C.D. Barnes and 0. Pornpeiaiio (Eds.) P r u u r e ~in~ Bruin Research. Vol. XN 0 1YY1 Elseviei Science Publishers B.V

47 CHAPTER 4

Afferent regulation of locus coeruleus neurons: anatomy, physiology and pharmacology

',

G. Aston-Jones M.T. Shipley 2 , G. Chouvet 3 , M. Ennis *, E. van Bockstaele V. Pieribone 4, R. Shiekhattar ',H. Akaoka G. Drolet B. Astier 5 , P. Charlity R.J. Valentino and J.T. Williams

',

'

',

3,

DiLision of Behacioral Neurobiology, Department of Mental Health Sciences, Hahnemann Uniaer.sity, Broad and Vine, Philadelphia, PA and Department of Anatomy and Cell Biology, Unicersity of Cincinnati, College of Medicine, Cincinnati, OH, U.S.A.; INSERM U 171, Centre Hospitalier Lyon-Sud, Pais. 4H, Chemin de Grand Reuoyet, 69310 Pierre-Benite, France; Department of Histology and Neurobiology, Karolinska Institutet, S-104 01 Stockholm, Sweden; -5 Laboratoire Neuropharmacologie, University Claude Bernard, Faculte de Pharmacie, 8 Are. Rockefeller, 69008 Lyon, France; Oregon Health Sciences Unicersity, Vollum Institute for Biomedical Research, 3181 SW Sam Jackson Park Road, Portland, OR, U.S.A.

'

Tract-tracing and electrophysiology studies have revealed that major inputs to the nucleus locus coeruleus (LC) are found in two structuies, the nucleus paragigantocellularis (PGi) and the perifascicular area of the nucleus prepositus hypoglossi (PrH), both located in the rostra1 medulla. Minor afferents to LC were found in the dorsal cap of the paraventricular hypothalamus and spinal lamina X. Recent studies have also revealed limited inputs from two areas nearby the LC, the caudal midbrain periaqueductal gray (PAG) and the ventromedial pericoerulear region. The pericoeruleus may provide a local circuit interface to LC neurons. Recent electron microscopic analyses have revealed that LC dendrites extend preferentially into the rostroniedial and caudal juxtaependymal pericoerulear regions. These extracoerulear LC dendrites may receive afferents in addition to those projecting to LC proper. However, single-pulse stimulation of inputs to such dendritic regions reveals little or no effect on LC neurons. Double-labeling studies have revealed that a variety of neurotransmitters impinging on LC neurons originate in its two major afferents, PGi and PrH. The LC is innervated by PGi neurons that stain for markers of adrenalin, enkephalin or corticotropin-releasing factor. Within PrH, large proportions of LC-projecting neurons stained for GABA o r met-enkephalin. Finally, in contrast to previous conclusions, the

-'

dorsal raphe does not provide the robust 5-HT innervation found in the LC. W e conclude that 5-HT inputs may derive from local 5-HT neurons in the pericoerulear area. Neuropharmacology experiments revealed that the PGi provides a potent excitatory amino acid (EAA) input to the LC, acting primarily at non-NMDA receptors in the LC. Other studies indicated that this pathway mediates certain sensory responses of LC neurons. NMDA-mediated sensory responses were also revealed during local infusion of magnesium-free solutions. Finally, adrenergic inhibition of LC from PGi could also be detected in nearly every LC neuron tested when the EAA-mediated excitation is first eliminated. In contrast to PGi, the PrH potently and consistently inhibited LC neurons via a GABAergic projection acting at GABA, receptors within LC. Such PrH stimulation also potently attenuated LC sensory responses. Finally, afferents to PGi areas that also contain LC-projecting neurons were identified. Major inputs were primarily autonomic in nature, and included the caudal medullary reticular formation, the parabrachial and Kolliker-Fuse nuclei, the PAG, NTS and certain hypothalamic areas. These results are interpreted to indicate that the LC may function in parallel to peripheral autonomic systems, providing a cognitive complement to sympathetic function.

Key words: excitatory amino acici, enkephalin, CRF, epinephrine, locus coeruleus, nucleus paragigantocellularis, nucleus prepositus hypoglossi

48

Introduction The noradrenergic nucleus locus coeruleus (LC) has been the subject of intense scrutiny and diverse functional hypotheses. Interest in this nucleus can be traced to the finding by Dahlstrijm and Fuxe (1964) that the LC provides the major source of norepinephrine (NE) to the telencephalon. Subsequent studies have revealed that the LC is the most ubiquitous of all neural projection systems in the CNS, providing innervation of all major levels of the neuraxis (reviewed in Foote et al., 1983). Many investigations have also examined the effects of NE on activity of neurons in LC target areas (described in more detail by Waterhouse et al., Woodward et al., McCormick et al., and Segal et al., in this volume), and still others have delineated the physiological and pharmacological characteristics of LC neurons from in uitro preparations (see Williams et al., and Christie, this volume) as well as discharge properties of LC neurons in behaving animals (see Aston-Jones et al., Jacobs et al., Sakai, and Sarah and Segal, this volume). Despite this intense research effort, our understanding of the NE-LC system has been hampered by a serious void; until recently, there has been no systematic input-output analysis of LC neurons. Whereas functional analysis of other brain areas is predicated upon a thorough understanding of afferent connections and the effects of such inputs on cellular discharge, most LC functional analysis has been predicated on the results of lesion and pharmacological experiments (see Aston-Jones et aL, 1984). However, a new approach to understanding the noradrenergic LC system is emerging, one based upon analysis of afferent circuits regulating the activity of LC neurons. While much remains obscure about the precise functions of the LC, recent findings concerning the connections, neurotransmitters, and functions of the brain areas that innervate the LC are yielding new insights into the stimuli and events that control the LC, and thus the role of the LC in brain function.

Our finding that the nucleus LC is strongly innervated by two medullary nuclei, the PGi and prepositus hypoglossi (PrH) (reviewed below) (Aston-Jones et al., 1986a), has been supported by several recent findings (McMahon and Wall, 1985; Deutch et al., 1986; Ennis and Aston-Jones, 1987,1989b; Guyenet and Young, 1987; Haselton and Guyenet, 1987; Saper, 1987; Grenhoff et al., 1988; Hajos and Engberg, 1988; Pieribone et al., 1988; Chen and Engberg, 1989; Engberg, 1989; Rasmussen and Aghajanian, 1989; Sesack et al., 1989; Svensson et al., 1989; Tung et al., 1989; Van Bockstaele et al., 1989b; Wallace et al., 1989; Pieribone and Aston-Jones, 1991). It is being recognized that a restricted set of afferents to the LC can make sense of many previous observations. As Saper states in a recent review (Saper, 1987): “These observations explain certain findings in earlier anterograde neuroanatomical tracing studies, in which most fibers from cell groups that were thought to project to the LC.. .were found to stop at the edge of the nucleus, curiously avoiding the core of the LC . . . the outstanding finding of a single main source for LC excitatory inputs.. .may make it much easier to assign a function to LC.” Merents to LC: tract-tracing studies Our recent review of LC afferent control details previous studies in this area (Aston-Jones et al., 1990b). Receptor binding, immunocytochemical, and pharmacological studies indicated that a multitude of transmitter systems impinge on this nucleus, implying that the LC received inputs from a wide variety of central sites (Foote et al., 1983). This belief was strongly reinforced by the fact that the LC projects throughout the neuraxis, and therefore it was felt that this nucleus was likely to be under similarly widespread afferent regulation. This viewpoint was further supported by tract-tracing studies published over a decade ago (Cedarbaum and Aghajanian, 1978; Clavier, 1979; Morgane and Jacobs, 1979) which indicated that the LC receives inputs from a wide array of brain

49

areas, including several telencephalic and other forebrain regions. However, our recent re-examination of LC afferents found that this view of multiple-source projections to LC was unwarranted (Aston-Jones et al., 1986a, 1990b; Pieribone et al., 1988, 1989; Pieribone and Aston-Jones, 1991). Using discrete iontophoretic injections of the sensitive tracttracer, WGA-HRP, into the LC proper, we found that only two areas contained numerous heavily labeled neurons: the nucleus PGi in the ventrolateral rostral medulla, and the area of medial PrH in the dorsomedial rostral medulla. Anterograde tracing from these areas confirmed that they provide major inputs to the LC. Two minor inputs (areas containing few cells weakly labeled) were also identified, in the dorsal cap of the paraventricular nucleus of the hypothalamus and in the intermediate zone of the cervical spinal cord. Similar results were also obtained using the retrograde tracer Fluoro-Gold (FG) (Pieribone and Aston-Jones, 1991). We used anterograde labeling to investigate discrepancies with earlier studies that had identified many more inputs to the LC. Injections into the central nucleus of the amygdala (CNA) (Aston-Jones et al., 1986a), prefrontal cortex (Chiang et al., 19871, dorsal raphe (Pieribone et al., 19891, dorsal spinal horn (Aston-Jones et al., 1986b), ventral tegmental area (VTA) (unpublished observations), or nucleus tractus solitarius (NTS) (Ennis and Aston-Jones, 1989b) yielded no fiber or terminal labeling in the LC but instead produced heavy labeling in adjacent, pericoerulear structures including parabrachial and pontine central grey areas. Furthermore, in the same studies single-pulse stimulation of these areas yielded no response in LC neurons, or responses that were weak and long in latency, consistent with the possibility that these areas had no direct input to the LC. In contrast, identical stimulation potently activated cells in pericoerulear areas that contained corresponding anterograde label (Aston-Jones et al., 1986a; Chiang et al., 1987; Ennis and Aston-Jones, 1989b). Thus, we surmised that

the previous reports of widespread afferents to the LC probably reflected retrograde labeling from injections that spread into pericoerulear areas that are specifically innervated by a host of structures that do not project to the LC. Indeed, when our injections encroached heavily on neighboring pericoerulear areas, retrogradely labeled neurons were found in many additional areas, similar to the previous reports. The conclusion from our anatomical and physiological investigations was that the major afferents to LC derive from the PGi and PrH, with possible minor inputs from the paraventricular hypothalamus and intermediate zone of the cervical spinal cord (Aston-Jones et al., 1986a). It should be noted that both of the retrograde tracers used, WGA-HRP and FG, yielded a substantial “halo” surrounding the injection site, so that analysis of possible input from pericoerulear areas was not feasible in our experiments, and we suggested that further study would be necessary to determine if local neurons in pericoerulear areas innervate LC (described below). It should also be noted that our more recent studies employing other tracers (WGA-apoHRP-Gold or choleratoxin) have revealed additional groups of cells retrogradely labeled from LC than were consistently seen with WGA-HRP (e.g., hypothalamus/preoptic area, Kolliker-Fuse nucleus, A5 area, B9 area). Additional studies are underway to determine the degree to which these additional areas innervate the LC nucleus vs. peri-LC regions, and their possible functional effect on LC neurons. Further analysis has revealed that the LC-projecting neurons in PrH and in PGi are not distributed equally throughout these two medullary nuclei, but rather that they reside in topographically restricted subdivisions (Aston-Jones et al., 1990b). As shown in Figures 1 and 2, LC afferent neurons are located in the very medial aspect of PrH; they extend throughout the length of this nucleus from the hypoglossal nucleus to the genu of the 7th nerve. Many cells also reside more ventrally, scattered along the lateral borders of the medial longitudinal fasciculus. As this area of

50

Fig. 1. Retrogradely labeled neurons in paragigantocellularis (PGi) (A) and prepositus hypoglossi (PrH) (B) following a WGAapoHRP-Gold injection into ipsilateral locus coeruleus (LC). Darkfield photomicrographs. A. Coronal section through PGi. Arrow marks ventral brain surface. B. Coronal section through PrH. Arrow marks floor of IVth ventricle at midline.

