Brain Research

Bulletin,

Vol. 4, pp. 519-534. Printed

in the

U.S.A.

Raphe Projections to the Locus Coeruleus in the Rat ly2 PETER J. MORGANE Worcester

AND M. S. JACOBS

Foundation for Experimental Biology, Shrewsbury, New York University, New York, NY

MA and

Received 21 April 1979 MORGANE, P. J. AND M. S. JACOBS. Raphe projections to the locus coeruleus in the rat. BRAIN RES. BULL. 4(4) 519534, 1979.-Afferent projections to the locus coeruleus from the various raphe nuclei, particularly of the midbrain (nuclei raphe dorsalis and medianus) and pons (nuclei raphe pontis and magnus), have been studied in the rat by retrograde transport methods using horseradish peroxidase (HRP). The locus coeruleus, in both its dorsomedial and ventrolateral divisions, and in its various anterior-posterior components, were injected with 0.05 ~1 of horseradish peroxidase following which various structures of the brainstem, particularly the raphe nuclei, were examined for HRP reactive cells. It was found that injections in most components of the locus coeruleus were associated with HRP positive cells in varying degrees of density in the nuclei raphe dorsalis, medianus, pontis, and magnus, with considerably sparser labelhng in the anterior aspects of the medullary raphe nuclei pallidus and obscurus. Labelled cells were also seen in the nuclei of the solitary tract, contralateral locus coeruleus, lateral reticular areas of the pons and midbrain, nuclei pontis oralis and caudahs, vestibular nuclei, mesencephalic nucleus of the trigeminal nerve, fastigial nuclei of cerebellum and medial parabrachial nuclei. These data, showing widespread innervation of the locus coeruleus from all raphe nuclei, as well as many other brainstem areas, in the rat support the general view of heavy innervation of the locus coeruleus from both extra-raphe and raphe nulcei. These latter raphe projections, probably serotonergic in nature, provide anatomical support for the various experiments indicating considerable regulation of locus coeruleus activities, such as phasic events of REM sleep, among others, by most of the raphe nuclei. Thus, various activities of the locus coeruleus could be modulated or regulated by widespread projections from most raphe nuclei as well as several other regions of the brainstem. Locus coeruleus afferents Raphe efferents Horseradish peroxidase Locus coeruleus Raphe nuclei REM sleep PGO spikes Retrograde transport Noradrenergic systems Serotonergic innervation Locus coeruleus-raphe interactions

of the interconnections between the raphe nuclei and locus coeruleus is important in determining the roles of these aminergic formations in a wide variety of behaviors. The general layout of fluorescence maps of aminergic pathways in the brain have to date not been of sufficient resolution to work out the specific details of chemical anatomy at a nuclear level. For these more intimate details of connectivity axon transport studies appear to be the best means of unravelling these relations. In the present study we have concentrated on the serotonin fiber systems projecting from several raphe nuclei of the midbrain and pons onto the locus coeruleus in the rat. Thus, horseradish peroxidase (HRP) has been injected into several areas of the locus coeruleus and the labelling of cells in the midbrain (nucleus raphe dorsahs and nucleus raphe medianus) and pontine raphe nuclei (nucleus raphe pontis and nucleus raphe magnus) studied. In recent years it has been put forward that oscillations in activity between these areas play vital roles in the regulation of sleep cycles [19,201 and studies by Simon et al. [%I, among others, have indicated that raphe inputs to the locus coeruleus control some key phasic events of REM sleep such as ponto-geniculo-occipital (PGO) spikes. Some continuing questions now under study in various AN understanding

laboratories

relate to the arrangement

of serotonergic

termi-

nals in the locus coeruleus with respect to organization of the different nuclei of the raphe system, especially seeking to determine the anatomical origin of the different kinds of serotonergic terminals observed in the locus coeruleus [14,291. If we consider the existence of several kinds of serotonergic terminals in the locus coeruleus then it is likely that different serotonergic mechanisms are to be found in the locus coeruleus. Considering, for example, the changes in tyrosine hydroxylase activity observed in the locus coeruleus and in the cerebral cortex after 5,6-dihydroxytryptamine administration, it seems likely that more than one regulatory process could occur in these conditions, giving some explanation of the two tyrosine hydroxylase activity peaks observed at the terminals. Thus, it has been hypothesized that the different kinds of serotonergic terminals could control different aspects of the functional activity of catecholamine cells of the locus coeruleus. Also, there might be some serotonergic control of the firing of some catecholaminergic-containing cells, which could be mediated by specific terminals, whereas other terminals may be involved in the regulation of cellular metabolism of catecholaminergic cells by some more diffuse neurohumoral actions. Neurophysiological studies provide considerable evidence for a physiological function for the serotonergiccatecholaminergic interactions. For example, impairment of

‘Supported by National Science Foundation Grant BNS-77-16512. “Reprint requests to: Dr. P. J. Morgane, Worcester Foundation for Experimental

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o 1979 ANKHO International

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catecholaminergic synthesis reverses the continuous desynchronization following destruction of the raphe system. Another major role for these interactions seems to depend on tonic endogenous variations in the activity of the two aminergic systems, which could possibly represent a basis for the production of tonic functional variations. in this regard we can consider the circadian aspects of modulation of the sleep-wake cycle, the problems posed by rebound following REM sleep deprivation, or certain pathological changes in sleep-waking regulation. The specificity of the regulatory mechanisms observed makes the hypothesis of a general balance between serotonin and catecholamine modulations doubtful. Accordingly. the interaction between serotonin and norepinephrine must be evaluated more in terms of a series of specific regulatory mechanisms. Considering the complexity of the anatomical circuitry interconnecting the different aminergic groups with other neuronal groups, it becomes easier to appreciate the difficulties of evaluating the interactions between different neurotransmitters in the brain. Such transmitter interaction studies have now become an essential approach in functional neurochemistry. As indicated above, interactions between serotonergic and noradrenergic neurons in the brain have been proposed on the basis of a wide variety of neurophysiological, neuropharmacolo~cai, and neurobiochemical studies. Some of these relevant to the present studies will be briefly reviewed. Conrad et ul. [IO], using anterograde labelling and degeneration studies, demonstrated that both the nucleus raphe dorsalis and nucleus raphe medianus project to the locus coeruieus in the rat. The reciprocal connections from the locus coeruleus to raphe were demonstrated by Loizou 1321, who lesioned the locus coeruleus in rats and traced fluorescence changes to the nucleus raphe dorsalis. Sakai Pt al. i.521, using retrograde transport techniques, traced various afferent fiber systems to the locus coeruleus complex in the cat. They studied most of the raphe nuclei following injection of horseradish peroxidase into the dorsomedial and ventrolateral divisions of the locus coeruleus. Strong HRP labelling was seen in the nucleus raphe dorsalis after injection of HRP into the principal or dorsomedial locus coeruleus. Even more HRP labelling was seen in the nucleus raphe dorsalis after injection of HRP into the ventrolateral Iocus coeruleus. The nucleus raphe pontis also was shown to project strongly to ventrolateral locus coeruleus but not at all to the principal (dorsomedial) locus coeruleus. The nucleus raphe magnus was found to project slightly to the ventrolatera1 but not dorsomedial locus coeruleus. The nucleus raphe medianus was not found to project to any part of the locus coeruleus complex in the cat. Bobillier et ul. [4], using anterograde axon transport methods in the cat, showed highly differential projections to the locus coeruleus from the nuclei raphe dorsalis, medianus, magnus, and pontis after selective injection of radiolabel into the different raphe nuclei. By these techniques they demonstrated that UN the midbrain and pontine raphe project to the locus coeruleus but they did not attempt to divide the locus coeruleus into component divisions. Bobillier et al. [Zj also studied the nucleus raphe medianus projections in the rat using anterograde axon transport methods. They found this midbrain raphe nucleus to project rather strongly to the nucleus locus coeruleus in its entire rostro-caudal extension. Sakai it al. [53,.541 later considerably extended their studies of locus coeruleus afferents in the cat by use of HRP injected into specific divisions of the coeruleus complex. Using these techniques with topographic

