that the cells in the medial PRF involved in the generation of REM sleep are not cholinergic. On the other hand, infusions of cholinergic agonists into the medial PRF readily trigger REM sleep [ 1,4-6, 15, 19,23, 31, 41-44, 511, and autoradiographic studies have demonstrated the presence of muscarinic receptors in this region [56]. Therefore, medial PRF neurons sensitive to acetylcholine may be responsible for REM sleep generation. The neurotransmitter identity of the cholinoceptive medial PRF neurons is not known. An examination of the distribution of catecholamines and serotonin, neurotransmitters which have traditionally been implicated in sleep and REM sleep along with acetylcholine, indicates that these neurons are not densely represented in the medial PRF [30,34]. Vasoactive intestinal peptide (VIP) which has been shown to coexist with acetylcholine in cholinergic neurons [26] and which also increases REM sleep [ 11,351 is not present in the medial PRF [12,25].

SHIROMAN

454 It is important to determine the source of the cholinergic input and the projection sites of the medial PRF neurons. Data from transection studies indicate that the neuronal network responsible for REM sleep resides within the PRF [22, 49. 501. One possibility is that ChAT containing cells in the

1 f;l .-\I

PPG and LDT may stimulate cholinoceptlve medial PRF neurons. A progressive buildup of activity in the medial PRF may then trigger the discrete neuronal cell groups responsible for the tonic and phasic components of REM sleep.

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