51

Fig. 2. LC afferent neurons in the ventrolateral rostral medulla (PGi area; panels A-D) and dorsomedial rostral medulla (PrH; panels E-H). Computer-aided plots showing retrogradely labeled neurons (filled squares) in the rostral medulla for an animal with an injection of Fluoro-Gold restricted to the nucleus LC proper. Sections (40 p m thickness) are ordered from caudal to rostral for PGi (A-D) and for PrH (E-H). Inserts in A and D. Low-power hemisections showing levels of plots in panels A and D. This injection did not incorporate peri-LC regions containing extranuclear LC dendrites.

PrH is immediately adjacent to the dorsal or lateral aspects of the medial longitudinal fasciculus, we have denoted it as the perifascicular PrH. LC-projecting cells in PGi are broadly distributed through the rostral ventrolateral medulla, but also exhibit significant topography (Pieribone and Aston-Jones, 1991). LC afferent neurons are preferentially located medially in rostral juxtafacial PGi, and are more laterally located in caudal aspects of PGi; very few neurons project to the LC from the niedial caudal PGi area (Fig. 2). This has significant implications for which afferents to PGi may be expected to contact LC-projecting neurons. For example, part of the projection from the ventrolateral PAG innervates the medial caudal aspect of PGi; this projection may not contact LC afferent cells. In contrast, fibers from the NTS or the ventromedial periaqueductal grey (supraoculomotor nucleus) terminate in regions that contain an abundance of LC-projecting neurons (Van Bockstaele et al., 1989a). Our recent investigations into the pathways taken by fibers projecting to the LC from PGi have yielded the unexpected result that at least three distinct pathways carry projections from the rostral ventrolateral medulla to the LC. Injections of PHA-L into the PGi labeled two distinct ascending fiber pathways (Van Bockstaele et al., 1989b). One was located laterally, proceeding rostrally just medial to the superior olive to pass through the Kolliker-Fuse and lateral parabrachialis areas before entering the LC from the lateral and rostral aspect. The second pathway was located medially in the medulla in the same vicinity as, but distinctly ventral to, the medullary adrenergic bundle (MB). The third pathway was revealed in our analysis of the ascending adrenergic innervation of the LC. Our immunohistochemistry (Pieribone et al., 1988; Astier et al., 1990; Pieribone and Aston-Jones, 1991), as well as that of others (e.g., Hokfelt et al., 1974, 1985; Astier et al., 1987), demonstrated that the prominent adrenergic input to LC uses the MB. We have recently confirmed this by showing that lesions of the MB decrease the number of adrenergic neu-

52

rons retrogradely labeled from the LC by 90% while decreasing the number of non-adrenergic neurons retrogradely labeled in the same area by only 48% (Astier et al., 1990). This further reveals that nearly all adrenergic projections utilize the MB, and that this pathway carries other inputs to the LC from PGi as well. Physiological characteristics of neurons antidromically identified from LC

Physiological properties of LC-projecting neurons in PGi. To confirm our anatomic results for major inputs to LC from the PGi, and to examine physiological properties of LC-projecting PGi neurons, we focally stimulated the LC and searched for antidromically activated neurons in the PGi of chloral hydrate-anesthetized rats. Twenty of 79 PGi neurons (25%) were antidromically activated from the LC (Ennis and AstonJones, 1987). The antidromic latencies for these neurons averaged 10 msec, but were bimodally distributed, reflecting slowly conducting and more rapidly conducting LC afferents. Other physiological attributes distinguished these two populations of LC afferents: the slowly conducting cells typically exhibited slow spontaneous activity and large positive spikes, while the more rapidly conducting cells were not spontaneously active and yielded small negative action potentials. In addition, the slowly conducting LC afferents were preferentially located in the ventromedial PGi. Finally, many LC-projecting PGi cells were potently activated by footpad stimulation at latencies (5-10 msec) consistent with our hypothesis that these cells mediate LC responses (typically 20 msec onset) to the same stimuli (see below). Physiological properties of LC-projecting neurons in PrH. Focal stimulation of the LC also produced antidromic activation in a high percentage of PrH neurons (17 of 61 cells, or 28%) (Ennis and Aston-Jones, 1989b). Typically these cells exhibited small, negative spikes and were spontaneously active (1-30 spikes/sec). In contrast, most

neurons detected in PrH recordings that were not antidromically activated from LC were not spontaneously active. Unlike PGi neurons, PrH cells that project to the LC do not respond to footpad stimulation. Thus, a high proportion of neurons in both the PGi and PrH were antidromically activated by focal LC stimulation, confirming the retrograde and anterograde tract-tracing results for major inputs to LC from these rostral medullary nuclei.

Attempts to antidromically activate neurons in other nuclei. Of 9 neurons in 11 penetrations through the NTS, none were antidromically activated by LC stimulation (Ennis and Aston-Jones, 1989b). This result is consistent with the findings in the above anatomic experiments that neurons in the NTS are not retrogradely labeled from the LC and is also consistent with our finding that lesions of the NTS do not alter the discharge of LC neurons. Seventeen neurons recorded in the ventral PAG, in the area of the dorsal raphe, exhibited large entirely positive spikes with impulse waveform durations of 2 to 3 msec, and a slow and regular spontaneous discharge rate ranging from 0.5 to 6 Hz. These physiological properties are similar to those described for dorsal and median raphe neurons (Aghajanian, 1978; Blier and de Montigny, 1987). None of these cells was antidromically activated from the LC, even with stimulation intensities of up to 1400 PA. In addition, none of 20 contralateral LC neurons were antidromically activated by LC stimulation (Ennis and Aston-Jones, 1989b), also consistent with our finding of no retrograde labeling between the loci coerulei. Finally, only two of 40 cells tested in the lateral reticular nucleus were found to be antidromically driven by focal LC stimulation (Ennis and Aston-Jones, 1989b). One of these was located in the rostral pole of this nucleus, at the caudal border of PGi and may be a member of this latter set of cells.

53

Inputs to the pericoerulear area: innervation of LC dendrites, local LC afferents or separate circuits?

It has been commonly thought that the regions surrounding the LC in the rat are largely composed of fiber tracts with few if any neurons. However, our recent work (Aston-Jones et af., 1986a, 1990b; Fu et al., 1988) along with that of several other groups, (e.g., Saper, 1982, 1987; Cechetto et al., 1985; Deutch et af., 1986; Sesack et af., 1989; Wallace et af., 1989), has demonstrated dense terminal fields from several brain areas to the pericoerulear region. Thus, there are dense inputs to the parabrachial area lateral to the LC from the NTS (Mantyh and Hunt, 19841, dorsal spinal horn (Cechetto et af., 1985; Standaert et al., 19861, and CNA (Aston-Jones et af., 1986a; Wallace et al., 1989) among others. The central grey medial or rostral to LC, on the other hand, receives inputs from the frontal cortex (Armten and Goldman, 1984; Chiang et af., 1987; Sesack et al., 1989), dorsal raphe (Conrad et al., 1974; Bobillier et al., 1979; Pieribone et af.,19891, CNA (Aston-Jones et af., 1986a; Wallace et al., 1989) and VTA (Deutch et af., 1986) (unpublished observations). These findings cast doubt on the view that the pericoerulear area is relatively cell-free, and prompted us to begin a systematic analysis of the neuropil in the region surrounding the LC nucleus. While these studies are fairly new, several interesting results have been obtained. In addition to Barrington’s nucleus and the laterodorsal tegmental nucleus, already well documented by others, we have found that the pericoerulear area is composed of large numbers of neurons which vary in size, shape and dendritic orientation as a function of location with respect to the LC (Fu et al., 1988). Such neurons are numerous on all borders of the LC nucleus. Of particular interest is the fact that some of these pericoerulear areas contain dense accumulations of dendrites from LC neurons (Fu et af., 1989) (described below) and are heavily targeted by fibers containing sev-

eral peptides. These pericoerulear areas have not been previously characterized, and we are presently using cytoarchitectonics and immunohistochemistry to parcellate this cell- and neurite-rich region. Although there are numerous neurons in the pericoerulear area, very little is known about whether pericoerulear neurons innervate the LC. Our previous studies using injections of retrograde tracers into the LC (Aston-Jones et af., 1986a) could not adequately address this issue because the injected tracer always produced an injection “halo” in the pericoerulear area, particularly in the rostromedial pericoerulear zone. We were able to tentatively rule out inputs from the parabrachial area (Aston-Jones et af., 1986a), but inputs from rostromedial pericoerulear areas are possible. Indeed, two recently developed anatomic techniques, PHA-L and WGA-apoHRP-Gold, have indicated that such pericoerulear neurons may innervate the LC. One region in which retrogradely labeled cells were often found, within the injection halo of WGA-HRP cases, was the caudal ventrolateral PAG, immediately rostral to the LC. Anterograde transport of PHA-L from the caudal ventrolateral PAG revealed prominent innervation of the central grey ventromedial to the LC, and scattered light innervation of the LC as well (Ennis et af., 1989, 1991). Additional electrophysiological experiments confirmed a minor input to the LC from this nearby PAG region (described below). Injections of the tracer WGA-apoHRP-Gold form discrete deposits which can easily be limited to only a subregion of the LC. In addition, this tracer produces no halo, making analysis of possible inputs from immediately adjacent pericoerulear areas feasible. In our initial use of this tracer with subtotal deposits into LC, we consistently found numerous retrogradely labeled neurons surrounding the LC, particularly in the ventromedial and rostral pericoerulear regions; some of these cells also stain immunohistochemically for 5-HT (see Fig. 8, described below). While it is

54

tempting to speculate that the pericoerulear area may provide numerous afferents to the LC area, it remains to be determined whether such local labeling is due to synaptic innervation of LC neurons, or dendrites that extend into the LC but do not innervate LC neurons.

Extranuclear dendrites of LC neurons Several reports have documented that processes of LC neurons extend outside the nucleus proper for a few hundred p m (Shimizu and Imamoto, 1970; Swanson, 1976; Shimizu et al., 1978; Grzanna and Molliver, 1980; Grzanna et al., 1980; Cintra et al., 1982). However, it has never been clear whether the extranuclear processes were dendrites, axons, or both. We recently adapted a gold-silver tone method (Gallyas et al., 1982) to intensify immunohistochemically stained processes. Using this method, our light microscopic examination of dopamine-P-hydroxylase (DPH)-positive LC neurons and dendrites revealed that extranuclear LC processes have a remarkable degree of spatial organization; the vast majority of processes ramified in two distinct, focal pericoerulear zones-rostromedial and caudal juxtaependymal peri-LC. As shown in Figure 3, light microscopic observations revealed that many processes in the medial pericoerulear area were clearly dendrites, but many others were very thin and beaded, and appeared to be axons (Fu et al., 1988). However, it was impossible on the basis of these observations to determine what proportion of these processes were axons and which were dendrites. To unambiguously identify the nature of such processes, we analyzed DPHand TH-stained material at the electron microscope level. Electron microscopic analysis indicated that out of more than 500 labelled processes in the rostromedial and caudal juxtaependymal peri-LC zones, all but three labelled processes were dendrites (the three exceptions were in the caudal zone) (Fu et al., 1989). In additional studies, we found that these extranuclear LC dendrites are heavily targeted by noncatecholaminergic afferent synapses. Thus, LC

Fig. 3. High magnification brightfield photo montage of a 25-gm-thick horizontal section through the LC and pericoerulear region stained with an antibody directed against dopamine-p-hydroxylase and Nissl counter-stained. Note profuse dendrites exiting the rostra1 edge of LC and forming a rostrally directed dendritic fascicle. Note also the fine, beaded nature of extranuclear dendrites in the rostromedial pericoerulear region. Many of these fibers resemble axons, but all were shown to be dendrites upon electron microscopic examination (Fu et al., submitted; Shipley et al., submitted). Orientation: Rostra1 is at the top, medial is to the right.

neurons have an appreciable postsynaptic surface that lies a considerable distance outside the confines of the nucleus proper. Moreover, this “re-

55

ceptive surface” is preferentially distributed in two discrete peri-LC zones. The fact that several brain areas project to these pericoerulear regions raises the possibility that some of these inputs synapse upon extranuclear dendrites of LC neurons. Both PGi and PrH project to these two pericoerulear zones as well as to the LC proper, and may terminate on LC neurons in both locations. However, at this time there are no data concerning the sources or nature of synaptic contacts onto the extranuclear dendrites of LC neurons. The rostromedial zone containing extranuclear dendrites, in particular, receives input from several brain areas that do not innervate the LC nucleus (frontal, insular and perirhinal cortices, amygdala, dorsal raphe, VTA). Although inputs to the soma-rich LC nucleus would presumably exert a greater influence on LC discharge than those impinging on distal dendrites, pericoerulear contacts onto LC processes could provide functionally important modulation of activity driven by PGi and PrH which do target the LC proper. While the foregoing histochemical analysis revealed properties of LC neurons viewed as a group, they do not distinguish possible differences among individual LC neurons. To address this issue, we have studied the morphologies and dendritic domains of individual, intracellularly filled LC neurons. These cells were filled with biocytin during the course of physiologic/ pharmacological studies in in vitro tissue slices taken in the horizontal plane (see Williams et al., this volume). Filled neurons were remarkably uniform in their morphology, with 4 or 5 major dendrites leaving the soma from all directions, and a single thin process (presumably the axon) often emanating from a proximal dendrite. As shown in Figure 4, dendrites of individual LC neurons could be seen to exit the LC nucleus for considerable distances, extending preferentially in the rostral, medial and caudal directions. This property also appears to be uniform for LC neurons; of the 20 filled cells examined to date, all have at least 1 dendrite that extends into the rostromedial peri-

lVth

Ventricle

ratrat\

\

Fig. 4. Camera-lucida drawing of an LC neuron that was intracellularly filled in a horizontal tissue slice (350 Fm-thick). Boundaries of the LC nucleus proper are indicated by the dashed lines. Note extranuclear dendrites extending rostrally and medially; a similar profile was obtained for all individual cells filled to date, with many cells exhibiting even longer extranuclear dendrites. Orientation as indicated.