preciseness. they demonstrated strong projrctiour to I he ventrolateral Iocus coeruleus from the nuclei raphe dorsaiis and pontis and light projections from the nucleus raphe magnus but could demonstrate no projections from the nucleus raphe medianus to the locus coerulells. The nucleus raphe dorsalis was the only raphe nucleus showing HRP reacti\;e neurons following injection of HRP into the dorsomedial locus coeruleus. These studies dcnlonstrated consid~r~~hlc hctc~rog~twify in the locus coeruleuy projection:, fiom X,Xious midbrain and pontine raphe nuzlci. Interestingly. as opposed to the findings of Bobillier V: trl. 141. no projections from the nucleus raphe medianus I(, either division of the locus coeruleus were found. To study retiprocitie\ of these connections Sakai et r/l. [ 511. in a retrograde transport study in the cat following injection of the nucleus rapho dorsalis with HRP, demonstrated projections f‘rom the principle Idorsomedial) and ventrolateral parts oi’ the locus cocrufeuc to the nucleus raphe dorsalis. Howt’\er. no attempts were made to specifically inject the other raphe nuclei to study the locus coeruleus projections to each nucleus. Finally. our group (Pasquier CJ/rrl. 1381). utilizing horseradish peroxidase tracing techniques, found intensive l~{beIIing in the nucieus raphe dorsalis in the rat after HRP injections centered on the dorsomedial part of the Iocus coeruleus. No reciprocal projection was clearly demonstrated, only one HRP reactive cell being seen in the locus coeruleus after injection of HRP into the nucleus raphe dorsalis. Pickel ct (11.139,401 have pointed out that light rmcruscopy cannot clearly distinguish axon terminals or demonstrate a specific neurotransmitter within labeled nxonc. Therefore, they sought to obtain direct ~Iltrast~tctLlrai etidence for serotonergic innervation of noradrenergic neurons of the nucleus locus coeruleus in the rat brain. This was accomplished by immunocytochemical localization by light and electron microscopy of antibodies to the neurotransmitter-synthesizing enzymes tyrosine hydroxylase and tryptophan hydroxylase. specific protein markers for catecholamine and serotonergic neurons. respectively. The immunocytochemical localization of tryptophan hydroxylase and tyrosine hydroxylase, specific biochemical markers for serotonin and ;atecholamine-containing neurons. respectively, demonstrated that noradrenergic neurons of the nucleus locus coeruleus are innervated by processes of serotonergic neurons. By fight and electron microscopy, tyrosine hydroxylase was IocaIized to the cytoplasm of the perikarya and to the proximal axonal and dendritic processes of intrinsic neurons. Tryptophan hvdroxylase, on the other hand, was contained only withln unmyelinated processes surrounding the perikarya and dendrites of catecholaminergic neurons. It was concluded that the neuronal processes in the locus coeruleus that contain tryptophan hydroxylase probably represent the axons and axon terminals of serotonergic neurons. since they are morphologically comparable to processes a~itoradio~raphica~Iy labeled for serotonergic neurons in cerebral cortex. These immLinocytochemj~1 demonstrations of innervatjon of the locus coeruleus by serotonergic axons provide ultrastructural evidence to support the contention that the activity of noradrenergic neurons is probably directly modulated b> serotonergic fibers. Leger and Descwrries [291 and Descarries and J_,eger [I4], using high resolution autoradiographic techniques for the identification of serotonin neuruns labelIed with exogenous tritiated serotonin, analyzed the distribution and fine structural features of serotonergic terminals in the rat as well as their intimate relationships with

LOCUS COERULEUS

AFFERENTS

norepinephrine neurons in the locus coeruleus. They found that both dorsal and ventral divisions of the rat locus coeruleus were equally innervated by serotonergic fibers, even though these two parts of the nucleus are composed of a different proportion of smaller fusiform versus larger multipolar nerve cell bodies. This finding precluded an exclusive innervation of either morphological type of noradrenergic neurons by serotonergic afferents. Precise estimation of the density of serotonergic innervation in the rat locus coeruleus can be determined only by more complete quantitative analysis of specimens taken from all parts of the nucleus. Nevertheless, preliminary figures, obtained from the dorsal division by Leger and Descarries, indicate that this serotonergic innervation is relatively dense. Thus, the serotonergic varicosities were shown to be apprxoimately 10 times more numerous in the locus coeruleus than in the fronto-parietal neocortex where they have also been counted. Moreover, the constituent nerve cell bodies and dendrites of rat locus coeruleus were shown to be compactly grouped, which increases even further the relative incidence of serotonergic varicosities per total number of nerve endings within this nucleus. In considering the intimate relationships of serotonergic afferents in the locus coeruleus, the question arises as to whether the small number of reactive varicosities exhibiting a synaptic junction is making such contacts with noradrenergic elements. Abundant light microscopic histofluroescence, immunohistochemical and radioautographic data support such an interpretation. In any event the studies of Leger and Descarries provide the first conclusive demonstration of synaptic junction between two types of monoaminergic neurons in the central nervous sytem. It also reveals that in the locus coeruleus axodendritic, but not axosomatic, synapses constitute a morphological basis for the control of noradrenergic neurons by serotonergic afferents. However, the relative paucity of morphologically defined synaptic junctions formed by the serotonergic varicosities in rat locus coeruleus suggests a possible alternative mechanism which might account for the modulation and/or transneuronal regulation of noradrenergic neurons by serotonergic afferents. As already known to occur in the peripheral autonomic nervous system, and previously proposed for non-synaptic serotonergic and noradrenergic varicosities in the neocortex or presumptive dopamine boutons in the neostriatum, the amine released from such nerve terminals could diffuse in tissue and exert an action even on relatively distant targets. Thus, in the locus coeruleus, serotonergic afferents might have a widespread influence independent of their synaptic connections [29]. Earlier studies had also made it increasingly clear that serotonergic afferents may be involved in a transneuronal regulation of norepinephrine metabolism in locus coeruleus neurons (Renaud et ul., 1975: Lewis IX al., 1976) and/or their territories of projection. Renaud et ul. [45] demonstrated that chemical lesioning of serotonergic terminals with 5,6dihydroxytryptamine induced activation of tyrosine hydroxylase within the locus coeruleus and in the coerulocortical noradrenergic neurons. They concluded that destruction of serotonin terminals suppresses their control of norepinephrine synthesis in the noradrenergic neurons of the locus coeruleus system and that changes in tyrosine hydroxylase activity represent one of the mechanisms involved in this regulatory process. Other evidence that catecholamine neurons in the locus coeruleus are controlled by serotonergic neurons of the anterior raphe nuclei was

521

provided by Lewis et al. [31]. They found that tyrosine hydroxylase activity in the locus coeruleus was augmented after selective lesions of the nucleus raphe dorsalis and nucleus raphe medianus but the percentage of this elevation was selective for each lesion, i.e., 30% increase with nucleus raphe dorsalis lesions and 81% increase with nucleus raphe medianus lesions. This disparity in elevation emphasizes the highly selective nature of the lesions and attests to their functional independence. The distance between the ventral extremity of the nucleus raphe dorsalis and the dorsal extremity of the nucleus raphe medianus is only 800 pm and histological control showed no overlap of one lesion onto the adjacent nucleus. Given that the lesions were anatomically specific to each raphe nucleus, it is important to ask whether tissue responses to lesioning were likewise specific. This appeared to be the case since: (1) there were non-equal physiological responses to tissue destruction in two extremely proximate areas; and (2) the systematically related DA neurons, as represented by groups A9 and AlO, were not affected as were the catecholaminergic neurons in the locus coeruleus. Tyrosine hydroxylase activity might also have been affected either by the destruction of cell bodies in the lesioned raphe nuclei having projections to the locus coeruleus or from the severing of axons projecting on the locus coeruleus and passing through the two raphe nuclei, or both. The electrolytic lesion studies of Lewis et al. [31] cannot resolve this question but may be interpreted in the light of other evidence, such as experiments in which whole classes of cell types are selectively destroyed. Thus, 5,6dihydroxytryptamine, which destroys serotonin neurons, caused an increase in tyrosine hydroxylase activity in the locus coeruleus which was maximal between 4 and 6 days after administration (Renaud et ul. [31]). Also, Pujol et al. [43) have shown that the destruction of the anterior part of the cat raphe system, including nuclei raphe dorsalis and raphe medianus, induces a significant increase in norepinephrine turnover in the projection fields of the locus coerulus. Viewing the findings of Lewis er al. [3 11 it seems likely that there is a direct or indirect control of tyrosine hydroxylase activity in the locus coeruleus by serotonergic fibers originating in the anterior raphe system. The different influence of the two lesions on tyrosine hydroxylase in the locus coeruleus is likely due to the destruction of separate and different raphe fiber systems. This is all the more reason for assessing the relative roles of the raphe nuclear projection to the locus coeruleus in regulating various activities in this nuclear complex. Pujol et al. [44], using biochemical methods, described two experimental models of reciprocal biochemical mechanisms of regulation between some noradrenergic and serotonergic neurons. A reciprocal interaction between the two aminergic systems was found by studying the effect of inactivation of the anterior part of the raphe system upon the rate of disappearance of norepinephrine after blockade of its synthesis by alpha-methyl-p-tyrosine. They made the following observations 48 hours after coagulation of the nuclei raphe dorsalis and raphe medianus: (1) the destruction of the anterior part of the raphe system did not change the noradrenergic concentrations measured in the different brain structures examined 48 hr after the lesion: and (2) at this time, 8 hr after administration of alpha-methyl-p-tyrosine, the noradrenergic concentrations measured in the cortex and cerebellum were significantly lower as compared to control animals. The rate of disappearance of norepinephrine was also higher in the mesencephalon but not in the other areas of

the brain. Pujol et (11. interpreted these results to indicate that surgical inactivation of the anterior raphe system induces a significant increase of norepinephrine turnover at the level of dorsal noradrenergic bundle terminals. These facts further suggest quite clearly the existence of interaction phenomena between the serotonin-contajning neurons of the raphe system and the dorsal noradrenergic neurons. Kostowski et (~1. [28] also provided biochemical evidence fol interactions between the raphe and locus coerulens. They provided data indicating that a two-way interaction between the raphe and the locus coeruleus exists. They found that bilateral lesions of the locus coeruleus affect the serotonergic system since a marked increase of forebrain S-HIAA concentration was seen 4 days after the lesion. Since only a slight decrease of hippocampal-cortical norepinephrine was found at this time, the increase of 5-HIAA does not seem to be correlated with the decrease of norepinephrine concentration. Furthermore, they postulated that some unknown compensatory mechanism takes place to restore the normal S-HIAA concentrations in the forebrain 10 days after the destruction of the locus coeruleus at which time the norepinephrine level is considerably reduced. The increase of S-HIAA in rats with lesions of noradrenergic pathways is in agreement with the findings of Jouvet [20], who reported that the destruction of the locus coeruleus or of ascending noradrenergic pathways in cats produced a signi~cant increase of both S-HJAA and tryptophan concentrations in the telencephalon. On the other hand, the increase of MOPEGSO, concentrations in rats lesioned in the raphe medianus also indicate changes in the metabolism of norepinephrine following the destruction of the serotonergic pathways. These various biochemical data provide further support fol the hypothesis of an interaction between the raphe and the locus coeruleus. Several functional models to demonstrate serotonergicnoradrenergic interactions have been setup in recent years with some emphasis on PGO spike release. Delorme c’t trl. [Ia] had first shown that reserpine- and PCPA-induced PGO-waves were diminished and even abolished by the serotonin precursor, 5hydroxytryptophan (5HTP). and they proposed the now widely accepted view that serotonin may physiologically inhibit the generation of PGO wases. The simplest model of such a system is that of Simon r? tri. [55] indicating direct raphe inhibition on a phasic PGO generator or pacemaker located at the pontine level. More elaborate models have subsequently been derived by Jalfre c’t trl. [IS] and Ruth-Monachon et cd. 146-501 indicating both serotonin and catecholaminergic inhibition of PGO activity. In this latter model the pacemaker and generators are separate from the tocus coeruleus and are themselves inhibited by locus coeruleus catecholaminergic cells. A monoamine gating model of PGO spike regulation was put forward by Brooks rf ~1. [5,6], They noted that it is more reasonable to believe that the changes in PGO wave activity induced by reserpine are a result of the drug’s ability to deplete monoamines. Although only serotonin levels were determined in this study, the depression of catecholamines is thought to follow a similar time course after reserpine treatment. Therefore, the results reported by them provide no direct indication regarding the relative importance of serotonin and catecholamines in the genesis of PGO spikes after reserpine. Other lines of evidence tend to implicate serotonin in this process. Thus. administration of the serotonin precursor, S-HTP, suppresses PGO waves in the reserpinized animal and selective depletion of serotonin by