LC region and many have a dendrite that also extends distally into the caudal juxtacoerulear zone as well. These properties were similar for LC neurons whose somata were located throughout the nucleus (though soma size and dendritic extent may be smaller for cells ’located caudally vs. rostrally in the nucleus). Thus, individual LC neurons often possess dendrites that extend into both of the preferred extranuclear dendritic zones; there do not appear to be separate subpopulations of LC neurons giving rise to extranuclear dendritic extensions. Neurochemical identity of afferents to LC

Several major neurotransmitter inputs to the LC have been systematically characterized by ourselves and others. Many of the neurotransmitters found in fibers in the LC or peri-LC are also found in cells in PGi, PrH or peri-LC, as indicated in Table 1. Using anatomic double-labeling techniques, we have identified sources of adren-

TABLE 1 Neurochemically characterized cells and fibers in the LC, peri-LC, PGi and PrH Transmitter

Fibers in LC

ACh

-

+

epinephrine

+

+

+

serotonin

+

+

+

+

+

* * Figs. 7 & 8; Bowker et al., 1981; Beitz, 1982; Hunt and Lovick, 1982; Steinbusch, 1984; Thor and Helke, 1988; Pieribone et al., 1989

excitatory amino acids

+

+

+

?

+

Ottersen and Storm-Mathisen, 1984a; Ottersen and Storm-Mathisen, 1984b; Ennis and Aston-Jones, 1986; Ennis and Aston-Jones, 1988; Aston-Jones and Ennis, 1988

GABA

+

+

+

+

* * Figs. 5 & 6; Ottersen and Storm-Mathisen, 1984a; Ottersen and Storm-Mathisen, 1984b; Mugnaini and Oertel, 1985; Shipley et a/., 1988; Ennis and Aston-Jones, 1989a; Ennis and Aston-Jones, 1989b

enkephalin

+

+

+

+

Hokfelt et al., 1977; Sar et al., 1978; Hokfelt et al., 1979; Uhl e t a / . , 1979; Miller and Pickel, 1980a; Miller and Pickel, 1980b; Watson et a/., 1980; Finley et al., 1981; Hunt and Lovick, 1982; Conrath-Verrier etal., 1983; Khachaturian et al., 1983; Guthrie and Basbaum, 1984; Lynch et al., 1984; Charnay et al., 1985; Fallon and Leslie, 1986; * * Cassini et al., 1989; Drolet et al., in press

substance P

+

+

+

+

Hokfelt et al., 1978; Ljungdahl et al., 1978; Nomura et al., 1982; Triepel etal., 1985; * * Cassini et al., 1989; Sutin and Jacobowitz, 1988

neurotensin

+

+

+

+

Uhl et al., 1979; * * Beitz, 1982; Jennes et al., 1982; Minagawa et al., 1983; Triepel et al., 1984; Papadopoulos et al., 1986

VIP

+

+

+

+

* * Eiden et al., 1982;Martin et al., 1987;Sutin and Jacobowitz, 1988; Wang and Aghajanian, 1989

somatostatin

+ +

+ +

+

+ +

Johansson et al., 1984;Vincent et al., 1985

CRF

Fibers in peri-LC

* * Unpublished observations by us.

Cells in PGi

+

+

Cells in PrH

Cells in peri-LC

References

* * Altschuler et al., 1984; Butcher and Woolf, 1984; Kimura et al., 1984; Sutin and Jacobowitz, 1988; Ruggerio et al., 1990 Hokfelt et al., 1974; Ross et al., 1981; Berod et al., 1984; Granata et al., 1985; Hokfelt et al., 1985; Kalia et al., 1985; Ruggiero et al., 1985; Astier et al., 1986; Astier et al., 1987; Haselton and Guyenet, 1987; Tucker et al., 1987; Pieribone et al., 1988; Thor and Helke, 1988; Pieribone and Aston-Jones, in press

+ +

* * Fig. 9; Bloom et al., 1982; Merchenthaler e t a / . , 1982; Olshcowka et al., 1982; Cummings et al., 1983; Swanson et al., 1983; Merchenthaler, 1984; Sakanaka et al., 1987

Fig. 5. Low-power darkfield photomicrographs of coronal sections taken from rostra1 (left) and caudal (right) LC, stained with an antibody against glutamate decarboxylase (GAD), a marker of GABAergic neurons. Note the dense GAD-positive terminal plexus within LC (white arrow), and surrounding the LC nucleus proper both rostrally and medially, in the region of the extranuclear dendrites (black arrow; see Figs. 3 and 4). Orientation: dorsal is at the top and medial (IVth ventricle) is at the left.

58

ergic and the GABAergic inputs to the LC, and preliminary studies also indicate sources of 5-HT, corticotropin-releasing factor (CRF) and enkephalin inputs. As summarized below, these neurochemically characterized inputs originate from PGi, PrH, or the pericoerulear area. Adrenergic afferents to the LC Using retrograde transport of FG combined with PNMT immunofluorescence, we (Pieribone et al., 1988; Pieribone and Aston-Jones, 1991) determined that most adrenergic afferents to the LC derive from the PGi. We calculated that PNMT-immunoreactive (PNMT-ir) neurons constitute 21% of LC-projecting cells from that area,

while a small number of adrenergic afferents from PrH also exist (4% of LC-projecting PrH neurons). Within the PGi, the proportion of LC afferent neurons that stained for PNMT varied topographically. In particular, although the number of LC-projecting neurons was smaller in rostral, juxtafacial PGi, approximately 80% of LC afferents from this subregion of PGi were PNMT-ir. In contrast, about 18% of LC afferents in more central PGi (e.g. , the subambigual, retrofacial aspect of this nucleus) also stain for PNMT. GABAergic afferents to L C Our physiological and pharmacological studies of the PrH-LC pathway have demonstrated a

Fig. 6 . Brightfield photomicrographs showing double-labeled GABAergic afferents to LC from PrH (examples indicated by arrows). Neurons in PrH exhibiting brown, diffuse, DAB reaction product for GAD (a marker of GABAergic neurons) immunohistochemistry and punctate black granules of WGA-apoHRP-Gold transported retrogradely from the LC. Note also numerous GAD-positive, non-LC afferent neurons. IVth ventricle is at the top, midline is to the right.

59

$

LC

Fig. 7. Computer-aided reconstruction of PHA-L-labeled fibers in the LC area after injections into the dorsal raphe. Note nearly total lack of labeling in LC proper, but robust innervation of the ventromedial pericoerulear region. Fiber labeling was also extensive in the rostra1 pericoerulear region.

strong GABAergic innervation of LC from this major afferent (Ennis and Aston-Jones, 1989a, 1989b) (described below). Immunohistochemistry for GABA or GAD through the LC area revealed a dense innervation of the LC nucleus by

GABAergic fibers and terminals (Shipley et at., 1988) (Fig. 5). Staining for GABA/GAD following colchicine treatments revealed numerous, labeled neuronal somata in the same area of PrH (medial, perifascicular) that also contains neurons that project to LC (Pieribone et al., 1990). Recent experiments using WGA-apoHRP-Gold as the retrograde tracer have demonstrated that a large number (more than 40%) of LC-projecting neurons from PrH are GAD- or GABA-positive (Pieribone et al., 1990) (Fig. 6). These findings provide anatomic confirmation of our physiological and pharmacological results (described below).

Serotonergic afferents to LC We have confirmed results of others that the LC is densely innervated by 5-HT fibers. How-

Fig. 8. High-power view of a 5-HT-positive neuron (as indicated by diffuse, opaque DAB reaction product) in the ventromedial pericoerulear area that also contains WGA-apoHRP-Gold transported from LC (black particles in somata). Most of the other WGA-apoHRP-Gold particles in this field are also contained in cells (apparent with DIC illumination) that are not 5-HT-positive.

60

61

ever, retrograde transport of WGA-HRP or FG from LC did not consistently label neurons in the dorsal raphe (Aston-Jones et al., 1986a), the previously presumed source of 5-HT to the LC. Anterograde transport of WGA-HRP or PHA-L from the dorsal raphe nucleus labeled fibers in the central grey medially adjacent to the LC, but not in the LC proper (Pieribone et af., 1989) (Fig. 7). In addition, extensive lesions of the dorsal raphe nucleus (including the lateral “wings”) failed to diminish the dense 5-HT fiber staining found in LC (Pieribone et af., 1989). Furthermore, brain hemisections in the frontal plane rostral or caudal to the LC, or LC hemi-island preparations (severing tissue around the LC on rostral, medial and caudal sides) did not decrease 5-HT staining in LC (Pieribone e f al., 1989). Most recently we have observed that 5-HT cells in the rostroventral pericoerulear region send processes into the LC (Pieribone et al., 1989). Such 5-HT neurons are also retrogradely labeled following discrete injections of WGA-apoHRP-Gold into the LC (Fig. 8). Therefore, 5-HT innervation of the LC may derive from this group of pericoerulear 5-HT neurons. Lesions of this area combined with 5-HT immunohistochemistry through the LC are needed to further substantiate this hypothesis.

CRF afferents to L C Using an antibody directed against rat/human CRF, we (Valentino et al., 1990) found numerous CRF-positive fibers in the LC and CRF-positive cells in areas that contain LC afferents (PGi, PrH, and the dorsal cap of the paraventricular hypothalamic nucleus; Fig. 9). Preliminary results using WGA-apoHRP-Gold retrograde tracing and

immunohistochemistry revealed doubly labeled neurons in the dorsal cap of the paraventricular hypothalamic nucleus and in the PGi. In addition, there are many CRF cells in the pericoerulear area (Fig. 91, raising the possibility that local, pericoerulear neurons also contribute to the CRF innervation of LC. These studies are in progress. Effects of CRF, and putative activation of CRF afferents, on LC activity, are described by Valentino and Curtis (this volume).