p-chlorophenylalanine induces wa\.c’r which resemble PC;0 waves following administration of reset-pine. it is possible that catecholamine depletion also is :I significant factor in the development of PGO waves following reserpine and Brooks c’t ai. IS.61 emphasized that further information is needed before the rote of individual monoamines in the regulation of PGO wave activity can be assessed. The temporal correlation between the development of PGO wave activity and the progressive fall in serotonin levels is consistent with the hq’pothesis that monoaminergic neurons regulate or gate REMtype PC0 waves. According to this hypothesis a group of’ monoam~nergic neurons, which are ~~)nicaily active during wakefulness and slow wave sleep. scr~t to inhibit REM-type PGO wave activity. Normally. the proposed neurons de-, crease in discharge rate only during REM sleep. permitting REM-type PGO waves to occur at that time. If this model is correct Brooks (‘/ crl. point out that reserpine may act hi depleting monoamines in the nerve terminals of the Sating neurons. When normal function of these neurons become< compromised, and their tonic inhibitory influence ii rcmoved, REM-type PGO wave activity would then appears The results of their infusion experiments with reserpine injected into the ventricles indicated that the influence of reserpine upon PGO waves is probably exerted at the levei of the brainstem caudal to the diencephafon. In thic regard. Bobillier t*f nl. 131, using autoradj~~g~phy in cat\, traced differential ascending projections of the nucleus raphe dor. salis and nucleus raphe medianus showing the nucleus raphe dorsalis to project directly to the lateral genicutatr nucleus (LGN). Still, given such a connection in the cat. the data of Brooks c’t trl. ].5,6] indicate that reserpine is not acting at the level of the LGN to decrease sctcttonin. thus releasing spikes. Rather, they concluded that reserpine act% at the brainstem level to induce PGO spikes and such ;I view is supported by regional serotonin measurements made aftciventricular infusion. In these experiments, PC0 spikes after reserpine appeared in the lateral geniculate nucleus and marginal gyrus at a time when the serotonin level was close to normal in both structures and s~gI~i~icanttydepressed only in the pans. The localization of reserpine’s action to the brainstem is of significance because it likely reveal, the site of which the normal regulation of REM-type PGO waves probably takes place. Viewed in terms of the n~~)no~~rnine gating hypothesis, the results suggesl that the gating neurons act upon some brainstem structtire. Both lesion and stimulation experiments tend to place the pacemaker which triggers REM-type PGO waves in the pontinc reticular forrnation at the level of the abducens nucleus 01 locus coeruleu~ corn-plex. They proposed that the tnon(~~i?nillergicneurons exert their tonic inhibitory influence directly upon this pacemaker. This would be consistent with the finding that stimulation of the pacemaker area normally evokes REM-type PGO waves only during REM sleep, but stirn~~~at~~~n of the same area evokes similar waves in slow wa~c sieep in reserpinized

animals.

The factors which normally

influence

the fiinction

of the proposed monoam~nergic gate are stili unknown pendingfurther knowledge of influences which act upon the WI%MS raphe nuclei projecting to the pacemaker area. Observations during recovery from reserpine, when gate function is gradually re-established, suggest that both arousal and the recent occurrence of REM sleep tend to close the gate and suppress REM-type PGO waves Simon cl r/f. 155\, in a compre~le~~~l~eanalysgs of the role of the raphe nuclei in the regulation fPGO waVe XtiuitY. follnd that raphe lesions produced an inCreXX iI1 [‘Go

LOCUS COERULEUS

AFF~RENTS

waves, nat only in REM sleep but in waking and slow wave sleep. Furthermore, the disturbance in regulation of PC0 waves was greatest in animals with large lesions involving se~~crul raphe nuclei. To us this latter observation appeared prima ,faci~ evidence that several raphe nuclei are involved in suppressing PGO waves and the general activity of the phasic and/or tonic generator(s) for REM sleep in the pons. Simon V? (11.found that unilateral parasagittal cuts, 1.5 mm lateral to the midline at the level of the raphe nuclei, caused changes in PGO wave activity similar to those following midline raphe lesions of comparable extent. They further noted that stimulation of the locus coeruleus (“pacemaker”) region in the dorsolateral pons, on the side ipsilateral to an extensive parasagittal cut, consistently evoked lateral geniculate responses during slow wave sleep as well as during REM sleep. Stimulation of the pacemaker on the opposite side of the pons evoked lateral genicutate responses only during REM sleep. Their results support the hypothesis that serotonin-containing neurons, having their perikarya in several midbrain and pontine raphe nuclei, play an important role in the normal regulation of REM-type PGO waves. Simon c’t (11. 155) also found that the results of both stimulation and lesion experiments indicate that the source or pacemaker of REM-type PC0 waves is in the pontine region of the brainstem. They emphasize that serotonin-cont~ning neurons act at the pontine level, since serotonin depletion in this region following infusion of reserpine into the fourth ventricle also induces PC0 waves. Therefore, they advanced a model for the regulation or gating of REM-type PGO waves in which neurons sending axons to end in the pacemaker area play the most important role. Some of their cats had caudal lesions, extending almost to the ventral surface of the brainstem which consistently damaged or destroyed the nucleus raphe medianus, nucleus raphe pontis, and the rostra1 half of the nucleus raphe magnus. The effect of these lesions upon PGO waves was similar in most respects to that of anterior raphe lesions except that waves occurred at a somewhat higher frequency without obvious grouping during recovery from anesthesia. Also, lateral geniculate PGO waves during slow wave sleep were more common than after anterior lesions. During slow wave sleep the waves occasionaliy occurred in groups, but more often they appeared at a relativety low frequency throllghout long periods of cortical synchrony. This tendency persisted despite the return of normal episodes of REM sleep by postoperative Day 6. Other animals had extensive lesions involving most anterior and middle groups of raphe nuclei. Chronic PGO wave activity was present in all these animals, the waves resembling REM-type PGO waves and they were present in both the cortex and the lateral geniculate nucleus. The frequency of the waves was relatively high and it did not diminish signi~cantiy during arousal. According to the model of Simon C? (I(. [55], the parasagittal cuts which spare the raphe nuclei but sever the axons passing laterally to the pacemaker area should have an effect upon PGO wave activity which is comparable to that of direct raphe lesions. To test this view, parasagittal cuts were made on one side of the brainstem similar in extent to the various midline lesions. In each of these experiments, the effect upon PGO wave activity was rather similar to that observed after the corresponding midline lesion. Thus, the change was most striking in the case of animals with extensive parasagittal cuts. Incessant PGO wave activity was present in the lateral geniculate nucleus of each of these animals for at least 24 hr, and in one case it continued for more than 4 days. The frequency of the