E n k e ~ ~ ~ a afferents Lin to LC Numerous fibers within the LC nucleus stain with an antibody raised against enkephalin. Enkephalin-positive fibers are even denser in the rostral and ventromedial pericoerulear regions containing extranuclear LC dendrites, raising the possibility that distal dendrites of LC neurons also receive enkephalin inputs. We have also observed numerous enkephalin-positive neurons in regions containing LC afferents in both PGi and PrH, as well as in the pericoerulear area. Finally, our retrograde tracing with WGA-apoHRP-Gold combined with enkephalin immunohistochemistry revealed a surprising number of doubly labeled neurons in both PGi and PrH, indicating that a substantial percentage of LC-projecting neurons in each of these rostral medullary regions contain enkephalin (Fig. 10; Drolet et al., 1990). The significance of these opioid peptide projections to the LC is the subject of ongoing research. Other neurotransmitters in PGi, PrH and L C We have examined neurons in PGi, PrH, and peri-LC to define the sources of other transmitter inputs to the LC. In addition to the neurons that stain for PNMT, 5-HT, GABA/GAD, CRF and

Fig. 9. Corticotropin-releasing factor-immunoreactive (CRF-IR) fibers and cells in the LC/peri-LC region, and in PGi. a. Photomicrograph of a coronal section showing fluorescent (rhodamine tagged) CRF-IR neurons immediately lateral to the LC nucleus proper. This animal was pretreated with colchicine to optimize cell-body staining of CRF. Arrow is in the IVth ventricle medially, and indicates the LC nucleus proper. b. CRF-IR neurons in the PGi of a colchicine-pretreated rat. Similar CRF labeling in animals with WGA-apoHRP-Gold injections in LC reveal that retrogradely labeled and CRF-IR neurons are interdigitated in PGi, and that some LC-projecting neurons in PGi are CRF-IR. Ventral brain surface visible at lower left. Dorsal is at the top and medial is to the right for both panels.

62

enkephalin reviewed above, we have identified neurons in the PGi and PrH that stain for ChAT, neurotensin, VIP, angler fish pancreatic polypeptide, dynorphin and substance P. In addition, fibers that stain for all of these putative neurotransmitter markers are densely located in periLC regions (Table l). Studies using retrograde transport combined with immunohistochemistry will determine which of these areas provides these neurochemically defined inputs to the LC. Cellular pharmacology of afferents to LC

Excitatory amino acid ( E M )pathway from PGi to LC Single pulse stimulation of PGi activated 78% of LC neurons (Ennis and Aston-Jones, 1986,

1988). Cholinergic antagonists had no effect on LC activation by PGi or sciatic nerve stimulation. This is consistent with recent observations that the LC appears to be nearly devoid of cholinergic fibers (Ruggiero et al., 1990). However, the EAA antagonists, kynurenic acid or D-glutamyl glycine, consistently blocked both PGi- and sciatic-induced activation of the LC (AstomJones and Ennis, 1988). Similar results have recently been obtained with local application of kynurenic acid or the non-NMDA antagonist CNQX (Fig. 11) (Ennis and Aston-Jones, submitted; Shiekhattar and Aston-Jones, 1991). The NMDA antagonists AP5 or AP7 were not effective on either response, indicating that PGi-induced EAA activation of LC neurons probably takes place at a non-NMDA receptor in the LC. These results

Fig. 10. Enkephalin-immunoreactive (Enk-IR) LC afferent neurons in the PGi. Brightfield photomicrograph of a coronal section through the PGi of a rat injected with WGA-apoHRP-Gold into the LC. Retrogradely transported WGA-apoHRP-Gold appears as black puncta inside neurons, whereas Enk-IR neurons contain the brown, diffuse diaminobenzidine reaction product. Neurons containing both labels are indicated by the solid arrows, while several sample Enk-IR cells that are not retrogradely labeled are indicated by the open arrows. Note the large percentage of LC-projecting neurons that are also Enk-IR. Ventral brain surface is at lower left; medial is at the right and dorsal is at the top.

63

30r

lull

680

I I I

, I

1360 Time (msec)

2040

'Or

f

kn

U

lot 0

During-CNOX

t

680

1360 Time (msec)

2040

30r Recovery

0

t

1360 Time ( m s e c )

680

20LO

Fig. 11. Locally applied CNQX blocks sensory responses of LC neurons. Cumulative peri-stimulus time histograms (PSTHs) revealing blockade of footpad nerve-evoked response in an LC neuron by local application of the specific nonNMDA antagonist CNQX. CNQX was applied from a composite dual-barrel micropipette as an 8 p M solution in artificial cerebrospinal fluid (ACSF). Similar blockade was observed for each of 12 cells tested. Footpad stimulation (to activate sciatic nerve) at arrows; 50 stimuli accumulated in each PSTH.

also suggested that sciatic-evoked excitation of LC neurons might be mediated through the PGiLC pathway. This was confirmed with experiments demonstrating that infusions of lidocaine, GABA or synaptic decouplers into PGi blocked or attenuated LC response to sciatic activation (described in Aston-Jones et al., this volume).

NMDA-receptor-mediated sensory response of L C neurons revealed by low-Mg++ infusion in vivo We (Shiekhattar et al., 1991) have developed a method to locally perfuse a small region of brain in vivo using slow microinfusion (30-60 nl/min) from a multibarrel micropipette. With this method, microinfusion of artificial cerebrospinal fluid (ACSF) lacking Mg++ ions onto LC neurons caused a significant increase (by about 36%) in footpad stimulation-evoked sensory responses of these cells (Fig. 12) (Shiekhattar and Aston-Jones, 1991). This increased responsiveness was prolonged, recovering about 3-5 min after termination of the Mg++-free ACSF infusion. This increase in sensory response magnitude was completely blocked by co-infusion with the specific NMDA antagonists AP5 (50 p M ; Fig. 12) or CGS 19755 (1 kM). There was no consistent effect of Mg++-free solutions on spontaneous LC discharge. These results demonstrate that NMDA receptor mechanisms exist for this sensory response of LC cells, but that they are normally occluded, presumably by Mg++ blockade of the NMDA channel. They also indicate a possible mechanism whereby EAA-mediated sensory responses may be augmented in the LC. Although little is known concerning variation of Mg++ concentrations in ciuo, these results indicate that decreased Mg could have profound effects on LC sensory responsiveness and, consequently, on noradrenergic neurotransmission throughout the brain. Finally, the finding that sensory input to LC accesses NMDA receptor mechanisms indicates that modulation of NMDA receptor activity may modulate LC function. + +

Adrenergic inhibition of L C from PGi Clear inhibitory effects of PGi stimulation on LC neurons appeared when the EAA-mediated activation of LC from the same stimulation sites in the PGi was eliminated. Under kynurenate blockade, an underlying inhibition from PGi was observed in 88% of LC cells (Ennis and AstonJones, 1988). We (Astier and Aston-Jones, 1989)

64

f

35

PRE

5

08. J .32'1

0

0

1360

2040

Time ( r n s e c )

g'

DUR

23 Q

0

I

680 1360 Time (rnsec)

2040

have also shown that systemic administration of the a 2 antagonist idazoxan (1 mg/kg) attenuates this inhibition in 80% of neurons tested (Fig. 13). A similar effect was seen for locally infused idazoxan (0.5-2.5 ng in 50 nl), whereas vehicle infusion was without effect. Several LC cells (15% of those tested) exhibited pure inhibition after PGi stimulation; systemic or local idazoxan was typically effective in antagonizing these responses as well. Furthermore, stimulation of the MB, which carries adrenergic fibers from the PGi to the LC, produced pure inhibition of LC activity. This response was also blocked by systemic or local idazoxan (Astier and Aston-Jones, 1989). These results provide physiological and pharmacological confirmation of our anatomic results for a prominent adrenergic projection to LC from C1 cells in PGi (described above), and indicate that this input uniformly inhibits activity of LC neurons.

**

T

0

100 -. ~

C

0

+ 0

cU X

Id

50 ACSF

Mg*+-f ree ACSF

Mg++-free

ACSFt AP5

Fig. 12. In ciuo perfusion of LC with MgCC-free ACSF reveals NMDA-mediated sensory response. A. PSTH of the response of a typical LC neuron to stimulation of the rear footpad (for sciatic nerve activation; at arrow) before infusion of Mg++-free ACSF.B. Additional late response to footpad stimulation is apparent during slow infusion (50 nl/min) of Mg++-free ACSF into the LC from a micropipette barrel adjacent to the recording barrel. 50 sweeps in each PSTH.C. Bar graph illustrating increase of footpad response magnitude for LC neurons during infusion of Mg++-free ACSF (ACSFM g f f ; **P < 0.01, paired t-test on absolute response magnitudes), and reversal by co-infused AP5 (ACSF-Mg + AP5), a specific NMDA-receptor antagonist. n = 12 cells for ACSF alone, 18 cells for Mg++-freeACSF, and 8 cells for Mg++-free ACSF plus AP5.

GABAergic inhibition of LC from PrH In contrast to the potent activation described above from PGi, low-frequency stimulation of the other major afferent to the LC, the PrH, potently inhibited 85% of LC neurons tested (Ennis and Aston-Jones, 1989a, 1989b). Idazoxan was without effect on this response, but picrotoxin (6 mg/kg, iv) consistently attenuated this inhibition. Bicuculline microinfused or microiontophoresed onto LC neurons during PrH stimulation also consistently antagonized this response, whereas locally applied strychnine was ineffective. Taken together, these results indicate a strong GABAergic projection from the PrH to the LC, consistent with the anatomic findings described above (Figs. 5 and 6). In addition to attenuating spontaneous LC discharge, stimulation of this GABAergic input from PrH also potently attenuated excitation of LC neurons evoked by sciatic nerve stimulation (Ennis and Aston-Jones, 1989b) (Fig. 14). Therefore, the PrH is able to significantly attenuate activation of LC neurons by their major excitatory input, the PGi.

65

Effect of activation of preuiously reported afferents on LC discharge The central nucleus of the amygdala (CNA) was previously reported to be the major afferent to the LC (Cedarbaum and Aghajanian, 19781, whereas our anatomical studies demonstrate a dense projection from the CNA to the lateral and rostromedial pericoerulear areas but no input to P G I Stim 15 I

A

I

PRE - KYN

1

Q

5 i

the LC proper (Aston-Jones et al., 1986a), consistent with other recent results (Wallace et al., 1989). As LC neurons extend dendrites into the extracoerulear neuropil, especially into the rostromedial pericoerulear zone, we considered the possibility that the CNA could influence LC neurons through contacts onto LC dendrites even though it does not innervate the LC proper. We used electrophysiological methods as an initial means of testing this possibility and discerning the functional impact of CNA on LC discharge (Aston-Jones et al., 1990b). High-intensity (2 mA)

5 t

0 V

=

P r H Stim

I

Time ( m s e c )

m

10

0

$

c

5

POST-KY N Tlrne ( rnsfc )

15

0

V

B FS

I

f

Time (msec)

m

10

i

oi

15r_

_ . 5

ldh

C

P O S T - K Y N 8. I D A

2 U 0

I

n

680

1360

2060

Time ( m s e c )

0 V

l5 T C

Time (msec )

Fig. 13. Local infusion of KYN into LC reveals underlying adrenergic inhibition from PGi. A. PSTH generated during single pulse, electrical stimulation of PGi (at arrow). Activation of PGi yields short latency, potent excitation of this typical LC neuron. B. PGi-evoked excitation of the cell shown in (A) is completely attenuated 1 min after infusion of 0.01 pmol of KYN into LC, as shown in (B) post-drug. Note that blockade excitation by KYN reveals a purely inhibitory response of this neuron to PGi stimulation. Stimulation intensity in (A and B) = 300 pA. C. The inhibition shown in (B) is completely attenuated 5 min after administration of the cr2 receptor antagonist idazoxan (0.2 mg/kg, iv). Similar results are obtained with local microinfusion of idazoxan into the LC. (From Astier and Aston-Jones, 1989.)

E

PrH Stim

& FS

1°1

1111 11111

11111

II

I

Uii I

Time ( r n s e c )

Fig. 14. A. PSTH showing inhibition of discharge of an LC neuron during single pulse electrical stimulation of PrH (300 FA, at arrow). B. PSTH, for the same LC neuron in (A), generated during footpad stimulation (FS). FS (20V, at arrow) yields robust excitation of this LC neuron. C. FS-evoked excitation of the same LC neuron is potently attenuated when FS (20 V) and PrH (300 p A ) stimuli are simultaneously delivered (at arrow). (From Ennis and Aston-Jones, 1989b.l

66

stimulation of CNA elicited only weak and inconsistent synaptic activation in 5/50 LC neurons (6 rats). Five other LC neurons were antidromically activated, as expected, because the LC densely innervates the CNA (unpublished results). In cbntrast, 19/30 neurons in the adjacent parabrachial area exhibited strong, short-latency synaptic activation from CNA stimulation (Aston-Jones et al., 1990b). The weak action of high intensity CNA stimulation on a few LC neurons may reflect a polysynaptic pathway (e.g., the CNA projection to the rostromedial pericoerulear area could innervate local neurons that may contact LC cells). It is also possible that CNA inputs to this pericoerulear area synapse upon distal dendrites of LC neurons; of course, both types of connections also may exist. Electron microscopic analysis is needed to decide conclusively among these possibilities. Similar results were obtained for the NTS (Ennis and Aston-Jones, 1989b) and the medial prefrontal cortex (Chiang et al., 1987). Our anterograde tract-tracing studies confirmed our retrograde transport studies, finding that the NTS strongly innervates the medial parabrachial area just lateral to the LC. Results for NTS stimulation resemble those for CNA stimulation, in that only weak, long-latency synaptic responses were observed in the LC while short-latency, potent excitation was observed in the adjacent parabrachial zone where we find fiber innervation from the NTS (Ennis and Aston-Jones, 1989b). The findings for the prefrontal cortex are similar; as reported by Aston-Jones et al. (this volume), train stimulation of cortex may reveal indirect but functionally important influences on LC activity. Finally, recent studies have found similar results for PAG projections to the LC area. Retrograde and anterograde tracing experiments revealed that the midbrain PAG projects heavily and focally to the rostromedial pericoerulear region. In contrast, the LC proper receives only sparse fibers from PAG (Aston-Jones et al., 1989b; Ennis et al., 1989, 1991; Van Bockstaele et al., 1991). Electrophysiological experiments were

PAG Stim

"1

A

Time (msec)

"1 5

m

--

B

:

Per1- LC

10-

0

1 .-

5

m

50 ~ / U I I U U O l ~ ~ ~ ~ ~ J . I ~ ~ _ 111.11110 ) 1 IL~lUllllllilllllllllJlll~ l~Unll 0 100 300 COO 500 600 Time ( m s e c

1

Fig. 15. A. PSTH showing weak synaptic activation of an LC neuron from PAG stimulation (1000 PA, at arrow). B. The same stimulation site yielded robust activation of neurons in the adjacent rostromedial pericoerulear region, as illustrated for a typical cell (stimulation at arrow, 1000 PA). PSTHs accumulated for 100 sweeps.

consistent with these anatomic results, finding that only 6/100 PAG neurons were antidromically activated from the LC and that most LC neurons were only weakly influenced by PAG stimulation (Fig. 15; Ennis et al., 1991). Overall, the mean activation of LC neurons from the lateral PAG was 32 spikes per 100 stimuli. In contrast, the majority of rostromedial pericoerulear neurons were robustly activated from the same PAG sites, yielding an average of 75 spikes per 100 stimuli (Fig. 15; Ennis et al., 1989, 1991). Latencies for responses in LC or peri-LC were similar, about 5-7 msec. These results indicate that while the PAG influences LC activity, the major impact of PAG on the dorsolateral pontine tegmentum is upon neurons in the rostromedial pericoerulear region rather than on LC neurons. Whether PAG-evoked responses of LC neurons are due to direct synaptic contacts onto LC neurons/dendrites, or are mediated via the

67

more responsive pericoerulear cells; requires further study. The results of the above studies underline the need for combined electrophysiological and electron microscopic studies of inputs to extranuclear dendrites and of possible pericoerulear connections to LC.

Effect3 of 5-HT and NE on LC acticity and responskity Although it has long been appreciated that the LC is densely innervated by 5-HT fibers, and interactions between brain NE and 5-HT systems have been implicated in a variety of clinically relevant phenomena (e.g., sleep-waking cycle control, depression), the effects of 5-HT on discharge of NE-LC neurons has been little investigated. Using microiontophoretic and micropressure techniques, we investigated the effects of directly applied 5-HT and other agents on LC discharge (Chouvet et al., 1988; Aston-Jones et al., 1991). lontophoretic 5-HT had no consistent effect on spontaneous LC activity, decreasing activity of many cells while the discharge of other neurons was unchanged or increased in the presence of this agent. However, similar iontophoretic application of 5-HT consistently and potently attenuated responses of LC neurons to iontophoretic glutamate (GIu). In contrast, 5-HT did not attenuate (and in some cases even potentiated) responses to ACh (Fig. 16). These results were not due to an artifact of iontophoresis as (i) there was no effect of iontophoresis at similar currents through an adjacent barrel containing saline, and (ii) similar effects were found for 5-HT directly applied to LC neurons by micropressure. Additional studies indicated that the 5-HT effects observed in the LC are primarily mediated by 5-HTl;, receptors (Aston-Jones et al., 1990a; Charlety et al., 1990). The 5-HTl, agonists 83 H D P A T and buspirone mimicked the effect of 5-HT by selectively attenuating responses of LC neurons to Glu. Iontophoretic application of the 5-HT,, agonist TFMPP weakly mimicked 5-HT,

NE 1 5 n A

Glu20nAs

Esxsxs3

s s s

s s s s s

0 s s s

s

- -

5-HT 1 O O n A

AChlOOnA

5-HT 1 0 0 n A

I III

NE 1 5 n A

m m

I I m IIII

Fig. 16. Comparison of norepinephrine (NE) and 5-HT effects on responses to glutamate (Glu) and ACh for the same LC neuron. Computer integrated activity-time histograms (5 sec bin width) for activity of a typical LC neuron during microiontophoresis, as indicated. NE applied with a long pulse of low current (at striped bars) inhibits basal activity but leaves responses evoked by Glu (applied at filled circles, upper trace) or ACh (applied at solid bars, lower trace) intact. In contrast, 5-HT applied in long pulses (at open bars) does not affect basal discharge rate, but markedly attenuates responses to Glu (upper trace). Note that although responses to Glu are attenuated by 5-HT, responses to ACh of the same neuron remain intact during application of 5-HT (lower trace). Both traces recorded from the same LC neuron. Calibration bar = 2 min. (From Aston-Jones et al., 1990).

indicating a possible minor 5-HT,, component to this response. In contrast, the 5-HT, agonist DO1 did not mimic 5-HT, and the 5-HT, antagonist ketanserin did not block 5-HT’s effects, leading us to conclude that a 5-HT, receptor is not significantly involved in this response. As the response of LC neurons to sciatic nerve activation appears to be mediated by an EAA pathway from the PGi, it seemed possible that this sensory response of LC neurons would be modulated by 5-HT inputs. Indeed, we have found that pretreatment of animals with parachlorophenylalanine (PCPA) to deplete 5-HT in nerve terminals significantly increased footpad-evoked responses of LC neurons, and that injection of the 5-HT precursor 5-hydroxytryptophan (5-HTP) to increase 5-HT in the brain reduces footpad

68

stimulation-evoked response magnitudes (Shiekhattar and Aston-Jones, unpublished observations). Along with the findings for PCPA administration in naive animals, these results indicate that 5-HT tonically modulates LC sensory responses. However, these systemic injections of PCPA and 5-HTP would be expected to affect 5-HT neurotransmission throughout the nervous system, and so their site of action for affecting LC responsiveness is unclear. To investigate whether direct 5-HT inputs to LC may modulate its sensory responses, we have initiated studies using direct application of 5-HT and 5-HT agonists in combination with footpad stimulation. Local application of 5-HT onto LC somata did not selectively attenuate footpad stimulation responses (Akaoka and Aston-Jones, unpublished results), in contrast to results anticipated by the effects of 5-HT on responses to directly applied Glu, described above. This may reflect inaccessibility of 5-HT applied at the soma to reach distant dendritic synapses mediating responses from PGi, or multiple EAA receptors on LC neurons, some of which (those responsive to footpad stimulation activation) are not sensitive to 5-HT. In contrast to these effects of 5-HT, iontophoresis of NE, or of the a 2 agonist clonidine, with modest currents markedly decreased spontaneous LC discharge but did not attenuate LC responses to either Glu or ACh (Fig. 16) (Chouvet et al., 1988; Aston-Jones et al., 1991); application of these agents with high currents attenuated evoked activity as well. Nonetheless, there was a selective sensitivity of basal discharge to inhibition by a 2 receptor activation. Also, in preliminary studies systemic administration of clonidine or of morphine produced a similar selective inhibition of spontaneous activity while response to footpad (sciatic) stimulation remained intact. When considered in terms of the ratio of phasic, evoked activity to tonic, basal activity of LC neurons (termed response contrast), markedly distinct effects were seen for 5-HT vs. for NE. By selectively attenuating Glu-evoked responses,

5-HT markedly decreased the response contrast of LC neurons for Glu-evoked activity, but did not change the ACh response contrast for these cells. NE, on the other hand, by selectively attenuating basal discharge without affecting responses to either Glu or ACh, markedly augmented response contrasts of LC neurons for both Glu and ACh.

merents to afferents: inputs to the nucleus paragigantocellularis Given that major afferents to LC arise in PGi and PrH, it is clear that knowledge of afferents to these medullary structures is critical to understanding the afferent control of LC. We have begun, therefore, to study the organization of afferents to these two medullary nuclei. Retrograde transport of WGA-HRP or FG, as well as anterograde transport of PHA-L, were used to identify afferents to the PGi (Van Bockstaele et aL, 1989,). As illustrated in Figure 17, major inputs found to the PGi were the caudal medullary reticular formation, raphe magnus area, KollikerFuse/ lateral parabrachialis, NTS, PAG, paraventricular hypothalamic nucleus, and a newly

Fig. 17. Diagram illustrating major afferents to retrofacial PGi in the rat. Abbreviations: amb, nucleus arnbiguus: CC, corpus callosum; CG, central gray; IC, inferior colliculus; KF, Kolliker, Fuse nucleus; LH, lateral hypothalamus: LPb, lateral parabrachial nucleus; LV, lateral ventricle; MdD, caudal medullary reticular formation dorsal to the lateral reticular nucleus; NTS, nucleus tractus solitarius; PVN, paraventricular nucleus of the hypothalamus; PGi, nucleus paragigantocellularis; RPa, raphe pallidus; V11, VIIth nerve nucleus; 4V, fourth ventricle.

69

described but very prominent input from the supraoculomotor nucleus of the central grey. Several of these afferents exhibited clear topography within the PGi. As seen with anterograde transport of PHA-L, for example, PAG projects primarily to the medial PGi while the NTS and the supraoculomotor nucleus project to specific, but distinctly different, areas of the PGi (Van Bockstaele et al., 1991). These results indicate that afferents to the PGi are diverse and located throughout the brainstem and spinal cord, but are generally associated with autonomic and integrative functions. Neurons that project to the LC are distributed diffusely, though with some topography, in PGi (Fig. 2). These results, together with the topography of afferent projections to PGi, indicate that all, or only a select few, of the afferents to the PGi may synapse upon LC-projecting neurons. At present we do not know which extrinsic or intrinsic afferents to PGi or PrH synapse upon neurons that project to LC. This is an important issue because knowing which inputs to the PGi or PrH contact LC-projecting cells will reveal functional circuits that most directly influence the LC system. Experiments to answer these questions are planned for both the PGi and PrH. Conclusions

Studies over the last 5 years have yielded a new perspective on the afferent regulation of the noradrenergic brain nucleus LC. Major afferents to the LC are found in two rostral medullary regions, the medial, perifascicular PrH in the dorsomedial medulla, and the PGi in the rostral ventral medulla. These major afferents utilize an EAA(s) (PGi) or GABA (PrH) as the most potent neurotransmitter agents in their projections to the LC. Thus, PGi activation predominantly excites LC neurons through a non-NMDA receptor-sensitive mechanism, while PrH potently inhibits LC neurons by a GABA, receptor in the LC.