523 waves throughout this period was comparable to that observed during REM sleep and after treatment with reserpine. Although the animals recovered consciousness within 8-10 hr after surgery and seemed to be aware of their environment during periods of wakefulness, PGO waves were not SUPpressed at these times. Within several days, however, suppression of wave activity during wakefulness became apparent in ail of the animals with extensive cuts and one week later such waves occurred only rarely. Nevertheless, waves resembling PGO waves of REM continued to appear shortly after the onset of cortical synchrony and were present throughout most long intervals of slow wave sleep. Although REM sleep returned within 2-5 days in all animals, the tendency for REM-type PGO waves to occur during slow wave sleep did not diminish. The use of large parasagittal cuts provided Simon et ul. with oppo~unities to test the gating model. Presumably, the waves which appeared throughout most periods of slow wave sleep for at least 2 weeks in these animals originated in the pacemaker on the lesioned side of the pons. The pacemaker on the opposite side of the brainstem should have remained under normal control, fully capable of evoking REM-type PGO waves only during REM sleep. In order to test the excitability of the two pacemakers, stimulating electrodes were implanted on each side of the pans in the cats which had extensive cuts, the electrodes being positioned in an area where stimulation normally was found to be effective in evoking REM-type PGO waves only during episodes of REM sleep. Throughout the l-4 day period immediately following the parasagittal cut, it was possible to evoke similar waves in the lateral geniculate nuclei by stimulating the brain on the lesioned side of the pons. During the first few post-operative days stimulation of the brainstem on the non-Iesioned side of the pons failed to evoke lateral geniculate responses. With regard to the localization of the serotonin gate it appears likely from the data of Simon pt a/. [55] that the neurons forming the serotonin gate Irave their perikarya scattered somewhat di’@.srly in the midline plane, rather than being localized in a single small region. This would explain the minor effect upon PGO wave activity exerted by small raphe lesions and the much greater effect exerte %y larger raphe lesions. The finding that parasagittal cuts wr;;e equally effective in altering PGO wave activity indicates that most of the perikarya probably lie close to the midline. The possibility that the majority of the perikarya lie more laterally, and give rise to axons which cross the midline to terminate in the contralateral pacemaker, was ruled out by the asymmetrical results in the stimulation experiments. All these findings are consistent with the hypothesis that the gating neurons have their perikarya within several nuclei of the raphe system. In considering the results of Simon it ctl. [551 a distinction should be made between transient and Iong lasting changes in PGO wave activity. The only permanent effect of the brainstem cuts and lesions seems to have been an impairment in the brain’s capacity to suppress REM-type PGO waves during slow wave sleep. This was most obvious in the animals with large parasagittal cuts, but was also apParent in the animals with the large raphe lesions. This suggests that the normal functionai role of the serotonin gate may be to prevent the occurrence of REM-type PGO waves during slow wave sleep. Thus, some additional regulatory mechanism may exist which was not permanently damaged in these experiments. This was termed the arousal gate which eventually suppressed REM-type PGO waves during wakefulness in all animals from which recordings were ob-

tained for at least 4-5 days. The transient increase in PGO wave activity, both immediately followjng surgery and during later periods of wakefulness, was interpreted by Simon ri (11.as a temporary inactivation of the arousal gate. Although the model of Simon ef nl. indicates the serotonin gating neurons as exerting their inhibitory influence directly upon the pacemaker region, it is also possible that this interaction takes place at some point along the pathway from the pacemaker to the lateral geniculate nucleus. It is improbable. however, that the serotonin gating neurons act directly at the lateral geniculate level, since unilateral parasagittal cuts exert a differential effect upon the ability of the two pontine pacemakers to evoke lateral geniculate responses during slow wave sleep. The findings of the study of Simon c’t ol. give support to the hypothesis that serotonin-containing neurons, having their perikarya in the nuclei of the raphe, exert a regulatory or gating influence upon REM-type PGO waves. According to the model these neurons should be tonicaJJy active during wakefulness and slow wave sleep, exerting an inhibitory influence upon the pacemaker system which may itself comprise the locus coeruleus complex or other pacemaker or generator cells {see Ruth-Monachon t*f rrl. model below). Thus, during REM sleep, the discharge rate of these neurons has been shown to diminish, thereby permitting the occurrence of REM-type PGO waves. Dement of ul. 1131 stressed the quantity and universality of phasic activity and suggested the consideration of not what causes these PGO spikes, but rather what prevents them from discharging all the time. Cessations in the firing of raphe units occur during REM sleep and in slow wave preceding lateral geniculate nucleus waves and, as noted by Dement et al. [13], the specificity and consistency of these findings suggest that the release of serotonin from dorsal raphe neurons normally suppresses LGN waves of REM sleep. In attempts to confirm this possibility they observed the effects of electrical stimulation of the raphe nuclei in cats. Electrodes were implanted in raphe dorsalis, pontis. and magnus and current applied to the nucleus raphe dorsalis during REM sleep, caused suppression of geniculate PGO waves occuring during the period of REM sleep. However. these same stimuli were ineffective in suppressing PC0 waves when delivered to nuclei raphe pontis and magnus. Stimulation of the nucleus raphe dorsalis in the waking state produced little or no discernible behavioral effects and. when delivered to the sleeping animal, did not cause awakening from slow wave sleep or from REM sleep. Kostowski (‘1 tri. [26] also found that stimulation of the nucleus raphe dorsalis in the cat inhibited PC0 wave activity induced by reserpine. They further reported that injection of serotonin directly into the locus coeruleus suppressed PGO waves induced by reserpine. He feels this to be sound additional evidence for direct inhibitory effects of the raphe on the PGO generating mechanism and possibly other classes of phasic events except, of course, during REM sleep. RuchMonachon et ul. [46-501, in a series of elaborate pharmacoIogicaJ experiments, developed an expanded PGO spike model. They pointed out that brain catecholamines and serotonin have been implicated in a great number of important activities of the central nervous system, especially in mood, drive, motivation, wakefulness, sleep and learning, as well as in motor coordination and in the control of various autonomic parameters such as temperature, cardiovascular functions and endocrine activity. They emphasized that most of these activities of the central nervous system are extremely complex, difficult to assess and depend on many

other variables besides the monoanunex. J‘hus. as Brook5 and others previously emphasized. PGO wave activity appears to be a more direct and quantifiable indicator of rhe activity of some brain monoamine systems, being an clectrophysiotogicat phenomenon originat-ing in a region of dense monoaminergic innervation and distributed to a few highfy circumscribed regions of the central nervous system. 4% opposed to the earlier views that serortrnin might be the only inhibitory transmitter blocking PGO waves. they presented strong evidence for an inhibitory influence of a noradreuergic system on the generation of PC0wave activity. However. this inhibition by norepinephl-any was less ~r~~r~(?~~~~~ed than that exerted by serotonin and ii marked reduction of noradrenergic transmission by itself did not produce PGO wave activity. But, when depletion r>f’norepin~phrinc was accompanied by a reduction of the ~-~lydroxytryptam~ne~gic activity, the inhibitory role of norepinephrine could clearly be demonstrated. Their conclusions :+rc not in accordance with the hypothesis that norepinephr~~~ neurons ofthe locrr~ coeruleus are playing the determinant part in the generation of PC0 waves by quanta1 release of a norepinephrine metabolite. In fact they found that $1 the pharmac~?t~~~ic~ll means used for reducing central noradrenergic transmission, either by inhibiting the synthexi\ or the ctorage ot norepinephrine or by blocking u-adrenoceptors, actttallq increased the density of PGO spikes induced by PCPG. Their results have led to development of a more complicated PGO spike model system involving multipfc neurotransmitter interactions. Relative to differential raphe effects on PGO activity. Cespuglio cl/ trl. [9] have found that localized cooling of the anterior and ventral part of the nucleus raphe dorsalis induces cortical synchronization and PGO waves folictwed sometimes by REM sleep. However, cooling the region of the raphe magnus produced cortical arousal. After depletion of brain serotonin by para-chlorophen~lalanine. cooling the raphe dorsalis resulted in muscle atonra and bursts of high frequency PGO waves but not in cortical synchronization. Following restoration of brain serotonin by int~~ven(;~ls atiministration of F-hydroxytryptophan. cooling again produced cortical synchronization and PGO waves as in the control experiments. As to differential effects of two raphe nuclei, these data would indicate less nucleus raphe magnus inhibition on PGO spikes and more inhibition on arousal mechanisms, thus pointing to selective raphe nucleus effects on the various components of REM sleep. Finally. Bobillier (jr a/. 121, in an autoradiographic study in the cat. provided clear evidence of close anatomical connections between the nucleus raphe medianus and several aminergic systems. They also illustrated the complexitv of the innumerabie anatomical links and the multipli;ity of the potential biochemical interactions that can occur at the level ofthe ceil bodies and their reciprocal terminaf Gelds following any kind of stimulation. Their data also raise \ome anatomical problems which remain a matter for specufation: thus. (1 I regarding the aminergic nature of the hetcrogenous population of neurons of the raphe, they considered whether their cronnectjons were direct or indirect: (2) considering the organization of specific subpopulations of neurons within each raphe nucleus. what are their neurochemical connections: (3) what is the topical organization of the projections of these systems: and (4) what are the relationships between the relative density of terminals and the functional activity? Bobillier i’( iii’. emphasize that. in view of the many behaviors associated with serotonergic systems. the h&erogenous and rlbiquitolls