However, the LC receives inputs from a variety of neurotransmitter systems, indicating that its afferent organization is more complex than such a synopsis might suggest. Indeed, we find that multiple neurotransmitters impinge on the LC from these two major afferent nuclei. Within the PGi, for example, there are not only EAA projections to the LC, but also prominent adrenergic inputs. When activation by EAAs is blocked pharmacologically, inhibition from these adrenergic projections becomes apparent. We also have recently obtained preliminary evidence for GABA and CRF inputs to the LC from the rostral ventrolateral medulla. These findings, taken together with other results reviewed here, indicate that functionally distinct subpopulations of PGi neurons may innervate the LC: (i> physiologically distinguishable subpopulations of PGi neurons are antidromically activated from the LC, and one of these subpopulations is predominantly located in the ventromedial aspect of PGi, (ii) there exist at least three distinct pathways for projections from the PGi to the LC, and (iii) projections from the PGi to the LC, and afferents to the PGi, are topographically distributed within the PGi. In addition, sciaticevoked LC activation appears to be preferentially mediated by neurons in the ventromedial PGi (Chiang and Aston-Jones, 1989; reviewed in AstonJones et al., this volume). The possibility of functionally distinct subpopulations of afferents to the LC from the PrH is presently less clear. As the PGi and the PrH are prominent in regulating LC activity, functions previously ascribed to these two medullary areas provide important insights into the functions of the NE-LC system. The PrH has been extensively studied in cats and primates as a preoculomotor nucleus, and many of its constituent neurons are important in the control of eye movements (Baker, 1977; McCrea et al., 1979). The close correspondence of the LC with increased attention to environmental stimuli and orienting behaviors (AstonJones and Bloom, 1981a,b) may derive in part from such oculomotor circuitry, as eye move-

70

ments are an important part of behavioral orienting responses that accompany increased attentiveness to external stimuli. The PGi area has been linked to cardiovascular, nociceptive and respiratory functions (Guyenet and Les Brown, 1986; Morrison et al., 1988; Sun et al., 1988). In particular, activation of this area broadly increases activity in the peripheral sympathetic nervous system (Ross et al., 1984). The strong excitatory input from this same area to the LC provides a neurobiological substrate for the fact that sympathoexcitatory stimuli are also very effective in activation of LC neurons (Elam et al., 1984, 1986), and is strongly supportive of the proposal that the NE-LC system serves as the cognitive limb of a globally conceived sympathetic nervous system (Aston-Jones, 1985; Aston-Jones et al., 1990b). The functional implications of the PGi and PrH as afferents to the LC, particularly as related to activity of LC neurons in behaving animals and the role of the noradrenergic LC system in vigilance, are discussed in more detail in Aston-Jones et al. (this volume). Important studies for the future include further examination of the role that extranuclear dendrites of LC neurons play in differential regulation of LC activity, and the extent and functional importance of LC innervation by ‘‘local’’ pericoerulear neurons. Sources of, and functional interactions between, the multitude of transrnitters that innervate the LC are also important aspects of our current and future research. Together, recent results concerning afferent regulation of the LC are revealing the neurobiological substrates by which activity in the globally projecting NE-LC system is regulated. These results lay the groundwork for further experimentation to provide a complete cellular anatomic, physiological and pharmacological understanding of the control of activity in these neurons. Such a cellular understanding is necessary for a comprehensive input-output analysis of the NE-LC system, which is essential to understanding the system’s function at a cellular and systems level.

Acknowledgements

This work was supported by PHS grants NS24698, NS20463, HL08097, DA06214, MH40008, MH42796, O N R contract N00014-86-0493, AFOSR grant 90-0147, the Phillippe Foundation, the Simone et Cino del Duca Foundation, INSERM, the Fonds de la Recherche en Sant6 du QuCbec. References Aghajanian, G.K. (1978) Feedback regulation of central monoaminergic neurons: Evidence from single-cell recording studies. In M.B.H. Youdim, W. Lnvenberg, D.F. Sharman and J.R. Lagnado (Eds.), Neurochemisrry und Neuropharmacology, Wiley, New York, pp. 1-32. Altschuler, R., Fex, J., Parakkal, M. and Eckenstein, F. (1984) Colocalization of enkephalin-like and ChAT-like immunoreactivities in olivocochlear neurons of the guinea pig. J. Histochem. Cytochem., 32: 839-843. Arnsten, A.F. and Goldman, R.P. (1984) Selective prefrontal cortical projections to the region of the locus coeruleus and raphe nuclei in the rhesus monkey. Brain Res., 306: 9-18. Astier, B. and Aston-Jones, G. (1989) Electrophysiological evidence for medullary adrenergic inhibition of rat locus coeruleus. Soc. Neurosci. Abstr., 15: 1012. Astier, B., Kitahama, K., Denoroy, L., BLrod, A., Jouvet, M. and Renaud, B. (1986) Biochemical evidence for an interaction between adrenaline and noradrenaline neurons in the rat brainstem. Brain Res., 397: 333-340. Astier, B., Kitahama, K., Denoroy, L., Jouvet, M. and Renaud, B. (1987) Immunohistochemical evidence for the adrenergic medullary longitudinal bundle as a major ascending pathway to the locus coeruleus. Neirrosci. Lett., 74: 132-138. Astier, B., Van Bockstaele, E.J., Aston-Jones, G . and Pierihone, V.A. (1990) Anatomic evidence for multiple pathways leading from the nucleus paragigantocellularis to the locus coeruleus in the rat. Neurosci. Lett. (In press). Aston-Jones, G. (1 985) Behavioral functions of locus coeruleus derived from cellular attributes. Physiol. Psych., 13: 118126. Aston-Jones, G. and Bloom, F.E. (1981a) Activity of norepinephrine-containing locus coeruleus neurons in hehaving rats anticipates fluctuations in the sleep-waking cycle. .I. Neurosci., 1: 876-86. Aston-Jones, G. and Bloom, F.E. (1981b) Norepinephrinecontaining locus coeruleus neurons in behaving rats exhibit pronounced responses to non-noxious environmental stimuli. J. Neurosci., 1: 887-900.

71 Aston-Jones, G. and Ennis, M. (1988) Sensory-evoked activation of locus coeruleus may be mediated by a glutamate pathway from the rostra1 ventrolateral medulla. In A. Cavalheiro, J. Lehmann and L. Turski (Eds.), Frontiers in Excitatory Amino Acid Research, A.R. Liss, Inc., New York. pp. 471-478. Aston-Jones, G., Foote, S.L. and Bloom, F.E. (1984) Anatomy and physiology of locus coeruleus neurons: Functional implications. In M. Ziegler and C.R. Lake (Eds.), Norepinephrine: Frontiers of Clinical Neuroscience, Vol. 2, Williams and Wilkins, Baltimore, pp. 92-1 16. Aston-Jones, G., Ennis, M., Pieribone, V.A., Nickell, W.T. and Shipley, M.T. (1986a) The brain nucleus locus coeruleus: Restricted afferent control of a broad efferent network. Science, 234: 734-737. Aston-Jones, G., Shipley, M.T., Nickell, W.T., Ennis, M. and Pieribone, V. (l986b) Afferents to locus coeruleus are largely restricted to two medullary nuclei: Anatomic and physiologic studies. Soc. Neurosci. Abstr., 12: 138. Aston-Jones, G., Van Bockstaele, E., Pieribone, V. and Shipley, V. (1989) Topographic projections from the periaqueductal gray (PAG) to the ventrolateral medulla (VLM) in the rat. Soc. Neurosci. Ahsrr., 15: 593. Aston-Jones, G., CharlCty, P., Akaoka, H., Shiekhattar, R. and Chouvet, G. (1990a) Serotonin acts at 5-HTla receptors to selectively attenuate glutamate-evoked responses of locus coeruleus neurons. Soc. Neurosci. Ahstr., 16: 799. Aston-Jones, G., Shipley, M.T., Ennis, M., Williams, J.T. and Pieribone, V.A. (1990h) Restricted afferent control of locus coeruleus neurons revealed by anatomic, physiologic and pharmacologic studies. In C.A. Marsden and D.J. Heal (Eds.), The Pharmacology of Noradrenaline in the Central Nervous System, Oxford University Press, G.B., Oxford, pp. 187-247. Aston-Jones, G., Akaoka, H., CharlCty, P. and Chouvet, G. (1991) Serotonin selectively attenuates glutamate-evoked activation of locus coeruleus neurons in ciuo. J. Neurosci., 11: 760-769. Baker, R. (1977) The nucleus prepositus. In B. Brooks and F. Bajandas (Eds.), Eye Movements, Plenum Press, New York, pp. 145-178. Beitz, A.J. (1982) The sites of origin of brainstem neurotensin and serotonin projections to the rodent nucleus raphe magnus. J. Neurosci., 2 (7): 829-842. BCrod, A,, Chat, M., Paut, L. and Tappaz, M. (1984) Catecholaminergic and GABAergic anatomical relationship in the rat substantia nigra, locus coeruleus, and hypothalamic median eminence: Immunocytochemical visualization of biosynthetic enzymes on serial semithin plastic-embedded sections. J. Histochem. Cytochem., 32: 1331-1338. Blier, P. and de Montigny, C. (1987) Modification of 5-HT ned administration of the 5neuron properties by su H T l A agonist gepirone: Electrophysiological studies in the rat brain. Synapse, 1: 470-480. Bloom, R.E., Battenberg, E.L.F., Rivier, J. and Vale, W. (1982) Corticotropin releasing factor (CRF): Immunoreactive neurones and fibers in rat hypothalamus. Reg. Peptides, 4: 43-48.

Bobillier, P., Seguin, S., Degueurce, A., Lewis, B.D. and Pujol, J.F. (1979) The efferent connections of the nucleus raphe centralis superior in the rat as revealed by autoradiography. Bruin Res., 166: 1-8. Bowker, R.M., Steinbusch, H.W.M. and Coulter, J.D. (1981) Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde H R P histochemical and immunocytochemical staining method. Bruin Res., 211: 412-417. Butcher, L. and Woolf, N.J. (1984) Histochemical distribution of acetylcholinesterase in the central nervous system: Clues to the localization of cholinergic neurons. In A. Bjorklund, T. Hokfelt and M.J. Kuhar (Eds.), Classical Transmitters and Transmitter Receptors in the CNS. Purt I I , Elsevier Science Publ. B.V., Amsterdam, pp. 1-45. Cassini, P., Ho, R.H. and Martin, G.F. (1989) The brainstem origin of enkephalin- and substance-P-like immunoreactive axons in the spinal cord of the North American opossum. Brain Behau. Evol., 34: 212-22. Cechetto, D.F., Standaert, D.G. and Saper, C.B. (1985) Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat. J. Comp. Neurol., 240: 153-160. Cedarbaum, J.M. and Aghajanian, G.K. (1978) Afferent projections to the rat locus coeruleus as determined by a retrograde tracing technique. J. Comp. Neurol., 178: 1-16. CharlCty, P.J., Akaoka, H., Aston-Jones, G. and Chouvet, G. (1990) In viuo pharmacological characterization of the serotonin receptors involved in the interaction with excitatory amino acids in the nucleus locus coeruleus of the rat. Eur. J. Pharm. Suppl., 3: 30. Charnay, Y., LCger, L., R o s i e r , J., Jouvet, M. and Dubois, P.M. (1985) Evidence for synenkephalin-like immunoreactivity in pontobulbar monoaminergic neurons of the cat. Brain Res, 335: 160-4. Chen, Z. and Engberg, G. (1989) The rat nucleus paragigantocellularis as a relay station to mediate peripherally induced central effects of nicotine. Neurosci. Lett., 101: 67-71. Chiang, C. and Aston-Jones, G. (1989) Microinjection of lidocaine, GABA or synaptic decouplers into the ventrolateral medulla blocks sciatic-evoked activation of locus coeruleus. Soc. Neurosci. Abstr., 15: 1012. Chiang, C., Ennis, M., Pieribone, V. and Aston-Jones, G. (1987) Effects of prefrontal cortex stimulation on locus coeruleus discharge. Soc. Neurosci. Abstr., 13: 912. Chouvet, G., Akaoka, H. and Aston-Jones, G. (1988) Serotonin selectively decreases responses of locus coeruleus neurons to glutamate. C.R. Acud. Sci. Paris, 290: D807D810. Cintra, L., Diaz-Cintra, S., Kemper, T. and Morgane, P.J. (1982) Nucleus locus coeruleus: A morphometric golgi study in rats of three age groups. Brain Res., 247: 17-28. Clavier, R.M. (1979) Afferent projections to the self-stimulation regions of the dorsal pons, including the locus coeruleus, in the rat as demonstrated by the horseradish peroxidase technique. Brain Res. Bull., 4: 497-504. Conrad, L.C.A., Leonard, C.M. and Pfaff, D.W. (1974) Connections of the median and dorsal raphe nuclei in the rat:

72 An autoradiographic and degeneration study. J. Comp. Neurol., 156: 179-206. Conrath-Varier, M., Dietl, M., Arluison, M., Cesselin, F., Bourgoin, S. and Hamon, M. (1983) Localization of Metenkephalin-like immunoreactivity within pain-related nuclei of cervical spinal cord, brainstem and midbrain in the cat. Bruin Res. Bull., 11: 587-604. Cummings, E., Elde, R., Ells, J. and Lindall, A. (1983) Corticotropin-releasing factor immunoreactivity is widely distributed within the central nervous system of the rat: An immunohistochemical study. J. Neurosci., 3: 1355-1368. Dahlstrom, A. and Fuxe, K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol. Scand., 62: 5-55. Deutch, A.Y., Goldstein, M. and Roth, R.H. (1986) Activation of the locus coeruleus induced by selective stimulation of the ventral tegmental area. Bruin Res., 363: 307-314. Drolet, G., Akaoka, H., Van Bockstaele, E.J., Aston-Jones, G. and Shipley, M.T. (1990) Opioid afferents to the locus coeruleus from the rostral medulla as detected by retrograde transport combined with immunohistochemistry. Soc. Neurosci. Abstr., 16: 1027. Eiden, L.E., Nilaver, G. and Palkovits, M. (1982) Distribution of vasoactive intestinal polypeptide (VIP) in the rat brain stem nuclei. Brain Res., 231: 472-7. Elam, M., Yao, T., Svensson, T.H. and ThorCn, P. (1984) Regulation of locus coeruleus neurons and splanchnic, sympathetic nerves by cardiovascular afferents. Bruin Res., 290: 281-7. Elam, M., Svensson, T.H. and ThorCn, P. (1986) Locus coeruleus neurons and sympathetic nerves: Activation by cutaneous sensory afferents. Bruin Res., 366: 254-261. Engberg, G. (1989) Nicotine induced excitation of locus coeruleus neurons is mediated via release of excitatory amino acids. Life Sci., 44: 1535-40. Ennis, M. and Aston-Jones, G. (1986) A potent excitatory input to the nucleus locus coeruleus from the ventrolateral medulla. Neurosci. Lett., 71: 299-305. Ennis, M. and Aston-Jones, G. (1987) Two physiologically distinct populations of neurons in the ventrolateral medulla innervate the locus coeruleus. Brain Res., 425: 275-282. Ennis, M. and Aston-Jones, G. (1988) Activation of locus coeruleus from nucleus paragigantocellularis: A new excitatory amino acid pathway in brain. J. Neurosci., 8: 36443657. Ennis, M. and Aston-Jones, G. (1989a) GABA-mediated inhibition of locus coeruleus from the dorsomedial rostral medulla. J. Neurosci., 9: 2973-2981. Ennis, M. and Aston-Jones, G. (1989b) Potent inhibitory input to locus coeruleus from the nucleus prepositus hypoglossi. Brain Res. Bull., 22: 793-803. Ennis, M., Behbehani, M., Van Bockstaele,.E.J., Aston-Jones, G. and Shipley, M.T. (1989) Influence of periaqueductal grey on rat locus coeruleus neurons. Soc. Neurosci. Abstr., 15: 1013. Ennis, M., Behbehani, M.M., Van Bockstaele, E.J., M.T., S. and Aston-Jones, G. (1991) Projections from the periaque-

ductal gray to nucleus locus coeruleus and the pericoerulear region: Anatomic and physiologic studies. J. Comp. Neurol., 306: 480-494. Fallon, J.H. and Leslie, F.M. (1986) Distribution of dynorphin and enkephalin peptides in the rat brain. J. Comp. Neurol., 249: 293-336. Finley, J.C.W., Maderdrut, J.L. and Petrusz, P. (1981) The immunocytochemical localization of enkephalin in the central nervous system of the rat. J. Comp. Neurol., 198: 541-565. Foote, S.L., Bloom, F.E. and Aston-Jones, G. (1983) Nucleus locus coeruleus: New evidence of anatomical and physiological specificity. Physiol. Reu., 63: 844-914. Fu, L., Shipley, M., Aston-Jones, G., Chiang, C. and Pieribone, V. (1988) Architecture of the pericoeruliar region in the rat. Soc. Neurosci. Abstr., 14: 404. Fu, L., Shipley, M.T. and Aston-Jones, G. (1989) Dendrites of rat locus coeruleus are asymmetrically distributed: Immunocytochemical LM and EM studies. SOC. Neurosci. Abstr., 15: 1013. Gallyas, F., Gorcs, T. and Merchenthaler, I. (1982) High-grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J. Histochem. Cytochem., 30: 183-184. Granata, A.R., Ruggiero, D.A., Park, D.H., Joh, T.H. and Reis, D.J. (1985) Brain stem area with C1 epinephrine neurons mediates baroreflex vasodepressor responses. Am. J. Physiol., 248: H247-H567. Grenhoff, J., Tung, C.S. and Svensson, T.H. (1988) The excitatory amino acid antagonist kynurenate induces pacemaker-like firing of dopamine neurons in rat ventral tegmental area in Lii'o. Acta Physiol. Scand., 134: 567-8. Grzanna, R. and Molliver, M.E. (1980) The locus coeruleus in the rat: An immunohistochemical delineation. Neuroscience, 5: 21-40. Grzanna, R., Molliver, M.E. and Coyle, J.T. (1980) Visualization of central noradrenergic neurons in thick sections by the unlabeled antibody method: A transmitter-specific Golgi image. Proc. Nutl. Acud. Sci. USA, 75: 2502-2506. Guthrie, J. and Basbaum, A.I. (1984) Colocalization of immunoreactive proenkephalin and prodynorphin products in medullary neurons of the rat. Neuropeptzdes, 4: 437-445. Guyenet, P.G. and Les Brown, D. (1986) Nucleus paragigantocellularis lateralis and lumbar sympathetic discharge in the rat. Am. J. Physiol., 250: R1981-R1094. Guyenet, P.G. and Young, B.S. (1987) Projections of nucleus paragigantocellularis lateralis to locus coeruleus and other structures in rat. Bruin Res., 406: 171-84. Hajos, M. and Engberg, G. (1988) Role of primary sensory neurons in the central effects of nicotine. Psychophurmucology (Berlin), 94: 468-70. Haselton, J.R. and Guyenet, P.G. (1987) PNMT neurons of the C1 cell group with projections to both locus coeruleus and spinal cord. Soc. Neurosci. Abstr., 13: 809. Hokfelt, T., Fuxe, K., Goldstein, M. and Johansson, 0. (1974) Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain. Bruin Res., 66: 235251.

73 Hokfelt, T., Ljungdahl, A,, Terenius, L., Elde, R. and Nisson, G. (1977) Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: Enkephalin and substance P. Proc. Natl. Acad. Sci. USA, 74: 30813085. Hokfelt, T., Ljungdahl, A., Steinbusch, H., Verhofstad, A., Nilsson, G., Brodin, E., Pernow, B. and Goldstein, M. (1978) lmmunohistochemical evidence of substance P-like immunoreactivity in some 5-hydroxytryptamine-containing neurons in the rat central nervous system. Neuroscience, 3: 517-538. Hokfelt, T., Terenius, L., Kuypers, H.G.J.M. and Dann, 0. (1979) Evidence for enkephalin immunoreactive neurons in the medulla oblongata projecting to the spinal cord. Neurosci. Lett., 14: 55-60. Hokfelt, T., Johansson, 0. and Goldstein, M. (1985) Central catecholamine neurons as revealed by immunohistochemistry with special reference to adrenaline neurons. In A. Bjoklund and T. Hokfelt (Eds.), Handbook of Chemical Neuroanatomy. Volume 2: Classical Transmitters in the CNS, Elsevier Science, New York, pp. 157-276. Hunt, S.P. and Lovick, T.A. (1982) The distribution of serotonin, met-enkephalin and beta-lipotropin-like immunoreactivity in neuronal perikarya of the cat brainstem. Neurosci. Lett., 30(2): 139-45. Jennes, L., Stumpf, W.E. and Kalivas, P.W. (1982) Neurotensin: Topographical distribution in rat brain by immunohistochemistry. J. Comp. Neurol., 210: 21 1-24. Johansson, O., Hokfelt, T. and Elde, R.P. (1984) Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience, 13: 265-339. Kalia, M., Fuxe, K. and Goldstein, M. (1985) Rat medulla oblongata. 111. adrenergic (C1 and C2) neurons, nerve fibers and presumptive terminal processes. J. Comp. Neurol.. 233: 333-349. Khachaturian, H., Lewis, M. and Watson, S.J. (1983) Enkephalin systems in the diencephalon and brainstem of the rat. J. Comp. Neurol., 220: 310-320. Kimura, H., McMeer, P.L. and Peng, J.-H. (1984) Choline acetyltransferase-containing neurons in the rat brain. In A. Bjorklund, T. Hokfelt and M.J. Kuhar (Ed.), Classical Transmitters and Transmitter Receptors in the CNS. Part II, Elsevier Science Publishers B.V., Amsterdam, pp. 51-65. Ljungdahl, A,, Hokfelt, T. and Nilsson, G. (1978) Distribution of substance P-like immunoreactivity in the central nervous system of the rat I. Cell bodies and nerve terminals. Neuroscience, 3: 63-80. Lynch, D.R., Strittmatter, S.M. and Snyder, S.H. (1984) Enkephalin convertase localization by [3H]guanidinoethylmercaptosuccinic acid autoradiography: Selective association with enkephalin-containing neurons. Proc. Natl. Acad. Sci. USA, 81: 6543-7. Mantyh, P.W. and Hunt, S.P. (1984) Neuropeptides are present in projection neurones at all levels in visceral and taste pathways: From periphery to sensory cortex. Brain Res., 299: 297-311. Martin, J.L., Dietl, M.M., Hof, P.R., Palacios, J.M. and Magistretti, P.J. (1987) Autoradiographic mapping of