LOCUS COERULEUS

AFFERENTS

nature of the raphe projections suggests that the activity of raphe neurons may be translated into behavioral terms not only in the raphe but also at the level of the post-synaptic target cells.

METHOD

Fifty-seven male Sprague-Dawley rats (Charles River Labs) weighing 250-300 g were used in these experiments. For placement of horseradish peroxidase (HRP) the rats were anesthetized with sodium pentobarbital(50 mgikg, IP) and mounted in a stereotaxic instrument. A 2.5 mm burr hole was drilled in the skull over the locus coeruleus centered 1.5 mm posterior and 1.3 mm lateral to the midline. The dura was opened and a glass pipette of 60 p tip diameter was lowered into the brain at a 30” angle through the cerebellum to a level of 5.6-5.9 mm below the skull surface so that the tip was centered in locus coeruleus. The locus coeruleus in the rat is small, approximately 1.5 mm in rostro-caudal extent, 0.5 mm in width and 0.7 mm in depth at its level of greatest cross-sectional diameter (compare with estimates of Cederbaum and Aghajanian [8]). Very minute corrections were made to focus the injections into the dorsomedial and ventrolateral areas of the nucleus at the level of the rostra1 and caudal poles. The use of a 60 p glass pipette allowed us to achieve dense deposits of HRP and minimized spread to adjacent structures. Injections were made slowly over a 30 minute period through the glass pipette attached to a microburet and a total injection volume of 0.05 ~1 was injected. Control injections were made in the cerebellar cortex dorsal to the locus coeruleus, in the lateral-most aspect of the mesencephalic nucleus of nerve V, in the reticular formation ventromedial to the locus coeruleus (but excluding the locus subcoeruleus area) and in the medial parabrachial region. After each injection the pipette was left in place 15 minutes and then withdrawn slowly and the wound closed with wound clips. After a survival time of 24 hours the rats were deeply anesthetized with sodium pentobarbital and perfused intra~ardially with 50 cc of 0.9% saline solution followed by 200 cc by fixitive solution (1% paraformaldehyde, 4% glutaraldehyde, 5% sucrose in 0.1 M phosphate buffer at pH 7.2). The brains were removed immediately and cut in the frontal or, in some cases, sagittal plane on a freezing microtome at 40 w thickness. The sections were processed for HRP reaction on the same day, as described in our previous papers (Pasquier et rrl. [37,38)1. Sections were collected and washed several times in a fresh buffer solution (Tris-HCl, pH 7) plus 5% sucrose. They were then processed in diamino-benzidine (3,3’,DAB) in Tris-HC1 buffer plus sucrose for one hour and transferred to a new DAB-Tris-Sucrose solution plus hydrogen peroxide for 30 min. Finally, the sections were washed in Tris without sucrose. The same day sections were mounted and left to dry overnight at 37°C. They were then lightly counterstained with cresyl violet and cover-slipped. The sections were examined under bright and dark-field iilumination. This latter mode of illumination allowed best for rapid scanning of the sections for HRP reactive cells, These were~identified by their content of coarse, highly refractile granules, which were clearly distinguished from vascular endothelial cells as well as non-vascular elements possessing endogenous peroxidase activity.

525 RESULTS

In 32 rats the HRP deposit was confined to the locus coeruleus. The center of the injection sites were rendered opaque by the dense HRP reaction product and varied from 50&800 p in transverse diameter and 300-900 CLin sagittal section at the nuclear center, tending to conform to the elongated contours of the locus coeruleus in this latter plane. In the present study we concentrated on examination for HRP reactive neurons in the midbrain raphe nuclei (raphe dorsalis and medianus) and pontine raphe (raphe magnus and pontis) and several surrounding areas, including the medial and lateral parabrachial nuclei, lateral reticular fo~ation of the midbrain and pans, mesencephalic and spinal nucleus of the trigeminal nerve, vestibular nuclei, lateral lemniscal area, nucleus of tractus solitarius, substantia nigra, and fastigial nucleus of the cerebellum. Most of these comprise areas or nuclear formations variously reported to project to the locus coeruleus. HRP reactive cells to unilateral injection of HRP into the locus coeruleus were found in the various raphe nuclei and other cell groups on both sides of the midline though varying slightly depending on the epicenter of locus coeruleus injection site. The specificity of the HRP injections to terminals within and immediately surrounding the locus coeruleus was maximized by use of the 60 w tip micropipette. Following the various locus coeruleus injections HRPpositive neurons were found over a considerable extent of the midbrain, pons and anterior medulla. The cell labelling varied greatly in different structures, indicating sparse or extensive projections from particular areas. Certain structures that were sparsely labelled in most brain areas did not show any HRP reactive neurons in some brains, though those showing heaviest labelling, such as some of the raphe nuclei, were labelled in all brains in which the injection site was on target in the locus coeruleus. Since in this study we have concentrated our attention on the midbrain and pontine raphe, we will divide our results into two groups for purposes of discussion, i.e., the raphe nuclei and other brainstem structures of the midbrain, pons, and anterior me&rlla. Ruphe Afferents

to Laws

Cuerdeus

Figures I, 2 and 3 indicate the area of densest HRP deposit at 3 different anterior-posterior levels of the locus coeruleus corresponding the anterior pole, middle third and posterior pole of the nucleus. In several instances the injection was centered to focus on either the dorsomedial aspect of the locus coeruleus or the ventrolateral aspect (Fig. 4). These latter correspond to the principle (dorsomedia1) locus coeruleus or the ventrolateral (locus coeruleus alpha) component in the cat where the nucleus is more divisible into discrete components than in the rat f51, 52, 53, 54). The most densely opaque region of the locus coeruleus in Figs. l-3 were approximately 500-900 p transversely and in sagittal section the epicenter of opaqueness corresponded to anterior, middle and posterior divisions though there was usually overlap of the anterior with the middle injection and overlap of the posterior with the middle injection. There was no overlap between the extreme anterior and posterior injection sites so that it is possible to compare the projection areas to these two polar regions thus disassociating any differential raphe or other fibers entering these zones.

526

FIG. 1. Photograph of HRP injection area in the locus coeruleus of the rat. This figure represents the epicenter of injection area in the anterior third of the locus coeruleus. The label extends slightly into the central gray and there is also some labelling of the cerebellar cortex above the locus coeruleus. Control HRP injections into cerebellar cortex above the locus coeruleus were not associated with labelling of any of the raphe nuclei Frontal section, Nissl counterstain, photo-reversal showing BRP injection as white zone indicated by amow at epicenter. Posteriorly in the locus coeruleus the label diffused into the middle third but not posterior third of the nucleus. In this and the subsequent two figures the arrow points to the center of the injection site in different anterior-posterior planes and, though diffusion to other aspects of the locus coeruleus cannot be ruled out. we did consistently observe a different raphe labelling pattern associated with injections in different anterior-posterior placements and depending on whether the injection wah more dorsomedial or ventrolateral in the locus coendeus (see text).

LOCUS COERULEUS

AFFERENTS

FIG. 2. Photograph of HRP injection area in the locus coeruleus of the rat. This figure represents the epicenter of injection area in the middle third of the locus coeruieus. The label extends into cerebellar cortex above locus coeruleus but control injections into this cortex were not associated with HRP reactive cells in any of the midbrain, pontine, or meduliary raphe nuclei. Frontal section, Nissl col~nterstain, photo-reversai showing HRP injection as white zone indicated by arrow at epicenter. Anteriorly and posteriorly the label extended into the anterior third and posterior third of the nucleus but did not involve the anterior or posterior poles of the locus coeruleus.

527

528

FIG. 3. Photograph of HRP injection area in locus coeruleus of the rat. This figure represents the epicenter of injection area in the posterior third of the locus coeruleu~. The label extends into cerebelfar cortex above iocus coeruleos but control injections into this cortex were not associated with HRP reactive cells in any of the raphe nuclei. Frontal section, Nissl counterstain, photo-reversal showing HRP injection as white zone indicated by arrow at epicenter. In this injection sites the label diffused into the middtc third of the locus coeruleus but not the anterior third.

LOCUS COERULEUS

529

AFFERENTS

FIG. 4. Photomicrograph of locus coeruleus injection site where injection epicenter was in the more dorsomedial sector of the nucleus. Rats with these type injections showed a different pattern of raphe labelling than when injections were centered in the more ventrolateral division of the locus coeruleus (see text). Arrow indicates epicenter of injection.

In animals injected in the anterior and middle locus coeruleus there was heavy labelling in the nucleus raphe dorsalis (+ t +) generally of equal dist~bution throughout the nucleus but with slightly more labelling of cells in the posterior pole of the nucleus. Both dorsomedial and ventrolateral injections in the locus coeruleus at these levels revealed heavy labelling in the nucleus raphe dorsalis, though ventrolateral injections gave a somewhat higher number of reactive celis (Fig. 5). injections centered on the posterior pole of the locus coeruleus were associated with fewer HRP positive cells in the nucleus raphe dorsalis and in these instances heavier labelling was seen in the posterior two-thirds of the nucleus. The cells labelled in the nucleus raphe dorsalis were mostly medium sized (25-35 p) multipoJar cells or cells that were oval in shape. With these same injection sites there was also some labelling of the nucleus raphe medianus though not to the extent of nucleus raphe dorsalis labelling. Thus, again, anterior locus coeruleus injections were associated with heaviest HRP reactive cells in the posterior two-thirds of nucleus raphe medianus with less labelling in the anterior third. This Jabelling was much more dense in the midhne portions of this nucleus in which medium sized multipolar and occasional fusiform cells were seen (Fig. 6). Considerably less labelling was seen in the peripheral part of nucleus raphe medianus. though an occasional small (10-15 p) oval cell was labelled in this zone. As in the case with nucleus raphe dorsaiis, the

more posterior locus coeruleus injections were associated with considerably less labelling in the entire nucleus raphe medianus. in the case of the pontine raphe nuclei pontis and magnus better labelling was seen in nucleus raphe points than in nucleus raphe magnus in all cases of locus coeruleus injections. Medium sized (25-35 F) multipolar and some round to pi~fo~ cells were the most common type of labelled cells seen (Fig. 7) in the nucleus raphe pontis. Small (IO-15 p) and medium (25-30 p) sized polygonal cells and a few scattered giant cells were labelled in nucleus raphe magnus. We could not distinguish in nucleus raphe magnus a distinct difference in labelling density following ventrolateral versus dorsomedial locus coeruleus injections though, in the case of nucleus raphe pontis, the ventrolateral locus coeruleus injection was clearly associated with much denser HRP labelling than following dorsomedi~ injections. Some labelled cells were seen in the medulla in nuclei raphe pallidus and obscurus associated with the more posterior locus coeruleus HRP injections. These raphe nuclei were not labelled following anterior locus coeruleus injections of HRP. Other Brainstem

A,fferents

to docks

Corrr,telts

At the level of the midbrain most HRP positive cells were muhipolar, small (10-18 p) to medium (25-35 CL)sized cells

530

MORGANE

FIG. 5. Darkfield illumination photomicrograph of a medium sized multipolar cell m the nucleus raphe dorsalis. This cell is filled with HRP granules following injection of HRP into the ventrolateral aspect of the locus coeruleus at approximately its middle third antero-posteriorly. Note that the entire soma and primary dendritic processes are densely stippled with granules of approximately uniform size. Mag. 800x.

FIG. 6. Low power darkfield illumination photomierograph of a group of HRP reactive multipolar and oval cell types in the nucleus raphe medianus following injection of HRP into the posterior polar area of the locus coeruleus. Mag. 200x.

AND

JACOBS

LOCUS COERULEUS

AFFERENTS

531

FIG. 7. Low power darkfield illumination photomicrograph of groups of HRP filled multipolar, round and an occasional oval cell in the nucleus raphe pontis following injection of HRP into the posterior polar regions of the locus coeruleus. Mag. 200x.

located largely in the ventral and ventro-lateral central gray substance mostly unilateral to the HRP injection in the locus coeruleus. The heaviest concentration of these were just beneath the aqueduct. Other cells were scattered in the floor and lateral wall of the aqueduct and were generally oval cells larger than cells in the central gray. Some HRP positive cells, often just 3-5 per animal, were seen in the contralateral locus coeruleus following all of the injections into the locus coeruleus. The ventrolateral locus coeruleus showed the greater density of reactive cells though, on some occasions, cells were seen in dorsomedial locus coeruleus. Several components of the solitary tract nuclei, both medial and lateral, were bilaterally labelled following locus coeruleus injections. The HRP injections into the more posterior area of the locus coeruleus were associated with the densest labelling in both lateral and medial solitary tract nuclei though some labelled cells were seen after HRP injections into the anterior portions of the locus coeruleus. The ventrolateral locus coeruleus injections appeared associated with the heaviest labelling of the solitary tract nuclei. Generally speaking the brainstem areas most consistently containing labelled HRP cells after locus coeruleus injection were the media1 parabrachial nuclei, all vestibular nuclei, the central gray, the mesencephalic nucleus of the trigeminal nerve, the lateral reticular formation of the pons and midbrain and upper medulla, the nucleus gigantocellularis, and the nucleus fastigius of the cerebellum. The more posterior and ventrolateral locus coeruleus injections gave the densest labelling in most of these structures. No labelling was seen in the substantia nigra or the lateral parabrachial nucleus. Control