[mono[l25I]iodo-TyrlO, MetOl7~asoactiveintestinal peptide binding sites in the rat brain. Neuroscience, 23: 539-65. McCrea, R.A., Baker, R. and Delgado-Garcia, J. (1979) Afferent and efferent organization of the prepositus hypoglossi nucleus. Prog. Brain Res., 50: 653-665. McMahon, S.B. and Wall, P.D. (1985) Electrophysiological mapping of the brainstem projections of spinal cord lamina I cells in the rat. Brain Res., 333: 19-26. Merchenthaler, I. (1984) Corticotropin releasing factor (CRF)-like immunoreactivity in the rat central nervous system. extrahypothalamic distribution. Peptides, 5: 53-69. Merchenthaler, I., Vigh, S., Petrusz, P. and Schally, A.V. (1982) Immunocytochemical localization of corticotropinreleasing factor (CRF) in the rat brain. Am. J. Anat., 165: 386-396. Miller, R.J. and Pickel, V.M. (1980a) The distribution and functions of the enkephalins. J. Histochem. Cytochem., 28: 903-17. Miller, R.J. and Pickel, V.M. (1980b) Immunohistochemical distribution of enkephalins: Interactions with catecholamine-containing systems. Adu. Biochem. Psychopharrnacol., 25: 349-59. Minagawa, H., Shiosaka, S., Inagaki, S., Sakanaka, M., Takatsuki, K., Ishimoto, I., Senba, E., Kawai, Y., Hard, Y., Matsuzaki, T. and Tohyama, M. (1983) Ontogeny of neurotensin-containing neuron system of the rat: Immunohistochemical analysis-11. Lower brainstem. Neuroscience, 8 (3): 467-486. Morgane, P.J. and Jacobs, M.S. (1979) Raphe projections to the locus coeruleus in the rat. Brain Res. Bull., 4: 519-34. Morrison, S.F., Milner, T.A. and Reis, D.J. (1988) Reticulospinal vasomotor neurons of the rat rostra1 ventrolateral medulla: Relationship to sympathetic nerve activity and the C1 adrenergic cell group. J. Neurosci., 8: 1286-1301. Mugnaini, E. and Oertel, W.H. (1985) An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In A. Bjorklund and T. Hokfelt (Eds.), Handbook of Chemical Neuroanatomy, Elsevier Science Publishers B.V., Amsterdam, pp. 436-608. Nomura, H., Shiosaka, S., Inagaki, S., Ishimoto, I., Senba, E., Sakanaka, M., Tekatsuki, K., Matsuzaki, T., Kubota, Y., Saito, H., Takase, S., Kogure, K. and Tohyama, M. (1982) Distribution of substance P-like immunoreactivity in the lower brainstem of the human fetus: An immunohistochemical study. Brain Res., 252 (2): 315-25. Olshcowka, J.A., O’Donohue, T.L., Mueller, G.P. and Jacobowitz, D.M. (1982) The distribution of corticotropin releasing factor-like immunoreactive neurons in rat brain. Peptides, 3: 995-1015. Ottersen, O.P. and Storm-Mathisen, J. (1984a) Glutamateand GABA-containing neurons in the mouse and rat brain, as demonstrated with a new immunocytochemical technique. J. Comp. Neurol., 229: 374-392. Ottersen, O.P. and Storm-Mathisen, J. (1984b) Neurons containing or accumulating transmitter amino acids. In A. Bjorklund, T. Hokfelt and M.J. Kuhar (Ed.), Classical Transmitters and Transmitter Receptors in the CNS. Part II, Elsevier Science Publishers B.V., Amsterdam, pp. 141-224.

74 Papadopoulos, G.C., Karamanlidis, A.N., Antonopoulos. J. and Dinopoulos, A. (1986) Neurotensin-like immunoreactive neurons in the hedgehog (Erinaceus europaeus) and the sheep (Ovis aries) central nervous system. J. Comp. Neurol., 244: 193-203. Pieribone, V.A. and Aston-Jones, G. (1991) Adrenergic innervation of the rat nucleus locus coeruleus arises from the C1 and C3 cell groups in the rostral medulla: An anatomic study combining retrograde transport and immunofluorescence . Neuroscience, 4 1 5 25 -542. Pieribone, V.A., Aston-Jones, G. and Bohn, M.C. (1988) Adrenergic and non-adrenergic neurons of the C1 and C3 areas project to locus coeruleus: A fluorescent double labeling study. Neurosci. Lett., 85: 297-303. Pieribone, V., Van Bockstaele, E., Shipley, M. and AstonJones, G. (1989) Serotonergic innervation of rat locus coeruleus derives from non-raphe brain areas. Soc. Neurosci. Abstr., 15: 420. Pieribone, V.A., Shipley, M.T., Ennis, M. and Aston-Jones, G. (1990) Anatomic evidence for GABA-ergic afferents to the rat locus coeruleus in the dorsal medial medulla: A immunocytochemical and retrograde transport study. Soc. Neurosci. Abstr., 16: 300. Rasmussen, K. and Aghajanian, G.K. (1989) Withdrawal-induced activation of locus coeruleus neurons in opiate-dependent rats: Attenuation by lesions of the nucleus paragigantocellularis. Brain Res, 505: 346-50. Ross, C., Armstrong, D., Ruggiero, D., Pickel, V., Joh, T. and Reis, D. (1981) Adrenaline neurons in the rostral ventrolateral medulla innervate thoracic spinal cord: A combined immunocytochemical and retrograde transport demonstration. Neurosci. Lett., 25: 257-262. Ross, C.A., Ruggiero, D.A., Park, D.H., Joh, T.H., Sved, A.F., Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J. (1984) Tonic vasomotor control by the rostral ventrolateral medulla: Effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J. Neurosci., 4: 474-494. Ruggiero, D.A., Ross, C.A., Anwar. M., Park, D.H., Joh, T.H. and Reis, D.J. (1985) Distribution of neurons containing phenylethanolamine N-methyltransferase in medulla and hypothalamus of rat. J. Comp. Neurol., 239: 127-154. Ruggiero, D.A., Giuliano, R., Anwar, M., Stornetta, R. and Reis, D.J. (1990) Anatomical substrates of cholinergic-autonomic regulation in the rat. J. Comp. Neurol., 292: 1-53. Sakanaka, M., Shibasaki, T. and Lederis, K. (1987) Corticotropin releasing factor-like immunoreactivity in the rat brain as revealed by a modified cobalt-glucose oxidase-diaminobenzidine method. J. Comp. Neurol., 260: 256-298. Saper, C.B. (1982) Reciprocal parabrachial-cortical connections in the rat. Brain Res., 242: 33-40. Saper, C.B. (1987) Function of the locus coeruleus. TINS, 10: 343-344. Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.J. and Cuatrecasas, P. (1978) Immunohistochemical localization of enkephalin in rat brain and spinal cord. J. Comp. Neurol., 182: 17-37.

Sesack, S.R., Deutch, A.Y., Roth, R.H. and Bunney. B.S. (1989) Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: An anterograde tract-tracing study with phaseolus vulgaris leucoagglutinin. J. Comp. Neurol., 290: 213-242. Shiekhattar, R. and Aston-Jones, G. (1991) NMDA-receptormediated responses of brain noradrenergic neurones are suppressed by extracellular magnesium. Synapse. (in press). Shiekhattar, R., Chiang, C. and Aston-Jones, G. (1991) Regulation of locus coeruleus discharge by extracellular calcium ion concentration. Synapse, (in press). Shimizu, N. and Imamoto, K. (1970) Fine structure of the locus coeruleus in the rat. Arch. Histol. Jap., 31: 229-246. Shimizu, N., Ohnishi, S., Satoh, K. and Tohyama, M. (1978) Cellular organization of locus coeruleus in the rat as studied by Golgi method. Arch. Histol. Jap., 41: 103-1 12. Shipley, M., Pieribone, V., Aston-Jones, G. and Ennis, M. (1988) GABA-ergic innervation of the rat locus coeruleus. Soc. Neurosci. Ahstr., 14: 406. Standaert, D.G., Watson, S.J., Houghten, R.A. and Saper, C.B. (1986) Opioid peptide immunoreactivity in spinal and trigeminal dorsal horn neurons projecting to the parabrachial nucleus in the rat. J. Neurosci., 6: 1220-6. Steinbusch, H.W.M. (1984) Serotonin-immunoreactive neurons and their targets in the CNS. In A. Bjorklund, T. Hokfelt and M.J. Kuhar (Eds.), Classical Transmitters and Transmitter Receptors in the CNS. Part 11, Elsevier Science Publishers B.V., Amsterdam, pp. 68-1 18. Sun, M., Hackett, J.T. and Guyenet, P.G. (1988) Sympathoexcitatory neurons of rostral ventrolateral medulla exhibit pacemaker properties in the presence of a glutamate-receptor antagonist. Bruin Res., 438: 23-40. Sutin, E.L. and Jacobowitz, D.M. (1988) Immunocytochemical localization of peptides and other neurochemicals in the rat laterodorsal tegmental nucleus and adjacent area. J. Comp. Neurol., 270: 243-70. Svensson, T.H., Engberg, G., Tung, C.S. and Grenhoff, J. (1989) Pacemaker-like firing of noradrenergic locus coeruleus neurons in r i m induced by the excitatory amino acid antagonist kynurenate in the rat. Acta fhysiol. Scand., 135: 421-2. Swanson, L.W. (1976) The locus coeruleus: A cytoarchitectonic, golgi and immunohistochemical study in the albino rat. Brain Res., 110: 39-56. Swanson, L.W., Sawchenko, P.E., Rivier, J. and Vale, W.W. (1983) Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: An immunohistochemical study. Neuroendocrinology, 36: 165186. Thor, K.B. and Helke, C.J. (1988) Catecholamine-synthesizing neuronal projections to the nucleus tractus solitarii of the rat. J. Comp. Neurol., 268: 264-280. Triepel, J., Mader, J., Weindl, A,, Heinrich, D., Forssmann, W.G. and Metz, J. (1984) Distribution of NT-IR perikarya in the brain of the guinea pig with special reference to cardiovascular centers in the medulla oblongata. Histochemistry, 81: 509-516. Triepel, J., Weindl, A,, Kiemle, I., Mader, J., Volz, H.P.,

75 Reinecke, M. and Forssmann, W.G. (1985) Substance Pimmunoreactive neurons in the brainstem of the cat related to cardiovascular centers. Cell Tissue Res., 241: 31 -41. Tucker, D.C., Saper, C.B., Ruggiero, D.A. and Reis, D.J. (1987) Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections to the hypothalamus and spinal cord. J . Comp. Neurol., 259: 59 1-603. Tung, C.S., Ugedo, L., Grenhoff, J., Engberg, G. and Svensson, T.H. (1989) Peripheral induction of burst firing in locus coeruleus neurons by nicotine mediated via excitatory amino acids. Synapse, 4: 313-8. Uhl, G.R., Goodman, R.R., Kuhar, M.J., Childers, S.R. and Snyder, S.H. (1979) Immunohistochemical mapping of enkephalin containing cell bodies. fibers and nerve terminals in the brainstem of the rat. Bruin Res., 166: 75-94. Valentino, R.J., Van Bockstaele, E.J. and Aston-Jones, G. (1990) Corticotropin-releasing factor-immunoreactive (CRF-IR) neurons are localized in nuclei which project to the locus coeruleus (LC). Soc. Neurosci. Abstr., 16: 519. Van Bockstaele, E.J., Pieribone, V.A. and Aston-Jones, G. (1989a) Diverse afferents converge on the nucleus paragigantocellularis in the rat ventrolateral medulla: Retrograde and anterograde tracing studies. J. Cornp. Neurol., 290: 561-584.

Van Bockstaele, E., Pieribone, V., Aston-Jones, G. and Shipley, M. (1989b) Multiple projection pathways from the ventrolateral medulla to locus coeruleus in rat. Soc. Neurosci. Abstr., 15: 1013. Van Bockstaele, E.J., Aston-Jones. G., Ennis, M. Shipley, M.T., and Pieribone, V.A. (1991) Subregions of the periaqueductal gray topographically innervate the rostra1 ventral medulla in the rat. J. Cornp. Neurol., 309: 1-23. Vincent, S.R., McIntosh, C.H., Buchan, A.M. and Brown, J.C. (1985) Central somatostatin systems revealed with monoclonal antibodies. J. Cornp. Neurol., 238(2): 169-186. Wallace, D.M., Magnuson, D.J. and Gray, T.S. (1989) The amygdala-brainstem pathway: Selective innervation of dopaminergic, noradrenergic and adrenergic cells in the rat. Neurosci. Lett., 97: 252-8. Wang, Y. and Aghajanian, G.K. (1989) Excitation of locus coeruleus neurons by vasoactive intestinal peptide: Evidence for a G-protein-mediated inward current. Bruin Res., 500: 107-118. Watson, S.J., Richard, C., Ciaranello, R.D. and Barchas, J.D. (1980) Interaction of opiate peptide and noradrenaline systems: Light microscopic studies. Peptides, 1: 23-30.

Afferent regulation of locus coeruleus neurons: anatomy, physiology and pharmacology.

Tract-tracing and electrophysiology studies have revealed that major inputs to the nucleus locus coeruleus (LC) are found in two structures, the nucle...
2MB Sizes 0 Downloads 0 Views