Injections

Following

the control injection in structures near the and in the cerebellar cortex and sub-cortex above the locus coeruleus no HRP labelling was observed in the nucleus raphe dorsalis, medianus, pontis and magnus though good HRP labelling of lateral reticular formation ceIIs was obtained. locus coeruleus

Injections ventral to the locus coeruleus in the reticular formation was not associated with HRP positive cells in the raphe nuclei though other areas labelled after locus coeruleus injections were clearly labelled in these extracoeruleus areas. Injections in the media1 parabrachial nuclei lateral to the mesencephalic nucleus of the trigeminal nerve gave good labelling of the locus coeruleus and most areas, including the raphe nuclei, that showed HRP positive cells after locus coeruleus injections. In addition, the substantia nigra, which was not labelled after locus coeruleus injections of HRP, was clearly labelled following these injections in the media1 parabrachial nuclei. DISCUSSION

We have reviewed the physiological, biochemical, pharmacological and anatomical evidence bearing on raphe-locus coeruleus interactions in cats and rats, particularly on the suppression of locus coeruleus activity by raphe afferents. Emphasis has been placed on a review of the various evidences of raphe regulation of locus coeruleus activity, particularly raphe effects on norepinephrine metabolism, sleep cycles, and regulation of phasic activities thought to be generated in the locus coeruleus area. These data clearly show that the raphe nuclei, on the basis of their multiple and diverse connections, are well able to play a strategic role in the modulation of many functions. In the present anatomical studies we have, by the use of small volumes (0.05 CL)of HRP injections into the nucleus locus coeruleus of rats, been able to retrogradely trace its afferent fiber systems to a wide variety of brainstem areas, including all of the midbrain and pontine raphe nuclei and the rostra1 parts of the medullary raphe nuclei. The density of projections, as indicated by the numbers of HRP labelled cells in different areas, varied greatly in the several brainstem structures examined. Some areas did not contain HRP reactive cells in all brains studied, though they did in some, thus indicating sparse or topographically limited proj-

ections whose terminal fields may not have been overlapped by the HRP injections in different animals. Some brainstem areas consistently showed only l-3 HRP reactive cells per section (or in rare cases per animal), again indicating a paucity of connections. With regard to the raphe projections, which examination was the main goal of this study, the density of the projections to the locus coeruleus varied between the several raphe nuclei, though all were found to project to the locus coeruieus. including the nucleus raphe medianus. This latter raphe nucleus was reported by Sakai rf N/. [52, 53, 541, using HRP methods, not to project to the locus coeruleus in the cat, though Bobillier et (11. [2,4], using anterograde transport methods, did demonstrate projections from the nucleus raphe medianus to the locus coeruleus in both the cat and rat. Our results in the rat, relative to the projections from the various raphe nuclei to the locus coeruleus, generally agree with those of Cederbaum and Aghajanian 181, though in the present studies we have carried out S&Y~;IY~ injections of different sectors of the locus coeruleus in the rat in order to conform, so far as possible, to the divisions of this nucleus as seen in the cat. By such differential injections of HRP into the dorsomedial and ventrolateral locus coeruleus and into its anterior and posterior poles we have been able to differentiate degrees of individual raphe nuclear relations with different sectors of the nucleus locus coeruleus. Our data provide the anatomical bases for widespread raphe effects on locus coeruleus activity and, in light of the work of Brooks and Gershon 151and Simon rt ~1. [SSl in cats, would indicate that all raphe nuclei may be involved in affecting phasic activities (including, in the cat, PGO activity) under all conditions other than in REM sleep when raphe controls are normally removed from the phasic generator(s). The fact that large parasagittal cuts between the raphe nuclei and the locus coeruleus area result in release of PGO spikes in cats, depending on the extent of the cuts, has led to the view that the more raphe fibers that are interrupted between the raphe nuclei and the PGO generator (which is possibly in the locus coeruleus) the more spikes are released due to removal of raphe inhibition [_551.Jn the case of the rat. where PGO spikes do not occur, it is possible these same connections are still inhibitory on other phasic generators in the dorso-lateral pons. Thus, serotonergic activity may act across midbrain, pontine and medually raphe nuclei to inhibit such activity except during REM sleep when such a serotonergic gate is opened thus releasing pontine generator systems. The “direction of flow” regarding locus coeruleus-raphe interactions appears largely from the raphe nuclear complex to the locus coeruleus, especially its more ventromedial components. Very few demonstrations of locus coeruleus to raphe projections appear reliable and in our studies of such possible connections, using HRP (381 or electrophysiological methods [l], we have not been able to conclusively demonstrate direct projections from locus coeruleus to the midbrain raphe. Likewise, Wang rt (11. [56] found that stimulation of the locus coeruleus in rats does not have a marked inhibitory effect on serotonin cells in the nucleus raphe dorsalis and that microiontophoretic application of noradrenaline directly

FIG. 8. Schematic representation of dit’tkse raphe projections from midbrain, pontine, and medullary raphe areas to the locus coeruleus in the rat. As noted in text, differential densities of projections are seen from each raphe region depending on the locus coeruleus injection site, including whether the injection is in the anterior. middle. or posterior levels of the nucleus or whether the injection is centered in the dorsomedial (DM) or ventrolateral (VL) sector of the nucleus. See text for detailed descriptions. Such a series of projections from the raphe nuclei extending from the midbrain to the medullary tevels provide anatomical support for the findings that interruptions of such pathways (as, for example, carried out in the cat) resldt in the release of phasic events of REM sleep such as PGO waves. the intensity of the release being dependent in the cat on bow matiy such connections are disrupted (see Simon PI ii/. [55ll.

into the serotonin cells does not consistently produce inhibition. They concluded that the locus coeruleus has a minor and possibly indirect influence on serotonin cells in the nucleus raphe dorsalis. The present studies indicating strong raphe inputs into the locus coeruleus again point up. particularly in relation to the many neurot~nsmitter models ‘of phasic activity controt in sleep-waking state, that more knowledge now needs to be gained regarding anatomical inputs to the raphe nuclei so we may determine what regulates raphe activity and, through it, the locus coeruleus. Since the noradrenergic neurons of the locus coeruleus project to widespread areas of the central nervous system they are in a strategic position to influence many activities, including the vigilance states. Obviously, the main inputs to this key structure, such as the serotonergic raphe systems, are of particular impo~ance in relation to how the locus coeruleus functions in its muItiple activities. The present studies indicate how important the entire raphe complex of nuclei (Fig. 8) may be in regulating the activities of the noradrenergic elements of the locus coeruleus and that the density of the innervation varies among the various raphe nuclei with regard to different component subareas of the locus coeruleus.

LOCUS COERULEUS

AFFERENTS

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